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How to Write a Research Paper Introduction (with Examples)

The research paper introduction section, along with the Title and Abstract, can be considered the face of any research paper. The following article is intended to guide you in organizing and writing the research paper introduction for a quality academic article or dissertation.

The research paper introduction aims to present the topic to the reader. A study will only be accepted for publishing if you can ascertain that the available literature cannot answer your research question. So it is important to ensure that you have read important studies on that particular topic, especially those within the last five to ten years, and that they are properly referenced in this section. 1 What should be included in the research paper introduction is decided by what you want to tell readers about the reason behind the research and how you plan to fill the knowledge gap. The best research paper introduction provides a systemic review of existing work and demonstrates additional work that needs to be done. It needs to be brief, captivating, and well-referenced; a well-drafted research paper introduction will help the researcher win half the battle.

The introduction for a research paper is where you set up your topic and approach for the reader. It has several key goals:

• Present your research topic
• Capture reader interest
• Summarize existing research
• Position your own approach
• Define your specific research problem and problem statement
• Highlight the novelty and contributions of the study
• Give an overview of the paper’s structure

The research paper introduction can vary in size and structure depending on whether your paper presents the results of original empirical research or is a review paper. Some research paper introduction examples are only half a page while others are a few pages long. In many cases, the introduction will be shorter than all of the other sections of your paper; its length depends on the size of your paper as a whole.

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Table of Contents

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The introduction in a research paper is placed at the beginning to guide the reader from a broad subject area to the specific topic that your research addresses. They present the following information to the reader

• Scope: The topic covered in the research paper
• Context: Background of your topic
• Importance: Why your research matters in that particular area of research and the industry problem that can be targeted

The research paper introduction conveys a lot of information and can be considered an essential roadmap for the rest of your paper. A good introduction for a research paper is important for the following reasons:

• It stimulates your reader’s interest: A good introduction section can make your readers want to read your paper by capturing their interest. It informs the reader what they are going to learn and helps determine if the topic is of interest to them.
• It helps the reader understand the research background: Without a clear introduction, your readers may feel confused and even struggle when reading your paper. A good research paper introduction will prepare them for the in-depth research to come. It provides you the opportunity to engage with the readers and demonstrate your knowledge and authority on the specific topic.
• It explains why your research paper is worth reading: Your introduction can convey a lot of information to your readers. It introduces the topic, why the topic is important, and how you plan to proceed with your research.
• It helps guide the reader through the rest of the paper: The research paper introduction gives the reader a sense of the nature of the information that will support your arguments and the general organization of the paragraphs that will follow. It offers an overview of what to expect when reading the main body of your paper.

What are the parts of introduction in the research?

A good research paper introduction section should comprise three main elements: 2

• What is known: This sets the stage for your research. It informs the readers of what is known on the subject.
• What is lacking: This is aimed at justifying the reason for carrying out your research. This could involve investigating a new concept or method or building upon previous research.
• What you aim to do: This part briefly states the objectives of your research and its major contributions. Your detailed hypothesis will also form a part of this section.

How to write a research paper introduction?

The first step in writing the research paper introduction is to inform the reader what your topic is and why it’s interesting or important. This is generally accomplished with a strong opening statement. The second step involves establishing the kinds of research that have been done and ending with limitations or gaps in the research that you intend to address. Finally, the research paper introduction clarifies how your own research fits in and what problem it addresses. If your research involved testing hypotheses, these should be stated along with your research question. The hypothesis should be presented in the past tense since it will have been tested by the time you are writing the research paper introduction.

The following key points, with examples, can guide you when writing the research paper introduction section:

• Highlight the importance of the research field or topic
• Describe the background of the topic
• Present an overview of current research on the topic

Example: The inclusion of experiential and competency-based learning has benefitted electronics engineering education. Industry partnerships provide an excellent alternative for students wanting to engage in solving real-world challenges. Industry-academia participation has grown in recent years due to the need for skilled engineers with practical training and specialized expertise. However, from the educational perspective, many activities are needed to incorporate sustainable development goals into the university curricula and consolidate learning innovation in universities.

• Reveal a gap in existing research or oppose an existing assumption
• Formulate the research question

Example: There have been plausible efforts to integrate educational activities in higher education electronics engineering programs. However, very few studies have considered using educational research methods for performance evaluation of competency-based higher engineering education, with a focus on technical and or transversal skills. To remedy the current need for evaluating competencies in STEM fields and providing sustainable development goals in engineering education, in this study, a comparison was drawn between study groups without and with industry partners.

• State the purpose of your study
• Highlight the key characteristics of your study
• Describe important results
• Highlight the novelty of the study.
• Offer a brief overview of the structure of the paper.

Example: The study evaluates the main competency needed in the applied electronics course, which is a fundamental core subject for many electronics engineering undergraduate programs. We compared two groups, without and with an industrial partner, that offered real-world projects to solve during the semester. This comparison can help determine significant differences in both groups in terms of developing subject competency and achieving sustainable development goals.

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Step 3: Fill in the specifics, such as your field of study, brief description or details you want to include, which will help the AI generate the outline for your Introduction.

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Step 5: Turn to Paperpal’s granular language checks to refine your content, tailor it to reflect your personal writing style, and ensure it effectively conveys your message.

You can use the same process to develop each section of your article, and finally your research paper in half the time and without any of the stress.

The purpose of the research paper introduction is to introduce the reader to the problem definition, justify the need for the study, and describe the main theme of the study. The aim is to gain the reader’s attention by providing them with necessary background information and establishing the main purpose and direction of the research.

The length of the research paper introduction can vary across journals and disciplines. While there are no strict word limits for writing the research paper introduction, an ideal length would be one page, with a maximum of 400 words over 1-4 paragraphs. Generally, it is one of the shorter sections of the paper as the reader is assumed to have at least a reasonable knowledge about the topic. 2 For example, for a study evaluating the role of building design in ensuring fire safety, there is no need to discuss definitions and nature of fire in the introduction; you could start by commenting upon the existing practices for fire safety and how your study will add to the existing knowledge and practice.

When deciding what to include in the research paper introduction, the rest of the paper should also be considered. The aim is to introduce the reader smoothly to the topic and facilitate an easy read without much dependency on external sources. 3 Below is a list of elements you can include to prepare a research paper introduction outline and follow it when you are writing the research paper introduction. Topic introduction: This can include key definitions and a brief history of the topic. Research context and background: Offer the readers some general information and then narrow it down to specific aspects. Details of the research you conducted: A brief literature review can be included to support your arguments or line of thought. Rationale for the study: This establishes the relevance of your study and establishes its importance. Importance of your research: The main contributions are highlighted to help establish the novelty of your study Research hypothesis: Introduce your research question and propose an expected outcome. Organization of the paper: Include a short paragraph of 3-4 sentences that highlights your plan for the entire paper

Cite only works that are most relevant to your topic; as a general rule, you can include one to three. Note that readers want to see evidence of original thinking. So it is better to avoid using too many references as it does not leave much room for your personal standpoint to shine through. Citations in your research paper introduction support the key points, and the number of citations depend on the subject matter and the point discussed. If the research paper introduction is too long or overflowing with citations, it is better to cite a few review articles rather than the individual articles summarized in the review. A good point to remember when citing research papers in the introduction section is to include at least one-third of the references in the introduction.

The literature review plays a significant role in the research paper introduction section. A good literature review accomplishes the following: Introduces the topic – Establishes the study’s significance – Provides an overview of the relevant literature – Provides context for the study using literature – Identifies knowledge gaps However, remember to avoid making the following mistakes when writing a research paper introduction: Do not use studies from the literature review to aggressively support your research Avoid direct quoting Do not allow literature review to be the focus of this section. Instead, the literature review should only aid in setting a foundation for the manuscript.

Remember the following key points for writing a good research paper introduction: 4

• Avoid stuffing too much general information: Avoid including what an average reader would know and include only that information related to the problem being addressed in the research paper introduction. For example, when describing a comparative study of non-traditional methods for mechanical design optimization, information related to the traditional methods and differences between traditional and non-traditional methods would not be relevant. In this case, the introduction for the research paper should begin with the state-of-the-art non-traditional methods and methods to evaluate the efficiency of newly developed algorithms.
• Avoid packing too many references: Cite only the required works in your research paper introduction. The other works can be included in the discussion section to strengthen your findings.
• Avoid extensive criticism of previous studies: Avoid being overly critical of earlier studies while setting the rationale for your study. A better place for this would be the Discussion section, where you can highlight the advantages of your method.
• Avoid describing conclusions of the study: When writing a research paper introduction remember not to include the findings of your study. The aim is to let the readers know what question is being answered. The actual answer should only be given in the Results and Discussion section.

To summarize, the research paper introduction section should be brief yet informative. It should convince the reader the need to conduct the study and motivate him to read further. If you’re feeling stuck or unsure, choose trusted AI academic writing assistants like Paperpal to effortlessly craft your research paper introduction and other sections of your research article.

1. Jawaid, S. A., & Jawaid, M. (2019). How to write introduction and discussion. Saudi Journal of Anaesthesia, 13(Suppl 1), S18.

2. Dewan, P., & Gupta, P. (2016). Writing the title, abstract and introduction: Looks matter!. Indian pediatrics, 53, 235-241.

3. Cetin, S., & Hackam, D. J. (2005). An approach to the writing of a scientific Manuscript1. Journal of Surgical Research, 128(2), 165-167.

4. Bavdekar, S. B. (2015). Writing introduction: Laying the foundations of a research paper. Journal of the Association of Physicians of India, 63(7), 44-6.

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• USC Libraries
• Research Guides

Organizing Your Social Sciences Research Paper

• 4. The Introduction
• 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 introduction leads the reader from a general subject area to a particular topic of inquiry. It establishes the scope, context, and significance of the research being conducted by summarizing current understanding and background information about the topic, stating the purpose of the work in the form of the research problem supported by a hypothesis or a set of questions, explaining briefly the methodological approach used to examine the research problem, highlighting the potential outcomes your study can reveal, and outlining the remaining structure and organization of the paper.

Key Elements of the Research Proposal. Prepared under the direction of the Superintendent and by the 2010 Curriculum Design and Writing Team. Baltimore County Public Schools.

Importance of a Good Introduction

Think of the introduction as a mental road map that must answer for the reader these four questions:

• What was I studying?
• Why was this topic important to investigate?
• What did we know about this topic before I did this study?
• How will this study advance new knowledge or new ways of understanding?

According to Reyes, there are three overarching goals of a good introduction: 1) ensure that you summarize prior studies about the topic in a manner that lays a foundation for understanding the research problem; 2) explain how your study specifically addresses gaps in the literature, insufficient consideration of the topic, or other deficiency in the literature; and, 3) note the broader theoretical, empirical, and/or policy contributions and implications of your research.

