methods used in a research study

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Research methods--quantitative, qualitative, and more: overview.

Quantitative Research
Qualitative Research
Data Science Methods (Machine Learning, AI, Big Data)
Text Mining and Computational Text Analysis
Evidence Synthesis/Systematic Reviews
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About Research Methods

This guide provides an overview of research methods, how to choose and use them, and supports and resources at UC Berkeley.

As Patten and Newhart note in the book Understanding Research Methods , "Research methods are the building blocks of the scientific enterprise. They are the "how" for building systematic knowledge. The accumulation of knowledge through research is by its nature a collective endeavor. Each well-designed study provides evidence that may support, amend, refute, or deepen the understanding of existing knowledge...Decisions are important throughout the practice of research and are designed to help researchers collect evidence that includes the full spectrum of the phenomenon under study, to maintain logical rules, and to mitigate or account for possible sources of bias. In many ways, learning research methods is learning how to see and make these decisions."

The choice of methods varies by discipline, by the kind of phenomenon being studied and the data being used to study it, by the technology available, and more. This guide is an introduction, but if you don't see what you need here, always contact your subject librarian, and/or take a look to see if there's a library research guide that will answer your question.

Suggestions for changes and additions to this guide are welcome!

START HERE: SAGE Research Methods

Without question, the most comprehensive resource available from the library is SAGE Research Methods. HERE IS THE ONLINE GUIDE to this one-stop shopping collection, and some helpful links are below:

SAGE Research Methods
Little Green Books (Quantitative Methods)
Little Blue Books (Qualitative Methods)
Dictionaries and Encyclopedias
Case studies of real research projects
Sample datasets for hands-on practice
Streaming video--see methods come to life
Methodspace- -a community for researchers
SAGE Research Methods Course Mapping

Library Data Services at UC Berkeley

Library Data Services Program and Digital Scholarship Services

The LDSP offers a variety of services and tools ! From this link, check out pages for each of the following topics: discovering data, managing data, collecting data, GIS data, text data mining, publishing data, digital scholarship, open science, and the Research Data Management Program.

Be sure also to check out the visual guide to where to seek assistance on campus with any research question you may have!

Library GIS Services

Other Data Services at Berkeley

D-Lab Supports Berkeley faculty, staff, and graduate students with research in data intensive social science, including a wide range of training and workshop offerings Dryad Dryad is a simple self-service tool for researchers to use in publishing their datasets. It provides tools for the effective publication of and access to research data. Geospatial Innovation Facility (GIF) Provides leadership and training across a broad array of integrated mapping technologies on campu Research Data Management A UC Berkeley guide and consulting service for research data management issues

General Research Methods Resources

Here are some general resources for assistance:

Assistance from ICPSR (must create an account to access): Getting Help with Data , and Resources for Students
Wiley Stats Ref for background information on statistics topics
Survey Documentation and Analysis (SDA) . Program for easy web-based analysis of survey data.

Consultants

D-Lab/Data Science Discovery Consultants Request help with your research project from peer consultants.
Research data (RDM) consulting Meet with RDM consultants before designing the data security, storage, and sharing aspects of your qualitative project.
Statistics Department Consulting Services A service in which advanced graduate students, under faculty supervision, are available to consult during specified hours in the Fall and Spring semesters.

Related Resourcex

IRB / CPHS Qualitative research projects with human subjects often require that you go through an ethics review.
OURS (Office of Undergraduate Research and Scholarships) OURS supports undergraduates who want to embark on research projects and assistantships. In particular, check out their "Getting Started in Research" workshops
Sponsored Projects Sponsored projects works with researchers applying for major external grants.
Next: Quantitative Research >>
Last Updated: Apr 25, 2024 11:09 AM
URL: https://guides.lib.berkeley.edu/researchmethods

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Knowledge Base
Methodology

Research Methods | Definition, Types, Examples

Research methods are specific procedures for collecting and analysing data. Developing your research methods is an integral part of your research design . When planning your methods, there are two key decisions you will make.

First, decide how you will collect data . Your methods depend on what type of data you need to answer your research question :

Qualitative vs quantitative : Will your data take the form of words or numbers?
Primary vs secondary : Will you collect original data yourself, or will you use data that have already been collected by someone else?
Descriptive vs experimental : Will you take measurements of something as it is, or will you perform an experiment?

Second, decide how you will analyse the data .

For quantitative data, you can use statistical analysis methods to test relationships between variables.
For qualitative data, you can use methods such as thematic analysis to interpret patterns and meanings in the data.

Methods for collecting data, examples of data collection methods, methods for analysing data, examples of data analysis methods, frequently asked questions about methodology.

Data are the information that you collect for the purposes of answering your research question . The type of data you need depends on the aims of your research.

Qualitative vs quantitative data

Your choice of qualitative or quantitative data collection depends on the type of knowledge you want to develop.

For questions about ideas, experiences and meanings, or to study something that can’t be described numerically, collect qualitative data .

If you want to develop a more mechanistic understanding of a topic, or your research involves hypothesis testing , collect quantitative data .

You can also take a mixed methods approach, where you use both qualitative and quantitative research methods.

Primary vs secondary data

Primary data are any original information that you collect for the purposes of answering your research question (e.g. through surveys , observations and experiments ). Secondary data are information that has already been collected by other researchers (e.g. in a government census or previous scientific studies).

If you are exploring a novel research question, you’ll probably need to collect primary data. But if you want to synthesise existing knowledge, analyse historical trends, or identify patterns on a large scale, secondary data might be a better choice.

Descriptive vs experimental data

In descriptive research , you collect data about your study subject without intervening. The validity of your research will depend on your sampling method .

In experimental research , you systematically intervene in a process and measure the outcome. The validity of your research will depend on your experimental design .

To conduct an experiment, you need to be able to vary your independent variable , precisely measure your dependent variable, and control for confounding variables . If it’s practically and ethically possible, this method is the best choice for answering questions about cause and effect.

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Your data analysis methods will depend on the type of data you collect and how you prepare them for analysis.

Data can often be analysed both quantitatively and qualitatively. For example, survey responses could be analysed qualitatively by studying the meanings of responses or quantitatively by studying the frequencies of responses.

Qualitative analysis methods

Qualitative analysis is used to understand words, ideas, and experiences. You can use it to interpret data that were collected:

From open-ended survey and interview questions, literature reviews, case studies, and other sources that use text rather than numbers.
Using non-probability sampling methods .

Qualitative analysis tends to be quite flexible and relies on the researcher’s judgement, so you have to reflect carefully on your choices and assumptions.

Quantitative analysis methods

Quantitative analysis uses numbers and statistics to understand frequencies, averages and correlations (in descriptive studies) or cause-and-effect relationships (in experiments).

You can use quantitative analysis to interpret data that were collected either:

During an experiment.
Using probability sampling methods .

Because the data are collected and analysed in a statistically valid way, the results of quantitative analysis can be easily standardised and shared among researchers.

Quantitative research deals with numbers and statistics, while qualitative research deals with words and meanings.

Quantitative methods allow you to test a hypothesis by systematically collecting and analysing data, while qualitative methods allow you to explore ideas and experiences in depth.

In mixed methods research , you use both qualitative and quantitative data collection and analysis methods to answer your research question .

A sample is a subset of individuals from a larger population. Sampling means selecting the group that you will actually collect data from in your research.

For example, if you are researching the opinions of students in your university, you could survey a sample of 100 students.

Statistical sampling allows you to test a hypothesis about the characteristics of a population. There are various sampling methods you can use to ensure that your sample is representative of the population as a whole.

The research methods you use depend on the type of data you need to answer your research question .

If you want to measure something or test a hypothesis , use quantitative methods . If you want to explore ideas, thoughts, and meanings, use qualitative methods .
If you want to analyse a large amount of readily available data, use secondary data. If you want data specific to your purposes with control over how they are generated, collect primary data.
If you want to establish cause-and-effect relationships between variables , use experimental methods. If you want to understand the characteristics of a research subject, use descriptive methods.

Methodology refers to the overarching strategy and rationale of your research project . It involves studying the methods used in your field and the theories or principles behind them, in order to develop an approach that matches your objectives.

Methods are the specific tools and procedures you use to collect and analyse data (e.g. experiments, surveys , and statistical tests ).

In shorter scientific papers, where the aim is to report the findings of a specific study, you might simply describe what you did in a methods section .

In a longer or more complex research project, such as a thesis or dissertation , you will probably include a methodology section , where you explain your approach to answering the research questions and cite relevant sources to support your choice of methods.

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Organizing Your Social Sciences Research Paper

6. The Methodology
Purpose of Guide
Design Flaws to Avoid
Independent and Dependent Variables
Glossary of Research Terms
Reading Research Effectively
Narrowing a Topic Idea
Broadening a Topic Idea
Extending the Timeliness of a Topic Idea
Academic Writing Style
Applying Critical Thinking
Choosing a Title
Making an Outline
Paragraph Development
Research Process Video Series
Executive Summary
The C.A.R.S. Model
Background Information
The Research Problem/Question
Theoretical Framework
Citation Tracking
Content Alert Services
Evaluating Sources
Primary Sources
Secondary Sources
Tiertiary Sources
Scholarly vs. Popular Publications
Qualitative Methods
Quantitative Methods
Insiderness
Using Non-Textual Elements
Limitations of the Study
Common Grammar Mistakes
Writing Concisely
Avoiding Plagiarism
Footnotes or Endnotes?
Further Readings
Generative AI and Writing
USC Libraries Tutorials and Other Guides
Bibliography

The methods section describes actions taken to investigate a research problem and the rationale for the application of specific procedures or techniques used to identify, select, process, and analyze information applied to understanding the problem, thereby, allowing the reader to critically evaluate a study’s overall validity and reliability. The methodology section of a research paper answers two main questions: How was the data collected or generated? And, how was it analyzed? The writing should be direct and precise and always written in the past tense.

Kallet, Richard H. "How to Write the Methods Section of a Research Paper." Respiratory Care 49 (October 2004): 1229-1232.

Importance of a Good Methodology Section

You must explain how you obtained and analyzed your results for the following reasons:

Readers need to know how the data was obtained because the method you chose affects the results and, by extension, how you interpreted their significance in the discussion section of your paper.
Methodology is crucial for any branch of scholarship because an unreliable method produces unreliable results and, as a consequence, undermines the value of your analysis of the findings.
In most cases, there are a variety of different methods you can choose to investigate a research problem. The methodology section of your paper should clearly articulate the reasons why you have chosen a particular procedure or technique.
The reader wants to know that the data was collected or generated in a way that is consistent with accepted practice in the field of study. For example, if you are using a multiple choice questionnaire, readers need to know that it offered your respondents a reasonable range of answers to choose from.
The method must be appropriate to fulfilling the overall aims of the study. For example, you need to ensure that you have a large enough sample size to be able to generalize and make recommendations based upon the findings.
The methodology should discuss the problems that were anticipated and the steps you took to prevent them from occurring. For any problems that do arise, you must describe the ways in which they were minimized or why these problems do not impact in any meaningful way your interpretation of the findings.
In the social and behavioral sciences, it is important to always provide sufficient information to allow other researchers to adopt or replicate your methodology. This information is particularly important when a new method has been developed or an innovative use of an existing method is utilized.

Bem, Daryl J. Writing the Empirical Journal Article. Psychology Writing Center. University of Washington; Denscombe, Martyn. The Good Research Guide: For Small-Scale Social Research Projects . 5th edition. Buckingham, UK: Open University Press, 2014; Lunenburg, Frederick C. Writing a Successful Thesis or Dissertation: Tips and Strategies for Students in the Social and Behavioral Sciences . Thousand Oaks, CA: Corwin Press, 2008.

Structure and Writing Style

I. Groups of Research Methods

There are two main groups of research methods in the social sciences:

The e mpirical-analytical group approaches the study of social sciences in a similar manner that researchers study the natural sciences . This type of research focuses on objective knowledge, research questions that can be answered yes or no, and operational definitions of variables to be measured. The empirical-analytical group employs deductive reasoning that uses existing theory as a foundation for formulating hypotheses that need to be tested. This approach is focused on explanation.
The i nterpretative group of methods is focused on understanding phenomenon in a comprehensive, holistic way . Interpretive methods focus on analytically disclosing the meaning-making practices of human subjects [the why, how, or by what means people do what they do], while showing how those practices arrange so that it can be used to generate observable outcomes. Interpretive methods allow you to recognize your connection to the phenomena under investigation. However, the interpretative group requires careful examination of variables because it focuses more on subjective knowledge.

II. Content

The introduction to your methodology section should begin by restating the research problem and underlying assumptions underpinning your study. This is followed by situating the methods you used to gather, analyze, and process information within the overall “tradition” of your field of study and within the particular research design you have chosen to study the problem. If the method you choose lies outside of the tradition of your field [i.e., your review of the literature demonstrates that the method is not commonly used], provide a justification for how your choice of methods specifically addresses the research problem in ways that have not been utilized in prior studies.

The remainder of your methodology section should describe the following:

Decisions made in selecting the data you have analyzed or, in the case of qualitative research, the subjects and research setting you have examined,
Tools and methods used to identify and collect information, and how you identified relevant variables,
The ways in which you processed the data and the procedures you used to analyze that data, and
The specific research tools or strategies that you utilized to study the underlying hypothesis and research questions.

In addition, an effectively written methodology section should:

Introduce the overall methodological approach for investigating your research problem . Is your study qualitative or quantitative or a combination of both (mixed method)? Are you going to take a special approach, such as action research, or a more neutral stance?
Indicate how the approach fits the overall research design . Your methods for gathering data should have a clear connection to your research problem. In other words, make sure that your methods will actually address the problem. One of the most common deficiencies found in research papers is that the proposed methodology is not suitable to achieving the stated objective of your paper.
Describe the specific methods of data collection you are going to use , such as, surveys, interviews, questionnaires, observation, archival research. If you are analyzing existing data, such as a data set or archival documents, describe how it was originally created or gathered and by whom. Also be sure to explain how older data is still relevant to investigating the current research problem.
Explain how you intend to analyze your results . Will you use statistical analysis? Will you use specific theoretical perspectives to help you analyze a text or explain observed behaviors? Describe how you plan to obtain an accurate assessment of relationships, patterns, trends, distributions, and possible contradictions found in the data.
Provide background and a rationale for methodologies that are unfamiliar for your readers . Very often in the social sciences, research problems and the methods for investigating them require more explanation/rationale than widely accepted rules governing the natural and physical sciences. Be clear and concise in your explanation.
Provide a justification for subject selection and sampling procedure . For instance, if you propose to conduct interviews, how do you intend to select the sample population? If you are analyzing texts, which texts have you chosen, and why? If you are using statistics, why is this set of data being used? If other data sources exist, explain why the data you chose is most appropriate to addressing the research problem.
Provide a justification for case study selection . A common method of analyzing research problems in the social sciences is to analyze specific cases. These can be a person, place, event, phenomenon, or other type of subject of analysis that are either examined as a singular topic of in-depth investigation or multiple topics of investigation studied for the purpose of comparing or contrasting findings. In either method, you should explain why a case or cases were chosen and how they specifically relate to the research problem.
Describe potential limitations . Are there any practical limitations that could affect your data collection? How will you attempt to control for potential confounding variables and errors? If your methodology may lead to problems you can anticipate, state this openly and show why pursuing this methodology outweighs the risk of these problems cropping up.

NOTE : Once you have written all of the elements of the methods section, subsequent revisions should focus on how to present those elements as clearly and as logically as possibly. The description of how you prepared to study the research problem, how you gathered the data, and the protocol for analyzing the data should be organized chronologically. For clarity, when a large amount of detail must be presented, information should be presented in sub-sections according to topic. If necessary, consider using appendices for raw data.

ANOTHER NOTE : If you are conducting a qualitative analysis of a research problem , the methodology section generally requires a more elaborate description of the methods used as well as an explanation of the processes applied to gathering and analyzing of data than is generally required for studies using quantitative methods. Because you are the primary instrument for generating the data [e.g., through interviews or observations], the process for collecting that data has a significantly greater impact on producing the findings. Therefore, qualitative research requires a more detailed description of the methods used.

YET ANOTHER NOTE : If your study involves interviews, observations, or other qualitative techniques involving human subjects , you may be required to obtain approval from the university's Office for the Protection of Research Subjects before beginning your research. This is not a common procedure for most undergraduate level student research assignments. However, i f your professor states you need approval, you must include a statement in your methods section that you received official endorsement and adequate informed consent from the office and that there was a clear assessment and minimization of risks to participants and to the university. This statement informs the reader that your study was conducted in an ethical and responsible manner. In some cases, the approval notice is included as an appendix to your paper.

III. Problems to Avoid

Irrelevant Detail The methodology section of your paper should be thorough but concise. Do not provide any background information that does not directly help the reader understand why a particular method was chosen, how the data was gathered or obtained, and how the data was analyzed in relation to the research problem [note: analyzed, not interpreted! Save how you interpreted the findings for the discussion section]. With this in mind, the page length of your methods section will generally be less than any other section of your paper except the conclusion.

Unnecessary Explanation of Basic Procedures Remember that you are not writing a how-to guide about a particular method. You should make the assumption that readers possess a basic understanding of how to investigate the research problem on their own and, therefore, you do not have to go into great detail about specific methodological procedures. The focus should be on how you applied a method , not on the mechanics of doing a method. An exception to this rule is if you select an unconventional methodological approach; if this is the case, be sure to explain why this approach was chosen and how it enhances the overall process of discovery.

Problem Blindness It is almost a given that you will encounter problems when collecting or generating your data, or, gaps will exist in existing data or archival materials. Do not ignore these problems or pretend they did not occur. Often, documenting how you overcame obstacles can form an interesting part of the methodology. It demonstrates to the reader that you can provide a cogent rationale for the decisions you made to minimize the impact of any problems that arose.

Literature Review Just as the literature review section of your paper provides an overview of sources you have examined while researching a particular topic, the methodology section should cite any sources that informed your choice and application of a particular method [i.e., the choice of a survey should include any citations to the works you used to help construct the survey].

It’s More than Sources of Information! A description of a research study's method should not be confused with a description of the sources of information. Such a list of sources is useful in and of itself, especially if it is accompanied by an explanation about the selection and use of the sources. The description of the project's methodology complements a list of sources in that it sets forth the organization and interpretation of information emanating from those sources.

Azevedo, L.F. et al. "How to Write a Scientific Paper: Writing the Methods Section." Revista Portuguesa de Pneumologia 17 (2011): 232-238; Blair Lorrie. “Choosing a Methodology.” In Writing a Graduate Thesis or Dissertation , Teaching Writing Series. (Rotterdam: Sense Publishers 2016), pp. 49-72; Butin, Dan W. The Education Dissertation A Guide for Practitioner Scholars . Thousand Oaks, CA: Corwin, 2010; Carter, Susan. Structuring Your Research Thesis . New York: Palgrave Macmillan, 2012; Kallet, Richard H. “How to Write the Methods Section of a Research Paper.” Respiratory Care 49 (October 2004):1229-1232; Lunenburg, Frederick C. Writing a Successful Thesis or Dissertation: Tips and Strategies for Students in the Social and Behavioral Sciences . Thousand Oaks, CA: Corwin Press, 2008. Methods Section. The Writer’s Handbook. Writing Center. University of Wisconsin, Madison; Rudestam, Kjell Erik and Rae R. Newton. “The Method Chapter: Describing Your Research Plan.” In Surviving Your Dissertation: A Comprehensive Guide to Content and Process . (Thousand Oaks, Sage Publications, 2015), pp. 87-115; What is Interpretive Research. Institute of Public and International Affairs, University of Utah; Writing the Experimental Report: Methods, Results, and Discussion. The Writing Lab and The OWL. Purdue University; Methods and Materials. The Structure, Format, Content, and Style of a Journal-Style Scientific Paper. Department of Biology. Bates College.

Writing Tip

Statistical Designs and Tests? Do Not Fear Them!

Don't avoid using a quantitative approach to analyzing your research problem just because you fear the idea of applying statistical designs and tests. A qualitative approach, such as conducting interviews or content analysis of archival texts, can yield exciting new insights about a research problem, but it should not be undertaken simply because you have a disdain for running a simple regression. A well designed quantitative research study can often be accomplished in very clear and direct ways, whereas, a similar study of a qualitative nature usually requires considerable time to analyze large volumes of data and a tremendous burden to create new paths for analysis where previously no path associated with your research problem had existed.

To locate data and statistics, GO HERE .

Another Writing Tip

Knowing the Relationship Between Theories and Methods

There can be multiple meaning associated with the term "theories" and the term "methods" in social sciences research. A helpful way to delineate between them is to understand "theories" as representing different ways of characterizing the social world when you research it and "methods" as representing different ways of generating and analyzing data about that social world. Framed in this way, all empirical social sciences research involves theories and methods, whether they are stated explicitly or not. However, while theories and methods are often related, it is important that, as a researcher, you deliberately separate them in order to avoid your theories playing a disproportionate role in shaping what outcomes your chosen methods produce.

Introspectively engage in an ongoing dialectic between the application of theories and methods to help enable you to use the outcomes from your methods to interrogate and develop new theories, or ways of framing conceptually the research problem. This is how scholarship grows and branches out into new intellectual territory.

Reynolds, R. Larry. Ways of Knowing. Alternative Microeconomics . Part 1, Chapter 3. Boise State University; The Theory-Method Relationship. S-Cool Revision. United Kingdom.

Yet Another Writing Tip

Methods and the Methodology

Do not confuse the terms "methods" and "methodology." As Schneider notes, a method refers to the technical steps taken to do research . Descriptions of methods usually include defining and stating why you have chosen specific techniques to investigate a research problem, followed by an outline of the procedures you used to systematically select, gather, and process the data [remember to always save the interpretation of data for the discussion section of your paper].

The methodology refers to a discussion of the underlying reasoning why particular methods were used . This discussion includes describing the theoretical concepts that inform the choice of methods to be applied, placing the choice of methods within the more general nature of academic work, and reviewing its relevance to examining the research problem. The methodology section also includes a thorough review of the methods other scholars have used to study the topic.

Bryman, Alan. "Of Methods and Methodology." Qualitative Research in Organizations and Management: An International Journal 3 (2008): 159-168; Schneider, Florian. “What's in a Methodology: The Difference between Method, Methodology, and Theory…and How to Get the Balance Right?” PoliticsEastAsia.com. Chinese Department, University of Leiden, Netherlands.

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Last Updated: May 9, 2024 11:05 AM
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Research Methodologies

What are research designs?
What are research methodologies?

What are research methods?

Quantitative research methods, qualitative research methods, mixed method approach, selecting the best research method.

Additional Sources

Research methods are different from research methodologies because they are the ways in which you will collect the data for your research project. The best method for your project largely depends on your topic, the type of data you will need, and the people or items from which you will be collecting data. The following boxes below contain a list of quantitative, qualitative, and mixed research methods.

Closed-ended questionnaires/survey: These types of questionnaires or surveys are like "multiple choice" tests, where participants must select from a list of premade answers. According to the content of the question, they must select the one that they agree with the most. This approach is the simplest form of quantitative research because the data is easy to combine and quantify.
Structured interviews: These are a common research method in market research because the data can be quantified. They are strictly designed for little "wiggle room" in the interview process so that the data will not be skewed. You can conduct structured interviews in-person, online, or over the phone (Dawson, 2019).

Constructing Questionnaires

When constructing your questions for a survey or questionnaire, there are things you can do to ensure that your questions are accurate and easy to understand (Dawson, 2019):

Keep the questions brief and simple.
Eliminate any potential bias from your questions. Make sure that they do not word things in a way that favor one perspective over another.
If your topic is very sensitive, you may want to ask indirect questions rather than direct ones. This prevents participants from being intimidated and becoming unwilling to share their true responses.
If you are using a closed-ended question, try to offer every possible answer that a participant could give to that question.
Do not ask questions that assume something of the participant. The question "How often do you exercise?" assumes that the participant exercises (when they may not), so you would want to include a question that asks if they exercise at all before asking them how often.
Try and keep the questionnaire as short as possible. The longer a questionnaire takes, the more likely the participant will not complete it or get too tired to put truthful answers.
Promise confidentiality to your participants at the beginning of the questionnaire.

Quantitative Research Measures

When you are considering a quantitative approach to your research, you need to identify why types of measures you will use in your study. This will determine what type of numbers you will be using to collect your data. There are four levels of measurement:

Nominal: These are numbers where the order of the numbers do not matter. They aim to identify separate information. One example is collecting zip codes from research participants. The order of the numbers does not matter, but the series of numbers in each zip code indicate different information (Adamson and Prion, 2013).
Ordinal: Also known as rankings because the order of these numbers matter. This is when items are given a specific rank according to specific criteria. A common example of ordinal measurements include ranking-based questionnaires, where participants are asked to rank items from least favorite to most favorite. Another common example is a pain scale, where a patient is asked to rank their pain on a scale from 1 to 10 (Adamson and Prion, 2013).
Interval: This is when the data are ordered and the distance between the numbers matters to the researcher (Adamson and Prion, 2013). The distance between each number is the same. An example of interval data is test grades.
Ratio: This is when the data are ordered and have a consistent distance between numbers, but has a "zero point." This means that there could be a measurement of zero of whatever you are measuring in your study (Adamson and Prion, 2013). An example of ratio data is measuring the height of something because the "zero point" remains constant in all measurements. The height of something could also be zero.

Focus Groups

This is when a select group of people gather to talk about a particular topic. They can also be called discussion groups or group interviews (Dawson, 2019). They are usually lead by a moderator to help guide the discussion and ask certain questions. It is critical that a moderator allows everyone in the group to get a chance to speak so that no one dominates the discussion. The data that are gathered from focus groups tend to be thoughts, opinions, and perspectives about an issue.

Advantages of Focus Groups

Only requires one meeting to get different types of responses.
Less researcher bias due to participants being able to speak openly.
Helps participants overcome insecurities or fears about a topic.
The researcher can also consider the impact of participant interaction.

Disadvantages of Focus Groups

Participants may feel uncomfortable to speak in front of an audience, especially if the topic is sensitive or controversial.
Since participation is voluntary, not every participant may contribute equally to the discussion.
Participants may impact what others say or think.
A researcher may feel intimidated by running a focus group on their own.
A researcher may need extra funds/resources to provide a safe space to host the focus group.
Because the data is collective, it may be difficult to determine a participant's individual thoughts about the research topic.

Observation

There are two ways to conduct research observations:

Direct Observation: The researcher observes a participant in an environment. The researcher often takes notes or uses technology to gather data, such as a voice recorder or video camera. The researcher does not interact or interfere with the participants. This approach is often used in psychology and health studies (Dawson, 2019).
Participant Observation: The researcher interacts directly with the participants to get a better understanding of the research topic. This is a common research method when trying to understand another culture or community. It is important to decide if you will conduct a covert (participants do not know they are part of the research) or overt (participants know the researcher is observing them) observation because it can be unethical in some situations (Dawson, 2019).

Open-Ended Questionnaires

These types of questionnaires are the opposite of "multiple choice" questionnaires because the answer boxes are left open for the participant to complete. This means that participants can write short or extended answers to the questions. Upon gathering the responses, researchers will often "quantify" the data by organizing the responses into different categories. This can be time consuming because the researcher needs to read all responses carefully.

Semi-structured Interviews

This is the most common type of interview where researchers aim to get specific information so they can compare it to other interview data. This requires asking the same questions for each interview, but keeping their responses flexible. This means including follow-up questions if a subject answers a certain way. Interview schedules are commonly used to aid the interviewers, which list topics or questions that will be discussed at each interview (Dawson, 2019).

Theoretical Analysis

Often used for nonhuman research, theoretical analysis is a qualitative approach where the researcher applies a theoretical framework to analyze something about their topic. A theoretical framework gives the researcher a specific "lens" to view the topic and think about it critically. it also serves as context to guide the entire study. This is a popular research method for analyzing works of literature, films, and other forms of media. You can implement more than one theoretical framework with this method, as many theories complement one another.

Common theoretical frameworks for qualitative research are (Grant and Osanloo, 2014):

Behavioral theory
Change theory
Cognitive theory
Content analysis
Cross-sectional analysis
Developmental theory
Feminist theory
Gender theory
Marxist theory
Queer theory
Systems theory
Transformational theory

Unstructured Interviews

These are in-depth interviews where the researcher tries to understand an interviewee's perspective on a situation or issue. They are sometimes called life history interviews. It is important not to bombard the interviewee with too many questions so they can freely disclose their thoughts (Dawson, 2019).

Open-ended and closed-ended questionnaires: This approach means implementing elements of both questionnaire types into your data collection. Participants may answer some questions with premade answers and write their own answers to other questions. The advantage to this method is that you benefit from both types of data collection to get a broader understanding of you participants. However, you must think carefully about how you will analyze this data to arrive at a conclusion.

Other mixed method approaches that incorporate quantitative and qualitative research methods depend heavily on the research topic. It is strongly recommended that you collaborate with your academic advisor before finalizing a mixed method approach.

How do you determine which research method would be best for your proposal? This heavily depends on your research objective. According to Dawson (2019), there are several questions to ask yourself when determining the best research method for your project:

Are you good with numbers and mathematics?
Would you be interested in conducting interviews with human subjects?
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What skills or experiences do you have that might help you with your research? Do you have any experiences from past research projects that can help with this one?
How much time do you have to complete the research? Some methods take longer to collect data than others.
What is your budget? Do you have adequate funding to conduct the research in the method you want?
How much data do you need? Some research topics need only a small amount of data while others may need significantly larger amounts.
What is the purpose of your research? This can provide a good indicator as to what research method will be most appropriate.
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Research Process

Choosing the Right Research Methodology: A Guide for Researchers

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Table of Contents

Choosing an optimal research methodology is crucial for the success of any research project. The methodology you select will determine the type of data you collect, how you collect it, and how you analyse it. Understanding the different types of research methods available along with their strengths and weaknesses, is thus imperative to make an informed decision.

Understanding different research methods:

There are several research methods available depending on the type of study you are conducting, i.e., whether it is laboratory-based, clinical, epidemiological, or survey based . Some common methodologies include qualitative research, quantitative research, experimental research, survey-based research, and action research. Each method can be opted for and modified, depending on the type of research hypotheses and objectives.

Qualitative vs quantitative research:

When deciding on a research methodology, one of the key factors to consider is whether your research will be qualitative or quantitative. Qualitative research is used to understand people’s experiences, concepts, thoughts, or behaviours . Quantitative research, on the contrary, deals with numbers, graphs, and charts, and is used to test or confirm hypotheses, assumptions, and theories.

Qualitative research methodology:

Qualitative research is often used to examine issues that are not well understood, and to gather additional insights on these topics. Qualitative research methods include open-ended survey questions, observations of behaviours described through words, and reviews of literature that has explored similar theories and ideas. These methods are used to understand how language is used in real-world situations, identify common themes or overarching ideas, and describe and interpret various texts. Data analysis for qualitative research typically includes discourse analysis, thematic analysis, and textual analysis.