A well-written introduction is important because, quite simply, you never get a second chance to make a good first impression. The opening paragraphs of your paper will provide your readers with their initial impressions about the logic of your argument, your writing style, the overall quality of your research, and, ultimately, the validity of your findings and conclusions. A vague, disorganized, or error-filled introduction will create a negative impression, whereas, a concise, engaging, and well-written introduction will lead your readers to think highly of your analytical skills, your writing style, and your research approach. All introductions should conclude with a brief paragraph that describes the organization of the rest of the paper.

Hirano, Eliana. “Research Article Introductions in English for Specific Purposes: A Comparison between Brazilian, Portuguese, and English.” English for Specific Purposes 28 (October 2009): 240-250; Samraj, B. “Introductions in Research Articles: Variations Across Disciplines.” English for Specific Purposes 21 (2002): 1–17; Introductions. The Writing Center. University of North Carolina; “Writing Introductions.” In Good Essay Writing: A Social Sciences Guide. Peter Redman. 4th edition. (London: Sage, 2011), pp. 63-70; Reyes, Victoria. Demystifying the Journal Article. Inside Higher Education.

Structure and Writing Style

I.  Structure and Approach

The introduction is the broad beginning of the paper that answers three important questions for the reader:

• What is this?
• Why should I read it?
• What do you want me to think about / consider doing / react to?

Think of the structure of the introduction as an inverted triangle of information that lays a foundation for understanding the research problem. Organize the information so as to present the more general aspects of the topic early in the introduction, then narrow your analysis to more specific topical information that provides context, finally arriving at your research problem and the rationale for studying it [often written as a series of key questions to be addressed or framed as a hypothesis or set of assumptions to be tested] and, whenever possible, a description of the potential outcomes your study can reveal.

These are general phases associated with writing an introduction: 1.  Establish an area to research by:

• Highlighting the importance of the topic, and/or
• Making general statements about the topic, and/or
• Presenting an overview on current research on the subject.

2.  Identify a research niche by:

• Opposing an existing assumption, and/or
• Revealing a gap in existing research, and/or
• Formulating a research question or problem, and/or
• Continuing a disciplinary tradition.

3.  Place your research within the research niche by:

• Stating the intent of your study,
• Outlining the key characteristics of your study,
• Describing important results, and
• Giving a brief overview of the structure of the paper.

NOTE:   It is often useful to review the introduction late in the writing process. This is appropriate because outcomes are unknown until you've completed the study. After you complete writing the body of the paper, go back and review introductory descriptions of the structure of the paper, the method of data gathering, the reporting and analysis of results, and the conclusion. Reviewing and, if necessary, rewriting the introduction ensures that it correctly matches the overall structure of your final paper.

II.  Delimitations of the Study

Delimitations refer to those characteristics that limit the scope and define the conceptual boundaries of your research . This is determined by the conscious exclusionary and inclusionary decisions you make about how to investigate the research problem. In other words, not only should you tell the reader what it is you are studying and why, but you must also acknowledge why you rejected alternative approaches that could have been used to examine the topic.

Obviously, the first limiting step was the choice of research problem itself. However, implicit are other, related problems that could have been chosen but were rejected. These should be noted in the conclusion of your introduction. For example, a delimitating statement could read, "Although many factors can be understood to impact the likelihood young people will vote, this study will focus on socioeconomic factors related to the need to work full-time while in school." The point is not to document every possible delimiting factor, but to highlight why previously researched issues related to the topic were not addressed.

Examples of delimitating choices would be:

• The key aims and objectives of your study,
• The research questions that you address,
• The variables of interest [i.e., the various factors and features of the phenomenon being studied],
• The method(s) of investigation,
• The time period your study covers, and
• Any relevant alternative theoretical frameworks that could have been adopted.

Review each of these decisions. Not only do you clearly establish what you intend to accomplish in your research, but you should also include a declaration of what the study does not intend to cover. In the latter case, your exclusionary decisions should be based upon criteria understood as, "not interesting"; "not directly relevant"; “too problematic because..."; "not feasible," and the like. Make this reasoning explicit!

NOTE:   Delimitations refer to the initial choices made about the broader, overall design of your study and should not be confused with documenting the limitations of your study discovered after the research has been completed.

ANOTHER NOTE : Do not view delimitating statements as admitting to an inherent failing or shortcoming in your research. They are an accepted element of academic writing intended to keep the reader focused on the research problem by explicitly defining the conceptual boundaries and scope of your study. It addresses any critical questions in the reader's mind of, "Why the hell didn't the author examine this?"

III.  The Narrative Flow

Issues to keep in mind that will help the narrative flow in your introduction :

• Your introduction should clearly identify the subject area of interest . A simple strategy to follow is to use key words from your title in the first few sentences of the introduction. This will help focus the introduction on the topic at the appropriate level and ensures that you get to the subject matter quickly without losing focus, or discussing information that is too general.
• Establish context by providing a brief and balanced review of the pertinent published literature that is available on the subject. The key is to summarize for the reader what is known about the specific research problem before you did your analysis. This part of your introduction should not represent a comprehensive literature review--that comes next. It consists of a general review of the important, foundational research literature [with citations] that establishes a foundation for understanding key elements of the research problem. See the drop-down menu under this tab for " Background Information " regarding types of contexts.
• Clearly state the hypothesis that you investigated . When you are first learning to write in this format it is okay, and actually preferable, to use a past statement like, "The purpose of this study was to...." or "We investigated three possible mechanisms to explain the...."
• Why did you choose this kind of research study or design? Provide a clear statement of the rationale for your approach to the problem studied. This will usually follow your statement of purpose in the last paragraph of the introduction.

IV.  Engaging the Reader

A research problem in the social sciences can come across as dry and uninteresting to anyone unfamiliar with the topic . Therefore, one of the goals of your introduction is to make readers want to read your paper. Here are several strategies you can use to grab the reader's attention:

• Open with a compelling story . Almost all research problems in the social sciences, no matter how obscure or esoteric , are really about the lives of people. Telling a story that humanizes an issue can help illuminate the significance of the problem and help the reader empathize with those affected by the condition being studied.
• Include a strong quotation or a vivid, perhaps unexpected, anecdote . During your review of the literature, make note of any quotes or anecdotes that grab your attention because they can used in your introduction to highlight the research problem in a captivating way.
• Pose a provocative or thought-provoking question . Your research problem should be framed by a set of questions to be addressed or hypotheses to be tested. However, a provocative question can be presented in the beginning of your introduction that challenges an existing assumption or compels the reader to consider an alternative viewpoint that helps establish the significance of your study. 
• Describe a puzzling scenario or incongruity . This involves highlighting an interesting quandary concerning the research problem or describing contradictory findings from prior studies about a topic. Posing what is essentially an unresolved intellectual riddle about the problem can engage the reader's interest in the study.
• Cite a stirring example or case study that illustrates why the research problem is important . Draw upon the findings of others to demonstrate the significance of the problem and to describe how your study builds upon or offers alternatives ways of investigating this prior research.

NOTE:   It is important that you choose only one of the suggested strategies for engaging your readers. This avoids giving an impression that your paper is more flash than substance and does not distract from the substance of your study.

Freedman, Leora  and Jerry Plotnick. Introductions and Conclusions. University College Writing Centre. University of Toronto; Introduction. The Structure, Format, Content, and Style of a Journal-Style Scientific Paper. Department of Biology. Bates College; Introductions. The Writing Center. University of North Carolina; Introductions. The Writer’s Handbook. Writing Center. University of Wisconsin, Madison; Introductions, Body Paragraphs, and Conclusions for an Argument Paper. The Writing Lab and The OWL. Purdue University; “Writing Introductions.” In Good Essay Writing: A Social Sciences Guide . Peter Redman. 4th edition. (London: Sage, 2011), pp. 63-70; Resources for Writers: Introduction Strategies. Program in Writing and Humanistic Studies. Massachusetts Institute of Technology; Sharpling, Gerald. Writing an Introduction. Centre for Applied Linguistics, University of Warwick; Samraj, B. “Introductions in Research Articles: Variations Across Disciplines.” English for Specific Purposes 21 (2002): 1–17; Swales, John and Christine B. Feak. Academic Writing for Graduate Students: Essential Skills and Tasks . 2nd edition. Ann Arbor, MI: University of Michigan Press, 2004 ; Writing Your Introduction. Department of English Writing Guide. George Mason University.

Writing Tip

Avoid the "Dictionary" Introduction

Giving the dictionary definition of words related to the research problem may appear appropriate because it is important to define specific terminology that readers may be unfamiliar with. However, anyone can look a word up in the dictionary and a general dictionary is not a particularly authoritative source because it doesn't take into account the context of your topic and doesn't offer particularly detailed information. Also, placed in the context of a particular discipline, a term or concept may have a different meaning than what is found in a general dictionary. If you feel that you must seek out an authoritative definition, use a subject specific dictionary or encyclopedia [e.g., if you are a sociology student, search for dictionaries of sociology]. A good database for obtaining definitive definitions of concepts or terms is Credo Reference .

Saba, Robert. The College Research Paper. Florida International University; Introductions. The Writing Center. University of North Carolina.

Another Writing Tip

When Do I Begin?

A common question asked at the start of any paper is, "Where should I begin?" An equally important question to ask yourself is, "When do I begin?" Research problems in the social sciences rarely rest in isolation from history. Therefore, it is important to lay a foundation for understanding the historical context underpinning the research problem. However, this information should be brief and succinct and begin at a point in time that illustrates the study's overall importance. For example, a study that investigates coffee cultivation and export in West Africa as a key stimulus for local economic growth needs to describe the beginning of exporting coffee in the region and establishing why economic growth is important. You do not need to give a long historical explanation about coffee exports in Africa. If a research problem requires a substantial exploration of the historical context, do this in the literature review section. In your introduction, make note of this as part of the "roadmap" [see below] that you use to describe the organization of your paper.

Introductions. The Writing Center. University of North Carolina; “Writing Introductions.” In Good Essay Writing: A Social Sciences Guide . Peter Redman. 4th edition. (London: Sage, 2011), pp. 63-70.

Yet Another Writing Tip

Always End with a Roadmap

The final paragraph or sentences of your introduction should forecast your main arguments and conclusions and provide a brief description of the rest of the paper [the "roadmap"] that let's the reader know where you are going and what to expect. A roadmap is important because it helps the reader place the research problem within the context of their own perspectives about the topic. In addition, concluding your introduction with an explicit roadmap tells the reader that you have a clear understanding of the structural purpose of your paper. In this way, the roadmap acts as a type of promise to yourself and to your readers that you will follow a consistent and coherent approach to addressing the topic of inquiry. Refer to it often to help keep your writing focused and organized.

Cassuto, Leonard. “On the Dissertation: How to Write the Introduction.” The Chronicle of Higher Education , May 28, 2018; Radich, Michael. A Student's Guide to Writing in East Asian Studies . (Cambridge, MA: Harvard University Writing n. d.), pp. 35-37.

• ~[123]~: May 9, 2024 11:05 AM
• ~[124]~: https://libguides.usc.edu/writingguide

• If you are writing in a new discipline, you should always make sure to ask about conventions and expectations for introductions, just as you would for any other aspect of the essay. For example, while it may be acceptable to write a two-paragraph (or longer) introduction for your papers in some courses, instructors in other disciplines, such as those in some Government courses, may expect a shorter introduction that includes a preview of the argument that will follow.  
• In some disciplines (Government, Economics, and others), it’s common to offer an overview in the introduction of what points you will make in your essay. In other disciplines, you will not be expected to provide this overview in your introduction.  
• Avoid writing a very general opening sentence. While it may be true that “Since the dawn of time, people have been telling love stories,” it won’t help you explain what’s interesting about your topic.  
• Avoid writing a “funnel” introduction in which you begin with a very broad statement about a topic and move to a narrow statement about that topic. Broad generalizations about a topic will not add to your readers’ understanding of your specific essay topic.  
• Avoid beginning with a dictionary definition of a term or concept you will be writing about. If the concept is complicated or unfamiliar to your readers, you will need to define it in detail later in your essay. If it’s not complicated, you can assume your readers already know the definition.  
• Avoid offering too much detail in your introduction that a reader could better understand later in the paper.
• picture_as_pdf Introductions

How to write an effective introduction for your research paper

Last updated

20 January 2024

Reviewed by

However, the introduction is a vital element of your research paper. It helps the reader decide whether your paper is worth their time. As such, it's worth taking your time to get it right.

In this article, we'll tell you everything you need to know about writing an effective introduction for your research paper.

• The importance of an introduction in research papers

The primary purpose of an introduction is to provide an overview of your paper. This lets readers gauge whether they want to continue reading or not. The introduction should provide a meaningful roadmap of your research to help them make this decision. It should let readers know whether the information they're interested in is likely to be found in the pages that follow.

Aside from providing readers with information about the content of your paper, the introduction also sets the tone. It shows readers the style of language they can expect, which can further help them to decide how far to read.

When you take into account both of these roles that an introduction plays, it becomes clear that crafting an engaging introduction is the best way to get your paper read more widely. First impressions count, and the introduction provides that impression to readers.

• The optimum length for a research paper introduction

While there's no magic formula to determine exactly how long a research paper introduction should be, there are a few guidelines. Some variables that impact the ideal introduction length include:

Field of study

Complexity of the topic

Specific requirements of the course or publication

A commonly recommended length of a research paper introduction is around 10% of the total paper’s length. So, a ten-page paper has a one-page introduction. If the topic is complex, it may require more background to craft a compelling intro. Humanities papers tend to have longer introductions than those of the hard sciences.

The best way to craft an introduction of the right length is to focus on clarity and conciseness. Tell the reader only what is necessary to set up your research. An introduction edited down with this goal in mind should end up at an acceptable length.

• Evaluating successful research paper introductions

A good way to gauge how to create a great introduction is by looking at examples from across your field. The most influential and well-regarded papers should provide some insights into what makes a good introduction.

Dissecting examples: what works and why

We can make some general assumptions by looking at common elements of a good introduction, regardless of the field of research.

A common structure is to start with a broad context, and then narrow that down to specific research questions or hypotheses. This creates a funnel that establishes the scope and relevance.

The most effective introductions are careful about the assumptions they make regarding reader knowledge. By clearly defining key terms and concepts instead of assuming the reader is familiar with them, these introductions set a more solid foundation for understanding.

To pull in the reader and make that all-important good first impression, excellent research paper introductions will often incorporate a compelling narrative or some striking fact that grabs the reader's attention.

Finally, good introductions provide clear citations from past research to back up the claims they're making. In the case of argumentative papers or essays (those that take a stance on a topic or issue), a strong thesis statement compels the reader to continue reading.

Common pitfalls to avoid in research paper introductions

You can also learn what not to do by looking at other research papers. Many authors have made mistakes you can learn from.

We've talked about the need to be clear and concise. Many introductions fail at this; they're verbose, vague, or otherwise fail to convey the research problem or hypothesis efficiently. This often comes in the form of an overemphasis on background information, which obscures the main research focus.

Ensure your introduction provides the proper emphasis and excitement around your research and its significance. Otherwise, fewer people will want to read more about it.

• Crafting a compelling introduction for a research paper

Let’s take a look at the steps required to craft an introduction that pulls readers in and compels them to learn more about your research.

Step 1: Capturing interest and setting the scene

To capture the reader's interest immediately, begin your introduction with a compelling question, a surprising fact, a provocative quote, or some other mechanism that will hook readers and pull them further into the paper.

As they continue reading, the introduction should contextualize your research within the current field, showing readers its relevance and importance. Clarify any essential terms that will help them better understand what you're saying. This keeps the fundamentals of your research accessible to all readers from all backgrounds.

Step 2: Building a solid foundation with background information

Including background information in your introduction serves two major purposes:

It helps to clarify the topic for the reader

It establishes the depth of your research

The approach you take when conveying this information depends on the type of paper.

For argumentative papers, you'll want to develop engaging background narratives. These should provide context for the argument you'll be presenting.

For empirical papers, highlighting past research is the key. Often, there will be some questions that weren't answered in those past papers. If your paper is focused on those areas, those papers make ideal candidates for you to discuss and critique in your introduction.

Step 3: Pinpointing the research challenge

To capture the attention of the reader, you need to explain what research challenges you'll be discussing.

For argumentative papers, this involves articulating why the argument you'll be making is important. What is its relevance to current discussions or problems? What is the potential impact of people accepting or rejecting your argument?

For empirical papers, explain how your research is addressing a gap in existing knowledge. What new insights or contributions will your research bring to your field?

Step 4: Clarifying your research aims and objectives

We mentioned earlier that the introduction to a research paper can serve as a roadmap for what's within. We've also frequently discussed the need for clarity. This step addresses both of these.

When writing an argumentative paper, craft a thesis statement with impact. Clearly articulate what your position is and the main points you intend to present. This will map out for the reader exactly what they'll get from reading the rest.

For empirical papers, focus on formulating precise research questions and hypotheses. Directly link them to the gaps or issues you've identified in existing research to show the reader the precise direction your research paper will take.

Step 5: Sketching the blueprint of your study

Continue building a roadmap for your readers by designing a structured outline for the paper. Guide the reader through your research journey, explaining what the different sections will contain and their relationship to one another.

This outline should flow seamlessly as you move from section to section. Creating this outline early can also help guide the creation of the paper itself, resulting in a final product that's better organized. In doing so, you'll craft a paper where each section flows intuitively from the next.

Step 6: Integrating your research question

To avoid letting your research question get lost in background information or clarifications, craft your introduction in such a way that the research question resonates throughout. The research question should clearly address a gap in existing knowledge or offer a new perspective on an existing problem.

Tell users your research question explicitly but also remember to frequently come back to it. When providing context or clarification, point out how it relates to the research question. This keeps your focus where it needs to be and prevents the topic of the paper from becoming under-emphasized.

Step 7: Establishing the scope and limitations

So far, we've talked mostly about what's in the paper and how to convey that information to readers. The opposite is also important. Information that's outside the scope of your paper should be made clear to the reader in the introduction so their expectations for what is to follow are set appropriately.

Similarly, be honest and upfront about the limitations of the study. Any constraints in methodology, data, or how far your findings can be generalized should be fully communicated in the introduction.

Step 8: Concluding the introduction with a promise

The final few lines of the introduction are your last chance to convince people to continue reading the rest of the paper. Here is where you should make it very clear what benefit they'll get from doing so. What topics will be covered? What questions will be answered? Make it clear what they will get for continuing.

By providing a quick recap of the key points contained in the introduction in its final lines and properly setting the stage for what follows in the rest of the paper, you refocus the reader's attention on the topic of your research and guide them to read more.

• Research paper introduction best practices

Following the steps above will give you a compelling introduction that hits on all the key points an introduction should have. Some more tips and tricks can make an introduction even more polished.

As you follow the steps above, keep the following tips in mind.

Set the right tone and style

Like every piece of writing, a research paper should be written for the audience. That is to say, it should match the tone and style that your academic discipline and target audience expect. This is typically a formal and academic tone, though the degree of formality varies by field.

Kno w the audience

The perfect introduction balances clarity with conciseness. The amount of clarification required for a given topic depends greatly on the target audience. Knowing who will be reading your paper will guide you in determining how much background information is required.

Adopt the CARS (create a research space) model

The CARS model is a helpful tool for structuring introductions. This structure has three parts. The beginning of the introduction establishes the general research area. Next, relevant literature is reviewed and critiqued. The final section outlines the purpose of your study as it relates to the previous parts.

Master the art of funneling

The CARS method is one example of a well-funneled introduction. These start broadly and then slowly narrow down to your specific research problem. It provides a nice narrative flow that provides the right information at the right time. If you stray from the CARS model, try to retain this same type of funneling.

Incorporate narrative element

People read research papers largely to be informed. But to inform the reader, you have to hold their attention. A narrative style, particularly in the introduction, is a great way to do that. This can be a compelling story, an intriguing question, or a description of a real-world problem.

Write the introduction last

By writing the introduction after the rest of the paper, you'll have a better idea of what your research entails and how the paper is structured. This prevents the common problem of writing something in the introduction and then forgetting to include it in the paper. It also means anything particularly exciting in the paper isn’t neglected in the intro.

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Research Paper Introduction Examples

Looking for research paper introduction examples? Quotes, anecdotes, questions, examples, and broad statements—all of them can be used successfully to write an introduction for a research paper. It’s instructive to see them in action, in the hands of skilled academic writers.

Let’s begin with David M. Kennedy’s superb history, Freedom from Fear: The American People in Depression and War, 1929–1945 . Kennedy begins each chapter with a quote, followed by his text. The quote above chapter 1 shows President Hoover speaking in 1928 about America’s golden future. The text below it begins with the stock market collapse of 1929. It is a riveting account of just how wrong Hoover was. The text about the Depression is stronger because it contrasts so starkly with the optimistic quotation.