Quantitative research methodology:

The goal of quantitative research is to test hypotheses, confirm assumptions and theories, and determine cause-and-effect relationships. Quantitative research methods include experiments, close-ended survey questions, and countable and numbered observations. Data analysis for quantitative research relies heavily on statistical methods.

Analysing qualitative vs quantitative data:

The methods used for data analysis also differ for qualitative and quantitative research. As mentioned earlier, quantitative data is generally analysed using statistical methods and does not leave much room for speculation. It is more structured and follows a predetermined plan. In quantitative research, the researcher starts with a hypothesis and uses statistical methods to test it. Contrarily, methods used for qualitative data analysis can identify patterns and themes within the data, rather than provide statistical measures of the data. It is an iterative process, where the researcher goes back and forth trying to gauge the larger implications of the data through different perspectives and revising the analysis if required.

When to use qualitative vs quantitative research:

The choice between qualitative and quantitative research will depend on the gap that the research project aims to address, and specific objectives of the study. If the goal is to establish facts about a subject or topic, quantitative research is an appropriate choice. However, if the goal is to understand people’s experiences or perspectives, qualitative research may be more suitable.

Conclusion:

In conclusion, an understanding of the different research methods available, their applicability, advantages, and disadvantages is essential for making an informed decision on the best methodology for your project. If you need any additional guidance on which research methodology to opt for, you can head over to Elsevier Author Services (EAS). EAS experts will guide you throughout the process and help you choose the perfect methodology for your research goals.

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Published by Nicolas at March 21st, 2024 , Revised On March 12, 2024

The Ultimate Guide To Research Methodology

Research methodology is a crucial aspect of any investigative process, serving as the blueprint for the entire research journey. If you are stuck in the methodology section of your research paper , then this blog will guide you on what is a research methodology, its types and how to successfully conduct one.

Table of Contents

What Is Research Methodology?

Research methodology can be defined as the systematic framework that guides researchers in designing, conducting, and analyzing their investigations. It encompasses a structured set of processes, techniques, and tools employed to gather and interpret data, ensuring the reliability and validity of the research findings.

Research methodology is not confined to a singular approach; rather, it encapsulates a diverse range of methods tailored to the specific requirements of the research objectives.

Here is why Research methodology is important in academic and professional settings.

Facilitating Rigorous Inquiry

Research methodology forms the backbone of rigorous inquiry. It provides a structured approach that aids researchers in formulating precise thesis statements , selecting appropriate methodologies, and executing systematic investigations. This, in turn, enhances the quality and credibility of the research outcomes.

Ensuring Reproducibility And Reliability

In both academic and professional contexts, the ability to reproduce research outcomes is paramount. A well-defined research methodology establishes clear procedures, making it possible for others to replicate the study. This not only validates the findings but also contributes to the cumulative nature of knowledge.

Guiding Decision-Making Processes

In professional settings, decisions often hinge on reliable data and insights. Research methodology equips professionals with the tools to gather pertinent information, analyze it rigorously, and derive meaningful conclusions.

This informed decision-making is instrumental in achieving organizational goals and staying ahead in competitive environments.

Contributing To Academic Excellence

For academic researchers, adherence to robust research methodology is a hallmark of excellence. Institutions value research that adheres to high standards of methodology, fostering a culture of academic rigour and intellectual integrity. Furthermore, it prepares students with critical skills applicable beyond academia.

Enhancing Problem-Solving Abilities

Research methodology instills a problem-solving mindset by encouraging researchers to approach challenges systematically. It equips individuals with the skills to dissect complex issues, formulate hypotheses , and devise effective strategies for investigation.

Understanding Research Methodology

In the pursuit of knowledge and discovery, understanding the fundamentals of research methodology is paramount.

Basics Of Research

Research, in its essence, is a systematic and organized process of inquiry aimed at expanding our understanding of a particular subject or phenomenon. It involves the exploration of existing knowledge, the formulation of hypotheses, and the collection and analysis of data to draw meaningful conclusions.

Research is a dynamic and iterative process that contributes to the continuous evolution of knowledge in various disciplines.

Types of Research

Research takes on various forms, each tailored to the nature of the inquiry. Broadly classified, research can be categorized into two main types:

Quantitative Research: This type involves the collection and analysis of numerical data to identify patterns, relationships, and statistical significance. It is particularly useful for testing hypotheses and making predictions.
Qualitative Research: Qualitative research focuses on understanding the depth and details of a phenomenon through non-numerical data. It often involves methods such as interviews, focus groups, and content analysis, providing rich insights into complex issues.

Components Of Research Methodology

To conduct effective research, one must go through the different components of research methodology. These components form the scaffolding that supports the entire research process, ensuring its coherence and validity.

Research Design

Research design serves as the blueprint for the entire research project. It outlines the overall structure and strategy for conducting the study. The three primary types of research design are:

Exploratory Research: Aimed at gaining insights and familiarity with the topic, often used in the early stages of research.
Descriptive Research: Involves portraying an accurate profile of a situation or phenomenon, answering the ‘what,’ ‘who,’ ‘where,’ and ‘when’ questions.
Explanatory Research: Seeks to identify the causes and effects of a phenomenon, explaining the ‘why’ and ‘how.’

Data Collection Methods

Choosing the right data collection methods is crucial for obtaining reliable and relevant information. Common methods include:

Surveys and Questionnaires: Employed to gather information from a large number of respondents through standardized questions.
Interviews: In-depth conversations with participants, offering qualitative insights.
Observation: Systematic watching and recording of behaviour, events, or processes in their natural setting.

Data Analysis Techniques

Once data is collected, analysis becomes imperative to derive meaningful conclusions. Different methodologies exist for quantitative and qualitative data:

Quantitative Data Analysis: Involves statistical techniques such as descriptive statistics, inferential statistics, and regression analysis to interpret numerical data.
Qualitative Data Analysis: Methods like content analysis, thematic analysis, and grounded theory are employed to extract patterns, themes, and meanings from non-numerical data.

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Choosing a Research Method

Selecting an appropriate research method is a critical decision in the research process. It determines the approach, tools, and techniques that will be used to answer the research questions.

Quantitative Research Methods

Quantitative research involves the collection and analysis of numerical data, providing a structured and objective approach to understanding and explaining phenomena.

Experimental Research

Experimental research involves manipulating variables to observe the effect on another variable under controlled conditions. It aims to establish cause-and-effect relationships.

Key Characteristics:

Controlled Environment: Experiments are conducted in a controlled setting to minimize external influences.
Random Assignment: Participants are randomly assigned to different experimental conditions.
Quantitative Data: Data collected is numerical, allowing for statistical analysis.

Applications: Commonly used in scientific studies and psychology to test hypotheses and identify causal relationships.

Survey Research

Survey research gathers information from a sample of individuals through standardized questionnaires or interviews. It aims to collect data on opinions, attitudes, and behaviours.

Structured Instruments: Surveys use structured instruments, such as questionnaires, to collect data.
Large Sample Size: Surveys often target a large and diverse group of participants.
Quantitative Data Analysis: Responses are quantified for statistical analysis.

Applications: Widely employed in social sciences, marketing, and public opinion research to understand trends and preferences.

Descriptive Research

Descriptive research seeks to portray an accurate profile of a situation or phenomenon. It focuses on answering the ‘what,’ ‘who,’ ‘where,’ and ‘when’ questions.

Observation and Data Collection: This involves observing and documenting without manipulating variables.
Objective Description: Aim to provide an unbiased and factual account of the subject.
Quantitative or Qualitative Data: T his can include both types of data, depending on the research focus.

Applications: Useful in situations where researchers want to understand and describe a phenomenon without altering it, common in social sciences and education.

Qualitative Research Methods

Qualitative research emphasizes exploring and understanding the depth and complexity of phenomena through non-numerical data.

A case study is an in-depth exploration of a particular person, group, event, or situation. It involves detailed, context-rich analysis.

Rich Data Collection: Uses various data sources, such as interviews, observations, and documents.
Contextual Understanding: Aims to understand the context and unique characteristics of the case.
Holistic Approach: Examines the case in its entirety.

Applications: Common in social sciences, psychology, and business to investigate complex and specific instances.

Ethnography

Ethnography involves immersing the researcher in the culture or community being studied to gain a deep understanding of their behaviours, beliefs, and practices.

Participant Observation: Researchers actively participate in the community or setting.
Holistic Perspective: Focuses on the interconnectedness of cultural elements.
Qualitative Data: In-depth narratives and descriptions are central to ethnographic studies.

Applications: Widely used in anthropology, sociology, and cultural studies to explore and document cultural practices.

Grounded Theory

Grounded theory aims to develop theories grounded in the data itself. It involves systematic data collection and analysis to construct theories from the ground up.

Constant Comparison: Data is continually compared and analyzed during the research process.
Inductive Reasoning: Theories emerge from the data rather than being imposed on it.
Iterative Process: The research design evolves as the study progresses.

Applications: Commonly applied in sociology, nursing, and management studies to generate theories from empirical data.

Research design is the structural framework that outlines the systematic process and plan for conducting a study. It serves as the blueprint, guiding researchers on how to collect, analyze, and interpret data.

Exploratory, Descriptive, And Explanatory Designs

Exploratory design.

Exploratory research design is employed when a researcher aims to explore a relatively unknown subject or gain insights into a complex phenomenon.

Flexibility: Allows for flexibility in data collection and analysis.
Open-Ended Questions: Uses open-ended questions to gather a broad range of information.
Preliminary Nature: Often used in the initial stages of research to formulate hypotheses.

Applications: Valuable in the early stages of investigation, especially when the researcher seeks a deeper understanding of a subject before formalizing research questions.

Descriptive Design

Descriptive research design focuses on portraying an accurate profile of a situation, group, or phenomenon.

Structured Data Collection: Involves systematic and structured data collection methods.
Objective Presentation: Aims to provide an unbiased and factual account of the subject.
Quantitative or Qualitative Data: Can incorporate both types of data, depending on the research objectives.

Applications: Widely used in social sciences, marketing, and educational research to provide detailed and objective descriptions.

Explanatory Design

Explanatory research design aims to identify the causes and effects of a phenomenon, explaining the ‘why’ and ‘how’ behind observed relationships.

Causal Relationships: Seeks to establish causal relationships between variables.
Controlled Variables : Often involves controlling certain variables to isolate causal factors.
Quantitative Analysis: Primarily relies on quantitative data analysis techniques.

Applications: Commonly employed in scientific studies and social sciences to delve into the underlying reasons behind observed patterns.

Cross-Sectional Vs. Longitudinal Designs

Cross-sectional design.

Cross-sectional designs collect data from participants at a single point in time.

Snapshot View: Provides a snapshot of a population at a specific moment.
Efficiency: More efficient in terms of time and resources.
Limited Temporal Insights: Offers limited insights into changes over time.

Applications: Suitable for studying characteristics or behaviours that are stable or not expected to change rapidly.

Longitudinal Design

Longitudinal designs involve the collection of data from the same participants over an extended period.

Temporal Sequence: Allows for the examination of changes over time.
Causality Assessment: Facilitates the assessment of cause-and-effect relationships.
Resource-Intensive: Requires more time and resources compared to cross-sectional designs.

Applications: Ideal for studying developmental processes, trends, or the impact of interventions over time.

Experimental Vs Non-experimental Designs

Experimental design.

Experimental designs involve manipulating variables under controlled conditions to observe the effect on another variable.

Causality Inference: Enables the inference of cause-and-effect relationships.
Quantitative Data: Primarily involves the collection and analysis of numerical data.

Applications: Commonly used in scientific studies, psychology, and medical research to establish causal relationships.

Non-Experimental Design

Non-experimental designs observe and describe phenomena without manipulating variables.

Natural Settings: Data is often collected in natural settings without intervention.
Descriptive or Correlational: Focuses on describing relationships or correlations between variables.
Quantitative or Qualitative Data: This can involve either type of data, depending on the research approach.

Applications: Suitable for studying complex phenomena in real-world settings where manipulation may not be ethical or feasible.

Effective data collection is fundamental to the success of any research endeavour.

Designing Effective Surveys

Objective Design:

Clearly define the research objectives to guide the survey design.
Craft questions that align with the study’s goals and avoid ambiguity.

Structured Format:

Use a structured format with standardized questions for consistency.
Include a mix of closed-ended and open-ended questions for detailed insights.

Pilot Testing:

Conduct pilot tests to identify and rectify potential issues with survey design.
Ensure clarity, relevance, and appropriateness of questions.

Sampling Strategy:

Develop a robust sampling strategy to ensure a representative participant group.
Consider random sampling or stratified sampling based on the research goals.

Conducting Interviews

Establishing Rapport:

Build rapport with participants to create a comfortable and open environment.
Clearly communicate the purpose of the interview and the value of participants’ input.

Open-Ended Questions:

Frame open-ended questions to encourage detailed responses.
Allow participants to express their thoughts and perspectives freely.

Active Listening:

Practice active listening to understand areas and gather rich data.
Avoid interrupting and maintain a non-judgmental stance during the interview.

Ethical Considerations:

Obtain informed consent and assure participants of confidentiality.
Be transparent about the study’s purpose and potential implications.

Observation

1. participant observation.

Immersive Participation:

Actively immerse yourself in the setting or group being observed.
Develop a deep understanding of behaviours, interactions, and context.

Field Notes:

Maintain detailed and reflective field notes during observations.
Document observed patterns, unexpected events, and participant reactions.

Ethical Awareness:

Be conscious of ethical considerations, ensuring respect for participants.
Balance the role of observer and participant to minimize bias.

2. Non-participant Observation

Objective Observation:

Maintain a more detached and objective stance during non-participant observation.
Focus on recording behaviours, events, and patterns without direct involvement.

Data Reliability:

Enhance the reliability of data by reducing observer bias.
Develop clear observation protocols and guidelines.

Contextual Understanding:

Strive for a thorough understanding of the observed context.
Consider combining non-participant observation with other methods for triangulation.

Archival Research

1. using existing data.

Identifying Relevant Archives:

Locate and access archives relevant to the research topic.
Collaborate with institutions or repositories holding valuable data.

Data Verification:

Verify the accuracy and reliability of archived data.
Cross-reference with other sources to ensure data integrity.

Ethical Use:

Adhere to ethical guidelines when using existing data.
Respect copyright and intellectual property rights.

2. Challenges and Considerations

Incomplete or Inaccurate Archives:

Address the possibility of incomplete or inaccurate archival records.
Acknowledge limitations and uncertainties in the data.

Temporal Bias:

Recognize potential temporal biases in archived data.
Consider the historical context and changes that may impact interpretation.

Access Limitations:

Address potential limitations in accessing certain archives.
Seek alternative sources or collaborate with institutions to overcome barriers.

Common Challenges in Research Methodology

Conducting research is a complex and dynamic process, often accompanied by a myriad of challenges. Addressing these challenges is crucial to ensure the reliability and validity of research findings.

Sampling Issues

Sampling bias:.

The presence of sampling bias can lead to an unrepresentative sample, affecting the generalizability of findings.
Employ random sampling methods and ensure the inclusion of diverse participants to reduce bias.

Sample Size Determination:

Determining an appropriate sample size is a delicate balance. Too small a sample may lack statistical power, while an excessively large sample may strain resources.
Conduct a power analysis to determine the optimal sample size based on the research objectives and expected effect size.

Data Quality And Validity

Measurement error:.

Inaccuracies in measurement tools or data collection methods can introduce measurement errors, impacting the validity of results.
Pilot test instruments, calibrate equipment, and use standardized measures to enhance the reliability of data.

Construct Validity:

Ensuring that the chosen measures accurately capture the intended constructs is a persistent challenge.
Use established measurement instruments and employ multiple measures to assess the same construct for triangulation.

Time And Resource Constraints

Timeline pressures:.

Limited timeframes can compromise the depth and thoroughness of the research process.
Develop a realistic timeline, prioritize tasks, and communicate expectations with stakeholders to manage time constraints effectively.

Resource Availability:

Inadequate resources, whether financial or human, can impede the execution of research activities.
Seek external funding, collaborate with other researchers, and explore alternative methods that require fewer resources.

Managing Bias in Research

Selection bias:.

Selecting participants in a way that systematically skews the sample can introduce selection bias.
Employ randomization techniques, use stratified sampling, and transparently report participant recruitment methods.

Confirmation Bias:

Researchers may unintentionally favour information that confirms their preconceived beliefs or hypotheses.
Adopt a systematic and open-minded approach, use blinded study designs, and engage in peer review to mitigate confirmation bias.

Tips On How To Write A Research Methodology

Conducting successful research relies not only on the application of sound methodologies but also on strategic planning and effective collaboration. Here are some tips to enhance the success of your research methodology:

Tip 1. Clear Research Objectives

Well-defined research objectives guide the entire research process. Clearly articulate the purpose of your study, outlining specific research questions or hypotheses.

Tip 2. Comprehensive Literature Review

A thorough literature review provides a foundation for understanding existing knowledge and identifying gaps. Invest time in reviewing relevant literature to inform your research design and methodology.

Tip 3. Detailed Research Plan

A detailed plan serves as a roadmap, ensuring all aspects of the research are systematically addressed. Develop a detailed research plan outlining timelines, milestones, and tasks.

Tip 4. Ethical Considerations

Ethical practices are fundamental to maintaining the integrity of research. Address ethical considerations early, obtain necessary approvals, and ensure participant rights are safeguarded.

Tip 5. Stay Updated On Methodologies

Research methodologies evolve, and staying updated is essential for employing the most effective techniques. Engage in continuous learning by attending workshops, conferences, and reading recent publications.

Tip 6. Adaptability In Methods

Unforeseen challenges may arise during research, necessitating adaptability in methods. Be flexible and willing to modify your approach when needed, ensuring the integrity of the study.

Tip 7. Iterative Approach

Research is often an iterative process, and refining methods based on ongoing findings enhance the study’s robustness. Regularly review and refine your research design and methods as the study progresses.

Frequently Asked Questions

What is the research methodology.

Research methodology is the systematic process of planning, executing, and evaluating scientific investigation. It encompasses the techniques, tools, and procedures used to collect, analyze, and interpret data, ensuring the reliability and validity of research findings.

What are the methodologies in research?

Research methodologies include qualitative and quantitative approaches. Qualitative methods involve in-depth exploration of non-numerical data, while quantitative methods use statistical analysis to examine numerical data. Mixed methods combine both approaches for a comprehensive understanding of research questions.

How to write research methodology?

To write a research methodology, clearly outline the study’s design, data collection, and analysis procedures. Specify research tools, participants, and sampling methods. Justify choices and discuss limitations. Ensure clarity, coherence, and alignment with research objectives for a robust methodology section.

How to write the methodology section of a research paper?

In the methodology section of a research paper, describe the study’s design, data collection, and analysis methods. Detail procedures, tools, participants, and sampling. Justify choices, address ethical considerations, and explain how the methodology aligns with research objectives, ensuring clarity and rigour.

What is mixed research methodology?

Mixed research methodology combines both qualitative and quantitative research approaches within a single study. This approach aims to enhance the details and depth of research findings by providing a more comprehensive understanding of the research problem or question.

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Home » Research Methodology – Types, Examples and writing Guide

Research Methodology – Types, Examples and writing Guide

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Research Methodology

Definition:

Research Methodology refers to the systematic and scientific approach used to conduct research, investigate problems, and gather data and information for a specific purpose. It involves the techniques and procedures used to identify, collect , analyze , and interpret data to answer research questions or solve research problems . Moreover, They are philosophical and theoretical frameworks that guide the research process.

Structure of Research Methodology

Research methodology formats can vary depending on the specific requirements of the research project, but the following is a basic example of a structure for a research methodology section:

I. Introduction

Provide an overview of the research problem and the need for a research methodology section
Outline the main research questions and objectives

II. Research Design

Explain the research design chosen and why it is appropriate for the research question(s) and objectives
Discuss any alternative research designs considered and why they were not chosen
Describe the research setting and participants (if applicable)

III. Data Collection Methods

Describe the methods used to collect data (e.g., surveys, interviews, observations)
Explain how the data collection methods were chosen and why they are appropriate for the research question(s) and objectives
Detail any procedures or instruments used for data collection

IV. Data Analysis Methods

Describe the methods used to analyze the data (e.g., statistical analysis, content analysis )
Explain how the data analysis methods were chosen and why they are appropriate for the research question(s) and objectives
Detail any procedures or software used for data analysis

V. Ethical Considerations

Discuss any ethical issues that may arise from the research and how they were addressed
Explain how informed consent was obtained (if applicable)
Detail any measures taken to ensure confidentiality and anonymity

VI. Limitations

Identify any potential limitations of the research methodology and how they may impact the results and conclusions

VII. Conclusion

Summarize the key aspects of the research methodology section
Explain how the research methodology addresses the research question(s) and objectives

Research Methodology Types

Types of Research Methodology are as follows:

Quantitative Research Methodology

This is a research methodology that involves the collection and analysis of numerical data using statistical methods. This type of research is often used to study cause-and-effect relationships and to make predictions.

Qualitative Research Methodology

This is a research methodology that involves the collection and analysis of non-numerical data such as words, images, and observations. This type of research is often used to explore complex phenomena, to gain an in-depth understanding of a particular topic, and to generate hypotheses.

Mixed-Methods Research Methodology

This is a research methodology that combines elements of both quantitative and qualitative research. This approach can be particularly useful for studies that aim to explore complex phenomena and to provide a more comprehensive understanding of a particular topic.

Case Study Research Methodology

This is a research methodology that involves in-depth examination of a single case or a small number of cases. Case studies are often used in psychology, sociology, and anthropology to gain a detailed understanding of a particular individual or group.

Action Research Methodology

This is a research methodology that involves a collaborative process between researchers and practitioners to identify and solve real-world problems. Action research is often used in education, healthcare, and social work.

Experimental Research Methodology

This is a research methodology that involves the manipulation of one or more independent variables to observe their effects on a dependent variable. Experimental research is often used to study cause-and-effect relationships and to make predictions.

Survey Research Methodology

This is a research methodology that involves the collection of data from a sample of individuals using questionnaires or interviews. Survey research is often used to study attitudes, opinions, and behaviors.

Grounded Theory Research Methodology

This is a research methodology that involves the development of theories based on the data collected during the research process. Grounded theory is often used in sociology and anthropology to generate theories about social phenomena.

Research Methodology Example

An Example of Research Methodology could be the following:

Research Methodology for Investigating the Effectiveness of Cognitive Behavioral Therapy in Reducing Symptoms of Depression in Adults

Introduction:

The aim of this research is to investigate the effectiveness of cognitive-behavioral therapy (CBT) in reducing symptoms of depression in adults. To achieve this objective, a randomized controlled trial (RCT) will be conducted using a mixed-methods approach.

Research Design:

The study will follow a pre-test and post-test design with two groups: an experimental group receiving CBT and a control group receiving no intervention. The study will also include a qualitative component, in which semi-structured interviews will be conducted with a subset of participants to explore their experiences of receiving CBT.

Participants:

Participants will be recruited from community mental health clinics in the local area. The sample will consist of 100 adults aged 18-65 years old who meet the diagnostic criteria for major depressive disorder. Participants will be randomly assigned to either the experimental group or the control group.

Intervention :

The experimental group will receive 12 weekly sessions of CBT, each lasting 60 minutes. The intervention will be delivered by licensed mental health professionals who have been trained in CBT. The control group will receive no intervention during the study period.

Data Collection:

Quantitative data will be collected through the use of standardized measures such as the Beck Depression Inventory-II (BDI-II) and the Generalized Anxiety Disorder-7 (GAD-7). Data will be collected at baseline, immediately after the intervention, and at a 3-month follow-up. Qualitative data will be collected through semi-structured interviews with a subset of participants from the experimental group. The interviews will be conducted at the end of the intervention period, and will explore participants’ experiences of receiving CBT.

Data Analysis:

Quantitative data will be analyzed using descriptive statistics, t-tests, and mixed-model analyses of variance (ANOVA) to assess the effectiveness of the intervention. Qualitative data will be analyzed using thematic analysis to identify common themes and patterns in participants’ experiences of receiving CBT.

Ethical Considerations:

This study will comply with ethical guidelines for research involving human subjects. Participants will provide informed consent before participating in the study, and their privacy and confidentiality will be protected throughout the study. Any adverse events or reactions will be reported and managed appropriately.

Data Management:

All data collected will be kept confidential and stored securely using password-protected databases. Identifying information will be removed from qualitative data transcripts to ensure participants’ anonymity.

Limitations:

One potential limitation of this study is that it only focuses on one type of psychotherapy, CBT, and may not generalize to other types of therapy or interventions. Another limitation is that the study will only include participants from community mental health clinics, which may not be representative of the general population.

Conclusion:

This research aims to investigate the effectiveness of CBT in reducing symptoms of depression in adults. By using a randomized controlled trial and a mixed-methods approach, the study will provide valuable insights into the mechanisms underlying the relationship between CBT and depression. The results of this study will have important implications for the development of effective treatments for depression in clinical settings.

How to Write Research Methodology

Writing a research methodology involves explaining the methods and techniques you used to conduct research, collect data, and analyze results. It’s an essential section of any research paper or thesis, as it helps readers understand the validity and reliability of your findings. Here are the steps to write a research methodology:

Start by explaining your research question: Begin the methodology section by restating your research question and explaining why it’s important. This helps readers understand the purpose of your research and the rationale behind your methods.
Describe your research design: Explain the overall approach you used to conduct research. This could be a qualitative or quantitative research design, experimental or non-experimental, case study or survey, etc. Discuss the advantages and limitations of the chosen design.
Discuss your sample: Describe the participants or subjects you included in your study. Include details such as their demographics, sampling method, sample size, and any exclusion criteria used.
Describe your data collection methods : Explain how you collected data from your participants. This could include surveys, interviews, observations, questionnaires, or experiments. Include details on how you obtained informed consent, how you administered the tools, and how you minimized the risk of bias.
Explain your data analysis techniques: Describe the methods you used to analyze the data you collected. This could include statistical analysis, content analysis, thematic analysis, or discourse analysis. Explain how you dealt with missing data, outliers, and any other issues that arose during the analysis.
Discuss the validity and reliability of your research : Explain how you ensured the validity and reliability of your study. This could include measures such as triangulation, member checking, peer review, or inter-coder reliability.
Acknowledge any limitations of your research: Discuss any limitations of your study, including any potential threats to validity or generalizability. This helps readers understand the scope of your findings and how they might apply to other contexts.
Provide a summary: End the methodology section by summarizing the methods and techniques you used to conduct your research. This provides a clear overview of your research methodology and helps readers understand the process you followed to arrive at your findings.

When to Write Research Methodology

Research methodology is typically written after the research proposal has been approved and before the actual research is conducted. It should be written prior to data collection and analysis, as it provides a clear roadmap for the research project.

The research methodology is an important section of any research paper or thesis, as it describes the methods and procedures that will be used to conduct the research. It should include details about the research design, data collection methods, data analysis techniques, and any ethical considerations.

The methodology should be written in a clear and concise manner, and it should be based on established research practices and standards. It is important to provide enough detail so that the reader can understand how the research was conducted and evaluate the validity of the results.

Applications of Research Methodology

Here are some of the applications of research methodology:

To identify the research problem: Research methodology is used to identify the research problem, which is the first step in conducting any research.
To design the research: Research methodology helps in designing the research by selecting the appropriate research method, research design, and sampling technique.
To collect data: Research methodology provides a systematic approach to collect data from primary and secondary sources.
To analyze data: Research methodology helps in analyzing the collected data using various statistical and non-statistical techniques.
To test hypotheses: Research methodology provides a framework for testing hypotheses and drawing conclusions based on the analysis of data.
To generalize findings: Research methodology helps in generalizing the findings of the research to the target population.
To develop theories : Research methodology is used to develop new theories and modify existing theories based on the findings of the research.
To evaluate programs and policies : Research methodology is used to evaluate the effectiveness of programs and policies by collecting data and analyzing it.
To improve decision-making: Research methodology helps in making informed decisions by providing reliable and valid data.

Purpose of Research Methodology

Research methodology serves several important purposes, including:

To guide the research process: Research methodology provides a systematic framework for conducting research. It helps researchers to plan their research, define their research questions, and select appropriate methods and techniques for collecting and analyzing data.
To ensure research quality: Research methodology helps researchers to ensure that their research is rigorous, reliable, and valid. It provides guidelines for minimizing bias and error in data collection and analysis, and for ensuring that research findings are accurate and trustworthy.
To replicate research: Research methodology provides a clear and detailed account of the research process, making it possible for other researchers to replicate the study and verify its findings.
To advance knowledge: Research methodology enables researchers to generate new knowledge and to contribute to the body of knowledge in their field. It provides a means for testing hypotheses, exploring new ideas, and discovering new insights.
To inform decision-making: Research methodology provides evidence-based information that can inform policy and decision-making in a variety of fields, including medicine, public health, education, and business.

Advantages of Research Methodology

Research methodology has several advantages that make it a valuable tool for conducting research in various fields. Here are some of the key advantages of research methodology:

Systematic and structured approach : Research methodology provides a systematic and structured approach to conducting research, which ensures that the research is conducted in a rigorous and comprehensive manner.
Objectivity : Research methodology aims to ensure objectivity in the research process, which means that the research findings are based on evidence and not influenced by personal bias or subjective opinions.
Replicability : Research methodology ensures that research can be replicated by other researchers, which is essential for validating research findings and ensuring their accuracy.
Reliability : Research methodology aims to ensure that the research findings are reliable, which means that they are consistent and can be depended upon.
Validity : Research methodology ensures that the research findings are valid, which means that they accurately reflect the research question or hypothesis being tested.
Efficiency : Research methodology provides a structured and efficient way of conducting research, which helps to save time and resources.
Flexibility : Research methodology allows researchers to choose the most appropriate research methods and techniques based on the research question, data availability, and other relevant factors.
Scope for innovation: Research methodology provides scope for innovation and creativity in designing research studies and developing new research techniques.

Research Methodology Vs Research Methods

About the author.

Muhammad Hassan

Researcher, Academic Writer, Web developer

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15 Types of Research Methods

types of research methods, explained below

Research methods refer to the strategies, tools, and techniques used to gather and analyze data in a structured way in order to answer a research question or investigate a hypothesis (Hammond & Wellington, 2020).

Generally, we place research methods into two categories: quantitative and qualitative. Each has its own strengths and weaknesses, which we can summarize as:

Quantitative research can achieve generalizability through scrupulous statistical analysis applied to large sample sizes.
Qualitative research achieves deep, detailed, and nuance accounts of specific case studies, which are not generalizable.

Some researchers, with the aim of making the most of both quantitative and qualitative research, employ mixed methods, whereby they will apply both types of research methods in the one study, such as by conducting a statistical survey alongside in-depth interviews to add context to the quantitative findings.

Below, I’ll outline 15 common research methods, and include pros, cons, and examples of each .

Types of Research Methods

Research methods can be broadly categorized into two types: quantitative and qualitative.