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“We in America today are nearer the final triumph over poverty than ever before in the history of any land.”—Herbert Hoover, August 11, 1928 Like an earthquake, the stock market crash of October 1929 cracked startlingly across the United States, the herald of a crisis that was to shake the American way of life to its foundations. The events of the ensuing decade opened a fissure across the landscape of American history no less gaping than that opened by the volley on Lexington Common in April 1775 or by the bombardment of Sumter on another April four score and six years later. (adsbygoogle = window.adsbygoogle || []).push({}); The ratcheting ticker machines in the autumn of 1929 did not merely record avalanching stock prices. In time they came also to symbolize the end of an era. (David M. Kennedy, Freedom from Fear: The American People in Depression and War, 1929–1945 . New York: Oxford University Press, 1999, p. 10)

Kennedy has exciting, wrenching material to work with. John Mueller faces the exact opposite problem. In Retreat from Doomsday: The Obsolescence of Major War , he is trying to explain why Great Powers have suddenly stopped fighting each other. For centuries they made war on each other with devastating regularity, killing millions in the process. But now, Mueller thinks, they have not just paused; they have stopped permanently. He is literally trying to explain why “nothing is happening now.” That may be an exciting topic intellectually, it may have great practical significance, but “nothing happened” is not a very promising subject for an exciting opening paragraph. Mueller manages to make it exciting and, at the same time, shows why it matters so much. Here’s his opening, aptly entitled “History’s Greatest Nonevent”:

On May 15, 1984, the major countries of the developed world had managed to remain at peace with each other for the longest continuous stretch of time since the days of the Roman Empire. If a significant battle in a war had been fought on that day, the press would have bristled with it. As usual, however, a landmark crossing in the history of peace caused no stir: the most prominent story in the New York Times that day concerned the saga of a manicurist, a machinist, and a cleaning woman who had just won a big Lotto contest. This book seeks to develop an explanation for what is probably the greatest nonevent in human history. (John Mueller, Retreat from Doomsday: The Obsolescence of Major War . New York: Basic Books, 1989, p. 3)

In the space of a few sentences, Mueller sets up his puzzle and reveals its profound human significance. At the same time, he shows just how easy it is to miss this milestone in the buzz of daily events. Notice how concretely he does that. He doesn’t just say that the New York Times ignored this record setting peace. He offers telling details about what they covered instead: “a manicurist, a machinist, and a cleaning woman who had just won a big Lotto contest.” Likewise, David Kennedy immediately entangles us in concrete events: the stunning stock market crash of 1929. These are powerful openings that capture readers’ interests, establish puzzles, and launch narratives.

Sociologist James Coleman begins in a completely different way, by posing the basic questions he will study. His ambitious book, Foundations of Social Theory , develops a comprehensive theory of social life, so it is entirely appropriate for him to begin with some major questions. But he could just as easily have begun with a compelling story or anecdote. He includes many of them elsewhere in his book. His choice for the opening, though, is to state his major themes plainly and frame them as a paradox. Sociologists, he says, are interested in aggregate behavior—how people act in groups, organizations, or large numbers—yet they mostly examine individuals:

A central problem in social science is that of accounting for the function of some kind of social system. Yet in most social research, observations are not made on the system as a whole, but on some part of it. In fact, the natural unit of observation is the individual person…  This has led to a widening gap between theory and research… (James S. Coleman, Foundations of Social Theory . Cambridge, MA: Harvard University Press, 1990, pp. 1–2)

After expanding on this point, Coleman explains that he will not try to remedy the problem by looking solely at groups or aggregate-level data. That’s a false solution, he says, because aggregates don’t act; individuals do. So the real problem is to show the links between individual actions and aggregate outcomes, between the micro and the macro.

The major problem for explanations of system behavior based on actions and orientations at a level below that of the system [in this case, on individual-level actions] is that of moving from the lower level to the system level. This has been called the micro-to-macro problem, and it is pervasive throughout the social sciences. (Coleman, Foundations of Social Theory , p. 6)

Explaining how to deal with this “micro-to-macro problem” is the central issue of Coleman’s book, and he announces it at the beginning.

Coleman’s theory-driven opening stands at the opposite end of the spectrum from engaging stories or anecdotes, which are designed to lure the reader into the narrative and ease the path to a more analytic treatment later in the text. Take, for example, the opening sentences of Robert L. Herbert’s sweeping study Impressionism: Art, Leisure, and Parisian Society : “When Henry Tuckerman came to Paris in 1867, one of the thousands of Americans attracted there by the huge international exposition, he was bowled over by the extraordinary changes since his previous visit twenty years before.” (Robert L. Herbert, Impressionism: Art, Leisure, and Parisian Society . New Haven, CT: Yale University Press, 1988, p. 1.) Herbert fills in the evocative details to set the stage for his analysis of the emerging Impressionist art movement and its connection to Parisian society and leisure in this period.

David Bromwich writes about Wordsworth, a poet so familiar to students of English literature that it is hard to see him afresh, before his great achievements, when he was just a young outsider starting to write. To draw us into Wordsworth’s early work, Bromwich wants us to set aside our entrenched images of the famous mature poet and see him as he was in the 1790s, as a beginning writer on the margins of society. He accomplishes this ambitious task in the opening sentences of Disowned by Memory: Wordsworth’s Poetry of the 1790s :

Wordsworth turned to poetry after the revolution to remind himself that he was still a human being. It was a curious solution, to a difficulty many would not have felt. The whole interest of his predicament is that he did feel it. Yet Wordsworth is now so established an eminence—his name so firmly fixed with readers as a moralist of self-trust emanating from complete self-security—that it may seem perverse to imagine him as a criminal seeking expiation. Still, that is a picture we get from The Borderers and, at a longer distance, from “Tintern Abbey.” (David Bromwich, Disowned by Memory: Wordsworth’s Poetry of the 1790s . Chicago: University of Chicago Press, 1998, p. 1)

That’s a wonderful opening! Look at how much Bromwich accomplishes in just a few words. He not only prepares the way for analyzing Wordsworth’s early poetry; he juxtaposes the anguished young man who wrote it to the self-confident, distinguished figure he became—the eminent man we can’t help remembering as we read his early poetry.

Let us highlight a couple of other points in this passage because they illustrate some intelligent writing choices. First, look at the odd comma in this sentence: “It was a curious solution, to a difficulty many would not have felt.” Any standard grammar book would say that comma is wrong and should be omitted. Why did Bromwich insert it? Because he’s a fine writer, thinking of his sentence rhythm and the point he wants to make. The comma does exactly what it should. It makes us pause, breaking the sentence into two parts, each with an interesting point. One is that Wordsworth felt a difficulty others would not have; the other is that he solved it in a distinctive way. It would be easy for readers to glide over this double message, so Bromwich has inserted a speed bump to slow us down. Most of the time, you should follow grammatical rules, like those about commas, but you should bend them when it serves a good purpose. That’s what the writer does here.

The second small point is the phrase “after the revolution” in the first sentence: “Wordsworth turned to poetry after the revolution to remind himself that he was still a human being.” Why doesn’t Bromwich say “after the French Revolution”? Because he has judged his book’s audience. He is writing for specialists who already know which revolution is reverberating through English life in the 1790s. It is the French Revolution, not the earlier loss of the American colonies. If Bromwich were writing for a much broader audience—say, the New York Times Book Review—he would probably insert the extra word to avoid confusion.

The message “Know your audience” applies to all writers. Don’t talk down to them by assuming they can’t get dressed in the morning. Don’t strut around showing off your book learnin’ by tossing in arcane facts and esoteric language for its own sake. Neither will win over readers.

Bromwich, Herbert, and Coleman open their works in different ways, but their choices work well for their different texts. Your task is to decide what kind of opening will work best for yours. Don’t let that happen by default, by grabbing the first idea you happen upon. Consider a couple of different ways of opening your thesis and then choose the one you prefer. Give yourself some options, think them over, then make an informed choice.

Using the Introduction to Map out Your Writing

Whether you begin with a story, puzzle, or broad statement, the next part of the research paper introduction should pose your main questions and establish your argument. This is your thesis statement—your viewpoint along with the supporting reasons and evidence. It should be articulated plainly so readers understand full well what your paper is about and what it will argue.

After that, give your readers a road map of what’s to come. That’s normally done at the end of the introductory section (or, in a book, at the end of the introductory chapter). Here’s John J. Mearsheimer presenting such a road map in The Tragedy of Great Power Politics . He not only tells us the order of upcoming chapters, he explains why he’s chosen that order and which chapters are most important:

The Plan of the Book The rest of the chapters in this book are concerned mainly with answering the six big questions about power which I identified earlier. Chapter 2, which is probably the most important chapter in the book, lays out my theory of why states compete for power and why they pursue hegemony. In Chapters 3 and 4, I define power and explain how to measure it. I do this in order to lay the groundwork for testing my theory… (John J. Mearsheimer, The Tragedy of Great Power Politics . New York: W. W. Norton, 2001, p. 27)

As this excerpt makes clear, Mearsheimer has already laid out his “six big questions” in the research paper introduction. Now he’s showing us the path ahead, the path to answering those questions.

At the end of the research paper introduction, give your readers a road map of what’s to come. Tell them what the upcoming sections will be and why they are arranged in this particular order.

Learn how to write an introduction for a research paper .

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How to Write a Research Introduction

Last Updated: December 6, 2023 Fact Checked

This article was co-authored by Megan Morgan, PhD . Megan Morgan is a Graduate Program Academic Advisor in the School of Public & International Affairs at the University of Georgia. She earned her PhD in English from the University of Georgia in 2015. There are 7 references cited in this article, which can be found at the bottom of the page. This article has been fact-checked, ensuring the accuracy of any cited facts and confirming the authority of its sources. This article has been viewed 2,653,113 times.

The introduction to a research paper can be the most challenging part of the paper to write. The length of the introduction will vary depending on the type of research paper you are writing. An introduction should announce your topic, provide context and a rationale for your work, before stating your research questions and hypothesis. Well-written introductions set the tone for the paper, catch the reader's interest, and communicate the hypothesis or thesis statement.

Introducing the Topic of the Paper

• In scientific papers this is sometimes known as an "inverted triangle", where you start with the broadest material at the start, before zooming in on the specifics. [2] X Research source
• The sentence "Throughout the 20th century, our views of life on other planets have drastically changed" introduces a topic, but does so in broad terms.
• It provides the reader with an indication of the content of the essay and encourages them to read on.

• For example, if you were writing a paper about the behaviour of mice when exposed to a particular substance, you would include the word "mice", and the scientific name of the relevant compound in the first sentences.
• If you were writing a history paper about the impact of the First World War on gender relations in Britain, you should mention those key words in your first few lines.

• This is especially important if you are attempting to develop a new conceptualization that uses language and terminology your readers may be unfamiliar with.

• If you use an anecdote ensure that is short and highly relevant for your research. It has to function in the same way as an alternative opening, namely to announce the topic of your research paper to your reader.
• For example, if you were writing a sociology paper about re-offending rates among young offenders, you could include a brief story of one person whose story reflects and introduces your topic.
• This kind of approach is generally not appropriate for the introduction to a natural or physical sciences research paper where the writing conventions are different.

Establishing the Context for Your Paper

• It is important to be concise in the introduction, so provide an overview on recent developments in the primary research rather than a lengthy discussion.
• You can follow the "inverted triangle" principle to focus in from the broader themes to those to which you are making a direct contribution with your paper.
• A strong literature review presents important background information to your own research and indicates the importance of the field.

• By making clear reference to existing work you can demonstrate explicitly the specific contribution you are making to move the field forward.
• You can identify a gap in the existing scholarship and explain how you are addressing it and moving understanding forward.

• For example, if you are writing a scientific paper you could stress the merits of the experimental approach or models you have used.
• Stress what is novel in your research and the significance of your new approach, but don't give too much detail in the introduction.
• A stated rationale could be something like: "the study evaluates the previously unknown anti-inflammatory effects of a topical compound in order to evaluate its potential clinical uses".

Specifying Your Research Questions and Hypothesis

• The research question or questions generally come towards the end of the introduction, and should be concise and closely focused.
• The research question might recall some of the key words established in the first few sentences and the title of your paper.
• An example of a research question could be "what were the consequences of the North American Free Trade Agreement on the Mexican export economy?"
• This could be honed further to be specific by referring to a particular element of the Free Trade Agreement and the impact on a particular industry in Mexico, such as clothing manufacture.
• A good research question should shape a problem into a testable hypothesis.

• If possible try to avoid using the word "hypothesis" and rather make this implicit in your writing. This can make your writing appear less formulaic.
• In a scientific paper, giving a clear one-sentence overview of your results and their relation to your hypothesis makes the information clear and accessible. [10] X Trustworthy Source PubMed Central Journal archive from the U.S. National Institutes of Health Go to source
• An example of a hypothesis could be "mice deprived of food for the duration of the study were expected to become more lethargic than those fed normally".

• This is not always necessary and you should pay attention to the writing conventions in your discipline.
• In a natural sciences paper, for example, there is a fairly rigid structure which you will be following.
• A humanities or social science paper will most likely present more opportunities to deviate in how you structure your paper.

Research Introduction Help

Community Q&A

• Use your research papers' outline to help you decide what information to include when writing an introduction. Thanks Helpful 0 Not Helpful 1
• Consider drafting your introduction after you have already completed the rest of your research paper. Writing introductions last can help ensure that you don't leave out any major points. Thanks Helpful 0 Not Helpful 0

• Avoid emotional or sensational introductions; these can create distrust in the reader. Thanks Helpful 50 Not Helpful 12
• Generally avoid using personal pronouns in your introduction, such as "I," "me," "we," "us," "my," "mine," or "our." Thanks Helpful 31 Not Helpful 7
• Don't overwhelm the reader with an over-abundance of information. Keep the introduction as concise as possible by saving specific details for the body of your paper. Thanks Helpful 24 Not Helpful 14

You Might Also Like

• ↑ https://library.sacredheart.edu/c.php?g=29803&p=185916
• ↑ https://www.aresearchguide.com/inverted-pyramid-structure-in-writing.html
• ↑ https://libguides.usc.edu/writingguide/introduction
• ↑ https://writing.wisc.edu/Handbook/PlanResearchPaper.html
• ↑ https://dept.writing.wisc.edu/wac/writing-an-introduction-for-a-scientific-paper/
• ↑ https://writing.wisc.edu/handbook/assignments/planresearchpaper/
• ↑ http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3178846/

About This Article

To introduce your research paper, use the first 1-2 sentences to describe your general topic, such as “women in World War I.” Include and define keywords, such as “gender relations,” to show your reader where you’re going. Mention previous research into the topic with a phrase like, “Others have studied…”, then transition into what your contribution will be and why it’s necessary. Finally, state the questions that your paper will address and propose your “answer” to them as your thesis statement. For more information from our English Ph.D. co-author about how to craft a strong hypothesis and thesis, keep reading! Did this summary help you? Yes No

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6 Examples of Research Paper Introductions to Hook Your Readers

Welcome to our guide on research paper introductions!

As you may already know, the introduction is a critical component of any research paper, as it sets the tone for the rest of the paper and establishes the reader’s expectations.

The introductory paragraph in a research paper should give an overview of what the reader can expect from the rest of the paper.

It should also establish a context for readers who are new to this topic. More importantly, when writing a research paper introduction, it’s best to focus on two key points: the problem being addressed and how this particular study (or studies) addresses it.

If those two points are clear then readers should be able to follow along as they read more into your paper.

In the following sections, we’ll provide you with examples of different types of research paper introductions to help you get started.

Types of Research Paper Introductions

General introductions.

A general introduction is suitable when you’re discussing a range of different topics or subjects within one paper. It provides the reader with an idea of where you stand in regard to the topic at hand and can be followed up with a sentence or two explaining your specific angle.

Topic: The Effect of Social Media on Mental Health

Social media has become an integral part of our daily lives, and its impact on mental health has become a subject of increasing concern in recent years. While social media platforms have provided a new means of communication and access to information, they have also been linked to negative mental health outcomes such as depression, anxiety, and low self-esteem. This research paper aims to explore the relationship between social media use and mental health outcomes. Specifically, we will investigate how social media affects individuals’ perceptions of themselves and their social relationships, as well as the potential impact on their emotional well-being. Through a thorough literature review and original research, we hope to shed light on the complex relationship between social media and mental health and provide insights into effective interventions for those who may be negatively impacted by social media use.

Topic: Formal Presentations at University

Most universities require their graduate-level courses to have some form of formal presentation such as a dissertation, master’s thesis, or doctoral dissertation. These presentations often take the form of an oral defense. Formal presentations provide both teachers and students with feedback in order to make future projects better. Teachers also use these projects to gauge student performance and provide guidance during group discussions throughout the course. What makes these oral defenses different than others is that they must adhere to strict guidelines set forth by the institution overseeing them. Failure to comply with these guidelines may result in expulsion from the university. Oral defenses typically contain three major parts: an opening statement, main body, and closing statement. However, some institutions may offer variations of this format. For instance, many institutions now require a written version of the presentation that includes all major aspects required by the institution.

Quote-Based Research Paper Introduction

This type of introduction is ideal for research papers that rely heavily on quotes to establish their point.

In these cases, quoting the most relevant piece of information is usually necessary because doing so will allow the reader to better understand what your discussion entails.

With that said, remember that every quote you include needs to add something significant and/or novel which hasn’t been covered before – otherwise it won’t be worth including in your paper!

Topic: International Student Success at the University Level

Shakespeare once said that “A rose by any other name would smell as sweet.” This line from Romeo and Juliet suggests that names don’t matter. Similarly, the fact that someone goes to school in a certain country does not affect the quality of education. Yet research has shown that international students perform worse academically than domestic ones in American colleges and universities. So, the question becomes: How do we foster success among international students? It is possible that differences in culture and socialization contribute to the observed gaps in academic achievement. Given that, our next step should be to examine the challenges faced by international students in an effort to find effective ways of addressing these challenges.

Topic: The Impact of Social Media on Mental Health

As Mark Twain once said, “Comparison is the death of joy.” In today’s world, social media platforms provide us with countless opportunities to compare our lives to those of others, often leading to feelings of inadequacy, anxiety, and depression. Research has shown that excessive social media use can have a negative impact on mental health, particularly among young adults. Given the increasing prevalence of social media in our daily lives, it’s important to explore this topic in more depth and understand the ways in which social media use can impact mental health outcomes.

Surprising Fact-based Introductions

A surprising fact-based introduction works well with scientific or medical topics where there is surprising new evidence that directly impacts how people view a certain issue. In such cases, it’s often necessary to give some background on what exactly is at stake in order to highlight why it’s important and relevant. Following that, you can introduce the key findings and what they mean.

Topic: The relationship between caffeine and weight loss

Caffeine is a well-known stimulant, which is consumed by millions of people on a daily basis in the form of coffee, tea, energy drinks, or pills. Although it’s unclear how much caffeine is too much for an individual to consume each day, it’s commonly believed that the positive effects of caffeine are canceled out by the increased appetite that results from drinking caffeinated beverages. But did you know that caffeine might actually help you lose weight?! Recent research conducted by a team of investigators from the Johns Hopkins Bloomberg School of Public Health and Harvard T.H. Chan School of Public Health found that the more caffeine an individual consumes, the lower their risk for obesity. That’s right: while it may sound counterintuitive, having one or two cups of coffee per day could actually decrease your chances of becoming overweight or obese.

Topic: The Benefits of Napping on Memory Retention

Did you know that a short nap can help boost your memory retention by up to 30%? Most people think of napping as something reserved for children or lazy adults, but recent research has shown that a short nap can actually be beneficial for cognitive function, particularly in regard to memory. In today’s fast-paced world, we often prioritize productivity over rest, but the science behind napping suggests that we should be doing the opposite. In this paper, we will explore the benefits of napping on memory retention and discuss how individuals and organizations can incorporate napping into their daily routines for optimal cognitive performance.

Explanatory research paper introductions

These types of research paper introductions are used when you’re introducing a new concept or idea that requires the audience to have knowledge of basic facts in order to follow along.

In this case, it’s critical that you provide the necessary background information and then follow up with a statement about why the rest of the research is interesting and/or relevant.

For example, you might say that the paper discusses a recent study on mindfulness and meditation.

In order to fully grasp the research and the claims being made, it’s necessary to first understand what mindfulness is and why it’s relevant. The following paragraphs would therefore serve as an introduction to both topics.

Topic: Mindfulness

Mindfulness is a mental state achieved by focusing one’s awareness on the present moment, while calmly acknowledging and accepting one’s feelings, thoughts, and bodily sensations. Regular practice of mindfulness can be highly beneficial to both mental and physical health. There are many different techniques for practicing mindfulness: Mindful walking, sitting with an open mind, eating without distractions, listening deeply to others, or simply noticing what you see in your environment. What’s most important is that you develop the ability to pay attention in a way that helps you connect with yourself and the world around you. And just like anything else, mindfulness takes practice. The best way to get started is by taking part in a course or program offered at your local community center, hospital, or online.

Topic: The Effectiveness of Online Learning in Higher Education

As more and more students turn to online learning to pursue their higher education, questions have arisen regarding the effectiveness of this type of education. While online learning offers a number of benefits, such as flexibility and convenience, it also presents unique challenges, including the lack of face-to-face interaction with instructors and classmates. This research paper aims to examine the effectiveness of online learning in higher education and compare it to traditional classroom-based learning. By conducting a literature review and analyzing data from surveys and interviews with students and instructors, this paper seeks to identify the advantages and disadvantages of online learning and provide recommendations for improving its effectiveness.

Narration-based paper introductions

This type of research paper introduction is used when you’re discussing a topic or event in detail.

You could talk about why it’s important, explain what happened and how it relates to your larger point, summarize what happened (as with a timeline), or present an example to prove your thesis.

Whatever you choose, it’s imperative that you make your readers want to keep reading.

This is done by telling a compelling story, using storytelling elements such as tension and conflict, or painting a vivid picture of the scene with sensory details.