Quantitative methods involve systematic empirical investigation of observable phenomena via statistical, mathematical, or computational techniques, providing an in-depth understanding of a specific concept or phenomenon (Schweigert, 2021). The strengths of this approach include its ability to produce reliable results that can be generalized to a larger population, although it can lack depth and detail.
Qualitative methods encompass techniques that are designed to provide a deep understanding of a complex issue, often in a specific context, through collection of non-numerical data (Tracy, 2019). This approach often provides rich, detailed insights but can be time-consuming and its findings may not be generalizable.

These can be further broken down into a range of specific research methods and designs:

Combining the two methods above, mixed methods research mixes elements of both qualitative and quantitative research methods, providing a comprehensive understanding of the research problem . We can further break these down into:

Sequential Explanatory Design (QUAN→QUAL): This methodology involves conducting quantitative analysis first, then supplementing it with a qualitative study.
Sequential Exploratory Design (QUAL→QUAN): This methodology goes in the other direction, starting with qualitative analysis and ending with quantitative analysis.

Let’s explore some methods and designs from both quantitative and qualitative traditions, starting with qualitative research methods.

Qualitative Research Methods

Qualitative research methods allow for the exploration of phenomena in their natural settings, providing detailed, descriptive responses and insights into individuals’ experiences and perceptions (Howitt, 2019).

These methods are useful when a detailed understanding of a phenomenon is sought.

1. Ethnographic Research

Ethnographic research emerged out of anthropological research, where anthropologists would enter into a setting for a sustained period of time, getting to know a cultural group and taking detailed observations.

Ethnographers would sometimes even act as participants in the group or culture, which many scholars argue is a weakness because it is a step away from achieving objectivity (Stokes & Wall, 2017).

In fact, at its most extreme version, ethnographers even conduct research on themselves, in a fascinating methodology call autoethnography .

The purpose is to understand the culture, social structure, and the behaviors of the group under study. It is often useful when researchers seek to understand shared cultural meanings and practices in their natural settings.

However, it can be time-consuming and may reflect researcher biases due to the immersion approach.

Example of Ethnography

Liquidated: An Ethnography of Wall Street by Karen Ho involves an anthropologist who embeds herself with Wall Street firms to study the culture of Wall Street bankers and how this culture affects the broader economy and world.

2. Phenomenological Research

Phenomenological research is a qualitative method focused on the study of individual experiences from the participant’s perspective (Tracy, 2019).

It focuses specifically on people’s experiences in relation to a specific social phenomenon ( see here for examples of social phenomena ).

This method is valuable when the goal is to understand how individuals perceive, experience, and make meaning of particular phenomena. However, because it is subjective and dependent on participants’ self-reports, findings may not be generalizable, and are highly reliant on self-reported ‘thoughts and feelings’.

Example of Phenomenological Research

A phenomenological approach to experiences with technology by Sebnem Cilesiz represents a good starting-point for formulating a phenomenological study. With its focus on the ‘essence of experience’, this piece presents methodological, reliability, validity, and data analysis techniques that phenomenologists use to explain how people experience technology in their everyday lives.

3. Historical Research

Historical research is a qualitative method involving the examination of past events to draw conclusions about the present or make predictions about the future (Stokes & Wall, 2017).

As you might expect, it’s common in the research branches of history departments in universities.

This approach is useful in studies that seek to understand the past to interpret present events or trends. However, it relies heavily on the availability and reliability of source materials, which may be limited.

Common data sources include cultural artifacts from both material and non-material culture , which are then examined, compared, contrasted, and contextualized to test hypotheses and generate theories.

Example of Historical Research

A historical research example might be a study examining the evolution of gender roles over the last century. This research might involve the analysis of historical newspapers, advertisements, letters, and company documents, as well as sociocultural contexts.

4. Content Analysis

Content analysis is a research method that involves systematic and objective coding and interpreting of text or media to identify patterns, themes, ideologies, or biases (Schweigert, 2021).

A content analysis is useful in analyzing communication patterns, helping to reveal how texts such as newspapers, movies, films, political speeches, and other types of ‘content’ contain narratives and biases.

However, interpretations can be very subjective, which often requires scholars to engage in practices such as cross-comparing their coding with peers or external researchers.

Content analysis can be further broken down in to other specific methodologies such as semiotic analysis, multimodal analysis , and discourse analysis .

Example of Content Analysis

How is Islam Portrayed in Western Media? by Poorebrahim and Zarei (2013) employs a type of content analysis called critical discourse analysis (common in poststructuralist and critical theory research ). This study by Poorebrahum and Zarei combs through a corpus of western media texts to explore the language forms that are used in relation to Islam and Muslims, finding that they are overly stereotyped, which may represent anti-Islam bias or failure to understand the Islamic world.

5. Grounded Theory Research

Grounded theory involves developing a theory during and after data collection rather than beforehand.

This is in contrast to most academic research studies, which start with a hypothesis or theory and then testing of it through a study, where we might have a null hypothesis (disproving the theory) and an alternative hypothesis (supporting the theory).

Grounded Theory is useful because it keeps an open mind to what the data might reveal out of the research. It can be time-consuming and requires rigorous data analysis (Tracy, 2019).

Grounded Theory Example

Developing a Leadership Identity by Komives et al (2005) employs a grounded theory approach to develop a thesis based on the data rather than testing a hypothesis. The researchers studied the leadership identity of 13 college students taking on leadership roles. Based on their interviews, the researchers theorized that the students’ leadership identities shifted from a hierarchical view of leadership to one that embraced leadership as a collaborative concept.

6. Action Research

Action research is an approach which aims to solve real-world problems and bring about change within a setting. The study is designed to solve a specific problem – or in other words, to take action (Patten, 2017).

This approach can involve mixed methods, but is generally qualitative because it usually involves the study of a specific case study wherein the researcher works, e.g. a teacher studying their own classroom practice to seek ways they can improve.

Action research is very common in fields like education and nursing where practitioners identify areas for improvement then implement a study in order to find paths forward.

Action Research Example

Using Digital Sandbox Gaming to Improve Creativity Within Boys’ Writing by Ellison and Drew was a research study one of my research students completed in his own classroom under my supervision. He implemented a digital game-based approach to literacy teaching with boys and interviewed his students to see if the use of games as stimuli for storytelling helped draw them into the learning experience.

7. Natural Observational Research

Observational research can also be quantitative (see: experimental research), but in naturalistic settings for the social sciences, researchers tend to employ qualitative data collection methods like interviews and field notes to observe people in their day-to-day environments.

This approach involves the observation and detailed recording of behaviors in their natural settings (Howitt, 2019). It can provide rich, in-depth information, but the researcher’s presence might influence behavior.

While observational research has some overlaps with ethnography (especially in regard to data collection techniques), it tends not to be as sustained as ethnography, e.g. a researcher might do 5 observations, every second Monday, as opposed to being embedded in an environment.

Observational Research Example

A researcher might use qualitative observational research to study the behaviors and interactions of children at a playground. The researcher would document the behaviors observed, such as the types of games played, levels of cooperation , and instances of conflict.

8. Case Study Research

Case study research is a qualitative method that involves a deep and thorough investigation of a single individual, group, or event in order to explore facets of that phenomenon that cannot be captured using other methods (Stokes & Wall, 2017).

Case study research is especially valuable in providing contextualized insights into specific issues, facilitating the application of abstract theories to real-world situations (Patten, 2017).

However, findings from a case study may not be generalizable due to the specific context and the limited number of cases studied (Walliman, 2021).

See More: Case Study Advantages and Disadvantages

Example of a Case Study

Scholars conduct a detailed exploration of the implementation of a new teaching method within a classroom setting. The study focuses on how the teacher and students adapt to the new method, the challenges encountered, and the outcomes on student performance and engagement. While the study provides specific and detailed insights of the teaching method in that classroom, it cannot be generalized to other classrooms, as statistical significance has not been established through this qualitative approach.

Quantitative Research Methods

Quantitative research methods involve the systematic empirical investigation of observable phenomena via statistical, mathematical, or computational techniques (Pajo, 2022). The focus is on gathering numerical data and generalizing it across groups of people or to explain a particular phenomenon.

9. Experimental Research

Experimental research is a quantitative method where researchers manipulate one variable to determine its effect on another (Walliman, 2021).

This is common, for example, in high-school science labs, where students are asked to introduce a variable into a setting in order to examine its effect.

This type of research is useful in situations where researchers want to determine causal relationships between variables. However, experimental conditions may not reflect real-world conditions.

Example of Experimental Research

A researcher may conduct an experiment to determine the effects of a new educational approach on student learning outcomes. Students would be randomly assigned to either the control group (traditional teaching method) or the experimental group (new educational approach).

10. Surveys and Questionnaires

Surveys and questionnaires are quantitative methods that involve asking research participants structured and predefined questions to collect data about their attitudes, beliefs, behaviors, or characteristics (Patten, 2017).

Surveys are beneficial for collecting data from large samples, but they depend heavily on the honesty and accuracy of respondents.

They tend to be seen as more authoritative than their qualitative counterparts, semi-structured interviews, because the data is quantifiable (e.g. a questionnaire where information is presented on a scale from 1 to 10 can allow researchers to determine and compare statistical means, averages, and variations across sub-populations in the study).

Example of a Survey Study

A company might use a survey to gather data about employee job satisfaction across its offices worldwide. Employees would be asked to rate various aspects of their job satisfaction on a Likert scale. While this method provides a broad overview, it may lack the depth of understanding possible with other methods (Stokes & Wall, 2017).

11. Longitudinal Studies

Longitudinal studies involve repeated observations of the same variables over extended periods (Howitt, 2019). These studies are valuable for tracking development and change but can be costly and time-consuming.

With multiple data points collected over extended periods, it’s possible to examine continuous changes within things like population dynamics or consumer behavior. This makes a detailed analysis of change possible.

a visual representation of a longitudinal study demonstrating that data is collected over time on one sample so researchers can examine how variables change over time

Perhaps the most relatable example of a longitudinal study is a national census, which is taken on the same day every few years, to gather comparative demographic data that can show how a nation is changing over time.

While longitudinal studies are commonly quantitative, there are also instances of qualitative ones as well, such as the famous 7 Up study from the UK, which studies 14 individuals every 7 years to explore their development over their lives.

Example of a Longitudinal Study

A national census, taken every few years, uses surveys to develop longitudinal data, which is then compared and analyzed to present accurate trends over time. Trends a census can reveal include changes in religiosity, values and attitudes on social issues, and much more.

12. Cross-Sectional Studies

Cross-sectional studies are a quantitative research method that involves analyzing data from a population at a specific point in time (Patten, 2017). They provide a snapshot of a situation but cannot determine causality.

This design is used to measure and compare the prevalence of certain characteristics or outcomes in different groups within the sampled population.

A visual representation of a cross-sectional group of people, demonstrating that the data is collected at a single point in time and you can compare groups within the sample

The major advantage of cross-sectional design is its ability to measure a wide range of variables simultaneously without needing to follow up with participants over time.

However, cross-sectional studies do have limitations . This design can only show if there are associations or correlations between different variables, but cannot prove cause and effect relationships, temporal sequence, changes, and trends over time.

Example of a Cross-Sectional Study

Our longitudinal study example of a national census also happens to contain cross-sectional design. One census is cross-sectional, displaying only data from one point in time. But when a census is taken once every few years, it becomes longitudinal, and so long as the data collection technique remains unchanged, identification of changes will be achievable, adding another time dimension on top of a basic cross-sectional study.

13. Correlational Research

Correlational research is a quantitative method that seeks to determine if and to what degree a relationship exists between two or more quantifiable variables (Schweigert, 2021).

This approach provides a fast and easy way to make initial hypotheses based on either positive or negative correlation trends that can be observed within dataset.

While correlational research can reveal relationships between variables, it cannot establish causality.

Methods used for data analysis may include statistical correlations such as Pearson’s or Spearman’s.

Example of Correlational Research

A team of researchers is interested in studying the relationship between the amount of time students spend studying and their academic performance. They gather data from a high school, measuring the number of hours each student studies per week and their grade point averages (GPAs) at the end of the semester. Upon analyzing the data, they find a positive correlation, suggesting that students who spend more time studying tend to have higher GPAs.

14. Quasi-Experimental Design Research

Quasi-experimental design research is a quantitative research method that is similar to experimental design but lacks the element of random assignment to treatment or control.

Instead, quasi-experimental designs typically rely on certain other methods to control for extraneous variables.

The term ‘quasi-experimental’ implies that the experiment resembles a true experiment, but it is not exactly the same because it doesn’t meet all the criteria for a ‘true’ experiment, specifically in terms of control and random assignment.

Quasi-experimental design is useful when researchers want to study a causal hypothesis or relationship, but practical or ethical considerations prevent them from manipulating variables and randomly assigning participants to conditions.

Example of Quasi-Experimental Design

A researcher wants to study the impact of a new math tutoring program on student performance. However, ethical and practical constraints prevent random assignment to the “tutoring” and “no tutoring” groups. Instead, the researcher compares students who chose to receive tutoring (experimental group) to similar students who did not choose to receive tutoring (control group), controlling for other variables like grade level and previous math performance.

Related: Examples and Types of Random Assignment in Research

15. Meta-Analysis Research

Meta-analysis statistically combines the results of multiple studies on a specific topic to yield a more precise estimate of the effect size. It’s the gold standard of secondary research .

Meta-analysis is particularly useful when there are numerous studies on a topic, and there is a need to integrate the findings to draw more reliable conclusions.

Some meta-analyses can identify flaws or gaps in a corpus of research, when can be highly influential in academic research, despite lack of primary data collection.

However, they tend only to be feasible when there is a sizable corpus of high-quality and reliable studies into a phenomenon.

Example of a Meta-Analysis

The power of feedback revisited (Wisniewski, Zierer & Hattie, 2020) is a meta-analysis that examines 435 empirical studies research on the effects of feedback on student learning. They use a random-effects model to ascertain whether there is a clear effect size across the literature. The authors find that feedback tends to impact cognitive and motor skill outcomes but has less of an effect on motivational and behavioral outcomes.

Choosing a research method requires a lot of consideration regarding what you want to achieve, your research paradigm, and the methodology that is most valuable for what you are studying. There are multiple types of research methods, many of which I haven’t been able to present here. Generally, it’s recommended that you work with an experienced researcher or research supervisor to identify a suitable research method for your study at hand.

Hammond, M., & Wellington, J. (2020). Research methods: The key concepts . New York: Routledge.

Howitt, D. (2019). Introduction to qualitative research methods in psychology . London: Pearson UK.

Pajo, B. (2022). Introduction to research methods: A hands-on approach . New York: Sage Publications.

Patten, M. L. (2017). Understanding research methods: An overview of the essentials . New York: Sage

Schweigert, W. A. (2021). Research methods in psychology: A handbook . Los Angeles: Waveland Press.

Stokes, P., & Wall, T. (2017). Research methods . New York: Bloomsbury Publishing.

Tracy, S. J. (2019). Qualitative research methods: Collecting evidence, crafting analysis, communicating impact . London: John Wiley & Sons.

Walliman, N. (2021). Research methods: The basics. London: Routledge.

Chris Drew (PhD)

Dr. Chris Drew is the founder of the Helpful Professor. He holds a PhD in education and has published over 20 articles in scholarly journals. He is the former editor of the Journal of Learning Development in Higher Education. [Image Descriptor: Photo of Chris]

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Chris Drew (PhD) https://helpfulprofessor.com/author/chris-drew-phd/ What is IQ? (Intelligence Quotient)
Chris Drew (PhD) https://helpfulprofessor.com/author/chris-drew-phd/ 5 Top Tips for Succeeding at University

Research Methods Guide: Research Design & Method

Introduction
Survey Research
Interview Research
Data Analysis
Resources & Consultation

Tutorial Videos: Research Design & Method

Research Methods (sociology-focused)

Qualitative vs. Quantitative Methods (intro)

Qualitative vs. Quantitative Methods (advanced)

FAQ: Research Design & Method

What is the difference between Research Design and Research Method?

Research design is a plan to answer your research question. A research method is a strategy used to implement that plan. Research design and methods are different but closely related, because good research design ensures that the data you obtain will help you answer your research question more effectively.

Which research method should I choose ?

It depends on your research goal. It depends on what subjects (and who) you want to study. Let's say you are interested in studying what makes people happy, or why some students are more conscious about recycling on campus. To answer these questions, you need to make a decision about how to collect your data. Most frequently used methods include:

Observation / Participant Observation
Focus Groups
Experiments
Secondary Data Analysis / Archival Study
Mixed Methods (combination of some of the above)

One particular method could be better suited to your research goal than others, because the data you collect from different methods will be different in quality and quantity. For instance, surveys are usually designed to produce relatively short answers, rather than the extensive responses expected in qualitative interviews.

What other factors should I consider when choosing one method over another?

Time for data collection and analysis is something you want to consider. An observation or interview method, so-called qualitative approach, helps you collect richer information, but it takes time. Using a survey helps you collect more data quickly, yet it may lack details. So, you will need to consider the time you have for research and the balance between strengths and weaknesses associated with each method (e.g., qualitative vs. quantitative).

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Last Updated: Aug 21, 2023 10:42 AM

Research Methods In Psychology

Saul Mcleod, PhD

Editor-in-Chief for Simply Psychology

BSc (Hons) Psychology, MRes, PhD, University of Manchester

Saul Mcleod, PhD., is a qualified psychology teacher with over 18 years of experience in further and higher education. He has been published in peer-reviewed journals, including the Journal of Clinical Psychology.

Learn about our Editorial Process

Olivia Guy-Evans, MSc

Associate Editor for Simply Psychology

BSc (Hons) Psychology, MSc Psychology of Education

Olivia Guy-Evans is a writer and associate editor for Simply Psychology. She has previously worked in healthcare and educational sectors.

Research methods in psychology are systematic procedures used to observe, describe, predict, and explain behavior and mental processes. They include experiments, surveys, case studies, and naturalistic observations, ensuring data collection is objective and reliable to understand and explain psychological phenomena.

Hypotheses are statements about the prediction of the results, that can be verified or disproved by some investigation.

There are four types of hypotheses :

Null Hypotheses (H0 ) – these predict that no difference will be found in the results between the conditions. Typically these are written ‘There will be no difference…’
Alternative Hypotheses (Ha or H1) – these predict that there will be a significant difference in the results between the two conditions. This is also known as the experimental hypothesis.
One-tailed (directional) hypotheses – these state the specific direction the researcher expects the results to move in, e.g. higher, lower, more, less. In a correlation study, the predicted direction of the correlation can be either positive or negative.
Two-tailed (non-directional) hypotheses – these state that a difference will be found between the conditions of the independent variable but does not state the direction of a difference or relationship. Typically these are always written ‘There will be a difference ….’

All research has an alternative hypothesis (either a one-tailed or two-tailed) and a corresponding null hypothesis.

Once the research is conducted and results are found, psychologists must accept one hypothesis and reject the other.

So, if a difference is found, the Psychologist would accept the alternative hypothesis and reject the null. The opposite applies if no difference is found.

Sampling techniques

Sampling is the process of selecting a representative group from the population under study.

A sample is the participants you select from a target population (the group you are interested in) to make generalizations about.

Representative means the extent to which a sample mirrors a researcher’s target population and reflects its characteristics.

Generalisability means the extent to which their findings can be applied to the larger population of which their sample was a part.

Volunteer sample : where participants pick themselves through newspaper adverts, noticeboards or online.
Opportunity sampling : also known as convenience sampling , uses people who are available at the time the study is carried out and willing to take part. It is based on convenience.
Random sampling : when every person in the target population has an equal chance of being selected. An example of random sampling would be picking names out of a hat.
Systematic sampling : when a system is used to select participants. Picking every Nth person from all possible participants. N = the number of people in the research population / the number of people needed for the sample.
Stratified sampling : when you identify the subgroups and select participants in proportion to their occurrences.
Snowball sampling : when researchers find a few participants, and then ask them to find participants themselves and so on.
Quota sampling : when researchers will be told to ensure the sample fits certain quotas, for example they might be told to find 90 participants, with 30 of them being unemployed.

Experiments always have an independent and dependent variable .

The independent variable is the one the experimenter manipulates (the thing that changes between the conditions the participants are placed into). It is assumed to have a direct effect on the dependent variable.
The dependent variable is the thing being measured, or the results of the experiment.

Operationalization of variables means making them measurable/quantifiable. We must use operationalization to ensure that variables are in a form that can be easily tested.

For instance, we can’t really measure ‘happiness’, but we can measure how many times a person smiles within a two-hour period.

By operationalizing variables, we make it easy for someone else to replicate our research. Remember, this is important because we can check if our findings are reliable.

Extraneous variables are all variables which are not independent variable but could affect the results of the experiment.

It can be a natural characteristic of the participant, such as intelligence levels, gender, or age for example, or it could be a situational feature of the environment such as lighting or noise.

Demand characteristics are a type of extraneous variable that occurs if the participants work out the aims of the research study, they may begin to behave in a certain way.

For example, in Milgram’s research , critics argued that participants worked out that the shocks were not real and they administered them as they thought this was what was required of them.

Extraneous variables must be controlled so that they do not affect (confound) the results.

Randomly allocating participants to their conditions or using a matched pairs experimental design can help to reduce participant variables.

Situational variables are controlled by using standardized procedures, ensuring every participant in a given condition is treated in the same way

Experimental Design

Experimental design refers to how participants are allocated to each condition of the independent variable, such as a control or experimental group.

Independent design ( between-groups design ): each participant is selected for only one group. With the independent design, the most common way of deciding which participants go into which group is by means of randomization.
Matched participants design : each participant is selected for only one group, but the participants in the two groups are matched for some relevant factor or factors (e.g. ability; sex; age).
Repeated measures design ( within groups) : each participant appears in both groups, so that there are exactly the same participants in each group.
The main problem with the repeated measures design is that there may well be order effects. Their experiences during the experiment may change the participants in various ways.
They may perform better when they appear in the second group because they have gained useful information about the experiment or about the task. On the other hand, they may perform less well on the second occasion because of tiredness or boredom.
Counterbalancing is the best way of preventing order effects from disrupting the findings of an experiment, and involves ensuring that each condition is equally likely to be used first and second by the participants.

If we wish to compare two groups with respect to a given independent variable, it is essential to make sure that the two groups do not differ in any other important way.

Experimental Methods

All experimental methods involve an iv (independent variable) and dv (dependent variable)..

Field experiments are conducted in the everyday (natural) environment of the participants. The experimenter still manipulates the IV, but in a real-life setting. It may be possible to control extraneous variables, though such control is more difficult than in a lab experiment.
Natural experiments are when a naturally occurring IV is investigated that isn’t deliberately manipulated, it exists anyway. Participants are not randomly allocated, and the natural event may only occur rarely.

Case studies are in-depth investigations of a person, group, event, or community. It uses information from a range of sources, such as from the person concerned and also from their family and friends.

Many techniques may be used such as interviews, psychological tests, observations and experiments. Case studies are generally longitudinal: in other words, they follow the individual or group over an extended period of time.

Case studies are widely used in psychology and among the best-known ones carried out were by Sigmund Freud . He conducted very detailed investigations into the private lives of his patients in an attempt to both understand and help them overcome their illnesses.

Case studies provide rich qualitative data and have high levels of ecological validity. However, it is difficult to generalize from individual cases as each one has unique characteristics.

Correlational Studies

Correlation means association; it is a measure of the extent to which two variables are related. One of the variables can be regarded as the predictor variable with the other one as the outcome variable.

Correlational studies typically involve obtaining two different measures from a group of participants, and then assessing the degree of association between the measures.

The predictor variable can be seen as occurring before the outcome variable in some sense. It is called the predictor variable, because it forms the basis for predicting the value of the outcome variable.

Relationships between variables can be displayed on a graph or as a numerical score called a correlation coefficient.

types of correlation. Scatter plot. Positive negative and no correlation

If an increase in one variable tends to be associated with an increase in the other, then this is known as a positive correlation .
If an increase in one variable tends to be associated with a decrease in the other, then this is known as a negative correlation .
A zero correlation occurs when there is no relationship between variables.

After looking at the scattergraph, if we want to be sure that a significant relationship does exist between the two variables, a statistical test of correlation can be conducted, such as Spearman’s rho.

The test will give us a score, called a correlation coefficient . This is a value between 0 and 1, and the closer to 1 the score is, the stronger the relationship between the variables. This value can be both positive e.g. 0.63, or negative -0.63.

Types of correlation. Strong, weak, and perfect positive correlation, strong, weak, and perfect negative correlation, no correlation. Graphs or charts ...

A correlation between variables, however, does not automatically mean that the change in one variable is the cause of the change in the values of the other variable. A correlation only shows if there is a relationship between variables.

Correlation does not always prove causation, as a third variable may be involved.

Interview Methods

Interviews are commonly divided into two types: structured and unstructured.

A fixed, predetermined set of questions is put to every participant in the same order and in the same way.

Responses are recorded on a questionnaire, and the researcher presets the order and wording of questions, and sometimes the range of alternative answers.

The interviewer stays within their role and maintains social distance from the interviewee.

There are no set questions, and the participant can raise whatever topics he/she feels are relevant and ask them in their own way. Questions are posed about participants’ answers to the subject

Unstructured interviews are most useful in qualitative research to analyze attitudes and values.

Though they rarely provide a valid basis for generalization, their main advantage is that they enable the researcher to probe social actors’ subjective point of view.

Questionnaire Method

Questionnaires can be thought of as a kind of written interview. They can be carried out face to face, by telephone, or post.

The choice of questions is important because of the need to avoid bias or ambiguity in the questions, ‘leading’ the respondent or causing offense.

Open questions are designed to encourage a full, meaningful answer using the subject’s own knowledge and feelings. They provide insights into feelings, opinions, and understanding. Example: “How do you feel about that situation?”
Closed questions can be answered with a simple “yes” or “no” or specific information, limiting the depth of response. They are useful for gathering specific facts or confirming details. Example: “Do you feel anxious in crowds?”

Its other practical advantages are that it is cheaper than face-to-face interviews and can be used to contact many respondents scattered over a wide area relatively quickly.

Observations

There are different types of observation methods :

Covert observation is where the researcher doesn’t tell the participants they are being observed until after the study is complete. There could be ethical problems or deception and consent with this particular observation method.
Overt observation is where a researcher tells the participants they are being observed and what they are being observed for.
Controlled : behavior is observed under controlled laboratory conditions (e.g., Bandura’s Bobo doll study).
Natural : Here, spontaneous behavior is recorded in a natural setting.
Participant : Here, the observer has direct contact with the group of people they are observing. The researcher becomes a member of the group they are researching.
Non-participant (aka “fly on the wall): The researcher does not have direct contact with the people being observed. The observation of participants’ behavior is from a distance

Pilot Study

A pilot study is a small scale preliminary study conducted in order to evaluate the feasibility of the key s teps in a future, full-scale project.

A pilot study is an initial run-through of the procedures to be used in an investigation; it involves selecting a few people and trying out the study on them. It is possible to save time, and in some cases, money, by identifying any flaws in the procedures designed by the researcher.

A pilot study can help the researcher spot any ambiguities (i.e. unusual things) or confusion in the information given to participants or problems with the task devised.

Sometimes the task is too hard, and the researcher may get a floor effect, because none of the participants can score at all or can complete the task – all performances are low.

The opposite effect is a ceiling effect, when the task is so easy that all achieve virtually full marks or top performances and are “hitting the ceiling”.

Research Design

In cross-sectional research , a researcher compares multiple segments of the population at the same time

Sometimes, we want to see how people change over time, as in studies of human development and lifespan. Longitudinal research is a research design in which data-gathering is administered repeatedly over an extended period of time.

In cohort studies , the participants must share a common factor or characteristic such as age, demographic, or occupation. A cohort study is a type of longitudinal study in which researchers monitor and observe a chosen population over an extended period.

Triangulation means using more than one research method to improve the study’s validity.

Reliability

Reliability is a measure of consistency, if a particular measurement is repeated and the same result is obtained then it is described as being reliable.

Test-retest reliability : assessing the same person on two different occasions which shows the extent to which the test produces the same answers.
Inter-observer reliability : the extent to which there is an agreement between two or more observers.

Meta-Analysis

A meta-analysis is a systematic review that involves identifying an aim and then searching for research studies that have addressed similar aims/hypotheses.

This is done by looking through various databases, and then decisions are made about what studies are to be included/excluded.

Strengths: Increases the conclusions’ validity as they’re based on a wider range.

Weaknesses: Research designs in studies can vary, so they are not truly comparable.

Peer Review

A researcher submits an article to a journal. The choice of the journal may be determined by the journal’s audience or prestige.

The journal selects two or more appropriate experts (psychologists working in a similar field) to peer review the article without payment. The peer reviewers assess: the methods and designs used, originality of the findings, the validity of the original research findings and its content, structure and language.

Feedback from the reviewer determines whether the article is accepted. The article may be: Accepted as it is, accepted with revisions, sent back to the author to revise and re-submit or rejected without the possibility of submission.

The editor makes the final decision whether to accept or reject the research report based on the reviewers comments/ recommendations.

Peer review is important because it prevent faulty data from entering the public domain, it provides a way of checking the validity of findings and the quality of the methodology and is used to assess the research rating of university departments.

Peer reviews may be an ideal, whereas in practice there are lots of problems. For example, it slows publication down and may prevent unusual, new work being published. Some reviewers might use it as an opportunity to prevent competing researchers from publishing work.

Some people doubt whether peer review can really prevent the publication of fraudulent research.

The advent of the internet means that a lot of research and academic comment is being published without official peer reviews than before, though systems are evolving on the internet where everyone really has a chance to offer their opinions and police the quality of research.

Types of Data

Quantitative data is numerical data e.g. reaction time or number of mistakes. It represents how much or how long, how many there are of something. A tally of behavioral categories and closed questions in a questionnaire collect quantitative data.
Qualitative data is virtually any type of information that can be observed and recorded that is not numerical in nature and can be in the form of written or verbal communication. Open questions in questionnaires and accounts from observational studies collect qualitative data.
Primary data is first-hand data collected for the purpose of the investigation.
Secondary data is information that has been collected by someone other than the person who is conducting the research e.g. taken from journals, books or articles.

Validity means how well a piece of research actually measures what it sets out to, or how well it reflects the reality it claims to represent.

Validity is whether the observed effect is genuine and represents what is actually out there in the world.

Concurrent validity is the extent to which a psychological measure relates to an existing similar measure and obtains close results. For example, a new intelligence test compared to an established test.
Face validity : does the test measure what it’s supposed to measure ‘on the face of it’. This is done by ‘eyeballing’ the measuring or by passing it to an expert to check.
Ecological validit y is the extent to which findings from a research study can be generalized to other settings / real life.
Temporal validity is the extent to which findings from a research study can be generalized to other historical times.

Features of Science

Paradigm – A set of shared assumptions and agreed methods within a scientific discipline.
Paradigm shift – The result of the scientific revolution: a significant change in the dominant unifying theory within a scientific discipline.
Objectivity – When all sources of personal bias are minimised so not to distort or influence the research process.
Empirical method – Scientific approaches that are based on the gathering of evidence through direct observation and experience.
Replicability – The extent to which scientific procedures and findings can be repeated by other researchers.
Falsifiability – The principle that a theory cannot be considered scientific unless it admits the possibility of being proved untrue.

Statistical Testing

A significant result is one where there is a low probability that chance factors were responsible for any observed difference, correlation, or association in the variables tested.

If our test is significant, we can reject our null hypothesis and accept our alternative hypothesis.

If our test is not significant, we can accept our null hypothesis and reject our alternative hypothesis. A null hypothesis is a statement of no effect.

In Psychology, we use p < 0.05 (as it strikes a balance between making a type I and II error) but p < 0.01 is used in tests that could cause harm like introducing a new drug.

A type I error is when the null hypothesis is rejected when it should have been accepted (happens when a lenient significance level is used, an error of optimism).

A type II error is when the null hypothesis is accepted when it should have been rejected (happens when a stringent significance level is used, an error of pessimism).

Ethical Issues

Informed consent is when participants are able to make an informed judgment about whether to take part. It causes them to guess the aims of the study and change their behavior.
To deal with it, we can gain presumptive consent or ask them to formally indicate their agreement to participate but it may invalidate the purpose of the study and it is not guaranteed that the participants would understand.
Deception should only be used when it is approved by an ethics committee, as it involves deliberately misleading or withholding information. Participants should be fully debriefed after the study but debriefing can’t turn the clock back.
All participants should be informed at the beginning that they have the right to withdraw if they ever feel distressed or uncomfortable.
It causes bias as the ones that stayed are obedient and some may not withdraw as they may have been given incentives or feel like they’re spoiling the study. Researchers can offer the right to withdraw data after participation.
Participants should all have protection from harm . The researcher should avoid risks greater than those experienced in everyday life and they should stop the study if any harm is suspected. However, the harm may not be apparent at the time of the study.
Confidentiality concerns the communication of personal information. The researchers should not record any names but use numbers or false names though it may not be possible as it is sometimes possible to work out who the researchers were.

Open access
Published: 11 May 2024

Low selection of HIV PrEP refills at private pharmacies among clients who initiated PrEP at public clinics: findings from a mixed-methods study in Kenya

Katrina F. Ortblad 1 ,
Alexandra P. Kuo 2 ,
Peter Mogere 3 ,
Stephanie D. Roche 1 ,
Catherine Kiptinness 3 ,
Njeri Wairimu 3 ,
Stephen Gakuo 3 ,
Jared M. Baeten 4 , 5 &
Kenneth Ngure 4 , 6

BMC Health Services Research volume 24 , Article number: 618 ( 2024 ) Cite this article

Metrics details

In Africa, the delivery of HIV pre-exposure prophylaxis (PrEP) at public healthcare clinics is challenged by understaffing, overcrowding, and HIV-associated stigma, often resulting in low PrEP uptake and continuation among clients. Giving clients the option to refill PrEP at nearby private pharmacies, which are often more convenient and have shorter wait times, may address these challenges and improve PrEP continuation.

This mixed methods study used an explanatory sequential design. At two public clinics in Kiambu County, Kenya, clients ≥ 18 years initiating PrEP were given the option to refill PrEP at the clinic where they initiated for free or at one of three nearby private pharmacies for 300 Kenyan Shillings (~ $3 US Dollars). The providers at these pharmacies (pharmacists and pharmaceutical technologists) were trained in PrEP service delivery using a prescribing checklist and provider-assisted HIV self-testing, both with remote clinician oversight. Clients were followed up to seven months, with scheduled refill visits at one, four, and seven months. The primary outcomes were selection of pharmacy-based PrEP refills and PrEP continuation. Following pilot completion, 15 in-depth interviews (IDIs) with clients who refilled PrEP were completed. We used descriptive statistics and thematic analysis to assess study outcomes.

From November 2020 to November 2021, 125 PrEP clients were screened and 106 enrolled. The majority (59%, 63/106) of clients were women and the median age was 31 years (IQR 26–38 years). Over 292 client-months of follow-up, 41 clients (39%) refilled PrEP; only three (3%) at a participating pharmacy. All clients who completed IDIs refilled PrEP at clinics. The reasons why clients did not refill PrEP at pharmacies included: a preference for clinic-delivered PrEP services (i.e., pre-existing relationships, access to other services), concerns about pharmacy-delivered PrEP services (i.e., mistrust, lower quality care, costs), and lack of knowledge of this refill location.

Conclusions

These findings suggest that clients who initiate PrEP at public clinics in Kenya may have already overcome barriers to clinic-delivered PrEP services and prefer PrEP access there. To reach new populations that could benefit from PrEP, a stand-alone model of pharmacy-delivered PrEP services may be needed.

Trial registration

ClinicalTrials.gov: NCT04558554 [registered: June 5, 2020].

Peer Review reports

Despite the provision of free highly effective HIV prevention interventions, including oral pre-exposure prophylaxis (PrEP) [ 1 , 2 ], the rates of HIV incidence persist above the level of epidemic control in many African countries [ 3 , 4 ]. Individuals that could benefit from PrEP services often face barriers to accessing PrEP at public healthcare clinics, which has resulted in low PrEP initiation and continuation (i.e., refills) in these settings [ 5 , 6 , 7 , 8 ]. Barriers to PrEP access include long travel distances to and wait times at the clinics, fears of stigma associated with visiting HIV care centers at the clinics, and limited hours of clinic operations [ 9 , 10 ]. The delivery of PrEP services (e.g., HIV testing, counseling, drug dispensing) at private pharmacies may address some of these barriers, as pharmacies are ubiquitous in many African countries, provide diverse services, and are often open for extended hours [ 11 , 12 , 13 ]. Additionally, private pharmacies are often the first places many people in low- and middle-income countries seek health services [ 14 , 15 , 16 ], as they already provide many sexual and reproductive health-related products (e.g., treatment for sexually transmitted infections), and clients are willing to purchase products at pharmacies that are available for free at public clinics (e.g., contraception) [ 17 ].

Recent pilot studies that tested different models of PrEP initiation at private pharmacies in Kenya found that uptake was high and direct delivery of PrEP in these settings may engage populations (e.g., men, unmarried people) who could benefit from PrEP and differ from those engaged in clinic-based PrEP services [ 18 , 19 , 20 ]. Additionally, these studies found that PrEP continuation at private pharmacies was comparable, or in some cases higher, than that at public clinics [ 18 , 19 ]. However, regulatory barriers (e.g., restrictions on cadres of workers who can prescribe antiretrovirals and complete HIV rapid diagnostic testing [RDT]) exist in Kenya and many similar settings that make the scale-up PrEP initiation at private pharmacies challenging.

Compared to PrEP initiation, PrEP refilling is less medically complex, and potentially more feasible to delivery in non-clinical settings, including private pharmacies. With PrEP refills, there are fewer concerns about dispensing PrEP during the acute HIV infection stage – as seroconversions are rare among regular PrEP users [ 21 ] – and the medical safety of PrEP has already been determined at initiation. Thus, policy makers may feel more comfortable having individuals interested in PrEP services initiate at a clinic and have the option to refill PrEP at a nearby pharmacy, so that at initiation an RDT-certified clinician or nurse with prescribing privileges is avaliable and any necessary laboratory testing (e.g., creatinine testing) can be completed. We used mixed methods to understand the feasibility of this differentiated PrEP service delivery model in Kenya and identify weak points for model refinement.

Study design and setting

We conducted a mixed methods study using an explanatory sequential design [ 22 ]. First, we implemented a one-arm, prospective pilot study testing a model of clinic-based PrEP initiation with the option of pharmacy-based PrEP refills (ClinicalTrials.gov: NCT04558554), then we completed in-depth interviews to better understand the pilot findings. The study was conducted in Kiambu County, Kenya, where the population-level HIV prevalence is 4% [ 23 ] and there are ~ 100 public clinics delivering PrEP (~ 10–15% of all clinics in the county) and > 6,000 registered private pharmacies [ 24 ].

For the pilot, we engaged two public clinics and three private pharmacies. The clinics selected for participation were ones our research team has previously engaged with on other PrEP implementation projects [ 1 , 21 , 25 , 26 , 27 , 28 ]; thus, they were familiar with participation in research activities. The pharmacies selected were registered with the Kenya Pharmacy and Poisons Board, had a full-time licensed pharmacist or pharmaceutical technologist, and had a private room for counseling and the provision of HIV testing services. All engaged clinicians completed a brief training on how to offer PrEP refills to new PrEP clients at nearby participating pharmacies. All engaged pharmacy providers (i.e., pharmacist and pharmaceutical technologists) completed a two-day virtual training on pharmacy-delivered PrEP services (implementation occurred during the COVID-19 pandemic) [ 19 ]. This training included counseling on HIV risk and prevention interventions, PrEP use and safety, provider-assisted HIV self-testing, drug dispensing, and record keeping; following training, on-site technical assistance was available, as needed.

Participants and procedures

With the help of trained healthcare providers, we recruited individuals initiating PrEP at the participating clinics and enrolled those ≥ 18 years old who were not pregnant or breastfeeding and willing to engage in research activities. At the clinics, participants received PrEP services in accordance with Kenya’s national PrEP delivery guidelines [ 29 ], which include counseling on HIV risk reduction and drug adherence, HIV rapid diagnostic testing, serum creatine testing (if available), syndromic assessment of sexually transmitted infections, and a one-month PrEP supply at initiation and three-month supply at follow-up visits. At the end of each PrEP initiation or follow-up visit, participants were given the option to return to the clinic where they initiated PrEP for free refills (i.e., 0 Kenyan Shilling [KES] client fee) or alternatively refill PrEP at one of three nearby pharmacies for a 300 KES fee (~ $3 US Dollars [USD]). To facilitate pharmacy-based PrEP refilling, participants were verbally given directions to and the contact numbers of participating pharmacies; information that was written and shared with participants upon request.

Participants who opted to refill PrEP at a pharmacy were attended by trained pharmacy providers who implemented a care pathway for pharmacy-delivered PrEP services our research team developed in collaboration with Kenyan stakeholders [ 30 ] (model details reported elsewhere [ 19 , 30 ]). Pharmacy providers attended to PrEP clients using a standardized prescribing checklist that guided them through conducting a behavioral HIV risk assessment, medical safety assessment, and HIV testing; clients who met the criteria on the checklist for PrEP continuation were dispensed a three-month PrEP supply. Any participants who did not meet the checklist criteria (i.e., because they reported a history of liver or kidney disease, or tested HIV-positive) were referred back to the public clinic where they initiated PrEP for further evaluation. If providers had any questions about clients’ PrEP eligibility or PrEP dispensing, remote clinicians were available for support 24/7 via phone and SMS. In this study, the HIV test kits and PrEP drugs used at pharmacies were donated by the Kenya Ministry of Health. Thus, the fee clients paid for pharmacy PrEP refills (determined after consultation with participating pharmacy providers) was to compensate providers for their time spent delivering PrEP services.

The study protocol was reviewed and approved by the Scientific Ethics Review Unit at the Kenya Medical Research Institute and the Human Subjects Division at the University of Washington. All procedures were followed in accordance with the relevant guidelines and regulations (e.g., Declaration of Helsinki). All participants provided written informed consent and received 500 KES (~ $5 USD) as compensation for their time completing research activities.

Data collection

Research assistants stationed at the clinics and pharmacies completed questionnaires with participants following each PrEP visit (questionnaires included in Additional files 1 & 2 ). The questionnaires collected information on clients’ socio-demographic characteristics, healthcare seeking behaviors, sexual behaviors, self-reported PrEP adherence (follow-up visits only), and experiences and perceptions of pharmacy-delivered PrEP services. At PrEP initiation, participants were asked to report their preferred location for accessing PrEP (i.e., private clinic, public clinic, or private pharmacy). Research assistants did not participate in the delivery of PrEP services; they were only engaged in research-related activities.

Utilization outcomes

Our primary pilot study outcomes were selection of pharmacy-based PrEP refills and PrEP continuation among enrolled participants. We defined selection of pharmacy-based refills as the percentage of participants that went to a pharmacy and were dispensed PrEP. We defined PrEP continuation as the percentage of participants due for a scheduled PrEP follow-up visit (at one, four, and seven months) who returned to a clinic or pharmacy and were dispensed PrEP [ 19 , 21 ]. Additionally, we measured PrEP adherence (secondary outcome) using a validated 100-point scale that averaged participants’ self-reported responses to three questions: their number of pills missed, ability to use PrEP, and frequency of PrEP use in the past month [ 31 ]. The latter two of these two adherence questions were assessed using 5-point Likert scales; all question responses were transformed to 100-point scales, with higher scores indicating better adherence [ 31 ].

Implementation outcomes

At each PrEP clinic or pharmacy visit, we assessed participants’ perceived acceptability of, appropriateness of, and willingness to pay for pharmacy-based PrEP refills. To assess acceptability, we asked participants how strongly they agreed (5-point Likert scale) with two statements that assessed if they liked or would recommend pharmacy-delivered PrEP services (based on the Theoretical Framework of Acceptability [ 32 , 33 ]). To assess appropriateness (only measured at follow-up visits), we asked participants how strongly they agreed (5-point Likert scale) with two statements that assessed how well pharmacy-delivered PrEP services fit or were a good match for their needs (based on the Intervention Appropriateness Measure [ 34 ]). We defined outcome success for these measures as > 80% of participants agreeing or strongly agreeing with a statement. When considering willingness to pay for pharmacy-based PrEP refills, participants were asked to consider a package of services that included counseling, a medical safety assessment, HIV testing, and a three-month PrEP supply.

Quantitative analyses

We used descriptive statistics to report all findings from the pilot study. For PrEP continuation, this outcome was only reported among participants eligible for PrEP follow-up visits; due to the short duration of the pilot study, not all enrolled participants were eligible for follow-up visits at four and seven months. To better understand PrEP continuation by participants’ preferred PrEP access location, we completed a subgroup analysis that assessed this outcome among participants who reported a preference for PrEP access at a clinic (private or public) versus a pharmacy (private) at enrollment. To determine if there were any significant differences ( p < 0.05) in PrEP continuation between these subgroups, we used a chi-squared test. We used StataSE 16 (College Station, USA) to complete all quantitative analyses.

We aimed to enroll 200 participants in this pilot study. Based on our experience conducting other pilot studies [ 20 , 35 ], this sample size was considered sufficient for generating preliminary data on our primary study outcomes. Additionally, this sample size was determined feasible given the time and budget constraints of the study.

Factors influencing PrEP refill location

Post-pilot completion, we opted to conduct in-depth interviews with participants to better understand why hardly any selected to refill PrEP at a pharmacy (see study Results). Our sampling frame included all participants who refilled PrEP at least once during the study period, but not at the pharmacy; capturing a population we know had interest in continuing PrEP services. Given the tightly scoped nature of our research question, we anticipated that 15 interviews would be sufficient to reach thematic saturation [ 36 ]. We therefore purposefully invited eligible participants until the 15 participants were interviewed. Using semi-structured interview guides (found in Additional file 3 ), an experienced Kenyan qualitative researcher solicited information about interviewees’ understanding of the pharmacy PrEP refill option, what motivated their decision to refill PrEP at the clinic instead of a pharmacy, and potential barriers to refilling PrEP at a pharmacy. All interviews were conducted in a private room (at the local research team’s office or the clinic where clients were receiving PrEP services) in the interviewee’s preferred language (English or Swahili). All interviews were audio recorded, transcribed verbatim, and translated (if applicable) to English.

We analyzed all interview transcripts using thematic analysis [ 37 ]. One author (AK) achieved data immersion by reading all transcripts multiple times, then created a codebook of facilitators to clinic-based and barriers to pharmacy-based PrEP refills. AK created one spreadsheet per code, collated relevant passages from each transcript, then read through each code’s contents in its entirety. We organized codes into primary reasons for opting to refill PrEP at the clinic (as opposed to a pharmacy) and checked these for face validity with the qualitative researcher who conducted the interviews (author NW). Lastly, we selected illustrative quotes for each reason identified. To complete our qualitative analyses, we used Microsoft Excel (Redmond, USA).

From November 2020 to November 2021, we screened 125 clients initiating PrEP at public clinics and enrolled 106 participants in the pilot study. We stopped enrollment prior to achieving our target sample size because selection of pharmacy PrEP refills was much lower than anticipated (described below). Among enrolled participants, 59% (63/106) were women and the median age was 31 years (interquartile range [IQR] 26 to 38 years), Table 1 . Most (67%, 71/106) participants were married and half (49%, 52/106) were in an HIV serodifferent relationship. When asked their preferred setting for PrEP refills at enrollment, clients were split among (private) pharmacies (45%, 48/106) and clinics (public: 42%, 44/106; private: 13%, 14/106).

Over the duration of the pilot study (292 months of total participant follow-up), 39% (41/106) of participants refilled PrEP at any point. PrEP continuation was greatest at one month (38%, 40/106), then decreased by four (31%, 16/51) and seven (31%, 8/26) months, Fig. 1 . Only three of the 41 participants (7%) that returned for any PrEP refills did so at a private pharmacy. Two of these participants only refilled PrEP once at a pharmacy (at one month and four months) and one refilled PrEP twice at a pharmacy (at four months and seven months); all these participants additionally refilled PrEP at a clinic either before ( n = 2) or after ( n = 2) they refilled PrEP at a pharmacy. Among the participants that continued PrEP, adherence was high. At one month, the median number of pills missed was 0 (IQR 1–2) and the ability to use and frequency of use scores were 100 (IQR 75–100); these findings remained consistent over PrEP follow-up visits.

PrEP location selection and continuation at one, four, and seven months following initiation among pilot participants. Percentages are calculated amongst those eligible to initiate or continue PrEP at each visit. Month (M). a Participants who refilled at the pharmacy ( n = 3 unique individuals) also refiled at a clinic at least once during the pilot. Only one such individual refilled twice at the pharmacy (at Months 4 and 7)

In our subgroup analysis where we assessed any PrEP continuation by participants’ stated preference for PrEP access location at enrollment, we did not find any significant differences in continuation among those who reported a preference for accessing PrEP at clinics (43%, 23/53) versus pharmacies (40%, 18/48; p = 0.82).

At PrEP initiation and follow-up visits, most participants (> 50%) indicated that they were “unsure” of the extent to which they agreed or disagreed with the statements on the acceptability and appropriateness of pharmacy-delivered PrEP services, Table 2 . At initiation, less than half of participants agreed or strongly agreed that they anticipated they would like (42%, 44/106) or recommend (43%, 46/106) pharmacy-delivered PrEP services; far below our a priori acceptability assessment threshold of > 80%. At follow-up visits, these findings remained consistent, with < 20% of participants agreeing or strongly agreeing with the acceptability assessment statements. Similarly, at one month (the first timepoint at which appropriateness was assessed), only 12% (5/41) of participants agreed or strongly agreed that pharmacy-delivered PrEP services fit their needs or was a good match for their needs; findings that remained consistent (and below our a priori appropriateness assessment threshold) at four and seven months.

Despite these findings, most participants (83%, 88/106 at initiation) reported that they would be willing to pay some amount for a package of pharmacy-delivered PrEP services, Table 2 . At PrEP initiation, the median amount participants were willing to pay was 200 KES (IQR 100–300 KES), which is equivalent to ~ $2 USD (IQR ~ $1 to $3 USD). This reported amount decreased slightly at one and four months, then increased at seven months among participants still engaged in PrEP services.

From our qualitative data, we identified five primary reasons why interviewees did not opt to refill PrEP at a pharmacy (Table 3 ): convenience, cost, desire for continuity of the client-provider relationship, quality of care concerns, and misunderstanding the pharmacy PrEP refill option. First, some interviewees reported that getting PrEP at clinics was more convenient, as they could access other health services needed while there. Second, some interviewees said that the cost of pharmacy-delivered PrEP services deterred them from seeking PrEP refills at a pharmacy. Third, a few interviewees explained that they had established a relationship with PrEP providers at the clinic during their initiation visit and wished to continue seeing the same providers for follow-up, rather than new providers at the pharmacy. Fourth, a handful of interviewees expressed concern that pharmacy-based PrEP services would be lower quality in terms of provider competency and forthrightness, worrying, for example, that pharmacy providers would not maintain confidentiality. Lastly, despite having enrolled in the pilot study, two interviewees said they were unaware of the pharmacy PrEP refill option.

Findings from this mixed-methods implementation study suggest that individuals who initiate PrEP at public clinics may prefer accessing services there and that giving such clients the option to refill PrEP at nearby private pharmacies may do little to improve PrEP continuation among this demographic. Although PrEP continuation among participants in this pilot was comparable to that observed in other clinic-based PrEP implementation studies [ 38 , 39 , 40 ], almost no participants opted to refill PrEP at nearby private pharmacies. Most participant expressed uncertainty as to whether they thought pharmacy-delivered PrEP services would be acceptable and appropriate, which may have been driven by their lack of first-hand experience with this new PrEP delivery model. Participants’ concerns around the cost and quality of pharmacy-delivered PrEP refills may have further driven their low selection of this refill option, as could have a general lack of knowledge of this option among some participants.

Few participants may have selected pharmacy PrEP refills in this study because those enrolled may have already overcome barriers to clinic-delivered PrEP services and did not want to reinitiate PrEP care in a new service delivery location. We also did not implement any demand generation strategies to entice clients who might otherwise not initiate clinic-based PrEP services without a pharmacy-based refill option. As indicated by some interviewees, the clients we enrolled might have had a preexisting preference for clinic-delivered services because they or their sexual partners (especially those living with HIV) were already accessing other services, such as family planning or antiretroviral therapy, there. Due to the relatively short duration of the pilot (13 months), it is also possible that participants might not have selected to refill PrEP at pharmacies because this option was time-limited, which would have required them to reengage in clinic-delivered PrEP services eventually if they had long-term PrEP continuation goals.

The low uptake of pharmacy-delivered PrEP refills could also be partially attributable to the novelty and limited scale of this PrEP delivery model. Because pharmacy-delivered PrEP services are not widespread in Kenya and only available at a handful of pharmacies participating in research studies, participants had reason to be skeptical about the quality of pharmacy PrEP services (e.g., pharmacy provider competency) delivered in this new setting – especially considering there is currently no governmental guidelines, formal training curriculum, or oversight for pharmacy providers to deliver PrEP services. Additionally, our training of clinic-based PrEP providers on the option of pharmacy PrEP refills might not have been adequate, resulting in some providers not being aware of or fully understanding how this option worked, which could have resulted in incomplete information on the option being relayed to potential participants.

When the findings from this study are compared with those from two recently completed pilot studies in Kenya, which tested a model in which trained pharmacy providers both initiated and continued clients on PrEP with great success [ 18 , 19 ], the evidence suggests that individuals who seek health services at public clinics may not be exchangeable with those who seek health services at private pharmacies. Modifications to our model where private pharmacies only dispense PrEP refills may be needed to potentially improve PrEP continuation outcomes, reach new PrEP clients, and decongest overburdened public clinics. Potential modifications could include adding demand generation strategies that target private pharmacies for the recruitment of new PrEP clients (who are then referred to public clinics for PrEP initiation), further subsidizing the cost of pharmacy PrEP refills (i.e., with client vouchers), or layering interventions that support linkage between clinics and pharmacies (e.g., client navigation services). These modifications may be needed because despite the demonstrated the feasibility of a stand-alone model of pharmacy-delivered PrEP initiation services [ 18 , 19 ], in many settings, including Kenya, current policies (e.g., pharmacy provider scope of practice) do not allow task shifting PrEP prescribing and HIV testing to pharmacy providers.

This study had some limitations. First, we only implemented the pilot at two public clinics and three private pharmacies in Kenya, thus limiting the generalizability of our findings to other settings within and outside of Kenya. Second, our verbal referral to nearby pharmacies for PrEP refills may have been too simplistic; clinic-based PrEP clients interested in pharmacy refills may have benefited from a more formalized referral process supported with structured forms or financial incentives. Third, since most study participants did not refill PrEP at pharmacies, our observed implementation outcomes measured participants’ perceived acceptability and appropriateness of a model they never experienced; perceptions are often subject to change after individuals experience an intervention unfamiliar to them [ 41 ]. Fourth, all PrEP adherence outcomes were self-reported and thus subject to social desirability bias; they also were primarily reflective of clinic-based PrEP service delivery because few participants selected pharmacy-based refills. Fifth, our qualitative findings only reflected the perspectives of those who refilled PrEP at a clinic and not those who never returned for clinic-based PrEP refills and may have preferred a pharmacy-based refill option. Finally, since much of the pilot implementation occurred during the COVID-19 pandemic, our outcomes on selected location for PrEP refilling could have been biased by perceptions participants had about their safety and potential risk of COVID infection in these settings.

The findings from this study emphasize the importance of developing client-centered models for PrEP service delivery that meet individuals who could potentially benefit from PrEP services where they are at and cater to their individual preferences. If individuals have a strong preference for the delivery of health services in a particular setting, instead of referring them elsewhere, we should continue to develop interventions that meet their needs and update supporting policies and supply chains to enable and facilitate these interventions. In Africa, private pharmacies are staffed by trained healthcare professionals and frequently deliver sexual and reproductive health services; pharmacy-based HIV prevention and treatment interventions could reach new populations who could benefit from these services and bring us closer to ending the AIDS epidemic [ 4 ].

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Abbreviations

Acquired immunodeficiency syndrome

Coronavirus Disease 2019

Human immunodeficiency virus

In-depth interview

Interquartile range

Kenyan Shilling

Pre-exposure prophylaxis

Rapid diagnostic testing

Short message service

United States dollar

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Acknowledgements

We are grateful for all the participants and providers who participated in this study. We would also like to thank the research staff at the Partners in Health and Research Development clinic for their time and contributions. We acknowledge the County Government of Kiambu for their collaboration and support of this research.

This research was supported by supported by the National Institute of Allergy and Infectious Disease (P30 AI027757, PI: Celum). KFO received additional support from the National Institute of Mental Health (R00 MH121166, PI: Ortblad and R34 MH120106, PI: Ortblad).

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KFO obtained funding for this research, with support from JMB. PM, CK, and KN oversaw implementation of the research in Kenya. APK, SG, and NW managed the quantitative and qualitative study data. APK and NW contributed to data analysis. KFO, APK, and SDR drafted the first version of this manuscript. All authors (KFO, APK, PM, SDR, CK, NW, SG, JMB, KN) reviewed the manuscript, added comments and edits for consideration, and approved the manuscript for publication.

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Ortblad, K.F., Kuo, A.P., Mogere, P. et al. Low selection of HIV PrEP refills at private pharmacies among clients who initiated PrEP at public clinics: findings from a mixed-methods study in Kenya. BMC Health Serv Res 24 , 618 (2024). https://doi.org/10.1186/s12913-024-10995-0

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Challenges and opportunities of English as the medium of instruction in diploma midwifery programs in Bangladesh: a mixed-methods study

Anna Williams 1 ,
Jennifer R. Stevens 2 ,
Rondi Anderson 3 &
Malin Bogren 4

BMC Medical Education volume 24 , Article number: 523 ( 2024 ) Cite this article

Metrics details

English is generally recognized as the international language of science and most research on evidence-based medicine is produced in English. While Bangla is the dominant language in Bangladesh, public midwifery degree programs use English as the medium of instruction (EMI). This enables faculty and student access to the latest evidence-based midwifery content, which is essential for provision of quality care later. Yet, it also poses a barrier, as limited English mastery among students and faculty limits both teaching and learning.

This mixed-methods study investigates the challenges and opportunities associated with the implementation of EMI in the context of diploma midwifery education in Bangladesh. Surveys were sent to principals at 38 public midwifery education institutions, and 14 English instructors at those schools. Additionally, ten key informant interviews were held with select knowledgeable stakeholders with key themes identified.

Surveys found that English instructors are primarily guest lecturers, trained in general or business English, without a standardized curriculum or functional English language laboratories. Three themes were identified in the key informant interviews. First, in addition to students’ challenges with English, faculty mastery of English presented challenges as well. Second, language labs were poorly maintained, often non-functional, and lacked faculty. Third, an alternative education model, such as the English for Specific Purposes (ESP) curriculum, has potential to strengthen English competencies within midwifery schools.

Conclusions

ESP, which teaches English for application in a specific discipline, is one option available in Bangladesh for midwifery education. Native language instruction and the middle ground of multilingualism are also useful options. Although a major undertaking, investing in an ESP model and translation of technical midwifery content into relevant mother tongues may provide faster and more complete learning. In addition, a tiered system of requirements for English competencies tied to higher levels of midwifery education could build bridges to students to help them access global evidence-based care resources. Higher levels might emphasize English more heavily, while the diploma level would follow a multilingualism approach, teach using an ESP curriculum, and have complementary emphasis on the mother tongue.

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Introduction

As the international language of science, English holds an important position in the education of healthcare professionals. Globally, most scientific papers are published in English. In many non-native English-speaking countries, English is used as the language of instruction in higher education [ 1 ]. The dominant status held by the English language in the sciences is largely considered to increase global access to scientific information by unifying the scientific community under a single lingua franca [ 2 ].

In Bangladesh, where the mother tongue is Bangla and midwifery diploma programs are taught in English, knowledge of English facilitates student and instructor access to global, continuously updated evidence-based practice guidance. This includes basic and scientific texts, media-based instructional materials (including on life-saving skills), professional journals, and proceedings of medical conferences. Many of these resources are available for free online, which can be particularly useful in healthcare settings that have not integrated evidence-based practice.

In addition to opportunity though, English instruction also creates several challenges. Weak student and faculty English competency may impede midwifery education quality in Bangladesh. Globally, literature has linked limited instructor competency in the language of instruction with reduced depth, nuance, and accuracy in conveying subject matter content [ 3 ]. This can lead to the perpetuation of patterns of care in misalignment with global evidence. In addition, students’ native language proficiency in their topic of study can decline when instruction is in English, limiting native language communication between colleagues on the job later on [ 4 , 5 ].

In this paper, we examine the current status of English language instruction within public diploma midwifery programs in Bangladesh. Midwifery students are not required to demonstrate a certain skill level in English to enter the program. However, they are provided with English classes in the program. Midwifery course materials are in English, while—for ease and practicality—teaching aids and verbal classroom instruction are provided in Bangla. Following graduation, midwifery students must pass a national licensing exam given in English to practice. Upon passing, some new midwives are deployed as public employees and are posted to sub-district health facilities where English is not used by either providers or clients. Others will seek employment as part of non-governmental organization (NGO) projects where English competency can be of value for interacting with global communities, and for participating in NGO-specific on-the-job learning opportunities. The mix of both challenge and opportunity in this context is complex.

Our analysis examines the reasons for the identified English competency gaps within midwifery programs, and potential solutions. We synthesize the findings and discuss solutions in the context of the global literature. Finally, we present a set of viable options for strengthening English competencies among midwifery faculty and students to enable better quality teaching and greater learning comprehension among students.