Topic: The impact of stress on memory and learning

It’s well known that stress can impair our ability to learn new things, but it also has an impact on our memories. Well, I have first-hand experience with this. The night before my midterm, I had a major fight with my partner. This was one of the worst arguments we’ve ever had and it went on for hours. Needless to say, it stressed me out and there’s no doubt that it hurt my performance on the test. The funny thing is that in the days following the argument, my partner and I were able to clear everything up, so now we’re good again. But this experience taught me one very important lesson: the impact of stress on memory and learning might seem negligible but it can actually have a long-term impact on academic performance.

Topic: The Effects of Social Media on Mental Health

As I sit here scrolling through my Instagram feed, I can’t help but feel a sense of envy and inadequacy wash over me. The seemingly perfect lives of influencers and friends alike are on full display, leaving me to question why I don’t have the same level of success and happiness. It’s no secret that social media has become a ubiquitous part of our daily lives, with billions of people using platforms like Instagram, Facebook, and Twitter to connect with others and share their lives. But what are the effects of this constant exposure to other people’s carefully curated images and narratives? Are we putting ourselves at risk for developing mental health issues like anxiety and depression? In this paper, I will explore the research that has been done on the topic of social media and mental health, examining both the positive and negative effects that social media can have on our psychological well-being. By the end of this paper, you will have a better understanding of how social media impacts mental health and what steps we can take to mitigate its negative effects.

Teaser-based research paper introductions

The most common types of research paper introductions are teasers.

This simply means that your first sentence is a hook: something to grab your reader’s attention and make them want to read more.

One way to create a compelling teaser is by addressing an interesting question or controversial topic, such as “Why do some people succeed and others fail?”

Another approach is to pose a problem that you plan to solve.

Here are examples of a teaser-based research paper introductions.

Topic: Why do some people succeed and others fail?

Failure is a part of life. We all fail at one time or another, it’s just something that happens. But why do some people fail more than others? And what about people who never seem to fail? You see them every day, they are in your office and everywhere else you go. These people always seem to be successful and highly driven. How do they do it? This is a question that has puzzled psychologists and other social scientists for years. In the past, a lot of psychologists believed that success was due to differences in abilities and intelligence. Others said it depended on luck. Still, others believed that family background determined whether someone succeeded or not. As you can imagine, these explanations led to considerable debate and disagreement among scholars.

Topic: The Future of Artificial Intelligence in Healthcare

Are we on the cusp of a revolution in healthcare? With the rise of artificial intelligence (AI), the answer might just be yes. From machine learning algorithms that can identify diseases faster and more accurately than humans to chatbots that can answer patient questions 24/7, AI is poised to transform every aspect of healthcare. But as with any technology, there are risks and challenges that must be addressed. In this paper, we’ll explore the ways in which AI is being used in healthcare today and the potential implications for patients, doctors, and the healthcare industry as a whole. Buckle up, because the future is here, and it’s powered by AI.

Final Remarks: Research Paper Introductions

Crafting an effective research paper introduction can be challenging, but with the right approach and attention to detail, you can create an introduction that captures the reader’s attention and sets the stage for the rest of the paper.

Whether you choose a general introduction, a quote-based introduction, a surprising fact-based introduction, or an anecdotal introduction, be sure to clearly state your topic and thesis, provide background information, and engage your readers in meaning.

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Home » Research Paper – Structure, Examples and Writing Guide

Research Paper – Structure, Examples and Writing Guide

Table of Contents

Research Paper

Definition:

Research Paper is a written document that presents the author’s original research, analysis, and interpretation of a specific topic or issue.

It is typically based on Empirical Evidence, and may involve qualitative or quantitative research methods, or a combination of both. The purpose of a research paper is to contribute new knowledge or insights to a particular field of study, and to demonstrate the author’s understanding of the existing literature and theories related to the topic.

Structure of Research Paper

The structure of a research paper typically follows a standard format, consisting of several sections that convey specific information about the research study. The following is a detailed explanation of the structure of a research paper:

The title page contains the title of the paper, the name(s) of the author(s), and the affiliation(s) of the author(s). It also includes the date of submission and possibly, the name of the journal or conference where the paper is to be published.

The abstract is a brief summary of the research paper, typically ranging from 100 to 250 words. It should include the research question, the methods used, the key findings, and the implications of the results. The abstract should be written in a concise and clear manner to allow readers to quickly grasp the essence of the research.

Introduction

The introduction section of a research paper provides background information about the research problem, the research question, and the research objectives. It also outlines the significance of the research, the research gap that it aims to fill, and the approach taken to address the research question. Finally, the introduction section ends with a clear statement of the research hypothesis or research question.

Literature Review

The literature review section of a research paper provides an overview of the existing literature on the topic of study. It includes a critical analysis and synthesis of the literature, highlighting the key concepts, themes, and debates. The literature review should also demonstrate the research gap and how the current study seeks to address it.

The methods section of a research paper describes the research design, the sample selection, the data collection and analysis procedures, and the statistical methods used to analyze the data. This section should provide sufficient detail for other researchers to replicate the study.

The results section presents the findings of the research, using tables, graphs, and figures to illustrate the data. The findings should be presented in a clear and concise manner, with reference to the research question and hypothesis.

The discussion section of a research paper interprets the findings and discusses their implications for the research question, the literature review, and the field of study. It should also address the limitations of the study and suggest future research directions.

The conclusion section summarizes the main findings of the study, restates the research question and hypothesis, and provides a final reflection on the significance of the research.

The references section provides a list of all the sources cited in the paper, following a specific citation style such as APA, MLA or Chicago.

How to Write Research Paper

You can write Research Paper by the following guide:

• Choose a Topic: The first step is to select a topic that interests you and is relevant to your field of study. Brainstorm ideas and narrow down to a research question that is specific and researchable.
• Conduct a Literature Review: The literature review helps you identify the gap in the existing research and provides a basis for your research question. It also helps you to develop a theoretical framework and research hypothesis.
• Develop a Thesis Statement : The thesis statement is the main argument of your research paper. It should be clear, concise and specific to your research question.
• Plan your Research: Develop a research plan that outlines the methods, data sources, and data analysis procedures. This will help you to collect and analyze data effectively.
• Collect and Analyze Data: Collect data using various methods such as surveys, interviews, observations, or experiments. Analyze data using statistical tools or other qualitative methods.
• Organize your Paper : Organize your paper into sections such as Introduction, Literature Review, Methods, Results, Discussion, and Conclusion. Ensure that each section is coherent and follows a logical flow.
• Write your Paper : Start by writing the introduction, followed by the literature review, methods, results, discussion, and conclusion. Ensure that your writing is clear, concise, and follows the required formatting and citation styles.
• Edit and Proofread your Paper: Review your paper for grammar and spelling errors, and ensure that it is well-structured and easy to read. Ask someone else to review your paper to get feedback and suggestions for improvement.
• Cite your Sources: Ensure that you properly cite all sources used in your research paper. This is essential for giving credit to the original authors and avoiding plagiarism.

Research Paper Example

Note : The below example research paper is for illustrative purposes only and is not an actual research paper. Actual research papers may have different structures, contents, and formats depending on the field of study, research question, data collection and analysis methods, and other factors. Students should always consult with their professors or supervisors for specific guidelines and expectations for their research papers.

Research Paper Example sample for Students:

Title: The Impact of Social Media on Mental Health among Young Adults

Abstract: This study aims to investigate the impact of social media use on the mental health of young adults. A literature review was conducted to examine the existing research on the topic. A survey was then administered to 200 university students to collect data on their social media use, mental health status, and perceived impact of social media on their mental health. The results showed that social media use is positively associated with depression, anxiety, and stress. The study also found that social comparison, cyberbullying, and FOMO (Fear of Missing Out) are significant predictors of mental health problems among young adults.

Introduction: Social media has become an integral part of modern life, particularly among young adults. While social media has many benefits, including increased communication and social connectivity, it has also been associated with negative outcomes, such as addiction, cyberbullying, and mental health problems. This study aims to investigate the impact of social media use on the mental health of young adults.

Literature Review: The literature review highlights the existing research on the impact of social media use on mental health. The review shows that social media use is associated with depression, anxiety, stress, and other mental health problems. The review also identifies the factors that contribute to the negative impact of social media, including social comparison, cyberbullying, and FOMO.

Methods : A survey was administered to 200 university students to collect data on their social media use, mental health status, and perceived impact of social media on their mental health. The survey included questions on social media use, mental health status (measured using the DASS-21), and perceived impact of social media on their mental health. Data were analyzed using descriptive statistics and regression analysis.

Results : The results showed that social media use is positively associated with depression, anxiety, and stress. The study also found that social comparison, cyberbullying, and FOMO are significant predictors of mental health problems among young adults.

Discussion : The study’s findings suggest that social media use has a negative impact on the mental health of young adults. The study highlights the need for interventions that address the factors contributing to the negative impact of social media, such as social comparison, cyberbullying, and FOMO.

Conclusion : In conclusion, social media use has a significant impact on the mental health of young adults. The study’s findings underscore the need for interventions that promote healthy social media use and address the negative outcomes associated with social media use. Future research can explore the effectiveness of interventions aimed at reducing the negative impact of social media on mental health. Additionally, longitudinal studies can investigate the long-term effects of social media use on mental health.

Limitations : The study has some limitations, including the use of self-report measures and a cross-sectional design. The use of self-report measures may result in biased responses, and a cross-sectional design limits the ability to establish causality.

Implications: The study’s findings have implications for mental health professionals, educators, and policymakers. Mental health professionals can use the findings to develop interventions that address the negative impact of social media use on mental health. Educators can incorporate social media literacy into their curriculum to promote healthy social media use among young adults. Policymakers can use the findings to develop policies that protect young adults from the negative outcomes associated with social media use.

References :

• Twenge, J. M., & Campbell, W. K. (2019). Associations between screen time and lower psychological well-being among children and adolescents: Evidence from a population-based study. Preventive medicine reports, 15, 100918.
• Primack, B. A., Shensa, A., Escobar-Viera, C. G., Barrett, E. L., Sidani, J. E., Colditz, J. B., … & James, A. E. (2017). Use of multiple social media platforms and symptoms of depression and anxiety: A nationally-representative study among US young adults. Computers in Human Behavior, 69, 1-9.
• Van der Meer, T. G., & Verhoeven, J. W. (2017). Social media and its impact on academic performance of students. Journal of Information Technology Education: Research, 16, 383-398.

Appendix : The survey used in this study is provided below.

Social Media and Mental Health Survey

• How often do you use social media per day?
• Less than 30 minutes
• 30 minutes to 1 hour
• 1 to 2 hours
• 2 to 4 hours
• More than 4 hours
• Which social media platforms do you use?
• Others (Please specify)
• How often do you experience the following on social media?
• Social comparison (comparing yourself to others)
• Cyberbullying
• Fear of Missing Out (FOMO)
• Have you ever experienced any of the following mental health problems in the past month?
• Do you think social media use has a positive or negative impact on your mental health?
• Very positive
• Somewhat positive
• Somewhat negative
• Very negative
• In your opinion, which factors contribute to the negative impact of social media on mental health?
• Social comparison
• In your opinion, what interventions could be effective in reducing the negative impact of social media on mental health?
• Education on healthy social media use
• Counseling for mental health problems caused by social media
• Social media detox programs
• Regulation of social media use

Thank you for your participation!