Study design

We employed a mixed-methods study design [ 6 ] in order to assess the quality of English instruction within education programs, and options for its improvement. Data collection consisted of two surveys of education institutes, a web-search of available English programs in Bangladesh, and key informant interviews. Both surveys followed a structured questionnaire with a combination of open- and closed-ended questions and were designed by the authors. One survey targeted the 38 institute principals and the other targeted 14 of the institutes’ 38 English instructors (those for whom contact information was shared). The web-search focused on generating a list of available English programs in Bangladesh that had viable models that could be tapped into to strengthen English competencies among midwifery faculty and students. Key informant interviews were unstructured and intended to substantiate and deepen understanding of the survey and web-search findings.

No minimum requirements exist for students’ English competencies upon entry into midwifery diploma programs. Students enter directly from higher secondary school (12th standard) and complete the midwifery program over a period of three years. Most students come from modest economic backgrounds having completed their primary and secondary education in Bangla. While English instruction is part of students’ secondary education, skill attainment is low, and assessment standards are not in place to ensure student mastery. To join the program, midwifery students are required to pass a multi-subject entrance exam that includes a component on English competency. However, as no minimum English standard must be met, the exam does not screen out potential midwifery students. Scoring, for instance, is not broken down by subject. This makes it possible to answer zero questions correctly in up to three of the subjects, including English, and pass the exam.

Processes/data collection

Prior to the first survey, principals were contacted by UNFPA with information about the survey and all provided verbal consent to participate. The survey of principals collected general information about the resources available for English instruction at the institutes. It was a nine-item questionnaire with a mix of Yes/No, multiple choice and write-in questions. Specific measures of interest were whether and how many English instructors the institutes had, instructors’ hiring criteria, whether institutes had language labs and if they were in use, and principals’ views on the need for English courses and their ideal mode of delivery (e.g., in-person, online, or a combination). This survey also gathered contact information of institute English instructors. These measures were chosen as they were intended to provide a high-level picture of institutes’ English resources such as faculty availability and qualifications, and use of language labs. To ensure questions were appropriately framed, a pilot test was conducted with two institute principals and small adjustments were subsequently made. Responses were shared via an electronic form sent by email and were used to inform the second survey as well as the key informant interviews. Of the 38 principals, 36 completed the survey.

The second survey, targeting English instructors, gathered information on instructors’ type of employment (e.g., institute faculty or adjunct lecturers); length of employment; student academic focus (e.g., midwifery or nursing); hours of English instruction provided as part of the midwifery diploma program; whether a standard English curriculum was used and if it was tailored toward the healthcare profession; use of digital content in teaching; education and experience in English teaching; and their views on student barriers to learning English. These measures were chosen to provide a basic criterion for assessing quality of English instruction, materials and resources available to students. For instance, instructors’ status as faculty would indicate a stronger degree of integration and belonging to the institute midwifery program than a guest lecturer status which allows for part time instruction with little job security. In addition, use of a standard, professionally developed English curriculum and integration of digital content into classroom learning would be indicative of higher quality than learning materials developed informally by instructors themselves without use of listening content by native speakers in classrooms. The survey was piloted with two English instructors. Based on their feedback, minor adjustments were made to one question, and it was determined that responses were best gathered by phone due to instructors’ limited internet access. Of the 14 instructors contacted, 11 were reached and provided survey responses by phone.

The web-search gathered information on available English language instruction programs for adults in Bangladesh, and the viability of tapping into any of them to improve English competency among midwifery students and faculty. Keywords Bangladesh + English courses , English training , English classes , study English and learn English were typed into Google’s search platform. Eleven English language instruction programs were identified. Following this, each program was contacted either by phone or email and further detail about the program’s offerings was collected.

Unstructured key informant interviews were carried out with select knowledgeable individuals to substantiate and enhance the credibility of the survey and web-search findings. Three in-country expert English language instructors and four managers of English language teaching programs were interviewed. In addition, interviews were held with three national-level stakeholders knowledgeable about work to make functional technologically advanced English language laboratories that had been installed at many of the training institutes. Question prompts included queries such as, ‘In your experience, what are the major barriers to Bangla-medium educated students studying in English at the university level?’, ‘What effective methods or curricula are you aware of for improving student English to an appropriate competency level for successful learning in English?’, and, ‘What options do you see for the language lab/s being used, either in their originally intended capacity or otherwise?’

Data analysis

All data were analyzed by the lead researcher. Survey data were entered into a master Excel file and grouped descriptively to highlight trends and outliers, and ultimately enable a clear description of the structure and basic quality attributes (e.g., instructors’ education, hours of English instruction, and curriculum development resources used). Web-search findings were compiled in a second Excel file with columns distinguishing whether they taught general English (often aimed at preparing students for international standard exams), Business English, or English for Specific Purposes (ESP). This enabled separation of standalone English courses taught by individual instructors as part of vocational or academic programs of study in other fields, and programs with an exclusive focus on English language acquisition. Key informant interviews were summarized in a standard notes format using Word. An inductive process of content analysis was carried out, in which content categories were identified and structured to create coherent meaning [ 7 ]. From this, the key overall findings and larger themes that grew from the initial survey and web-search results were drawn out.

The surveys (Tables 1 and 2 ) found that English instructors are primarily long-term male guest lecturers employed at each institute for more than two years. All principal respondents indicated that there is a need for English instruction—18 of the 19 reported that this is best done through a combination of in-person and computer-based instruction. Ten institutes reported that they have an English language lab, but none were used as such. The other institutes did not have language labs. The reported reasons for the labs not being in use were a lack of trained staff to operate them and some components of the technology not being installed or working properly. The findings from the instructors’ survey indicated that English instructors typically develop their own learning materials and teach general English without tailoring content to healthcare contexts. Only two mentioned using a standard textbook to guide their instruction and one described consulting a range of English textbooks to develop learning content. None reported using online or other digital tools for language instruction in their classrooms. Most instructors had an advanced degree (i.e., master’s degree) in English, and seven had received training in teaching English. Interviews with instructors also revealed that they themselves did not have mastery of English, as communication barriers in speaking over the phone appeared consistently across 10 of the 11 instructor respondents.

The web-search and related follow up interviews found that most English instruction programs (10 out of the 11) were designed for teaching general English and/or business English. The majority were offered through private entities aiming to reach individuals intending to study abroad, access employment that required English, or improve their ability to navigate business endeavors in English. One program, developed by the British Council, had flexibility to tailor its structure and some of its content to the needs of midwifery students. However, this was limited in that a significant portion of the content that would be used was developed for global audiences and thus not tailored to a Bangladeshi audience or to any specific discipline. One of the university English programs offered a promising ESP model tailored to midwifery students. It was designed by BRAC University’s Institute of Language for the university’s private midwifery training program.

Three themes emerged from the other key informant interviews (Table 3 ). The first was that, in addition to students’ challenges with English, faculty mastery of English presented challenges as well. Of the 34 faculty members intending to participate in the 2019–2020 cohort for the Dalarna master’s degree, half did not pass the prerequisite English exam. Ultimately, simultaneous English-Bangla translation was necessary for close to half of the faculty to enable their participation in the master’s program. English language limitations also precluded one faculty member from participating in an international PhD program in midwifery.

The second theme highlighted the language labs’ lack of usability. The language labs consisted of computers, an interactive whiteboard, audio-visual equipment, and associated software to allow for individualized direct interactions between teacher and student. However, due to the lack of appropriately trained staff to manage, care for and use the language lab equipment, the investment required to make the labs functional appeared to outweigh the learning advantages doing so would provide. Interviews revealed that work was being done, supported by a donor agency, on just one language lab, to explore whether it could be made functional. The work was described as costly and challenging, and required purchasing a software license from abroad, thus likely being impractical to apply to the other labs and sustain over multiple years.

The third theme was around the ESP curriculum model. The program developers had employed evidence-informed thinking to develop the ESP learning content and consulted student midwives on their learning preferences. Due to the student input, at least 80% of the content was designed to directly relate to the practice of midwifery in Bangladesh, while the remaining 10–20% references globally relevant content. This balance was struck based on students’ expressed interest in having some exposure to English usage outside of Bangladesh for their personal interest. For conversation practice, the modules integrated realistic scenarios of midwives interacting with doctors, nurses and patients. Also built into written activities were exercises where students were prompted to describe relevant health topics they are concurrently studying in their health, science or clinical classes. Given the midwifery students’ educational backgrounds and intended placements in rural parts of Bangladesh, an ESP curriculum model appeared to be the most beneficial existing program to pursue tapping into to strengthen English competencies within midwifery programs. This was because the content would likely be more accessible to students than a general English course by having vocabulary, activities and examples directly relevant to the midwifery profession.

The study findings demonstrate key weaknesses in the current model of English instruction taught in public midwifery programs. Notably, the quantitative findings revealed that some English instructors do not have training in teaching English, and none used standard curricula or online resources to structure and enhance their classroom content. In addition, weak mastery of English among midwifery faculty was identified in the qualitative data, which calls into question faculty’s ability to fully understand and accurately convey content from English learning materials. Global literature indicates that this is not a unique situation. Many healthcare faculty and students in low-resource settings, in fact, are faced with delivering and acquiring knowledge in a language they have not sufficiently mastered [ 8 ]. As a significant barrier to knowledge and skill acquisition for evidence-based care, this requires more attention from global midwifery educators [ 9 ].

Also holding back students’ English development is the finding from both the quantitative and qualitative data that none of the high-tech language labs were being used as intended. This indicates a misalignment with the investment against the reality of the resources at the institutes to use them. While setting up the costly language labs appears to have been a large investment with little to no return, it does demonstrate that strengthening English language instruction in post-secondary public education settings is a priority that the Bangladesh government is willing to invest in. However, scaling up access to an ESP curriculum model tailored to future midwifery practitioners in Bangladesh may be a more worthwhile investment than language labs [ 10 ].

The ESP approach teaches English for application in a specific discipline. It does this by using vocabulary, examples, demonstrations, scenarios and practice activities that are directly related to the context and professions those studying English live and work (or are preparing to work) in. One way ESP has been described, attributed to Hutchinson and Waters (1987), is, “ESP should properly be seen not as any particular language product but as an approach to language teaching in which all decisions as to content and method are based on the learner’s reason for learning” [ 11 ]. It is proposed by linguistic education researchers as a viable model for strengthening language mastery and subject matter comprehension in EMI university contexts [ 12 ].

Though it did not arise as a finding, reviewing the literature highlighted that Bangla language instruction may be an additional, potentially viable option. Linguistic research has long shown that students learn more thoroughly and efficiently in their mother tongue [ 12 ]. Another perhaps more desirable option may be multilingualism, which entails recognizing native languages as complementary in EMI classrooms, and using them through verbal instruction and supplemental course materials. Kirkpatrick, a leading scholar of EMI in Asia, suggests that multilingualism be formally integrated into EMI university settings [ 13 ]. This approach is supported by evidence showing that the amount of native language support students need for optimal learning is inversely proportional to their degree of English proficiency [ 14 ].

Ultimately, despite the language related learning limitations identified in this study, and the opportunities presented by native language and multilingualism approaches, there remains a fundamental need for members of the midwifery profession in Bangladesh to use up-to-date guidance on evidence-based midwifery care [ 11 ]. Doing that currently requires English language competence. Perhaps a tiered system of requirements for English competencies that are tied to diploma, Bachelor’s, Master’s and PhD midwifery programs could build bridges for more advanced students to access global resources. Higher academic levels might emphasize English more heavily, while the diploma level could follow a multilingualism approach—teaching using an ESP curriculum and integrating Bangla strategically to support optimal knowledge acquisition for future practice in rural facilities. Ideally, scores on a standard English competency exam would be used to assess students’ language competencies prior to entrance in English-based programs and that this would require more stringent English skill development prior to entering a midwifery program.

Methodological considerations

One of the limitations of this study is that it relied on self-reports and observation, rather than tested language and subject matter competencies. Its strengths though are in the relatively large number of education institutes that participated in the study, and the breadth of knowledge about faculty and student subject matter expertise among study co-authors. It was recognized that the lead researcher might be biased toward pre-determined perceptions of English competencies being a barrier to teaching and learning held by the lead institution (UNFPA). It was also recognized that due to the inherent power imbalance between researcher and participants, the manner of gathering data and engaging with stakeholders may contribute to confirmation bias, with respondents primarily sharing what they anticipated the researcher wished to hear (e.g., that English needed strengthening and the lead agency should take action to support the strengthening). The researcher thus engaged with participants independently of UNFPA and employed reflexivity by designing and carrying out the surveys to remotely collect standard data from institutes, as well as casting a wide net across institutes to increase broad representation. In addition, while institutes were informed that the surveys were gathering information about the English instruction within the institutes, no information was shared about potential new support to institutes. Finally, the researcher validated and gathered further details on the relevant information identified in the surveys through key informant interviews, which were held with stakeholders independent of UNFPA.

Adapting and scaling up the existing ESP modules found in this study, and integrating Bangla where it can enhance subject-matter learning, may be a useful way to help midwifery students and faculty improve their knowledge, skills, and critical thinking related to the field of midwifery. Given the educational backgrounds and likely work locations of most midwives in Bangladesh and many other LMICs, practitioners may want to consider investing in more opportunities for local midwives to teach and learn in their mother tongue. This type of investment would ideally be paired with a tiered system in which more advanced English competencies are required at higher-levels of education to ensure integration of global, evidence-based approaches into local standards of care.

Declarations.

Data availability

The datasets used and analyzed during the current study are available from the corresponding author upon reasonable request.

Abbreviations

Bangladesh Rehabilitation Assistance Committee

English medium instruction

English for Specific Purposes

Low- and Middle-Income Countries

Ministry of Health and Family Welfare

United Nations Population Fund

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Acknowledgements

The authors acknowledge Farida Begum, Rabeya Basri, and Pronita Raha for their contributions to data collection for this assessment.

This project under which this study was carried out was funded by funded by the Foreign Commonwealth and Development Office.

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This study was part of a larger project in Bangladesh approved by the Ministry of Health and Family Welfare (MOHFW) with project ID UZJ31. The MOHFW project approval allows data collection of this type, that is carried out as part of routine program monitoring and improvement, including informed verbal consent for surveys and key informant interviews.

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Williams, A., Stevens, J., Anderson, R. et al. Challenges and opportunities of English as the medium of instruction in diploma midwifery programs in Bangladesh: a mixed-methods study. BMC Med Educ 24 , 523 (2024). https://doi.org/10.1186/s12909-024-05499-8

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Published: 09 June 2023

Genomics of cold adaptations in the Antarctic notothenioid fish radiation

Iliana Bista ORCID: orcid.org/0000-0002-6155-3093 1 , 2 , 3 , 4 ,
Jonathan M. D. Wood ORCID: orcid.org/0000-0002-7545-2162 1 ,
Thomas Desvignes 5 ,
Shane A. McCarthy 1 , 2 ,
Michael Matschiner ORCID: orcid.org/0000-0003-4741-3884 6 , 7 ,
Zemin Ning 1 ,
Alan Tracey ORCID: orcid.org/0000-0002-4805-9058 1 ,
James Torrance 1 ,
Ying Sims 1 ,
William Chow 1 ,
Michelle Smith 1 ,
Karen Oliver 1 ,
Leanne Haggerty 8 ,
Walter Salzburger ORCID: orcid.org/0000-0002-9988-1674 9 ,
John H. Postlethwait ORCID: orcid.org/0000-0002-5476-2137 5 ,
Kerstin Howe ORCID: orcid.org/0000-0003-2237-513X 1 ,
Melody S. Clark ORCID: orcid.org/0000-0002-3442-3824 10 ,
H. William Detrich III ORCID: orcid.org/0000-0002-0783-4505 11 ,
C.-H. Christina Cheng 12 ,
Eric A. Miska ORCID: orcid.org/0000-0002-4450-576X 1 , 3 &
Richard Durbin ORCID: orcid.org/0000-0002-9130-1006 1 , 2

Nature Communications volume 14 , Article number: 3412 ( 2023 ) Cite this article

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Adaptive radiation
Comparative genomics
Evolutionary biology
Evolutionary genetics

Numerous novel adaptations characterise the radiation of notothenioids, the dominant fish group in the freezing seas of the Southern Ocean. To improve understanding of the evolution of this iconic fish group, here we generate and analyse new genome assemblies for 24 species covering all major subgroups of the radiation, including five long-read assemblies. We present a new estimate for the onset of the radiation at 10.7 million years ago, based on a time-calibrated phylogeny derived from genome-wide sequence data. We identify a two-fold variation in genome size, driven by expansion of multiple transposable element families, and use the long-read data to reconstruct two evolutionarily important, highly repetitive gene family loci. First, we present the most complete reconstruction to date of the antifreeze glycoprotein gene family, whose emergence enabled survival in sub-zero temperatures, showing the expansion of the antifreeze gene locus from the ancestral to the derived state. Second, we trace the loss of haemoglobin genes in icefishes, the only vertebrates lacking functional haemoglobins, through complete reconstruction of the two haemoglobin gene clusters across notothenioid families. Both the haemoglobin and antifreeze genomic loci are characterised by multiple transposon expansions that may have driven the evolutionary history of these genes.

The genomic timeline of cichlid fish diversification across continents

Genomic insights into the secondary aquatic transition of penguins

Draft genome assembly and transcriptome data of the icefish Chionodraco myersi reveal the key role of mitochondria for a life without hemoglobin at subzero temperatures

Introduction.

The suborder Notothenioidei is a textbook example of a marine fish adaptive radiation, with notothenioids being the dominant fish group of the Southern Ocean, both in terms of species richness and biomass, comprising between 130–140 species 1 , 2 , 3 (Fig. 1a ). The establishment and initial diversification of the notothenioids is closely linked to the separation of the Antarctic continent from surrounding land masses and the subsequent establishment of the Antarctic Circumpolar Current (ACC) 4 (Fig. 1b ). Formation of the ACC exacerbated the isolation of the Antarctic continent and contributed to cooling of the surrounding waters, glaciation of the continent, and appearance of sea ice 5 . These events in turn extirpated most of the original temperate fish fauna, and notothenioids expanded to fill the abandoned niches as they evolved adaptations to life in the isolated, cold, and highly oxygenated waters of the Southern Ocean 6 , 7 . Since notothenioids include species occurring in cool-temperate non-Antarctic regions 8 , as well as species occurring in icy, freezing higher latitudes (known as the “Antarctic clade” or Cryonotothenioidea (cryonotothenioids)) 9 , they represent a powerful model for the study of the genomic origins of extremophiles. Adaptations to cold include the presence of antifreeze glycoproteins (AFGPs) 10 , the lack of the classic heat shock response 11 , and the presence of giant muscle fibres in some notothenioids 12 . Further, a striking respiratory phenotype arose in the derived family Channichthyidae (“icefishes”), including the complete loss of functional haemoglobins in all of its species and the loss of cardiac myoglobin in six of them 13 . While haemoglobin loss was not lethal in the oxygenated waters of the cold Southern Ocean, these losses were likely not without fitness consequences, as indicated by numerous compensatory cardiovascular adaptations, including enlarged hearts, and increased vascular bores 4 .

a Notothenioid families and a number of species contained in each family based on the species list by ref. 3 are shown in parentheses, except for the Nototheniidae, which are paraphyletic, containing the Nototheniinae, G. gibberifrons and N. rossi . The target species sequenced in the present study are listed next to the tree. b Map of Antarctica and the Southern Ocean showing the geographic distribution of the five notothenioid species sequenced with PacBio. Colours correspond to the respective families on the tree. Coloured circles on the map indicate the sampling location. The blue belt around Antarctica indicates the region of the Antarctic Circumpolar Current (ACC), and the thin blue line the Antarctic polar front. The map was generated using ArcGIS (GIS Software, Version 10.0). Data for the geographic distribution of each species were derived from the Scientific Committee for Antarctic Research (SCAR), Antarctic Biodiversity Portal ( https://www.biodiversity.aq/ ), comprising occurrence records from multiple databases. Source data are provided as a Source Data file.

The in-depth characterisation of notothenioid genomes has been hampered in the past by their complex genome characteristics, such as high levels of repeats and heterozygosity, that have hindered the accuracy of genome assemblies based on short-read data. Furthermore, the few available high-quality notothenioid genome assemblies 14 , 15 cover only a small portion of the diversity in this group. The Vertebrate Genomes Project (VGP) ( https://vertebrategenomesproject.org/ ) 16 has demonstrated that long-read sequencing technologies can generate highly contiguous genome assemblies even for the most technically difficult species.

In this work, we achieve a step-change increase in Antarctic notothenioid genome resources for the broader community. We apply the VGP pipeline and standards to five selected notothenioid species representing key points in the radiation, and use other sequencing approaches, such as Illumina and linked reads to generate a total of 24 new genomes. Collectively these assemblies cover six of the eight notothenioid families (all except two non-Antarctic single-species families) (Fig. 1a ), including the five families that comprise the Antarctic radiation, and a non-Antarctic family. We use these new genome assemblies to address previously unresolved questions about the evolutionary history of the radiation. First, we present a new time-calibrated phylogeny, and with it a new time estimate for the expansion of the radiation. Next, we identify a significant genome size expansion and investigate the role of transposable elements (TEs) in cold adaptation throughout this adaptive radiation. Finally, we investigate the evolutionary history of two adaptively important gene families, the antifreeze genes, which were essential for survival in icy waters, and the haemoglobin genes, which were ultimately lost in icefish, the only vertebrate lacking functional haemoglobins.

Results and discussion

Genome sequencing, assembly, and annotation.

We generated and analysed reference genome assemblies for 24 notothenioid fish species across the radiation using a variety of sequencing technologies (Fig. 1 , Table 1 , Supplementary Data 1 ). Genomes of five species — Cottoperca gobio (synonymised by many to Cottoperca trigloides 3 ) , Trematomus bernacchii, Harpagifer antarcticus, Gymnodraco acuticeps, Pseudochaenichthys georgianus — each representing a different notothenioid family, were assembled using Pacific Biosciences (PacBio) long reads, in combination with 10X Genomics linked reads and Hi-C data (Methods). Briefly, we used Falcon-unzip 17 to generate each primary assembly from the PacBio reads and then applied 10X Genomics Chromium data for scaffolding and polishing. The genomes of C. gobio , H. antarcticus and P. georgianus were further scaffolded using Bionano hybrid scaffolding. In addition, for C. gobio and P. georgianus we also used Hi-C data (ARIMA and Dovetail respectively) for scaffolding with SALSA2 18 . To further improve the quality of these genomes, we performed manual curation using the Genome Evaluation Browser (gEVAL) 19 to remove mis-assemblies, false duplications, and sequencing contaminations such as symbionts and adapter sequences, and to merge scaffolds based on supporting evidence 20 (Supplementary Data 2 ). The genomes of C. gobio and P. georgianus , were assigned to 24 chromosomes, consistent with their known karyotypes 21 , 22 (Supplementary Fig. 1a–b ), with scaffold N50 25 Mb and 43 Mb, respectively. The reference assembly for C. gobio (fCotGob3.1, GCA_900634415.1) was previously described in ref. 23 . The other PacBio genomes were assembled to scaffolds, T. bernacchii (N50 8.8 Mb), H. antarcticus (N50 5.0 Mb), and G. acuticeps (N50 1.9 Mb). Genomes of 11 more species were sequenced with 10X Genomics using a single linked-read library for each and assembled with Supernova v2.0 24 with an average N50 of 2.6 Mb ( Supplementary Data 2 ). Genomes of eight additional species were sequenced using only Illumina HiSeq reads and assembled using a reference-guided approach. For these eight species, a primary assembly was generated with SOAPdenovo2 25 , and scaffolding was done using the closest PacBio genome as reference (except for Bathydraco marri for which scaffolding with a reference assembly failed) (Methods, Supplementary Data 3 ). This approach generally improved the N50 and BUSCO completeness for these species compared to the primary assembly. All 19 assemblies were also manually curated to remove external contamination, and false duplications (the latter in Supernova assemblies). We observed a smaller genome size in short read assemblies, compared to the PacBio and linked read ones from the same families (Supplementary Data 2 ). We attribute this to short read assemblers tending to collapse repeat regions during the assembly process 16 .

For the PacBio assemblies, BUSCO 26 gene completeness averages 95% (Supplementary Fig. 1c ), for 10X assemblies BUSCO averages 86%, and for short read assemblies 65%. Gene prediction for all PacBio assemblies was performed via the Ensembl Gene Annotation system 27 (Methods). The BUSCO completeness of the gene annotation averages 92% (Supplementary Fig. 1d ). Approximately 23–24,000 genes were annotated for the four cryonotothenioid species (24,373 for T. bernacchii , 23,146 for H. antarcticus , 24,091 for G. acuticeps , and 23,222 for P. georgianus (Supplementary Data 4 )), around 2000 genes more than the non-Antarctic species C. gobio (Ensembl genes: 21,662 23 ), suggesting that cold adaptation was accompanied by an expansion in the number of genes in notothenioids (Supplementary Fig. 1e–f ).

A new time-calibrated phylogeny for notothenioids

Our multi-species dataset affords the opportunity to establish a new time-calibrated phylogeny for the notothenioid radiation based on genome-wide data, to help resolve controversies about the timing of the diversification of the notothenioids relative to the chilling of the Southern Ocean. Most of the previously published phylogenetic hypotheses for notothenioids were based on limited numbers of genes 6 , RAD-seq 28 , 29 , or exome capture data 30 . By analysing genome-wide data extracted from BUSCO single copy genes from 41 percomorph fish species, including the 24 new and eight previously published notothenioid genomes 31 , 32 we provide the most comprehensive phylogenomic analyses of notothenioids to date, with taxonomic coverage of most of their sub-groups. Based on this analysis, calibrated using established teleost divergence dates 33 , the onset of diversification of the Cryonotothenioidea, which are characterised by the presence of AFGPs, is estimated at around 10.7 million years ago (MYA) (highest posterior density interval: 14.1–7.8 MYA) (Fig. 2a ). Previous work estimated this time at ~22 MYA 6 , 34 . While the appearance of AFGPs was previously estimated at 42–22 MYA, which would predate the major cooling of the Southern Ocean 6 , our new analysis indicates that the emergence of AFGPs occurred between 26.3–10.7 MYA. This period includes the Middle Miocene Climate Transition at 15–13 MYA and the subsequently increased Antarctic glaciation 35 . Furthermore, our analysis highlights that many speciation events in each major family took place within the last 5 million years (Fig. 2a ).

a Time-calibrated phylogeny of 41 percomorph fish species, including 31 species of notothenioids and 10 outgroup fish species, generated with BEAST2 39 . Branch length corresponds to time in million years (MYA) and grey rectangles show 95% highest posterior density intervals for node age estimates. All nodes received full support (Bayesian posterior probability = 1) except where noted. Species in bold were sequenced in this study. Branches for the Antarctic clade are highlighted in blue (cryonotothenioids), and non-Antarctic notothenioid species are marked in green. b Diversification of notothenioid species and temperature variation through time. Tree based on notothenioid species from panel a. The scatterplot shows data based on deep-sea δ 18 O benthic records which inversely reflect temperature with higher δ 18 O benthic corresponding to lower temperatures (green) and lower δ 18 O corresponding to higher temperatures (orange). The oxygen benthic is expressed as a ratio of two concentrations of oxygen isotopes 36 ; blue line shows moving average (Generalised Additive Model). Source data can be found in the Dryad repository at https://doi.org/10.5061/dryad.80gb5mktn .

To investigate climatic events that might have driven diversification in derived notothenioid clades we examined paleoclimate data, represented by δ 18 O records (derived from the ratio of 18 O/ 16 O stable isotopes), which reflect variations in the temperature of seawater 36 . These data indicate substantial fluctuations in global mean sea levels (GMSL) during the early Pliocene, and a sustained temperature drop in Antarctica ~3 MYA, which led to the rapid formation of large sea ice sheets 36 . Variations in sea ice formation may have played an important role in isolating populations, leading to further diversification of the cryonotothenioids (Fig. 2b ). A similar influence of cooling events has been suggested in other non-Antarctic cold-adapted radiations (e.g. aquatic crustacea and orchids) 37 , 38 , where diversification has been linked to past changes in global temperature.

Furthermore, our BEAST2 39 analyses support the monophyly of dragonfishes (family Bathydraconidae, represented here by Vomeridens infuscipinnis, Akarotaxis nudiceps, Bathydraco marri , and G. acuticeps ), as indicated by morphology and by RADseq data 29 , while other methods and previous studies 30 , 40 suggested that they are paraphyletic. This result was also found with maximum likelihood phylogeny reconstruction using IQ-TREE 41 when using the “strict” set of alignments (see Methods for definition). However, we note that when using the”permissive” or full alignment sets, concatenated IQ-TREE found the alternative, paraphyletic phylogeny that places G. acuticeps with the Channnichthyidae, albeit with relatively weak bootstrap values of 82 and 70, respectively. All notothenioid nodes apart from this node were found in all analyses, and had bootstrap values of at least 97.0. ASTRAL 42 analysis of the IQTREE gene trees also place G. acuticeps with the Channnichthyidae with Bayesian posterior probability from 0.38 to 0.62 depending on the filtering level (see Methods). In contrast to previous studies 29 , 34 , none of our phylogenetic analyses group together the neutrally buoyant Pleuragramma antarcticum and Dissostichus spp. We therefore suggest that neutral buoyancy evolved independently in these two lineages.

Transposon expansion is driving genome size evolution in notothenioids

Transposable element dynamics are increasingly recognised as major drivers of evolutionary innovation, and their analysis is greatly facilitated by the use of long-read sequencing technologies 43 , 44 . For example, the location of TE insertions can influence the expression of nearby genes and induce phenotypic variation 45 , 46 . The diversity of transposons varies substantially between organisms, with teleost fish genomes containing greater TE diversity compared to other vertebrates, such as mammals 43 , 47 . In teleosts, genome size tends to correlate with transposon abundance, while a reduction in genome size does not necessarily correspond to lower transposon diversity, but is more commonly caused by reduced copy numbers of TEs 48 , 49 . Here, we use a set of long read and linked read assemblies, together with high-quality de novo annotations, to investigate the expansions of transposable elements in notothenioids in relation to their genome sizes. We also investigate the timing of these expansions with respect to major lineage diversification events in the radiation.