Applications of Research Paper

Research papers have several applications in various fields, including:

• Advancing knowledge: Research papers contribute to the advancement of knowledge by generating new insights, theories, and findings that can inform future research and practice. They help to answer important questions, clarify existing knowledge, and identify areas that require further investigation.
• Informing policy: Research papers can inform policy decisions by providing evidence-based recommendations for policymakers. They can help to identify gaps in current policies, evaluate the effectiveness of interventions, and inform the development of new policies and regulations.
• Improving practice: Research papers can improve practice by providing evidence-based guidance for professionals in various fields, including medicine, education, business, and psychology. They can inform the development of best practices, guidelines, and standards of care that can improve outcomes for individuals and organizations.
• Educating students : Research papers are often used as teaching tools in universities and colleges to educate students about research methods, data analysis, and academic writing. They help students to develop critical thinking skills, research skills, and communication skills that are essential for success in many careers.
• Fostering collaboration: Research papers can foster collaboration among researchers, practitioners, and policymakers by providing a platform for sharing knowledge and ideas. They can facilitate interdisciplinary collaborations and partnerships that can lead to innovative solutions to complex problems.

When to Write Research Paper

Research papers are typically written when a person has completed a research project or when they have conducted a study and have obtained data or findings that they want to share with the academic or professional community. Research papers are usually written in academic settings, such as universities, but they can also be written in professional settings, such as research organizations, government agencies, or private companies.

Here are some common situations where a person might need to write a research paper:

• For academic purposes: Students in universities and colleges are often required to write research papers as part of their coursework, particularly in the social sciences, natural sciences, and humanities. Writing research papers helps students to develop research skills, critical thinking skills, and academic writing skills.
• For publication: Researchers often write research papers to publish their findings in academic journals or to present their work at academic conferences. Publishing research papers is an important way to disseminate research findings to the academic community and to establish oneself as an expert in a particular field.
• To inform policy or practice : Researchers may write research papers to inform policy decisions or to improve practice in various fields. Research findings can be used to inform the development of policies, guidelines, and best practices that can improve outcomes for individuals and organizations.
• To share new insights or ideas: Researchers may write research papers to share new insights or ideas with the academic or professional community. They may present new theories, propose new research methods, or challenge existing paradigms in their field.

Purpose of Research Paper

The purpose of a research paper is to present the results of a study or investigation in a clear, concise, and structured manner. Research papers are written to communicate new knowledge, ideas, or findings to a specific audience, such as researchers, scholars, practitioners, or policymakers. The primary purposes of a research paper are:

• To contribute to the body of knowledge : Research papers aim to add new knowledge or insights to a particular field or discipline. They do this by reporting the results of empirical studies, reviewing and synthesizing existing literature, proposing new theories, or providing new perspectives on a topic.
• To inform or persuade: Research papers are written to inform or persuade the reader about a particular issue, topic, or phenomenon. They present evidence and arguments to support their claims and seek to persuade the reader of the validity of their findings or recommendations.
• To advance the field: Research papers seek to advance the field or discipline by identifying gaps in knowledge, proposing new research questions or approaches, or challenging existing assumptions or paradigms. They aim to contribute to ongoing debates and discussions within a field and to stimulate further research and inquiry.
• To demonstrate research skills: Research papers demonstrate the author’s research skills, including their ability to design and conduct a study, collect and analyze data, and interpret and communicate findings. They also demonstrate the author’s ability to critically evaluate existing literature, synthesize information from multiple sources, and write in a clear and structured manner.

Characteristics of Research Paper

Research papers have several characteristics that distinguish them from other forms of academic or professional writing. Here are some common characteristics of research papers:

• Evidence-based: Research papers are based on empirical evidence, which is collected through rigorous research methods such as experiments, surveys, observations, or interviews. They rely on objective data and facts to support their claims and conclusions.
• Structured and organized: Research papers have a clear and logical structure, with sections such as introduction, literature review, methods, results, discussion, and conclusion. They are organized in a way that helps the reader to follow the argument and understand the findings.
• Formal and objective: Research papers are written in a formal and objective tone, with an emphasis on clarity, precision, and accuracy. They avoid subjective language or personal opinions and instead rely on objective data and analysis to support their arguments.
• Citations and references: Research papers include citations and references to acknowledge the sources of information and ideas used in the paper. They use a specific citation style, such as APA, MLA, or Chicago, to ensure consistency and accuracy.
• Peer-reviewed: Research papers are often peer-reviewed, which means they are evaluated by other experts in the field before they are published. Peer-review ensures that the research is of high quality, meets ethical standards, and contributes to the advancement of knowledge in the field.
• Objective and unbiased: Research papers strive to be objective and unbiased in their presentation of the findings. They avoid personal biases or preconceptions and instead rely on the data and analysis to draw conclusions.

Advantages of Research Paper

Research papers have many advantages, both for the individual researcher and for the broader academic and professional community. Here are some advantages of research papers:

• Contribution to knowledge: Research papers contribute to the body of knowledge in a particular field or discipline. They add new information, insights, and perspectives to existing literature and help advance the understanding of a particular phenomenon or issue.
• Opportunity for intellectual growth: Research papers provide an opportunity for intellectual growth for the researcher. They require critical thinking, problem-solving, and creativity, which can help develop the researcher’s skills and knowledge.
• Career advancement: Research papers can help advance the researcher’s career by demonstrating their expertise and contributions to the field. They can also lead to new research opportunities, collaborations, and funding.
• Academic recognition: Research papers can lead to academic recognition in the form of awards, grants, or invitations to speak at conferences or events. They can also contribute to the researcher’s reputation and standing in the field.
• Impact on policy and practice: Research papers can have a significant impact on policy and practice. They can inform policy decisions, guide practice, and lead to changes in laws, regulations, or procedures.
• Advancement of society: Research papers can contribute to the advancement of society by addressing important issues, identifying solutions to problems, and promoting social justice and equality.

Limitations of Research Paper

Research papers also have some limitations that should be considered when interpreting their findings or implications. Here are some common limitations of research papers:

• Limited generalizability: Research findings may not be generalizable to other populations, settings, or contexts. Studies often use specific samples or conditions that may not reflect the broader population or real-world situations.
• Potential for bias : Research papers may be biased due to factors such as sample selection, measurement errors, or researcher biases. It is important to evaluate the quality of the research design and methods used to ensure that the findings are valid and reliable.
• Ethical concerns: Research papers may raise ethical concerns, such as the use of vulnerable populations or invasive procedures. Researchers must adhere to ethical guidelines and obtain informed consent from participants to ensure that the research is conducted in a responsible and respectful manner.
• Limitations of methodology: Research papers may be limited by the methodology used to collect and analyze data. For example, certain research methods may not capture the complexity or nuance of a particular phenomenon, or may not be appropriate for certain research questions.
• Publication bias: Research papers may be subject to publication bias, where positive or significant findings are more likely to be published than negative or non-significant findings. This can skew the overall findings of a particular area of research.
• Time and resource constraints: Research papers may be limited by time and resource constraints, which can affect the quality and scope of the research. Researchers may not have access to certain data or resources, or may be unable to conduct long-term studies due to practical limitations.

About the author

Muhammad Hassan

Researcher, Academic Writer, Web developer

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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 ,
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• Michael Matschiner   ORCID: orcid.org/0000-0003-4741-3884 6 , 7 ,
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• 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 ,
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• 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.

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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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Contributions

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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Analysis of COF-300 synthesis: probing degradation processes and 3D electron diffraction structure

a XStruct, Department of Chemistry, Ghent University, Krijgslaan 281–S3, 9000 Ghent, Belgium, b COMOC – Center for Ordered Materials, Organometallics and Catalysis – Department of Chemistry, Ghent University, Krijgslaan 281–S3, 9000 Ghent, Belgium, c Rigaku Corporation, Haijima, Tokyo, Japan, and d Rigaku Europe SE, Neu-Isenburg, Germany * Correspondence e-mail: [email protected]

Although COF-300 is often used as an example to study the synthesis and structure of (3D) covalent organic frameworks (COFs), knowledge of the underlying synthetic processes is still fragmented. Here, an optimized synthetic procedure based on a combination of linker protection and modulation was applied. Using this approach, the influence of time and temperature on the synthesis of COF-300 was studied. Synthesis times that were too short produced materials with limited crystallinity and porosity, lacking the typical pore flexibility associated with COF-300. On the other hand, synthesis times that were too long could be characterized by loss of crystallinity and pore order by degradation of the tetrakis(4-aminophenyl)methane (TAM) linker used. The presence of the degradation product was confirmed by visual inspection, Raman spectroscopy and X-ray photoelectron spectroscopy (XPS). As TAM is by far the most popular linker for the synthesis of 3D COFs, this degradation process might be one of the reasons why the development of 3D COFs is still lagging compared with 2D COFs. However, COF crystals obtained via an optimized procedure could be structurally probed using 3D electron diffraction (3DED). The 3DED analysis resulted in a full structure determination of COF-300 at atomic resolution with satisfying data parameters. Comparison of our 3DED-derived structural model with previously reported single-crystal X-ray diffraction data for this material, as well as parameters derived from the Cambridge Structural Database, demonstrates the high accuracy of the 3DED method for structure determination. This validation might accelerate the exploitation of 3DED as a structure determination technique for COFs and other porous materials.

Keywords: 3D electron diffraction ; 3DED ; microcrystal electron diffraction ; microED ; covalent organic frameworks ; Cambridge Structural Database ; porous organic solids ; crystallization and crystal growth .

CCDC reference: 2321626

In Figs. S6–S8, the effect of time on I 65°C (as discussed earlier) is compared with the effect on the other samples. Note that, as expected, I RT is slower to form a crystalline material compared with I 65°C due to the reduced error correction at room temperature, with reflections appearing after 1 d, and fully developed crystallinity after 5 d. However, the pore structure never fully establishes, as indicated by the broad, late and small second step in the N 2 -sorption isotherm. The appearance of crystallinity in C 65°C is even more delayed, with no crystalline reflections observed after 1 d of reaction time, indicating the superiority of the intermediate-assisted procedure. Here, maximal crystallinity is observed after 5 d, as peaks start to broaden significantly after 7 d. Surprisingly, the best N 2 -sorption behaviour was observed for the 7 d sample, indicating that the relationship between crystallinity and porosity is not always straightforward. Finally, using the conditions of C RT, we were unable to form any crystalline material, even after 7 d of reaction time. We also checked if the scale of the synthesis had any influence on the material. Therefore, a sample (I 65°C ×5) was prepared in an identical way to I 65°C but with every quantity used multiplied by 5. The resulting PXRD patterns and the N 2 -sorption isotherms are presented in Fig. S9 and show no significant influence on the crystallinity and a small decrease of porosity (with a BET surface area of 1180 m 2  g −1 and V p of 0.71 obtained for I 65°C ×5).

The response of COF-300 to an intermediate-assisted synthesis protocol was studied by careful evaluation of the evolution of both crystallinity and porosity as functions of reaction time and temperature. Kinetic studies among four different synthesis conditions revealed three distinct stages in the synthesis of COF-300, namely a network build-up phase at short synthetic times (≤1 d) with low crystallinity and no pore flexibility, followed by an optimal stage (3 d) characterized by high crystallinity and porosity before partial breakdown by TAM degradation (≥5 d). This degradation process could be confirmed in both control experiments as well as the obtained COF materials and can easily be estimated by the observation of magenta-coloured reaction mixtures. As a pronounced influence of this degradation reaction on both crystallinity and porosity was observed and most 3D COFs are based on the TAM linker, knowledge of TAM degradation in a acidic environment is of utmost importance for the synthesis of high-quality 3D COFs. Knowledge of this degradation process might help to increase the synthetic toolbox for 3D COFs (which are mainly based on the TAM linker), which is still lacking compared with 2D COFs. However, using the optimized conditions, a reliable crystal structure of COF-300 could be readily obtained via 3DED analysis, indicating single crystallinity of the synthesized materials. The structure model obtained showed high completeness and comparable resolution and R values. Comparison with an SCXRD structure model as well as with data for similar chemical functionalities in the CSD database showed no significant differences, supporting that 3DED is a reliable and fast technique for the structure solution of COFs. As SCXRD structure solution is hardly possible and PXRD models often show ambiguity in structure determination, 3DED might play an important role in the future of COFs with better accessibility of 3DED diffraction equipment and improving dynamic refinement algorithms.

Crystal structure: contains datablock 1. DOI: https://doi.org/10.1107/S2052252524003713/vq5005sup1.cif

Structure factors: contains datablock 1. DOI: https://doi.org/10.1107/S2052252524003713/vq5005sup2.hkl

Supporting Information - revised - highlighted. DOI: https://doi.org/10.1107/S2052252524003713/vq5005sup3.pdf

Acknowledgements

The authors thank Karen Leus for the XPS measurements and Dieter Buyst for the solid-state NMR measurements.

Funding information

LB acknowledges Ghent University (UGent) for funding. PVDV acknowledges financial support through UGent concerted action (grant No. 01G01017) and the Fonds Wetenschappelijk Onderzoek (FWO)–Vlaanderen project (grant Nos. 3G020521 awarded to PVDV; 1275221N awarded to SB and KVH). Gas sorption and powder X-ray diffraction were made possible through UGent (grant Nos. 01B00215; BOF20/BAS/015 awarded to PVDV). The spectrometer electronics, magnet and accessories used for solid-state NMR measurements, including the BBI and high-gradient diffusion probe, were funded by the Hercules foundation (grant No. AUGE/09/2006); the solid-state (CP-MAS) and HR-MAS expansion were made possible by FWO (grant No. I006920N).

This is an open-access article distributed under the terms of the Creative Commons Attribution (CC-BY) Licence , which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited.

Top 10 Companies in Artificial intelligence (AI) and Big Data in Food Industry Market in 2024

Introduction 

In today's digitally transforming food industry, the integration of Artificial Intelligence (AI) and Big Data has become a game-changer. This blog explores the top 10 companies at the forefront of this innovation, leveraging technology to revolutionize the way we produce, distribute, and consume food. These companies have earned their positions based on market share, revenue, innovation, reputation, and profound impact on the industry.

Artificial intelligence (AI) and Big Data are revolutionizing the food industry by optimizing supply chain management, enhancing food safety and quality control, improving customer experiences through personalized interactions, enabling predictive analytics for demand forecasting, and fostering sustainable practices in agriculture. AI-driven technologies automate tasks like crop monitoring and quality assurance, while Big Data analytics provide insights into consumer preferences, supply chain transparency, and food safety, ultimately transforming how food is produced, distributed, and consumed globally.

The rising integration of Internet of Things (IoT) devices in the food industry is generating vast amounts of data, driving revenue growth by enabling improved traceability, food safety, and supply chain accountability. AI algorithms analyze IoT data to optimize equipment performance, monitor shipments, and ensure optimal storage conditions. Big data further refines marketing strategies, product innovation, and competitive intelligence, with technologies like RFID and GPS enhancing distribution efficiency and reducing waste. Strategic initiatives by major companies, such as Givaudan's Customer Foresight platform, leverage AI and big data to co-create innovative food solutions, though the high implementation costs and data security concerns pose challenges to market growth.

The worldwide AI and big data in food sector market size was worth USD 6.86 billion in 2022, and it is predicted to grow at a high revenue CAGR of 44.4% over the forecast period. Rising need for AI and big data technologies to improve overall efficiency in food production and distribution is the key driver of market revenue development.

World’s Prominent Companies Offering Artificial intelligence (AI) and Big Data in Food Industry; Top 10 by Revenue

• Intel Corporation
• SAS Institute Inc.
• Cargill, Incorporated
• Nutrien Ag Solutions, Inc.

Top 10 Globally Leading Companies in The Artificial intelligence (AI) and Big Data in Food Industry Market

Cargill, incorporated [annual revenue: usd 176.7 billion].

Cargill, Incorporated is a global leader in the food and agriculture industry, with a rich history dating back to 1865. The company specializes in commodity trading, food processing, and supply chain management, offering a diverse range of products from agricultural commodities to food ingredients and animal nutrition. Cargill is known for its commitment to sustainability and innovation, leveraging AI and Big Data technologies for supply chain optimization, quality control, and crop management. With a focus on quality, safety, and global reach, Cargill continues to be a key player in shaping the future of the food industry.

IBM [Annual Revenue: USD 62.07 Billion]

IBM , a global technology leader, offers cutting-edge AI and Big Data solutions for the food industry. Their Watson AI platform enables personalized nutrition and supply chain optimization, while IBM Food Trust ensures transparency and safety through blockchain technology. IBM's data analytics tools empower decision-making with deep insights into food quality and consumer trends. With a legacy of innovation, IBM continues to drive efficiency and sustainability in the food sector through advanced technology and industry expertise.

Intel Corporation [Annual Revenue: USD 54.2 Billion]

Intel Corporation , founded in 1968, is a leading technology company known for semiconductor innovations. Their product range includes microprocessors, SoCs, and software. In recent years, Intel has focused on AI and Big Data solutions, offering specialized hardware like Intel® Nervana™ Neural Network Processors (NNP) and software for diverse applications. In the food industry, Intel's AI and Big Data technologies enable quality control through computer vision, optimize supply chains, enhance predictive analytics for inventory management, support smart agriculture, and provide valuable consumer insights. Intel's commitment to AI and Big Data aligns with industry demands for efficient, sustainable, and consumer-centric food production and distribution.

Oracle [Annual Revenue: USD 52.51 Billion]

Oracle Corporation is a leading technology company known for its database software and enterprise solutions. Founded in 1977, Oracle has evolved to offer a comprehensive suite of products, including the Oracle Autonomous Database and Oracle Cloud Infrastructure, which incorporate AI and Big Data capabilities. For the food industry, Oracle provides specialized software solutions for point-of-sale, inventory management, and supply chain optimization. Leveraging AI and machine learning, Oracle enables predictive analytics, demand forecasting, and personalized customer experiences, making it a strategic partner for businesses seeking advanced technology solutions in the food sector.

Nutrien Ag Solutions, Inc. [Annual Revenue: USD 29 Billion]

Nutrien Ag Solutions, Inc. is a leading global provider of agricultural products and services, formed in 2018 through the merger of Agrium Inc. and Potash Corporation of Saskatchewan Inc. They offer a comprehensive suite of solutions including seeds, fertilizers, crop protection chemicals, precision agriculture technologies, and digital farming tools, leveraging AI and big data for optimized farming practices. Notable for their commitment to sustainability, Nutrien provides farmers with data-driven insights and predictive analytics to enhance productivity and environmental stewardship, making them a key player in advancing digital agriculture globally.

Capgemini [Annual Revenue: USD 22.5 Billion]

Capgemini is a global leader in consulting, technology services, and digital transformation, founded in 1967. With a comprehensive suite of offerings including strategy consulting, digital solutions, and industry-specific services, Capgemini excels in AI and Big Data applications for the food industry. Their expertise enables businesses to leverage AI-driven analytics for demand forecasting, supply chain optimization, and personalized customer experiences, making them a valuable partner for enhancing efficiency and competitiveness in food industry operations.

SAP. [Annual Revenue: USD 17.3 Billion]

SAP (Systems, Applications & Products in Data Processing) is a global enterprise software company founded in 1972, known for its ERP solutions and business applications. Key offerings include ERP, CRM, SCM, and BI tools. Notable achievements include SAP HANA, a groundbreaking in-memory database platform. In the food industry, SAP's AI and Big Data capabilities enable predictive analytics for demand forecasting, quality control through image recognition, and enhanced traceability using blockchain, empowering companies to optimize operations and ensure compliance across the supply chain. This integration of technology underscores SAP's commitment to driving digital transformation in the food sector.

SAS Institute Inc. [Annual Revenue: USD 3.2 Billion]

SAS Institute Inc. is a renowned analytics and AI software company founded in 1976. It specializes in providing advanced analytics, machine learning, and big data solutions to industries like the food sector. SAS's offerings enable food businesses to optimize supply chains, ensure food safety, and derive customer insights from data, aiding in targeted marketing and product development. With a strong focus on innovation and industry-specific applications, SAS remains a key player in leveraging analytics for business success in the food industry.

FoodLogiQ [Annual Revenue: USD 9.8 Billion]

FoodLogiQ is a leading provider of food safety and supply chain management solutions, founded in 2006. They offer cloud-based software tailored to the food industry, specializing in traceability, supplier management, and quality incident handling. Recent developments include integrating AI and big data analytics for predictive insights into food safety risks and supply chain optimization. FoodLogiQ's comprehensive solutions and industry leadership make them a trusted partner for major food brands globally, ensuring transparency, compliance, and efficiency throughout the supply chain.

io [Annual Revenue: USD 8.1 Billion]

IO in AI and Big Data for the food industry revolutionizes operations by integrating advanced analytics and AI technologies tailored for agriculture, production, distribution, and consumer insights. Notable achievements include precision agriculture, automated quality control, and personalized marketing strategies driven by predictive analytics. Recent developments include AI-driven robotics for harvesting, IoT sensor applications in transportation, and NLP for consumer sentiment analysis. IO's unique selling points lie in its scalable, end-to-end solutions focusing on sustainability and efficiency improvements across the food value chain.

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