We identified substantial variation in assembled genome size across the notothenioid phylogeny with the smallest genomes identified in the non-Antarctic temperate-water family Bovichtidae (0.6 Gb), which form a sister group to all other extant notothenioids, and the largest genomes in the high-latitude icefish species of the derived family Channichthyidae (1.1 Gb) (Fig. 3 , Table 1 , Supplementary Data 2 ). This observation is consistent with earlier estimates of large genome sizes in icefish based on flow cytometry 50 . The variation in genome size is nearly completely accounted for by changes in the total repeat content, suggesting that it is driven by TE expansions (Fig. 3c ). Such expansions are found in diverse members of the Antarctic cryonotothenioids, including Dissostichus , the sister lineage to all other cryonotothenioids, indicating that the onset of TE expansion was associated with the radiation of the clade (Fig. 3a, b ). This potentially indicates that the onset of the TE expansion coincided with, or possibly predated, the first diversification event in cryonotothenioids. TE expansion continued in the more derived clades (e.g., dragonfishes and icefishes), consistent with lineage-dependent expansion characterised by multiple young insertions as seen in a TE landscape analysis (Fig. 3b ). Further, we found that this increase in TE content is due to the simultaneous amplification of multiple TE families, including both DNA transposons and retrotransposons, with the proportion in overall coverage remaining fairly stable throughout the phylogeny (Fig. 3c , Supplementary Fig. 2 , Supplementary Data 5 ). Overall, the bulk of the expansion seems to have resulted from the activation of existing TE families, as several TE families present in the Antarctic clade are also present at low copy numbers in the Bovichtidae. Few TE families were observed exclusively within individual clades, although some unclassified TE elements remained in the dataset even after extensive manual curation.

a Species analysed, including 16 species sequenced in this study, and two previously published genomes ( E. maclovinus 32 , and D. mawsoni 57 ). b Repeat landscape plots showing the distribution of transposable element copies as percentage of divergence from consensus repeat models ( x -axis, Kimura divergence) versus genome coverage ( y -axis). Colours represent different TE classes. The red arrow indicates the timing of the earliest TE expansion identified in our analysis. c Correlation of repeat content with genome size (Pearson Correlation Coefficient, n = 16, R = 0.95, two-sided p = 1.647e-08, slope = 0.99), an increase of repeat fraction with genome size, and increase of DNA, LINE, and LTR TE classes with genome size. The shaded zone indicates 95% confidence interval. The plot was generated using package ggplot2 and function ggpubr 99 . Double forward slashes in the time axis indicate a cropped line in the tree branches. Source data are provided as a Source Data file.

In notothenioids, the capacity of transposons to generate evolutionary novelty and shape the evolutionary potential of whole lineages 44 could be linked to the development of the adaptive features that characterise this radiation. To assess the influence of these transposition events on the genomic evolution of notothenioids, we selected the antifreeze glycoprotein ( afgp ) and the haemoglobin genomic loci as representative models for in-depth examinations.

Evolution of the antifreeze glycoprotein gene family

The appearance of the antifreeze glycoprotein genes ( afgp) in Antarctic notothenioid fishes was probably the most important innovation enabling survival in the sub-zero waters of the Southern Ocean. AFGPs prevent organismal freezing by binding to ice crystals that enter the body, thereby arresting ice growth 51 , 52 . The multigene afgp family encodes an array of AFGP size isoforms, whereby each gene is composed of two exons, exon 1 (E1) encoding a signal peptide, and exon 2 (E2) encoding an AFGP polyprotein 10 , 53 (Supplementary Fig. 3a ). The long polyprotein precursor comprises many AFGP molecules composed of varying numbers of repeats of a tripeptide (Thr-Ala-Ala), linked by conserved three-residue spacers (mostly Leu-X-Phe), which on post-translational removal yield the mature AFGPs 10 , 53 , 54 . Taken together, the tandemly arrayed afgp copies with their extremely repetitive coding sequences present formidable bioinformatic challenges, precluding accurate sequence assemblies and reconstructions of the entire antifreeze gene locus from genomic data until now. The most comprehensive representation of the locus to date was assembled from Sanger-based sequencing of BAC clones for D. mawsoni , although this still contains gaps and uncertainties 55 . Furthermore, some aspects of the evolutionary derivation of afgp from its trypsinogen-like protease ( tlp ) ancestral gene have not yet been fully resolved. Other uncertainties include why copies of the chimeric afgp/tlp genes, proposed to be evolutionary intermediates 53 , 56 , persist in extant genomes of the cryonotothenioids, the origin of the three-residue linker sequence in the AFGP polyprotein, and finally, the mechanism of expansion of afgp copies 55 .

Using long read data, we assembled the entire afgp locus into a single contiguous genomic sequence for a derived icefish species ( P. georgianus ). We also assembled the region corresponding to this locus in a species from a clade that separated prior to the appearance of afgps ( C. gobio ) (Fig. 4a.1,3 ). For comparison, we reanalysed the afgp locus of D. mawsoni (Fig. 4a.2 ), which represents one of the earlier diverging lineages of cryonotothenioids after afgp emergence 55 . In addition, we annotated afgp genes in three more genomes representing three different cryonotothenioid families ( H. antarcticus, T. bernacchii, G. acuticeps ) and located them in multiple scaffolds (Supplementary Fig. 4 ). Manual reassembly to resolve the breaks in these gene clusters in these three species was not possible due to the lack of sufficiently rich long-range data. The assembly of the afgp locus in P. georgianus , which spans more than 1 Mb in length (1074 kb), was manually curated to correct mis-assemblies and to verify gene completeness (Methods). The P. georgianus locus is approximately ten times the length of the corresponding region (113 kb) in C. gobio , and more than twice the length of the intermediate D. mawsoni locus (467 kb). Overall we observe a remarkable locus size expansion that appears to have accelerated in icefishes (Fig. 4a.3 ).

a Reconstructed physical map of the antifreeze locus for three notothenioid species: (1) C. gobio , which represents the ancestral state of the locus, (2) D. mawsoni , and (3) P. georgianus , which represent derived loci. The C. gobio and D. mawsoni loci are shown at the same scale, and the P. georgianus locus is shown in half scale and reverse orientation. Coloured triangles represent different genes and lilac rectangles represent afgp copies (see gene index). afgp : antifreeze glycoprotein genes, tlp : trypsinogen-like protease, tryp1 : trypsinogen1, tryp3 : trypsinogen3 (both tryp1 and tryp3 are prss59 homologues), tomm40 : translocase of outer mitochondrial membrane 40 homolog, hsl : hormone sensitive lipase (lipeb), afgp/tlp: chimeric afgp and tlp gene. b Cladogram of the three species analysed, with total length of locus, repeat content (%), number of afgp gene copies, and number of transposon copies annotated in each species locus (including DNA, LINE, LTRs, and SINE elements). Colours represent different TE classes as shown in legend. Source data are provided as a Source Data file.

Annotation of the afgp genes could not be extended to the short read assemblies due to limitations of the length of the sequencing data and the complexity of the locus. The afgp genes typically contain >1 kb to ~7 kb of extremely repetitive sequence due to the nine-nucloetide repeats encoding the many AFGP molecules in the polyprotein (tripeptide Thr-Ala-Ala cds) (Supplementary Fig. 3b ), and this precludes correct assembly using 150 bp Illumina data. Even if the overall repeat coverage of the region is estimated, this still does not allow for an accurate estimate of the gene copy number due to the length variability of individual genes 55 . We therefore focused on species with long read data for copy number estimation (Supplementary Fig. 4 ), and those with structurally complete loci for additional analysis (Fig. 4 ).

We identified 15 afgp genes in P. georgianus (Fig. 4a.3 ), of which eight are structurally intact and expected to be functional, while the other seven contain various mutations and are thus potentially pseudogenes. In addition, there is one chimeric afgp/tlp gene, previously regarded as a putative evolutionary intermediate of afgp genes. This also appears to be a pseudogene, because it lacks the signal peptide exon-1, possesses a premature termination codon in the AFGP-coding exon-2, and exon-2 would encode a single long run of 722 Thr-Ala-Ala tripeptide repeats (~6.5 kb) without any of the conserved, cleavable tripeptide linker sequences of the functional afgp polyprotein genes (Supplementary Fig. 3b ) . Re-annotation of the D. mawsoni afgp region identified 14 afgp copies, and three chimeric afgp/tlp genes, one of which appears to be complete in terms of reading frame and cleavage sites, and therefore potentially functional (Fig. 4a.2 ).

The large size discrepancy between the P. georgianus and D. mawsoni afgp locus cannot be explained by the expansion of afgp genes alone, as only one extra copy was found in the icefish species, but instead seems to be primarily due to an expansion of TEs. The repeat content of the locus substantially exceeds the average TE content of the respective genomes ( D. mawsoni : 53.6% compared to 40.1% genome average; P. georgianus: 74.6% compared to 54.3%), consistent with a bias towards TE insertion or retention. We further found evidence of multiple new TE insertions in the region that include representatives of seven LTR, six LINE, and 18 DNA TE families, as well as large expansions of Gypsy, L2, hAT-ac, and Kolobok-T2 copy numbers (Fig. 4b , Supplementary Table 1 , Supplementary Fig. 5 ). Hence, in addition to transposon copy number being increased by segmental tandem duplication with the afgp genes, there has also been active transposition into this region, with the new TE copies potentially involved in local rearrangements.

The physical map of the afgp locus presented here for P. georgianus represents the most complete reconstruction to date for any icefish species (Fig. 4a.3 ). Previous attempts to map the locus using short read generated assemblies 31 identified only four to eight copies of the antifreeze genes in various notothenioid genomes. A long-read assembly of Chaenocephalus aceratus (blackfin icefish) revealed 11 afgps 15 . The locus is also found to be highly fragmented in a recent chromosome-level genome assembly of D. mawsoni 57 , once more demonstrating the challenge of assembling this region. Our assemblies also include finer mapping of co-localised, and potentially co-evolving, gene families such as the trypsinogen genes, and tracking of the evolution of the chimeric intermediate gene ( afgp/tlp ). Finally, we observed an inversion of the locus in the icefish P. georgianus in comparison to the D. mawsoni haplotype 1 reconstruction 55 .

Two important points emerge regarding the evolutionary progression of the afgp gene locus. First, the presence of varying numbers of apparently functional as well as multiple pseudogene copies of afgp genes indicates that the locus remains evolutionarily dynamic. We suggest that the maintenance of functional copies in a protein gene family is driven by the strength of selective pressures exerted by the environment. P. georgianus is distributed in the considerably milder lower latitudes of the Southern Ocean, around the North of the West Antarctic Peninsula and the Scotia Arc Islands 58 (Fig. 1b ), but has never been found in the colder high-latitude waters. Thus, degeneration of previously functional copies would be consistent with a relaxation of selection on maintaining a large functional copy number and an energetically costly high level of protein production. Second, the chimeric afgp/tlp gene, which was earlier considered to be an evolutionary intermediate state of the early afgp genes, can still be found in the icefish (Fig. 4 ). Whilst one chimeric copy remains, it has sustained premature protein truncation and mutation (Supplementary Fig. 3B ). This is in contrast to the presence of multiple chimeric genes and apparently at least one functional copy in D. mawsoni 55 . The chimeric gene in P. georgianus is clearly a pseudogene on its way to extinction. First, it lacks the signal peptide coding sequence, thus could not produce a secreted protein even if functional. Second, the very long run of coding sequence of afgp tripeptide repeats (722 repeats; ~6.5 kb) without any of the conserved cleavable 3-residue linker sequences in functional afgp polyprotein genes is indistinguishable from simple sequence repeats of nine nucleotides. This suggests that once independent functional afgp copies were formed and with independent tlp already present, the maintenance of a chimeric copy may have become unnecessary in the less selective lower latitude habitat ranges that P. georgianus colonised.

The evolutionarily dynamic nature of the notothenioid afgp gene family can also be gleaned from the sequence of T. bernacchii , which is a species that resides in the most severe conditions at the southernmost limit for marine life in the Southern Ocean (McMurdo Sound, 78 o S). Even though its afgp locus assembly lacks contiguity, its annotation presents the largest set of afgp gene copies of all analysed species (24 copies, of which at least 11 are apparently functional), and also maintains three chimeric genes (Supplementary Fig. 4 ). Future efforts in assembling the challenging afgp loci to contiguity for cryonotothenioids across latitudinal clines will inform on the evolutionary dynamics of the adaptive afgp trait as driven by environmental selective strength.

Gene gains and losses drive haemoglobin evolution in notothenioids

The haemoglobin gene family has also been under strong selective pressure in notothenioids 59 . Haemoglobin is essential for oxygen transport, and the evolution of haemoglobin genes has been fuelled by duplications that enabled diversification of paralogous genes, as well as adaptation to changing environments through alterations in expression patterns 60 , 61 . In teleosts, haemoglobins are organised in two clusters, each containing both α and β globin genes: the larger MN cluster (flanked by genes kank2 and nprl3 ), and the smaller LA cluster (flanked by rhbdf1b and aqp8 ) 62 . Relaxed selection on haemoglobins and red blood cells in the cold, oxygen-rich Southern Ocean has led to moderate to severe anaemia in multiple notothenioid lineages 63 . The icefish family (Channichthyidae), often called “white blooded” icefishes due to their translucent white blood, are the only known vertebrates that completely lack haemoglobin and do not produce mature erythrocytes. Instead, they rely on oxygen physically dissolved in the blood plasma, which is possible because of the high oxygen saturation level at the freezing temperatures of the Southern Ocean 64 . The mechanisms underlying the loss of haemoglobin genes in the icefish are still a mystery, partly because the reconstruction of complete haemoglobin loci was not possible with previous fragmented genome assemblies. Here we describe the most complete reconstruction of the haemoglobin loci in notothenioids to date, allowing us to evaluate their evolution, track the loss of haemoglobins in the icefish, and identify the potential involvement of transposable elements in this process.

Using five new long-read assemblies, we achieved the contiguous assembly of both LA and MN haemoglobin gene clusters for most of the species, including their flanking genes 62 , 65 , and compared these with four published assemblies for three notothenioids ( Eleginops maclovinus 32 , D. mawsoni 57 , and C. aceratus 15 ) and one temperate non-notothenioid perciform ( Perca flavescens 66 ) (Fig. 5a , Supplementary Fig. 6 ). The two loci were found to be distinct 67 and located on two different chromosomes (LA: chr19, MN: chr8) that originated in the teleost genome duplication 68 . We devised and use here a new naming system for teleost haemoglobin genes independent of the life stage of expression (Methods). In the icefish species examined, we confirm the complete loss of functional haemoglobin genes in both clusters, with a single pseudogenized gene copy remaining in the LA locus. Synteny analysis suggests that the loss of haemoglobins potentially occurred as a single event for each locus. Furthermore, we find that the remaining length of the MN locus in P. georgianus is characterised by multiple transposon insertions (Fig. 5b ). No other coding genes are found in the region, and the total length of the remaining genomic region is approximately the same size as that of red-blooded cryonotothenioids (30-60 kb). The most common transposon insertions include LINE/L2 elements, which account for ~20% of the total length of the P. georgianus MN locus (Supplementary Table 2 , Supplementary Fig. 7 ). Similar transposon insertions are found in the LA locus, where the only haemoglobin remnant is the third exon of the pseudogenized α-globin.2 (Fig. 5b ), as previously identified 15 , 67 , 69 .

a Species analysed and syntenic reconstruction of LA and MN haemoglobin gene clusters. b Transposon insertions in the MN region of H. antarcticus , G. acuticeps , and P. georgianus genomes. Red: α -globin genes, blue: β-globin genes, grey: flanking genes, purple: transposon insertions, yellow: TAT-like repeats. Pseudogenes are marked with asterisks. Breaks in the assembly are indicated with double forward slashes. Bold face indicates species sequenced in the present study. Arrows show locus orientation and total lengths of MN locus in different species are given in brackets (kb, at right). Source data are provided as a Source Data file.

In contrast, in red-blooded species, haemoglobin loci are characterised by several local duplications (Fig. 5 ). For the LA cluster, in the common ancestor of E. maclovinus and cryonotothenioids we find a previously unidentified duplication of the β-globin.1 gene that gave rise to two β-globin copies ( β-globin1.1 and 1.2 ) (Fig. 5a ). Subsequently, one duplicated copy was repeatedly lost or deleteriously altered in other cryonotothenioid species, while the other copy was retained throughout, until completely lost in the icefish. In the larger MN cluster, the number of haemoglobin gene copies varies considerably across species, with five to 11 α-globin and five to 13 β -globin genes, while containing multiple lineage-specific tandem duplications of the α-globin.1 and β - globin.1 gene pair in every lineage analysed (α-globin and β-globin genes, Fig. 5a ). We retrieved a lower number of copies in the MN locus of the one species for which short read data were used for assembly ( E. maclovinus 32 ), potentially due to incomplete assembly or misassembly. Several cryonotothenioid species display lineage-specific frame-shifts or premature stop codons predicted to cause loss of function. The nature of the pseudogenisation of these gene copies suggests multiple independent pseudogenisation events. Only the loss of the first α-globin.1 copy appears to be shared across cryonotothenioids. At the opposite end of the cluster, the α-globin.2 and β-globin.2 pair was retained with, however, the notable loss of β-globin.2 in H. antarcticus , and in G. acuticeps . The case of G. acuticeps is particularly interesting, as here we identify a massive expansion of haemoglobin genes in the MN cluster, comprising more than twice as many haemoglobin genes as in other cryonotothenioid species. G. acuticeps is a member of the Bathydraconidae (dragonfishes), the group most closely related to the haemoglobin-lacking icefish. Furthermore, we find that each haemoglobin gene pair is preceded by a TAT-like repeat insertion, consistent with non-homologous recombination between these repeats underlying the tandem expansion of gene pairs (Fig. 5b ). In other globin genes, such as myoglobins, TAT-like insertions have been shown to interfere with transcription regulation 70 , 71 raising the possibility that these insertions as well as overall copy number are affecting expression levels. Further analysis of haemoglobin expression levels would be needed to understand how these TAT-like insertions influence haemoglobin transcription regulation in notothenioids.

By assembling the most complete reconstruction of notothenioid haemoglobin loci to date, we track the patterns of loss and gain of haemoglobin copies across the radiation and identify transposon insertions that may have influenced their evolution, while syntenic analysis suggests that the loss of haemoglobins in icefish could be potentially attributed to a single deletion event. Though it has been suggested that the lack of erythrocytes may reduce blood viscosity, the energy required to pump high volumes of blood and the need for additional physiological adaptations put into question whether the loss of haemoglobins in icefishes is indeed an adaptive trait 4 , 59 . Perhaps the lack of intense niche competition aided the establishment of this phenotype in the highly oxygenated waters of the Southern Ocean in the past. However, relying on an oxygen absorption system that depends on oxygen diffusion leaves the icefish intensely vulnerable to rising temperatures and thus decreased dissolved oxygen in the future 64 .

In conclusion, we present the most extensive effort to date to investigate the genomic evolution underlying the iconic notothenioid fish radiation, via the generation of a set of 24 new genome assemblies encompassing representatives from almost all notothenioid families. We demonstrate that the use of high-quality genome assemblies over a wide taxonomic breadth can help to decipher the evolutionary history of this recent vertebrate radiation. We identify critical steps in the evolution of key gene families that involve large genomic rearrangements in repetitive regions that could only be reconstructed with the aid of long-read assemblies. We show that the evolutionary history of the remarkable notothenioid radiation was associated with, and potentially driven by, transposon proliferation, which could have affected the evolutionary advantage of these species during freezing events occurring in the Southern Ocean. In particular, TE expansion events can be linked to the structure of characteristic repetitive gene families such as the haemoglobin and antifreeze genes. Beyond these direct insights, our work provides an extensive resource for the future study of notothenioid genomic evolution, enabling further research to advance our understanding of the notothenioid radiation, and of genomic adaptations to extreme environments more widely. As the fate of notothenioid diversity is linked to very narrow margins of temperature tolerance, studying their adaptability is particularly relevant now with the unfolding climate crisis and the warming of the Southern Ocean 72 .

Sample processing and sequencing

Tissue samples were collected in compliance with all relevant ethical regulations and either frozen immediately at −80 ˚C, or preserved in ethanol and then frozen to preserve the quality of genomic DNA. Tissue preservation can influence the quality of extracted DNA 73 , and flash freezing is the optimal preservation method for use with long-read sequencing. High molecular weight DNA (HMW DNA) was extracted using the Bionano Agarose plug extraction protocol 74 or a modified version of the MagAttract kit (Qiagen) (Supplementary Data 1 ). The quantity of extracted HMW DNA was evaluated with the HS Qubit DNA kit and the fragment profile and overall quality was assessed with the Femto Pulse instrument (Agilent). Pacific Biosciences (PacBio) sequencing was performed with CLR (Continuous Long Reads) SMRT cells. PacBio libraries were made using the SMRTbell Template Prep Kit 1.0, following the PacBio protocol. A size selection was performed on the BluePippin instrument (Sage Science), with a 15 kb cut off and sequence data were generated on the Sequel instrument using Seq kit v2/Binding Kit v2.0 with a 10 h movie. Illumina sequencing paired-end (PE) libraries were generated for eight species and sequenced by multiplexing two species per lane on Illumina HiSeqX (150 bp PE). Linked reads 10X Genomics Chromium sequencing was performed for 17 species (Table 1 ). Linked-read libraries were prepared using the Chromium Genome Reagent Kit, and the Chromium Genome Library Kit & Gel Bead Kit, according to the manufacturer’s instructions 75 , 76 , with standard DNA input (1 ng). Hi-C libraries were generated with a Dovetail kit for P. georgianus and with ARIMA Genomics kit for C. gobio , and each was sequenced on the Illumina HiSeq4000 platform. Bionano Irys optical mapping was used for scaffolding the assembly of H. antarcticus , as well as that of C. gobio 23 .

Total RNA for RNAseq was extracted using the RNeasy extraction kit (Qiagen), from ~20–40 mg of tissue. The RNA quality was assessed with the Qubit HS RNA kit and Agilent Bioanalyzer Nano chips, and only extracts with RIN value > 8 were used for sequencing. Illumina 150 bp PE libraries were prepeared and sequenced on the HiSeq4000 platform. For C. gobio four tissues were used including brain, muscle, and ovary from one individual, preserved in RNAlater, and frozen spleen from a different individual. For T. bernacchii four RNAlater preserved tissue types were used including brain, muscle, and ovary, and testis from a second individual, For G. acuticeps two tissues were used, including brain and ovary from one individual, preserved in RNAlater. All sample processing and sequencing was performed at the Wellcome Sanger Institute, UK.

Genome assembly and curation

For genome assembly we used a combination of different sequencing technologies, which were either used in conjunction (hybrid assemblies) or individually. Our genome assemblies were generated as follows.

For C. gobio the assembly was generated based on 75x PacBio Sequel data, 54x Illumina HiSeqX data generated from a 10X Genomics Chromium library, Bionano Saphyr two-enzyme data (Irys) and 145x coverage HiSeqX data from a Hi-C library (for Hi-C, tissue from a different individual was used), as described in ref. 23 . An initial PacBio assembly was generated with Falcon-unzip without repeat-masking during overlap detection. The primary contigs were first scaffolded using a wtdbg 77 assembly as a guide, then scaffolded further using the 10X data with scaff10x and then with Bionano two-enzyme hybrid scaffolding. After using the PacBio data to gap-fill with PBJelly and polish with Arrow, the assembly was polished again using the 10X Illumina data and freebayes. Contiguity was then further increased by filling gaps with the contigs from a wtdgb assembly made from Canu 78 corrected PacBio reads. The assembly was then re-polished with Arrow and freebayes, and retained haplotigs were identified with purge_haplotigs 79 and scaffolded to chromosomes using Arima Hi-C.

For P. georgianus the assembly was based on 93x PacBio, 56x 10X Genomics Chromium, and Dovetail Hi-C data. An initial assembly was generated using Falcon-unzip, retained haplotig identification with purge_haplotigs, with 10X based scaffolding with scaff10x, BioNano hybrid-scaffolding, Hi-C based scaffolding with SALSA2 18 , Arrow polishing, and two rounds of FreeBayes 80 polishing. This assembly comprises 24 chromosomes, which were numbered in correspondence to the medaka HdR1 assembly ( Oryzias latipes , GCA_002234675.1), as for the C. gobio genome 23 .

The assembly for H. antarcticus was based on 67x PacBio data, 40x of 10X Genomics Chromium data, and Bionano Irys data. An initial PacBio assembly was generated with Falcon-unzip, retained haplotig identification with purge_haplotigs 79 , 10X based scaffolding with scaff10x, Bionano hybrid-scaffolding, Arrow polishing, and two rounds of FreeBayes polishing. For T. bernacchii and G. acuticeps the assemblies were based on PacBio and 10X data. For T. bernacchii , we used 46x PacBio data and 53x of 10X Genomics Chromium data, while the assembly for G. acuticeps was based on 31x PacBio data and 41.8x of 10X Genomics Chromium data. These were assembled using Falcon-unzip, 10X based scaffolding with scaff10x, Arrow polishing, and two rounds of FreeBayes polishing. Purge_dups 81 was run on the curated assemblies to further remove retained duplications.

Finally, to improve the quality of the PacBio assemblies we performed manual curation to remove mis-assemblies, duplications, and sequencing contamination, and to merge scaffolds based on supporting evidence, which has been shown to substantially improve the continuity and accuracy of genome assemblies 20 . Each assembly was manually curated using the Genome Evaluation Browser (gEVAL) 19 . Scaffold integrity was confirmed with PacBio read mapping and enhanced with 10X illumina read mapping, read information and contig end sequence overlaps. While for H. antarcticus scaffold integrity was further confirmed using Bionano BssSI optical maps, visualised in Bionano Access, breaking and re-joining where necessary. For P. georgianus a 2D map was built using Hi-C reads, allowing further scaffold correction and super-scaffolding to bring the assembly to chromosome scale. Artificially retained haplotypic duplications were removed with purge_dups 81 (Supplementary Table 3 ).

For 19 more species only 10X Chromium or Illumina HiSeqX were used for assembly (Table 1 ). For 11 species sequenced with 10X Genomics Chromium data, genome assembly was performed using Supernova 2.0 (Supernova 2.0 Software). After initial assembly, retained haplotigs were identified using purge_haplotigs 79 . For the remaining eight species which were only sequenced with Illumina HiSeqX, a primary assembly was generated with a reference guided approach using SOAPdenovo2 25 . The short insert reads were initially base error corrected using BFC ( https://github.com/lh3/bfc ). After this step, larger kmer sizes (e.g. 70), may be applied to improve assembly. SOAPdenovo 25 was used to process the cleaned short insert reads, followed by GapCloser for contig gap filling. The scaffolds were further enhanced by the use of cross_genome, a tool which maps genome synteny to merge scaffolds (Phusion2 - Browse /cross_genome), as has been previously applied in genome assemblies such as Tasmanian devil 82 and grass carp 83 . To finalise these assemblies, decontamination methods were used to remove contaminants from sequencing (e.g., adapter sequences) or symbionts. Metrics for sequencing data and assemblies are provided in Supplementary Data 3 and 6 , and Supplementary Table 4 .

Gene annotation

Gene annotation was generated for all five PacBio assemblies, using the Ensembl Gene Annotation system as follows 27 . Annotation was created primarily through alignment of short read RNAseq data to the genome. Gaps in the annotation were filled via protein-to-genome alignments of a select set of vertebrate proteins from UniProt 84 , which had experimental evidence for existence at the protein or transcript level.

At each locus, the data were collapsed and consolidated, with priority given to models derived from the RNAseq data, producing a set of final gene models along with their associated non-redundant transcript set. To help differentiate between true isoforms and fragments, the likelihood of each Open Reading Frame (ORF) was assessed in relation to known vertebrate proteins. Low-quality transcript models, e.g. those with evidence of a fragmented ORF, were removed. In loci where the RNAseq data were fragmented or missing, homology data took precedence, with preference given to longer transcripts that had strong intron support from the short-read data.

Gene models from the above process were classified into three main types: protein-coding, pseudogene, and long non-coding. Models with hits to known proteins, and few structural abnormalities were classified as protein-coding. Models with hits to known proteins that also display abnormalities such as the absence of a start codon, non-canonical splicing, unusually small intron structures (<75 bp) or excessive repeat coverage, were reclassified as pseudogenes. Single-exon models with a corresponding multi-exon copy elsewhere in the genome were classified as processed (retrotransposed) pseudogenes. If a model failed to meet the criteria of any of the previously described categories, did not overlap with a protein-coding gene, and had been constructed from transcriptomic data, then it was considered as a potential lncRNA. Potential lncRNAs were additionally filtered to remove single-exon loci due to the unreliability of such models.

Putative miRNAs were predicted via a BLAST of miRBase 85 against the genome, before passing the results to RNAfold 86 . Other small non-coding loci were identified by scanning Rfam 87 against the genome (described in more detail in ref. 27 ) and passing the results into Infernal 88 .

Gene annotations are available on the Ensembl server under GCA_900634415.1 (database version 9.31 81 ) for C. gobio , and on Ensembl Rapid Release ( https://rapid.ensembl.org/ ) for G. acuticeps , P. georgianus , H. antarcticus , and T. bernacchii (Supplementary Data 4 ). Comparison of orthologous clusters was performed with OrthoVenn2 89 . For antifreeze and haemoglobin genes further manual curation was undertaken as described below.

Transposable element annotation and analysis

De novo annotation of transposable elements was performed using RepeatModeler v.2.0 90 and RepeatMasker v.4.0.1 91 . For each of the five PacBio assemblies ( C. gobio, T. bernacchii, H. antarcticus, G. acuticeps , and P. georgianus ) a de novo repeat library was generated using RepeatModeler2 90 . To enhance the detection of LTR retrotransposons, the programs LTRharvest 92 and LTR_retriever 93 were run as part of the RepeatModeler2 pipeline. To improve the quality of the annotation, the identified elements from each genome were further curated manually using the “BLAST, extend, extract” process 94 to remove false assignments and achieve complete length elements. The consensus TE sequences that were identified by RepeatModeler2 were blasted against the genomes (BLAST+ 95 ), and the sequences of the 50 best hits were extracted along with a 1-kb long flanking sequence on each side. Multiple sequence alignments were generated for each set of top hits, using MUSCLE 96 . Each multiple sequence alignment was visualised with belvu 97 and then manually inspected to confirm TE element completeness. TEs that appeared to be extending beyond alignment boundaries were subjected to additional rounds of curation, until the complete sequence was recovered. Finally, new consensus sequences were extracted from the multiple alignments using hmmer ( http://hmmer.org/ ). Overall a total of ~2000 elements generated by RepeatModeler2 were manually curated. A custom TE library was created combining all the curated element outputs, and all genomes were masked with RepeatMasker (with options -rmblast -s).

To analyse the repeat content of each genome we used a Perl script to parse the RepeatMasker.out and.align files 98 . This was used to calculate the total amount of DNA of the genome and different categories of TEs (e.g. class, family), and the % of divergence from the consensus (Kimura divergence). The amount of DNA was then split in bins and plotted against the coverage to generate repeat landscape plots, which were made using ggplot2 99 in R. The % divergence indicates the age of TEs, with lower percentage divergence from the consensus sequence indicating younger TEs (Fig. 3a ). To examine the effect of TE expansions in genome size variation we correlated the total amount of DNA in TEs vs. assembly size using a linear regression model (Pearson correlation coefficient) (Fig. 3b ). To plot annotated TEs colocalized with gene copies (Fig. 5b ) we used the DNA Features Viewer library in python. Finally, TE copy numbers were calculated using another Perl script parsing the RepeatMasker output 100 (Fig. 4b ).

Phylogenetic analysis

Phylogenetic analysis was performed using single copy ortholog genes identified with BUSCO 26 , for the 24 newly sequenced notothenioid genomes and 17 previously published genomes of seven notothenioids and ten further species of percomorph fishes. The species and assembly versions used are listed in Supplementary Table 5 . BUSCO (v2) was run with lineage “actinopterygii_odb9”, and the sequences of single copy orthologs identified in each assembly and extracted for use in further analysis.

We used MAFFT v.7.453 101 to align 266 selected BUSCO genes that were single copy in our annotated gene sets. The 266 alignments were inspected by eye, and apparently misaligned sequence regions were set to missing data. A total of 1,141,524 amino acids were set to missing out of 6,410,688, including nine alignments that were excluded completely, leaving 257 alignments for further analysis. We then aligned nucleotide sequences of the same BUSCO genes according to the amino-acid alignments, ensuring that regions corresponding to the removed sequences were again set to missing data in the nucleotide sequence alignments. Sites with high entropy (entropy-like score > 0.5) or high proportion of missing data (gap rate >0.2) were removed with BMGE v.1.1 102 and alignments with more than three completely missing sequences, a minimum length below 500 bp, or a standard deviation of among-sequence GC-content variation >0.03 were excluded. These filters were passed by 228 alignments. For each alignment we performed gene-tree analyses using BEAST2 v.2.6.0 39 with a Markov-chain Monte Carlo chain length of 25 million iterations, assuming the Yule model of diversification 103 and the uncorrelated lognormal relaxed clock model 104 , and averaging over substitution models with the bModelTest add-on package 105 . These gene trees were time-calibrated by arbitrarily constraining their root age to 100 million years (with a standard deviation of 0.1). Chain convergence was suggested by effective sample sizes (ESS) per parameter >200.

We identified the most suitable alignments for further phylogenomic analyses based on the minimum ESS value per alignment and estimates for the mutation rate and its among-species variation. We compiled a “strict” set of alignments that included all those that had a mean mutation rate estimate below 0.002 per bp per million year, a mutation rate standard deviation (relative to the mean estimate) below 0.9, and a minimum ESS value >100; this set was a subset of a second, “permissive” set of alignments in which we placed those that had a mean mutation rate estimate below 0.00025 per bp per million years, a mutation rate standard deviation below 1.1, and a minimum ESS value >50. The strict and permissive sets contained 140 and 200 alignments, respectively.

For the strict set of 140 alignments, the permissive set of 200 alignments, and the “full” set of 257 alignments, we performed maximum-likelihood phylogenetic analyses with IQ-TREE v.1.7 41 after alignment concatenation, maintaining separate partitions with unlinked instances of the GTR+Gamma substitution model for each of the original alignments. Node support was assessed with 1000 ultrafast bootstrap replicates 106 . Each of the three analyses was complemented with an estimation of gene- and site-specific concordance factors, and the three resulting sets of gene trees were used for separate species-tree analyses with ASTRAL v.5.7.3 42 .

Finally, we estimated the phylogeny and the divergence times of notothenioid species with BEAST2 from a concatenated alignment combining all alignments of the strict set. To avoid potentially saturated sites, we excluded all third codon positions from this analysis, and to reduce its computational demand we grouped 280 original data blocks (separating first and second codon positions for each of the 140 original alignments of the strict set) into 12 partitions selected with the cluster algorithm of PartitionFinder v.2.1.1 107 , assuming linked branch lengths, equal weights for all model parameters, a minimum partition size of 5000 bp, and the GTR+Gamma substitution model. The same substitution model was also assumed in the BEAST2 analysis, together with the birth-death model of diversification 108 and the uncorrelated lognormal relaxed clock model 104 . Time calibration of the phylogeny was based on four age constraints defined according to a recent timeline of teleost evolution inferred from genome and fossil information 33 , at the most recent common ancestors of clades: Eupercaria, around 97.47 MYA (2.5–97.5 inter-percentile range: 91.3–104.0 MYA); the clade combining Eupercaria, Ovalentaria, and Anabantaria—around 101.79 MYA (95.4–109.0 MYA); the clade combining these four groups with Syngnatharia and Pelagiaria—around 104.48 MYA (97.3–112.0 MYA); and the clade combining those six groups with Gobiaria—around 107.08 MYA (100.0–114.0 MYA). All constraints were implemented as lognormal prior distributions with mean values as specified above and a standard deviation between 0.033 and 0.036. In addition, we constrained the unambiguous 33 , 109 , 110 , 111 monophyly of the groups Notothenioidei, Perciformes, Ovalentaria, Anabantaria, and the clade combining the latter two groups. We performed six replicate BEAST2 analyses with 330 million MCMC iterations, and convergence among MCMC chains was confirmed by ESS values >120 for all model parameters and >270 for the likelihood and the prior and posterior probabilities. The posterior tree distribution was summarised in the form of a maximum-clade credibility tree with TreeAnnotator v.2.6.0 112 . We attempted to repeat the BEAST2 analyses with the permissive and full datasets, but these proved too computationally demanding to complete, so that even after 330 million MCMC iterations and run times of several months, some of the ESS values remained below 100. Nevertheless, the preliminary results from these analyses supported the same tree topology as the analyses with the strict dataset.

To place the time calibrated phylogeny in the context of historic ocean temperature variation, we used estimated benthic oxygen values previously published in ref. 36 . The moving average was plotted with geom_smooth in R using a generalised additive model (GAM: (y ~ s(x, bs = “cs”)).

afgp/tlp locus annotation and reconstruction

The location of the afgp locus in each genome assembly was initially identified with BLAST+ 95 searches, using as queries a copy of afgp and other gene sequences annotated in the previously published D. mawsoni locus (accession HQ447059.1, haplotype1) 55 . Specifically, within the locus the following genes were used as queries: Antifreeze glycoprotein H1-A2 ( afgp ), Trypsinogen H1-1d ( tryp1 ), Trypsinogen H1-3a ( tryp3 ), Trypsinogen-like protease 1 ( tlp ), Translocase of outer mitochondrial membrane 40 ( tomm40 ), hormone sensitive lipase HSL ( lipeb ; for this gene a full length transcript was obtained independently and used as query) 55 . The exact location of each gene copy was confirmed and annotated manually, to identify numbers and sizes of exons. Each afgp copy was manually inspected for the presence of frame shifts and gaps to identify complete genes and pseudogenized copies.

All five genomes sequenced with PacBio contained copies of all six genes used for BLAST+ analyses ( afgps and flanking genes) (Fig. 4 , Supplementary Fig. 4 ). To further improve the assembly of the afgp locus on the P. georgianus genome, we mapped Falcon-corrected PacBio reads to the diploid assembly using minimap2 113 , and then filtered the mapped reads to remove secondary alignments (samtools view -F 256). We used GAP5 114 to inspect and manually curate the mapped reads. Reads that mapped to more than one location were linked in GAP5, and by further inspecting these links and extending soft-clipped sequence, it was possible to merge contigs, resulting in a complete representation of the whole afgp gene locus. Finally, the reassembled sequence was polished using Racon 115 .

New naming system for teleost haemoglobin genes

The current haemoglobin gene naming system in fish mostly relies on the zebrafish laboratory model species and on the expression pattern of each of its haemoglobin genes during embryonic and/or adult phases. While informative for zebrafish research, using a naming system based on embryonic or adult expression for species in which expression dynamics of haemoglobin genes cannot be assessed may lead to misinterpretations, especially because expression patterns of haemoglobin genes are known to be influenced by local organisation of the genomic region that may not be conserved across species 65 , 116 . Therefore, designating orthologous haemoglobin genes across species needs a nomenclature system that is independent of an expression pattern that may not be evolutionarily conserved. We thus propose here a novel naming system based on genomic organisation rather than expression data.

First, the established haemoglobin alpha and beta denominations (i.e., hba and hbb ) are conserved due to clear sequence conservation. Second, the presence of each gene in the LA or the MN cluster is added as a suffix (e.g., hbala and hbamn ). Third, a final numeral suffix is added to reflect the relative positioning of the gene within each cluster. The orientation of the most upstream hba gene determines the orientation of the cluster and is arbitrarily named hbala1 and hbamn1 for the LA and MN clusters, respectively. Thus, the nomenclature reflects position but not necessarily orthology. The neighbouring hbb gene is called hbbla1 and hbbmn1 for the LA and MN clusters, respectively. The names of genes further to the conventional right of the locus are suffixed with incremental numbers following the orientation of the cluster. Tandem duplicated genes (e.g., hbamn1.1 and hbamn1.2 ) and pseudogene (e.g., hbamn1.3p ) nomenclatures follow the Zebrafish nomenclature guidelines established by the Zebrafish Information Network (ZFIN) 117 .

Haemoglobin gene locus annotation and reconstruction

To study haemoglobin genes in notothenioid species we focused on the five species ( C. gobio, T. bernacchii, H. antarcticus, G. acuticeps , and P. georgianus ) assembled with PacBio data, along with three previously published assemblies ( D. mawsoni 57 , E. maclovinus 32 , and C. aceratus 15 ) to provide as good a clade coverage as possible. The reference genome assembly of the yellow perch (Perca flavescens ) 66 , a close relative to notothenioids within the order Perciformess 9 , was used as a reference to determine haemoglobin gene exon boundaries. We used flanking genes to confirm orthology between genes and clusters across species, and each exon of each gene was retrieved and their exact positions in the corresponding genome assembly was recorded. Using the T. bernacchii assembly as reference species we performed mVISTA 118 alignments of genomic regions with LAGAN 119 or Shuffle-LAGAN 120 . Protein sequences were aligned with MUSCLE 96 and phylogenetic trees were reconstructed with RAxML-NG 121 using the best-fitting substitution model according to ModelFinder based on Bayesian information criterion (BIC) 122 , 50 parsimony and 50 random starting trees, and 200 bootstraps or bootstopping at a default cut-off of 0.03 (protein alignments in Supplementary Data 7 ).

We used self-alignments with dotter 123 to visualise the complete reconstruction of each haemoglobin cluster and we manually inspected each locus to identify possible gaps or mis-assemblies. We can confirm complete gapless assembly of the MN haemoglobin locus for each PacBio species, with the exception of one gap identified in the G. acuticeps genome, which could not be corrected with the available data (Fig. 5 ). Furthermore, the P. georgianus chromosomal assembly MN haemoglobin locus was also manually curated using GAP5 114 , as described above for the afgp locus.

Detailed information on all the tools and versions used for each analysis are provided in Supplementary Table 6 .

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

The genome assemblies generated in this study have been deposited on NCBI under BioProject PRJEB53202 and the following accessions: C. gobio : GCA_900634415.1 (alt. hap. GCA_900634435.1 ), T. bernacchii GCA_902827165.1 (alt. hap. GCA_902827105.1 ), H. antarcticus GCA_902827135.1 (alt. hap. GCA_902827095.1 ), G. acuticeps GCA_902827175.1 (alt. hap. GCA_902827185.1 ), P. georgianus GCF_902827115.1 and GCA_902827115.2 (alt. hap GCA_902827155.1 ), B. diacanthus GCA_943590825.1 , B. variegatus GCA_943593645.1 , T. loennbergii GCA_943590855.1 , L. larseni GCA_943594155.1 , L. squamifrons GCA_943593335.1 , T. hansoni GCA_943593355.1 , T. scotti GCA_943590805.1 , L. nudifrons GCA_943590975.1 , G. gibberifrons GCA_943591055.1 , N. rossii GCA_943590865.1 , D. longedorsalis GCA_943591025.1 , H. velifer GCA_943590885.1 , A. nudiceps GCA_943590845.1 , B. marri GCA_943591095.1 , V. infuscipinnis GCA_943590875.1 , C. wilsoni GCA_943593825.1 , C. antarcticus GCA_943590835.1 , P. macropterus GCA_943590895.1 , C. dewitti GCA_943594065.1 . Gene annotation for species C. gobio is available on Ensembl [ www.ensembl.org ], and for T. bernacchii, H. antarcticus, G. acuticeps, P. georgianus gene annotations are available on Ensembl Rapid Release [ https://rapid.ensembl.org/ ]. RefSeq annotations for C. gobio , T. bernacchii, G. acuticeps , and P. georgianus can be found on NCBI under assembly accession numbers. All raw sequencing data are available on NCBI (accessions listed in Supplementary Data 1 ). Source data are provided as source data file. Data used for phylogenetic analysis along with alignments and phylogenetic trees are available on Dryad: https://doi.org/10.5061/dryad.80gb5mktn . Source data are provided with this paper.

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Acknowledgements

We thank the Wellcome Sanger Institute Scientific Operations for help with sequencing data production. We thank Laura Gerrish for help with generating an ArcGIS map of Antarctica and the Southern Ocean. I.B., S.A.M., and R.D. were supported by Wellcome grants WT207492 and WT206194; I.B. and E.A.M. by Wellcome grants 104640 and 092096; C.H.C.C. by US National Science Foundation grant ANT11-42158; M.M. by a mobility fellowship from the Norwegian Research Council (FRIPRO 275869); T.D., J.H.P. by NSF OPP-1543383 and OPP-1947040; H.W.D. by US National Science Foundation grants OPP-0132032, PLR-1444167, and OPP-1955368, and the Marine Science Centre at Northeastern University (publication number 427), M.S.C. by NERC-UKRI core funding to the British Antarctic Survey; W.S. by Swiss National Science Foundation (176039). J.M.D.W., Y.S., J.T., W.C., and K.H. by Wellcome WT206194; L.H. by WT108749 and WT222155. For the purpose of open access, the authors have applied a CC BY public copyright licence to any Author Accepted Manuscript version arising from this submission.

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I.B., R.D., E.A.M., H.W.D., J.H.P., C.H.C.C., M.S.C., T.D., and W.S. designed the study and selected species for sequencing. I.B. led design, data generation, and analysis. H.W.D., CH.C.C., J.H.P., T.D., and M.S.C. contributed samples. I.B., M.S., and K.O. generated sequencing data. I.B., S.A.M., and Z.N. assembled genomes. J.M.D.W., A.T., Y.S., J.T., W.C., and K.H. performed genome curation. I.B., T.D., M.M., C.H.C.C., and J.M.D.W. performed data analyses. I.B. performed transposon annotation. L.H. performed gene annotation. I.B. and J.T. prepared data for submission. R.D. and E.A.M. provided computational resources and funding. I.B. wrote the manuscript, with edits from R.D., C.H.C.C., J.M.D.W., M.S.C., T.D., M.M. and comments from all authors. All authors reviewed the final manuscript and approved it.

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Bista, I., Wood, J.M.D., Desvignes, T. et al. Genomics of cold adaptations in the Antarctic notothenioid fish radiation. Nat Commun 14 , 3412 (2023). https://doi.org/10.1038/s41467-023-38567-6

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Identification of geothermal potential based on land surface temperature derived from remotely sensed data

Research Article
Published: 14 September 2023
Volume 30 , pages 104726–104741, ( 2023 )

Cite this article

Jianyu Liu 1 na1 ,
Jiangqin Chao 1 , 2 na1 ,
Zhifang Zhao 3 , 4 , 5 , 6 ,
Fei Zhao 3 ,
Shiguang Xu 7 ,
Zhibin Lai 3 ,
Haiying Yang 3 ,
Qi Chen 3 &
Youle Tu 1

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With the continuous development of thermal infrared remote sensing technology and the maturation of remote sensing inversion algorithms based on surface temperatures, identifying high-temperature anomalous areas by inverting surface temperatures has become an crucial approach to finding geothermal potential areas. The eastern region of Longyang in western Yunnan Province is renowned for geothermal resources, though the distribution area of geothermal potential remains unknown. Therefore, this study used Landsat-8 TIRS data and four surface temperature inversion algorithms, namely, mono-window algorithm, single-channel algorithm, Du split window algorithm (SWD), and Jiménez-Muñoz split window algorithm (SWJ), to explore the astern region of Longyang. The inversion results were compared with Moderate Resolution Imaging Spectroradiometer Land Surface Temperature (MODIS LST) results for analysis and cross-validation to select the optimal algorithm. A multi-view remote sensing temperature anomaly information extraction method was adopted. Moreover, the overall threshold method, the fracture structure buffer method, and the joint analysis of diurnal temporal data were combined for the reduction of the thermal anomaly area as well as for comprehensively defining the geothermal prospective area in the study area. The results demonstrated that the mono-window algorithm had the highest accuracy with a Pearson coefficient of 0.77, which is more suitable for the surface temperature inversion in Longyang area. Furthermore, three geothermal anomalies (A, B, and C) were identified in the study area, with larger thermal anomaly in A and C, but a smaller one in B. All three areas had hot spring points verified, with A and C exhibiting more significant development potential. The research results provide a reliable methodological basis for the development of geothermal resources in the region.

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Detecting geothermal anomalies using Landsat 8 thermal infrared remote sensing data in the Ruili Basin, Southwest China

Land Surface Temperature Retrieval of the Geothermal Area in Eastern Liaoning, China, Based on Thermal Infrared Remotely Sensed Data of MODIS

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Acknowledgements

We acknowledge the National Aeronautics and Space Administration (NASA) and Geoscientific Data and Discovery Publishing System for providing the satellite data. We would like to thank the editors and all the reviewers who participated in the review.

This research is supported by “the National Natural Science Foundation of China” (Grant No. 41961064), “the Joint Fund of Science Technology Department of Yunnan Province and Yunnan University” (Grant No.2018FY001(-019)), “Science and Technology Plan Project of Yunnan Province Science and Technology Department” (Grant No.202101BA070001-145), and “the Second Professional Degree Postgraduate Practice Innovation Project of Yunnan University”(Grant No. ZC-22223197).

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Jianyu Liu and Jiangqin Chao contributed equally to this work.

Authors and Affiliations

Institute of International Rivers and Eco-Security, Yunnan University, Kunming, 650500, China

Jianyu Liu, Jiangqin Chao & Youle Tu

School of Geographical Sciences and Tourism, Zhaotong University, Zhaotong, 657000, China

Jiangqin Chao

School of Earth Sciences, Yunnan University, Kunming, 650500, China

Zhifang Zhao, Fei Zhao, Zhibin Lai, Haiying Yang & Qi Chen

Engineering Research Center of Domestic High-Resolution Satellite Remote Sensing Geology for Universities of Yunnan Province, Kunming, 650500, China

Zhifang Zhao

Yunnan Key Laboratory of Sanjiang Metallogeny and Resources Exploration and Utilization, Kunming, 650500, China

Key Laboratory of Sanjiang Metallogeny and Resources Exploration and Utilization, MNR, Kunming, 650500, China

Yunnan Geology and Mineral Explorating & Engineering Corp. (Group), Kunming, 650041, China

Shiguang Xu

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Conceptualization, J. L. and Z. Z.; data curation, J. L. and J. C.; formal analysis, Q. C.; funding acquisition, Z. Z.; investigation, F. Z. and S. X.; methodology, J. L., J. C., and Z. L.; resources, Z. Z.; software, J. L., J. C., Y. T., and Z. L.; validation, H. Y., Q. C., and Y. T.; visualization, Q. C.; writing—original draft, J. L. and J. C.; writing—review and editing, Z. Z., H. Y., and Q. C. All authors have read and agreed to the published version of the manuscript.

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Liu, J., Chao, J., Zhao, Z. et al. Identification of geothermal potential based on land surface temperature derived from remotely sensed data. Environ Sci Pollut Res 30 , 104726–104741 (2023). https://doi.org/10.1007/s11356-023-29678-0

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Published : 14 September 2023

Issue Date : October 2023

DOI : https://doi.org/10.1007/s11356-023-29678-0

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It has also highlighted the role of waterways such as the Zambezi River in the spread of the disease with almost four times the population estimated to live in areas suitable for malaria for up to nine months of the year than was previously thought.

The research entitled "Future malaria environmental suitability in Africa is sensitive to hydrology" was funded by the Natural Environment Research Council and is published today (9 May 2024) in the journal Science .

Dr Mark Smith an Associate Professor in Water Research in the Leeds' School of Geography and lead author of the study said: "This will give us a more physically realistic estimate of where in Africa is going to become better or worse for malaria.

"And as increasingly detailed estimates of water flows become available, we can use this understanding to direct prioritisation and tailoring of malaria interventions in a more targeted and informed way. This is really useful given the scarce health resources that are often available."

Malaria is a climate-sensitive vector-borne disease that caused 608,000 deaths among 249 million cases in 2022.

95% of global cases are reported in Africa but reductions in cases there have slowed or even reversed in recent years, attributed in part to a stall in investments in global responses to malaria control.

The researchers predict that the hot and dry conditions brought about by climate change will lead to an overall decrease in areas suitable for malaria transmission from 2025 onwards.

The new hydrology-driven approach also shows that changes in malaria suitability are seen in different places and are more sensitive to future greenhouse gas emissions than previously thought.

For example, projected reductions in malaria suitability across West Africa are more extensive than rainfall-based models suggested, stretching as far east as South Sudan, whereas projected increases in South Africa are now seen to follow watercourses such as the Orange River.

Co-author of the study Professor Chris Thomas from the University of Lincoln said: "The key advancement is that these models factor in that not all water stays where it rains, and this means breeding conditions suitable for malaria mosquitoes too can be more widespread -- especially along major river floodplains in the arid, savannah regions typical of many regions in Africa.

"What is surprising in the new modelling is the sensitivity of season length to climate change -- this can have dramatic effects on the amount of disease transmitted."

Simon Gosling, Professor of Climate Risks & Environmental Modelling at the University of Nottingham, co-authored the study and helped to coordinate the water modelling experiments used in the research. He said: "Our study highlights the complex way that surface water flows change the risk of malaria transmission across Africa, made possible thanks to a major research programme conducted by the global hydrological modelling community to compile and make available estimates of climate change impacts on water flows across the planet.

"Although an overall reduction in future risk of malaria might sound like good news, it comes at a cost of reduced water availability and a greater risk of another significant disease, dengue."

The researchers hope that further advances in their modelling will allow for even finer details of waterbody dynamics which could help to inform national malaria control strategies.

Dr Smith added: "We're getting to the point soon where we use globally available data to not only say where the possible habitats are, but also which species of mosquitoes are likely to breed where, and that would allow people to really target their interventions against these insects."

Infectious Diseases
Pests and Parasites
Environmental Issues
Environmental Awareness
Global climate model
Climate model
Infiltration (hydrology)
Pest (animal)
IPCC Report on Climate Change - 2007
Consensus of scientists regarding global warming
Climate engineering

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Materials provided by University of Leeds . Note: Content may be edited for style and length.

Journal Reference :

Mark W. Smith, Thomas Willis, Elizabeth Mroz, William H. M. James, Megan J. Klaar, Simon N. Gosling, Christopher J. Thomas. Future malaria environmental suitability in Africa is sensitive to hydrology . Science , 2024; 384 (6696): 697 DOI: 10.1126/science.adk8755

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The impact of biochar addition on morpho-physiological characteristics, yield and water use efficiency of tomato plants under drought and salinity stress

Ghulam Murtaza 1 ,
Muhammad Usman 2 ,
Javed Iqbal 3 ,
Muhammad Nauman Tahir 1 ,
Mohamed S. Elshikh 4 ,
Jawaher Alkahtani 4 ,
Monika Toleikienė 5 ,
Rashid Iqbal 6 ,
M. Irfan Akram 7 &
Nazim S. Gruda 8

BMC Plant Biology volume 24 , Article number: 356 ( 2024 ) Cite this article

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Metrics details

The use of saline water under drought conditions is critical for sustainable agricultural development in arid regions. Biochar is used as a soil amendment to enhance soil properties such as water-holding capacity and the source of nutrition elements of plants. Thus, the research was carried out to assess the impact of biochar treatment on the morphological and physiological characteristics and production of Solanum lycopersicum in greenhouses exposed to drought and saline stresses. The study was structured as a three-factorial in split-split-plot design. There were 16 treatments across three variables: (i) water quality, with freshwater and saline water, with electrical conductivities of 0.9 and 2.4 dS m − 1 , respectively; (ii) irrigation level, with 40%, 60%, 80%, and 100% of total evapotranspiration (ETC); (iii) and biochar application, with the addition of biochar at a 3% dosage by (w/w) (BC 3% ), and a control (BC 0% ). The findings demonstrated that salt and water deficiency hurt physiological, morphological, and yield characteristics. Conversely, the biochar addition enhanced all characteristics. Growth-related parameters, such as plant height, stem diameter, leaf area, and dry and wet weight, and leaf gas exchange attributes, such rate of transpiration and photosynthesis, conductivity, as well as leaf relative water content were decreased by drought and salt stresses, especially when the irrigation was 60% ETc or 40% ETc. The biochar addition resulted in a substantial enhancement in vegetative growth-related parameters, physiological characteristics, efficiency of water use, yield, as well as reduced proline levels. Tomato yield enhanced by 4%, 16%, 8%, and 3% when irrigation with freshwater at different levels of water deficit (100% ETc, 80% ETc, 60% ETc, and 40% ETc) than control (BC 0% ). Overall, the use of biochar (3%) combined with freshwater shows the potential to enhance morpho-physiological characteristics, support the development of tomato plants, and improve yield with higher WUE in semi-arid and arid areas.

Peer Review reports

Introduction

Tomato ( Solanum lycopersicum ) is widely recognized as one of the most commonly consumed vegetables globally [ 1 ]. Ensuring a substantial crop yield of tomatoes is crucial to address the growing need for food in Pakistan [ 2 ]. Tomato is abundant in minerals and antioxidants, including vitamin C, lycopene, and phenols [ 3 ]. Drought and salinity are the principal abiotic stresses that significantly restrict crop growth and yield on a global scale [ 4 ]. Bahawalpur is renowned for its arid climate, making it one of the most parched regions in Pakistan. Approximately 70% of water reserves are utilized for agricultural purposes, alongside several other elements that impact agricultural practices [ 5 ]. Much of the soil in Bahawalpur consists of sandy-loam and sand-based soils, characterized by a limited ability to retain water, a rapid rate of water penetration and a low level of clay. Consequently, these soils require meticulous treatment.

Irrigated agriculture consumes over two-thirds of global freshwater usage, making it the primary consumer of this resource [ 6 ]. Meeting the need for nutritious food for a growing global population while optimizing water usage for crop irrigation poses a significant challenge in agriculture nowadays [ 7 ]. The modern approach to water conservation focuses on enhancing water use efficiency while maintaining productivity levels [ 8 ]. Solanum lycopersicum plants, when exposed to drought stress, decrease the leaf area and photosynthetic level. This final result in a reduction in the accumulation of biomass and yield [ 9 ]. Pappula-Reddy et al. [ 10 ] found that water stress can result in yield reductions ranging from 13 to 94%, contingent upon the duration and intensity of the drought stress. Alza et al. [ 11 ] observed a 16% decrease in Solanum lycopersicum yield when exposed to a water deficit of 75% ETc compared to full irrigation. Nevertheless, drought-induced stress commonly decreases crop productivity and enhances water use efficiency, as demonstrated by [ 12 ].

To meet the growing demand for food due to population increase, it becomes essential to cultivate crops in soil with high salt content or irrigate them using water with high salt concentrations. This is particularly important in regions where water resources are frequently scarce [ 13 ]. Soil salinization is a highly detrimental abiotic stress that affects numerous cultivated crops globally [ 14 ]. It impacts about 20% of the cultivated area globally, leading to reduced plant growth and thus decreasing crop production [ 15 ]. The expansion of the salt-affected regions primarily arises from a blend of natural and human-induced factors, including insufficient and inadequate precipitation, elevated temperatures, excessive evapotranspiration, and subpar water and quality irrigation management [ 16 ]. Soil salinity substantially negatively impacts crop productivity, especially in vegetable crops. This is because vegetable crops often exhibit a limited capacity to withstand the adverse effects of salt stress [ 17 ]. Karimzadeh et al. [ 18 ] discovered that the combined influence of drought and salinity had a detrimental impact on the morpho-physiological characteristics of tomato seedlings. Salinity and drought conditions trigger the production of reactive oxygen species in cellular compartments, including mitochondria, peroxisomes, and chloroplasts. In addition, reactive oxygen species play a significant role in causing suboptimal plant growth and reduced productivity due to the oxidation of lipids in cellular membranes and the degradation of nucleic acids and enzyme proteins [ 19 ].

Biochar could promote long-term production and improve fertilizer and water utilization efficiency [ 20 ]. International Biochar Initiative defined biochar as a finely textured organic substance with significant carbon. It is generated by the process known as pyrolysis, which includes thermal decomposition of feedstock at temperatures ranging from 300 to 600 °C in the presence of limited or no oxygen [ 21 ]. Biochar utilization in agricultural systems has garnered attention in recent years because of its potential advantages in enhancing crop productivity and environmental conditions [ 22 ]. According to Haddad et al. [ 23 ], using fertilizers and biochar are also primary methods for improving water use efficiency, soil fertility, and crop yields in water-limited regions. These methods help mitigate the detrimental effects of water stress. Furthermore, biochar improves soil physical characteristics, including water retention capacity, bulk density, porosity, and fertility [ 24 ]. Biochar enhances soil moisture retention, decreasing osmotic and oxidative stress, promoting plant growth and facilitating water absorption via plants [ 25 ]. Biochar utilization enhances soil water availability by modifying soil composition and augmenting its water retention capacity [ 26 ]. Biochar can enhance the health of sandy soil impacted by salt under arid environments, resulting in increased growth and yield of vegetation and improved water use efficiency in tomatoes [ 27 ]. Zahedifar et al. [ 28 ] reported that biochar addition positively influenced low-quality soil, enhancing the growth characteristics, biomass, and yield of crops under water and salt-induced stress. According to another research, when biochar was applied at a dosage of 4.8 t/ha, it caused a rise in the quantity of leaves, flowers, and the size of tomato fruits [ 29 ]. However, this increase was insufficient to compensate for the decrease in fruit production and the elevated sodium ion levels accumulated in the root system due to salt stress [ 29 ]. The main aim of utilizing biochar is contingent upon various elements, including soil composition, the quantity of biochar incorporated into soil and physical and chemical attributes of the biochars, which largely rely on pyrolysis parameters and the feedstock material used [ 30 ].

Many studies focused on investigating the impacts of either salinity or drought stress. However, only a limited number of studies examined the combined effects of both salinity and drought stress, and these studies revealed conflicting results, particularly about the utilization of various types of biochars.

Hence, the main aims of this research were to examine the impact of drought and salt-induced stress on the morphological and physiological characteristics, water use efficiency, and tomatoes yield. Additionally, the study aimed to determine if applying biochar derived from sesame residue might mitigate the adverse impacts of salt and water stresses.

Materials and methods

Research site and experimental design.

The experiment was carried out from October 2022 to July 2023 in a greenhouse at Islamia University of Bahawalpur located in Bahawalpur, Punjab, Pakistan (29° 23’ 44.5956’’ N and 71° 41’ 0.0024’’ E). The weather pattern in District Bahawalpur is marked by scorching and arid summers, accompanied by prevailing dry and chilly conditions in winter. The maximum temperature increases to 48ºC, while the minimum temperature decreases to 7ºC. Summer often has numerous wind and dust storms. The region receives an average annual rainfall of 200 mm.

A three-factorial experiment was conducted with two water quality treatments. Sodium chloride (NaCl) was added to achieve salinity levels of 0.9 and 2.4 dS m − 1 . Four deficit irrigation levels 40% ETc, 60 ETc, 80 ETc, and 100% of total evapotranspiration (ETc) were investigated. Additionally, biochar was applied at a rate of 3% (w/w) (equivalent to 2.20 kg m − 2 ) (BC3%), while untreated soil served as the control (BC0%). The control treatment involved complete irrigation (100% of full irrigation) without the addition of biochar or salinity.

Experiments were designed as a randomized complete block (split-split-plot design) with three replicates. Water quality was identified as the primary factor, with irrigation levels serving as sub-factors within it. Correspondingly, biochar addition located in sub-sub-plots, The overall experiment was consisted of 48 experimental units, distributed as follows; 2 water quality × 4 irrigation levels × 2 biochar × 3 replicates. The experimental unit consists of a line 6 m length and 1 m width, with emitters spaced 0.4 m (15 plants) and 1 m between the experimental units. The control was full irrigation (100% ETc) without salinity and biochar.

The research was conducted using the commercial tomato variety ‘NIAB tomato-21’ in a greenhouse environment. Tomato seeds were planted in foam pots filled with a mixture of vermiculite and peat moss (in a 1:1 ratio by volume) on October 20, 2022. In a controlled environment within a fiberglass greenhouse, seedlings are grown using standard procedures at a daytime temperature of 25 °C and an at-night temperature of 20 °C to protect them from cold weather. After four weeks the seedlings were moved to the control greenhouse, where they were standardized to consistent size with five leaves. The control greenhouse maintained a temperature of 26 °C during the day, 19 °C at night, and a relative humidity of 75%. The agricultural practices commonly advised for commercial tomato production in greenhouses were utilized, which encompassed soil sterilization, insect management, and fertilizer. The local farmers followed the required application rates of 238 kg potassium, 142 kg phosphorus, and 285 kg nitrogen per acre for fertilizers during the growing seasons [ 17 ]. The greenhouse was the designated location for installing the surface drip irrigation system. The irrigation levels were selected based on the daily evapotranspiration and crop coefficient (Kc) standards. These levels were set at 40%, 60%, 80%, and 100% of the crop’s water requirement (ETc). The ETc was determined using the following formula (Allen et al. [ 77 ]

The variables in the equation are Eo, which represents the evaporation from pan A in millimeters; Kp, which represents the pan coefficient; and Kc, which represents the crop coefficient.

Soil and water analysis

Before the experiment, water and soil samples were collected from the greenhouse. A sample of sandy soil was air dried, passed through a 2 mm sieve, and a saturated soil paste extract was prepared. Analyzes of the water and soil samples, including the pH and EC, were performed using a pH (CG 817) and an EC (Test Kit Model 1500-20, Cole and Parmer) meter. Water-soluble sodium (Na + ), magnesium (Mg 2+ ), potassium (K + ), calcium (Ca 2+ ), and chloride (Cl − ) were measured using an ion chromatography device (ICS-5000, Thermo Fisher Scientific, Waltham, MA, USA). Bicarbonate (HCO 3 − ) and soluble carbonate (CO 3 2− ) were measured using a titration method [ 31 ]. The soil and water undergo chemical analysis; the results are displayed in Table 1 .

Production of Biochar

The biochar utilized in this experiment was derived from sesame residue. Biochar production involved heating the material to pyrolysis at a temperature of 550 °C for 2 h. The physico-chemical characteristics of biochar have been examined after its manufacture and are presented in Table 2 .

Measurement of growth-related parameters and physiological aspects

Plant growth-related attributes were assessed, such as the plant’s height, leaf area index, and diameter of stem, as well as the plant’s dry and fresh weight (containing both stems and leaves). Dry weight was measured using a digital balance assessment after the sample was desiccated at a temperature of 70 °C until a uniform dry weight was achieved, employing a convection oven. Leaf tissues were utilized to determine the leaf’s relative water content, which was defined as follows: the discs of leaves were collected to assess fresh weight, and then they were immersed in deionized water for a maximum of 4 h to get turgid weight. Dry mass was measured by placing the leaves in oven drying at around 85 °C till they attained a consistent weight. Leaf’s relative water content was determined by applying the methodology described by Smart and Bingham [ 32 ].

Measurement of LAI

The LAI values were measured throughout the growing period starting at 40 days after transplanting in 3 replications in each treatment at 10-day intervals and lasted until 4 times using the AccuPAR ceptometer LP-80, Decagon Devices Germany. The ceptometer is a battery-operated menu driven device, which is used to measure light interception in plant canopies to calculate LAI. Its main components are an integrated microprocessor-driven data logger and a probe with 80 sensors. Data were collected from menu screen by inserting the probe into canopy. For determining LAI in a destructive way, all leaves were removed separately from randomly selected 3 plants from each treatment at the last sampling date. The collected leaves (tomato, 10–12 per plant) were placed into a rectangular sketch of a white paper.

Three completely matured leaves from the uppermost layer of plants were chosen per each experimental part to determine the transpiration and photosynthetic rate and conductivity. The photosynthetic rate measurement was estimated in a closed system of infrared gas analyzer Li-Cor 6400 Portable Photosynthesis system. Before warming and calibrating the portable photosynthesis system. In the first step, the initial zeroing process for the built-in flow meter and the second step for the infra-red gas analyzer were observed. The measurements were used for optimal cuvette conditions such as 1000 Kumol photosynthetically active radiation (PAR), 400 µmol/ mol carbon dioxide, 30 °C leaf temperature, and 60% relative humidity with air flow rate of 500 cm³/min. The measurements of gas exchange were carried out between 9:00 to 11:00 a.m. The leaf surfaces were cleaned and dried before enclosed in the leaf cuvette. Data for photosynthesis rate and transpiration rate were simultaneously recorded. The spectrophotometric determination of chlorophyll a and b, carotenoids, and total chlorophyll is conducted using the method described by [ 33 ]. Leaf chlorophyll content was determined by using the Coombs method [ 33 ]. Leaves were gnawed using cock borer to get four sample areas of 1 cm² per gnawing. Samples were put into a vial, and 20 ml of 80% (v/v) acetone was poured into a vial and covered with aluminum foil. These samples were kept in a dark place for about three to seven days until extraction of all chlorophyll from leaves. Chlorophyll content was then determined using Spectrophotometer (Model UV 3101 PC) at wavelengths of 664 nm and 647 nm. The values for Chlorophyll a and b, carotenoids, and total chlorophyll were determined using the below Eq.

O.D. the extract’s optical density at the specified wavelength and V represents the extract’s volume, measured in milliliters (mL). W: mass of the leaves when they are freshly harvested (g) [ 34 ]. The amounts of proline in leaves were estimated using Clausen’s technique [ 35 ].

Water use efficiency and total yield

The total yield and each fruit weight were determined with digital balances during the entire harvesting period, measured in kilograms per square meter. The water use efficiency (WUE) was determined by dividing total fresh fruit yield (TFFY in kilograms) by the cumulative quantity of the water provided (CIW, in cubic meters) to the tomato plants over the entire planting season, as stated by Lovelli et al. [ 36 ].

Yield decline (YR %) and saved water (%) were calculated with Eqs. ( 8 ), ( 9 ) from the study conducted by [ 37 ]. The enhancement in WUE was computed by applying Eq. ( 10 ), as per the study conducted by [ 38 ].

WCC represents the use of water by control group, determined in m 3 /m 2 . WCT represents the water consumption of the treatment group, also determined in m 3 /m 2 .

Statistical evaluation

Data was statistically analysed by applying ANOVA with SAS software. The revised least significant difference (LSD) test was conducted at a confidence level of 0.05, as stated by Steel and Torrie [ 39 ].

Morphological characteristics of tomato plants

High salinity levels and water stress adversely impact several plant growth-related parameters, such as plant height, stem diameter, leaf area, and dry and wet weight. Conversely, using biochar enhanced all plant growth components (Table 3 ). In this concern, tomato plants under salt stress had lower ( P ≤ 0.05) the aforementioned parameters than fresh watered plants by 15.1%, 19.4%, 91.8%, 21.0%, and 12.8%, respectively. A similar decrease was seen when the deficiency irrigation levels were applied in comparison to fully irrigated plants. The irrigation stress leads to a notable reduction in most of the morphological traits, depending upon the period and level of the stress [ 3 ]. Our results revealed that, the most reduction was achieved at irrigation levels of 60% or 40% applied, lowered the previously indicated growth-related parameters by 22.7 or 29.6%, 23.7 or 40.2%, 17.5 or 28.8%, 26.5 or 37.0% and 16.0 or 28.9%, orderly, compared to full 100% irrigated plants. The plant’s vegetative growth properties were affected by the presence of saline water, which resulted in a nutritional imbalance. Furthermore, a high salt content caused poor plant growth, mainly due to ion toxicity and osmotic stress [ 8 ]. Conversely, biochar (BC 3 %) significantly improved tomato growth-related parameters in the current study’s region, increasing plant height by 6.8%, stem diameter by 7.6%, leaf area by 9.0%, dry weight by 6.3%, and wet weight by 5.3% in comparison to non-added biochar (BC 0 %).

The interaction among water stress, biochar, and salinity substantially impacted plant height, stem diameter, leaf area index, and fresh and dry weights, as shown in Table 4 . The biochar addition positively affected the vegetative growth characteristics across all irrigation levels, mainly when fresh water was used for irrigation. It’s interesting to note that the highest growth improvements in plant height (7.4% and 7.3%), stem diameter (5.7% and 13.6%), leaf area index (4.7% and 7.8%), fresh weight (6.2% and 4.8%), and dry weight (8.8% and 6.5%), respectively, were observed under irrigation level at 100% ETc and received biocahr at (BC 3 %), compared to non-added (BC 0 %) biochar plants. This was observed when comparing fresh water (0.9 dS m − 1 ) and saline water (2.4 dS m − 1 ), orderly. The positive impacts of biochar on vegetative growth characteristics are ascribed to its ability to stimulate microbial activity within the root zone and improve the soil’s capacity to retain water [ 11 ]. Furthermore, the biochar exhibits a substantial concentration of minerals, including calcium, magnesium, and inorganic carbon, which provide beneficial impacts on the growth of plants [ 12 ]. The application of biochar resulted in an improvement in the water status of the soil and a reduction in ion concentration in the presence of salt stress, so creating a conducive environment for the growth of plants. Additionally, the incorporation of biochar led to enhanced vegetative growth as a result of mitigating oxidative and osmotic stressors [ 13 ]. Conversely, the biochar addition with saltwater led to reduced vegetative growth attributes, particularly when the Solanum lycopersicum crop was exposed to drought-induced stress at 40% ETc and 60% ETc (Table 4 ). As it would be proposed, exactly how much biochar is used determines how much improvement it may achieve. For this reason, the adverse reaction to saline water could be due to the low biochar dosage (BC3%) treatment (Thomas et al., 2013).

Physiological parameters

The gas exchange of leaf attributes (photosynthesis, rate of transpiration conductivity, and leaf relative water content) were significantly decreased by drought, salt, and stresses, especially when the irrigation was 60 and 40% compared to 80 and 100%. When compared to fresh water, there was a significant drop in the aforementioned leaf gas exchange properties by 15.8%, 21.4%, 4.3%, and 10.7% when the tomato plant was irrigated with low quality (2.4 dS m − 1 ) water. Accordingly, the tomato plant was exposed to 60% ETc or 40% ETc, respectively, resulting in a significant decrease of 23.1% or 6.5%, 26.1% or 39.6%, 28.4% or 45.4%, and 13.3% or 21.2% of the aforementioned leaf gas exchange properties as compared to full irrigated (100% ETc) plants. The proline concentration in the leaves increased with salt and water deficit irrigation (2.4 dS m − 1 and at 60% or 40 ETc, orderly), comparatively to the control, Table ( 5 ). Under such circumstances, proline increased by 33.6% due to saline (2.4 dS m − 1 ) water, and in response to water deficit irrigation (i.e. 60% or 40 ETc, respectively), by 63.7% or 79.8%, relative to control plants. On the other hand, the incorporation of biochar at a concentration of 3% led to the most favorable leaf gas exchange characteristics, LRWC, augmenting by 5.2%, 4.8%, 9.9%, 2.5%, respectively, and the least amount of proline (dropped by 3.6%), in the tomato leaves as compared to plants that were not treated (Table 5 ). The incorporation of 3% biochar into freshwater resulted in the most significant improvements in leaf gas exchange characteristics across all water deficit treatments and when 100% ETc was added, as compared to the untreated plants without biochar. Conversely, the combination of salinity and deficit with 40% ETc and 60% ETc had a negative impact on all leaf gas exchange traits (Fig. 1 A–C). The findings depicted in Fig. 1 D demonstrate that the leaves of tomatoes cultivated under biochar with saline water exhibited the highest proline level at the maximum water deficit of 40% ETc. Conversely, the leaves watered with fresh water at 100% ETc displayed the lowest proline content. The irrigation levels with biochar and freshwater yielded the greatest LRWC values, surpassing those of the untreated plants (without biochar). In contrast, the lowest values for LRWC were found with biochar and irrigation with saline water under the highest water deficits of 40% and 60% ETc (Fig. 1 E).

Combined impacts of biochar, deficit irrigation, and salinity on various parameters of tomato leaf rate of photosynthesis (µmolCO 2 m − 2 S − 1 ) ( A ), rate of transpiration (mmolH 2 OCO 2 m − 2 S − 1 ) ( B ), conductivity (mmolH 2 OCO 2 m − 2 S − 1 ) ( C ), proline levels (mg/g − 1 FW) ( D ), and leaf relative water content (%) ( E )

Photosynthetic pigments

The photosynthetic pigments feature, including the leaf index (SPAD), total chlorophyll, chlorophyll a, chlorophyll b, and carotenoids decreased in tomato exposed to salinity and drought stress, as shown in Table 6 . Drought and salt stress have been shown to reduce the content of photosynthetic pigments. It’s found that, irrigating tomato with low quality of water (2.4 dS m − 1 ), reduced the aforementioned photosynthetic attributes by 18.2%, 11.4%, 11.1%, 18.3%, and 14.1%, respectively, in comparison to fresh water (0.9 dS m − 1 ) control. In respect deficit treatments, the most significant reduction was attained when exposed tomato plants to the most severe drought of 60% ETc or 40% ETc levels, resulting in decreases by 25.7 or 37.0%, 21.7 or 27.9%, 14.8 or 24.7%, 20.3 or 25.4%, and 14.7 or 26.5% for the correspondingly photosynthetic characteristics, compared to those plant received 100% ETc of water capacity.

Conversely, the incorporation of biochar significantly increased the leaf green index by 7.1%, total chlorophyll by 3.0%, chlorophyll a and b (by 7.9% and 5.8%, orderly), and carotenoids by 4.7%, in comparison to the plants that were not treated (BC 0 %) (Table 6 ). Plants that were treated with biochar and irrigated with fresh water at 100% of ETc exhibited the highest values for leaf pigment traits. In contrast, plants irrigated with saline water, particularly under the maximum water deficit of 40% ETc, had the lowest values (Table 7 ). The experimental findings demonstrate that the utilization of biochar leads to an enhancement in the rate of photosynthesis, hence suggesting a boost in the concentration of chlorophyll.

Water Use Efficiency and Fruit Yield

The biochar addition, water quality (fresh and saline water), and irrigation deficiency all impact the total yield and water use efficiency (WUE) of tomatoes. These effects are summarized in Table 8 . The findings revealed that incorporating biochar led to a significant increase in overall crop production and water use efficiency. By incorporating 3% biochar with fresh water, the tomato plants’ yield was enhanced by 4.6%, 16.7%, 8.6%, and 2.9% for 100%, 80%, 60%, and 40% ETc irrigation treatments, respectively, in comparison to the untreated plants (BC 0% ).

The water use efficiency (WUE) of tomato plants was observed to rise by 98% when they were exposed to biochar treatment and freshwater irrigation under a deficit irrigation of 40% ETc, as compared to full irrigation (Fig. 2 ). In comparison, the incorporation of biochar resulted in a decrease of 42% in tomato production when underwent saline water irrigation under the most extreme stress conditions (40% ETc), as depicted in Fig. 2 .

Combined impacts of biochar, water deficit, and salinity on the total fruit yield (kg m − 2 ) ( A ) and water usage efficiency (kg m − 3 ) ( B ) of Solanum lycopersicum

Water Use Efficiency (WUE) improvement and Irrigation Water savings

The findings presented in Table 8 demonstrate that the use of salt water resulted in a 15% decrease in yield and a 16% decrease in WUE. The findings displayed in Table 8 indicate that a 40% ETc irrigation deficit resulted in a 28% decrease in tomato yield, but a 79% improvement in water use efficiency (WUE) compared to the control group with 100% ETc. The application of biochar at the stated rate (BC 3% ) resulted in a 2.9% increase in the yield and a 1.17% increase in the water use efficiency (WUE) of tomato plants. The observed enhancement in crop yield and water use efficiency (WUE) can be ascribed to the soil-based biochar behavior, which facilitates the growth of roots within the soil.

Abiotic stressors such as salinity can greatly affect plant growth, morphological characteristics, and biochemical and physiological features. If stress arises during the sensitive phases of plant life, it might reduce crop yield. Stress caused by salinity is a significant issue, especially in developing nations where people rely heavily on agriculture. Accumulated salts in the soil solution can create osmotic pressure, limiting water availability to plants [ 11 ]. Moreover, excessive accumulation of chloride and sodium can lead to an imbalance of ionic levels and toxicity to ions, which can hinder the uptake of other mineral nutrients through plants. Moreover, it can stimulate the production of abscisic acid and inhibit growth promoters [ 7 ]. The most common damages caused by salinity include imbalances in the ionic and water levels of the plant, reduced photosynthesis, and stomatal closure [ 2 ]. The imposition of irrigation stress resulted in a substantial reduction in the majority of morphological traits, contingent upon the intensity and duration of the stress [ 40 ]. The presence of saline water negatively affected the vegetative growth characteristics of plants, mainly caused by an imbalance in nutrition [ 41 ]. In addition, an elevated salt concentration resulted in insufficient plant growth, caused primarily by ion toxicity and osmotic stress [ 42 ]. The biochar addition led to an augmentation of nutrient availability, potentially improving plant morphology [ 43 ]. In addition, applying biochar in the soil increased water availability, hence mitigating the effects of osmotic stress [ 44 ]. Biochar supplementation mainly improves soil water retention and water holding capacity in soils, whereas it enhances water infiltration and drainage (saturated hydraulic conductivity) in fine-textured soils. Biochar application in soils considerably improves root systems and excellent roots, which enhances plants’ ability to bind soil particles. Biochar is crucial in soil and water conservation in dry and semi-dry areas [ 45 ]. The beneficial impacts of rice-derived biochar addition on the vegetative growth characteristics are ascribed to the expansion of the microbe’s growth in root areas and the improved capacity of soil for holding water [ 45 ]. Furthermore, biochar is rich in minerals, including inorganic carbon, magnesium, and calcium, which positively affect plant development [ 46 ]. Biochar incorporation improved the moisture level of the soil and reduced the concentration of ions in the presence of salinity stress, creating optimal conditions for the growth of plants [ 47 ]. In addition, the biochar addition enhanced plant growth by reducing osmotic and oxidative stressors [ 48 ]. Drought and salinization induce ionic and osmotic stress, thereby eliciting cellular stress responses and fostering the generation of reactive oxygen species (ROS) disrupting regular cellular activities. Abscisic acid (ABA) levels regulate the generation of reactive oxygen species (ROS), serving as a crucial chemical signal for plants to detect environmental stress and control crop development [ 43 ].

Several studies have shown the adverse impacts of drought and salinity on photosynthetic characteristics, leaf relative water content (LRWC), and growth of plants [ 49 ]. Increased salt content was observed to reduce gas exchange in Solanum lycopersicum seedling leaves, as observed by Abbas et al. [ 50 ]. Our results correlate with Rodrigues et al. [ 51 ], who found that the proline level was significantly enhanced by deficit irrigation and that a surge in proline percentage was linked to both salinity and drought [ 47 ]. On the other hand, when compared to plants that were not treated, Solanum lycopersicum leaves with 3% biochar added showed the most significant levels of leaf-relative water contents and the lowest levels of proline (Table 5 ). The increase in both gas exchange and LRWC, and the decrease proline content was due to the increasing water availability in the soil and salt leaching from the root zone. This reduces osmotic stress and enhances water uptake by the plant [ 52 ]. When 3% biochar was added to freshwater, the leaf gas exchange attributes showed the highest values in all drought treatments than control. Conversely, when drought and salinity were combined with 40% and 60% irrigation, all the leaf gas exchange characteristics were negatively impacted (Fig. 1 A-C).

The findings shown in Fig. 1 -D demonstrate that leaves of Solanum lycopersicum planted under biochar with salted water at maximum water stress (deficit) of 40% irrigation had the highest level of proline. In comparison, leaves watered with fresh water at 100% irrigation had the lowest level of proline. Compared to the control, the highest leaf-relative water content levels were recorded for all irrigation treatments using biochar-derived from sesame and fresh-water. Conversely, under the highest water shortages of 40% irrigation and 60% irrigation, saline irrigation and biochar produced the lowest values for leaf-relative water content (Fig. 1 E). Massimi et al. [ 53 ] found that increasing the salt concentration decreased the transpiration rate by 70.55%, the stomatal conductance by 7.13%, and the photosynthetic rate by 72.34% in the leaves of tomato seedlings. According to the findings presented by Yang et al. [ 54 ], the addition of biochar significantly increased the photosynthetic rate (Ph), the relative water content (RWC), and recorded the lowest proline content in tomato plants exposed to a water deficit. Similarly, Rodrigues et al. [ 51 ] observed that adding biochar to stressed and unstressed tomato plants significantly improved the photosynthetic and transpiration rates. Additionally, the use of biochar improved the leaf gas exchange and LWRC under salinity and drought stress conditions, indicating that biochar helped the plants retain firm leaves under abiotic stresses [ 17 ]. In this study, the decreased chlorophyll could be due to damage to the thylakoid membranes, as a result of the destructive effect of reactive oxygen species (ROS) on chloroplasts [ 55 ]. The generation of reactive oxygen species (ROS) was significantly increased due to salinity and water deficiencies [ 50 ]. Another explanation for the observed reduction in chlorophyll concentration may be attributed to the detrimental effects of osmotic stress on the chloroplast layers, which leads to an increase in membrane permeability [ 56 , 74 ]. For instance, previous studies have demonstrated that salt stress and drought can lead to a decrease in the concentration of photosynthetic pigments in tomato leaves [ 57 , 58 ]. However, the incorporation of biochar led to a notable augmentation in the leaf green index, chlorophyll a, chlorophyll b, total chlorophyll, and carotenoids as compared to the plants that were not treated (BC 0% ) (Table 6 ). The findings were consistent with the results reported in references [ 59 , 60 ]. Specifically, they observed that the biochar application increased chlorophyll levels when Solanum lycopersicum was exposed to drought and salt-induced conditions. Biochar’s effect on chlorophyll and carotenoid levels under salt stress is associated with increased antioxidant activity and the development of antioxidant capacity. Biochar supplementation stimulates the uptake of magnesium, an essential component in chlorophyll production [ 29 ]. Nazarideljou et al. [ 65 ] also discovered that the utilization of a 5% amendment (biochar) enhanced the productivity and growth attributes of Solanum lycopersicum cultivated in a salt-induced environment. According to the findings of our study, the utilization of biochar enhances the rate of photosynthesis, which is an indicator of elevated chlorophyll levels. Biochar enhances photosynthetic pigments by influencing nutrient intake and availability (potassium, phosphorus, magnesium, calcium, and sulfur) and enhancing soil’s physiochemical and biological characteristics [ 62 , 63 ]. Biochar addition significantly enhanced the antioxidant activities, protecting the photosynthetic apparatus and pigments of plants from oxidative damage caused by salt stress [ 61 ].

The growth and yield of plants are negatively affected by salt and water stress, as evidenced by the findings of [ 64 , 66 ]. Previous research has revealed that biochar incorporation can enhance plant growth, boost crop yields, and improve water use efficiency [ 67 ]. In a study carried out by Wang et al. [ 68 ], it was discovered that the incorporation of 50 tons ha − 1 of biochar resulted in a 55% increase in Solanum lycopersicum yield and a 45% improvement in water use efficiency than the control. Biochar treatment prevents membrane damage by reducing Na + level and boosting K + , therefore enhancing leaf hydration status during salt stress. Biochar enhances leaf water status by elevating potassium (K) concentration, which is a crucial osmoprotectant in plant tissues. Applying biochar to plants enhances their leaf Relative Water Content, leading to an improved Water Use Efficiency by the plants [ 69 ].

When 3% biochar was added to freshwater, Solanum lycopersicum yield increased by 4.6%, 16.7%, 8.6%, and 2.9% for irrigation levels of 100, 80, 60, and 40%, respectively, then control. The water use efficiency (WUE) of Solanum lycopersicum plants increased by 97% when they were exposed to a deficit irrigation of 40% and supplemented with biochar. At the same time, they were supplied with fresh water. This is in comparison to Solanum lycopersicum plants that underwent full irrigation (Fig. 2 ). The increase in yield and WUE with the biochar might be explained by its ability to retain water, improve porosity, and provide nutrients to the plant under water stress conditions. The increase in WUE with deficit irrigation could be attributed to reductions in the transpiration rate (TR) and stomatal closure in response to salt and water stress [ 69 ].Conversely, the biochar addition resulted in a 42% decrease in Solanum lycopersicum yield when watered with saltwater under extreme stress-induced conditions (40%), as shown in Fig. 2 , then control. It should be concluded that the negative effects from the biochar addition on the tomato yield in this study were most likely related to physiological drought resulting from the interaction between the biochar, saline water, and water deficit, and the high pH of biochar. As a result, the root absorption of water was more incomprehensible, leading to a decrease in the yield [ 70 ]. A high pH can affect the nutrient release into the soil, resulting in a decrease in the yield [ 71 , 75 , 76 ]. According to Bahadur et al. [ 72 ] the addition of biochar to the soil improved some vegetative growth attributes, but did not mitigate the negative effects of salt stress on tomato fruit yield. Table 8 shows that the use of saline water resulted in a 14% decrease in Solanum lycopersicum and a 15% decrease in water use efficiency. The data displayed in Table 8 indicates that exposing the Solanum lycopersicum plants to an irrigation deficit of 40% resulted in a 28% decrease in yield while simultaneously enhancing the water use efficiency by 79% than control. By incorporating biochar at a rate of 3%, the Solanum lycopersicum plants exhibited a 2.9% increase in yield and a 1.17% increase in water use efficiency. This increase in the yield and WUE can be attributed to biochar behavior in the soil, promoting root growth in the soil. Similar results were reported by Vajjiravel et al. [ 73 ] in the study on pepper plants grown in greenhouse, which indicated that the addition of biochar improved the WUE and irrigation water savings.

Salinity and drought led to decreases in tomato’s growth-related characteristics, physiological parameters and productivity, due to damage from these stresses. In this sense, plant water status, photosynthetic efficiency, and corresponding chlorophyll pigment were all impacted by this outcome. Consequently, significant changes in plant development and growth by disrupting molecular, physiological, and biochemical processes are realised. Our findings indicating that, to effectively cultivate Solanum lycopersicum cultivars in semi-arid and arid areas with sandy soils and low agricultural productivity, it is necessary to add various amendments that help combat the detrimental impacts of drought and salinity. In this concern, the damage from these stresses can be ameliorated by the incorporation of 3% biochar, through positively improve the physio-morphological and functional traits and the water use efficiency of Solanum lycopersicum cultivated in a greenhouse environment. Biochar addition to sandy soil is a suggested technique to enhance the growth and production of tomatoes under drought and salinity conditions without any interaction between these two factors. Moreover, applying biochar enhances membrane stability, nutrient absorption, and nutrient balance, improving plant performance in salinity and drought stress conditions. Biochar limited the entry of harmful Na + and enhanced the entry of K + in response to salinity stress, which helps regulate stomata motions and enhance leaf gas exchange properties. In order to successfully adapt to the changing global climate, our results may help to develop strategies of the future applications to satisfying growth and yield with higher WUE of tomato under conditions impacted by salt and inadequate irrigation, particularly in arid and semiarid areas in Pakistan.

Data availability

The datasets analysed during this study are included in this manuscript.

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Acknowledgements

The authors extend their appreciation to the Researchers supporting project number (RSP2024R193), King Saud University, Riyadh, Saudi Arabia.

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School of Agriculture and Biology, Shanghai Jiao Tong University, 800 Dongchuan Road, Minghang District, Shanghai, 200240, China

Muhammad Usman

Department of Botany, Bacha Khan University, Charsadda, Khyber Pakhtunkhwa, 24420, Pakistan

Javed Iqbal

Department of Botany and Microbiology, College of Science, King Saud University, Riyadh, 11451, Saudi Arabia

Mohamed S. Elshikh & Jawaher Alkahtani

Institute of Agriculture, Lithuanian Research Centre for Agriculture and Forestry, Instituo Al. 1, Akademija, Kedainiai, LT- 58344, Lithuania

Monika Toleikienė

Department of Agronomy, Faculty of Agriculture and Environment, The Islamia University of Bahawalpur, Bahawalpur, 63100, Pakistan

Rashid Iqbal

Department of Entomology, Faculty of Agriculture and Environment, The Islamia University of Bahawalpur, Bahawalpur, 63000, Pakistan

M. Irfan Akram

Institute of Plant Sciences and Resource Conservation, Division of Horticultural Sciences, University of Bonn, 53115, Bonn, Germany

Nazim S. Gruda

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Contributions

Conceptualization, G.M. and M.U.; methodology, M.U.; software, J.I and M.T.; validation, M.N.T., M.S.E. and J.A.; formal analysis, R.I.; investigation, M.U.; resources, R.I. and M.T.; data curation, M.S.E., M.I.A.; writing—original draft preparation, G.M.; reviewing and editing N.S.G.; writing—review and editing, all authors; visualization, R.I., and M.I.A.; supervision, R.I.; project administration, G.M.; funding acquisition, M.S.E., N.S.G. and M.T. All authors have read and agreed to the published version of the manuscript.

Corresponding authors

Correspondence to Ghulam Murtaza , Rashid Iqbal or Nazim S. Gruda .

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Murtaza, G., Usman, M., Iqbal, J. et al. The impact of biochar addition on morpho-physiological characteristics, yield and water use efficiency of tomato plants under drought and salinity stress. BMC Plant Biol 24 , 356 (2024). https://doi.org/10.1186/s12870-024-05058-9

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