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Biology Worksheets, Notes, and Quizzes (PDF and PNG)

This is a collection of free biology worksheets, notes, handouts, slides, study guides and quizzes. Most content targets high school, AP biology, genetics, anatomy/physiology, immunology, and biology 101 and 102 in college. There is also biochemistry and physics for biologists. However, some resources are at the grade school and middle school level.

The files are PDF, PNG, JPG, and formats using Google Apps for Google Classroom. Most of the time, these formats are interchangeable. So, if you see something you like, but want a different format, just let us know. Print these resources, make transparencies and slides, etc.

In the interest of quick load time, not all of the images are shown. If you’d rather see them all, just contact us!

Biochemistry

[ Google apps worksheet ][ worksheet PDF ][ answers PDF ][ worksheet PNG ][ answers PNG ]

Enzymes Definitions

[ Google Slides worksheet ][ worksheet PDF ][ answers PDF ][ worksheet PNG ][ answers PNG ]

• 20 Amino Acids [ PNG ][ PDF ]
• Amino Acid Side Chains [ PNG ][ PDF ]
• Identifying Type of Biological Macromolecules [ Google Slides worksheet ][ worksheet PDF ][ answers PDF ][ worksheet PNG ][ answers PNG ]
• Disaccharide Examples [ PNG ]
• Products of Photosynthesis [ JPG ]
• Anabolism vs Catabolism [ PNG ]
• 3 Parts of a Nucleotide [ PNG ]
• Fermentation Definition and Examples [ PNG ]

General and Cell Biology

Organelles and Their Functions

Parts of a Plant Cell

Label Parts of a Chloroplast

[ Google Apps worksheet ][ worksheet PDF ][ answers PDF ][ worksheet PNG ][ answers PNG ]

Label Parts of a Mitochondria

Label the Animal Cell

[ Google Apps worksheet ][ worksheet PDF ][ worksheet PNG ][ answers PNG ]

Prokaryotes vs Eukaryotes Worksheet

Steps of the Cell Cycle

Steps of Mitosis

Membrane Transport Terms and Definitions

Membrane Transport Worksheet #2

The Plasma Membrane

Label a Bacterial Cell

• Label a Bacteriophage [ Google Apps worksheet ][ worksheet PDF ][ worksheet PNG ][ answers PNG ]
• Evidence of Evolution Worksheet [ Google Apps worksheet ][ Worksheet PDF ][ Worksheet PNG ][ Answers PNG ]
• Evolutionary Processes Worksheet [ worksheet Google Apps ][ worksheet PDF ][ worksheet PNG ][ answers PNG ]
• Major Receptor Families [ Google Apps worksheet ][ worksheet PDF ][ worksheet PNG ][ answers PNG ]
• Label a Bacterial Cell Membrane ( E. coli ) [ Google Apps worksheet ][ worksheet PDF ][ worksheet PNG ][ answers PNG ]

Anatomy and Physiology

These worksheets are only a portion of the available anatomy and physiology worksheets. Human anatomy and physiology worksheets have their own section.

Label the Heart

Label the Eye

[ Google Apps worksheet ][ worksheet PDF ][ answers PDF ][ worksheet PNG ]

Types of Blood Cells

[ worksheet Google Apps ][ worksheet PDF ][ worksheet PNG ][ answers PNG ]

Label the Muscles

[ worksheet PDF ][ worksheet PNG ][ answers PNG ]

Label the Ear

[ Google Apps worksheet ][ Worksheet PDF ][ Worksheet PNG ][ Answers PNG ]

Label the Lungs

Label the Kidney

Label the Liver

Label the Large Intestine

Label the Stomach

[ Google Apps worksheet ] [Worksheet PDF ][ Worksheet PNG ][ Answers PNG ]

External Nose Anatomy

[ Worksheet PDF ][ Worksheet Google Apps ][ Worksheet PNG ][ Answers PNG ]

Parts of the Nose

Label Bones of the Skeleton

Label the Lymph Node

Label the Parts of the Brain

Label the Lobes of the Brain

Brain Anatomical Sections

Arteries of the Brain

Label the Pancreas

Label the Spleen

Label the Digestive System

Label the Respiratory System

Parts of a Neuron

Label the Lips

Label the Skin

Label the Circulatory System

The Urinary Tract

[ Worksheet PDF ][ Worksheet Google Apps ][ Worksheet PNG ][ Answer Key PNG ]

The Bladder

• The Female Reproductive System [ worksheet PDF ][ worksheet Google Apps ][ worksheet PNG ][ answers PNG ]

Parts of a Flower

Label the Orchid Plant

[ Worksheet PDF ][ Worksheet Google Apps ][ Worksheet PNG ] [Answer Key PNG ]

Parts of an Orchid Flower

Parts of a Monocot Seed

Parts of a Fern

Parts of a Tree Trunk

Parts of a Tree

[ worksheet PDF ][ worksheet Google Apps ][ worksheet PNG ][ answers PNG ]

Parts of a Mushroom

Label the Shark

Label the Fish

Parts of a Bird

Bird Anatomy

Frog Life Cycle

Parts of a Mosquito (Insect)

Bones of the T. rex Skull

[ worksheets PDF ][ worksheet Google Slides ][ worksheet PNG ][ answers PNG ]

Holes of the T. rex Skull

• Label the T. rex Skeleton [ worksheets PDF ][ worksheet Google Slides ][ worksheet PNG ][ answers PNG ]
• Label Human Teeth [ Worksheet PDF ][ Worksheet Google Apps ][ Worksheet PNG ][ Answer Key PNG ]
• Monocot vs Dicot Seeds [ worksheet PDF ][ worksheet Google Slides ][ worksheet PNG ][ answers PNG ]
• Label the Moss [ worksheet PDF ][ worksheet Google Slides ][ worksheet PNG ][ answers PNG ]
• Diagram of the Human Eye [ JPG ]

Use a completed worksheet as a study guide.

Cells of the Immune System

Immune Cell Functions

[ worksheet Google Apps ][ worksheet PDF ][ worksheet PNG #1][ answers PNG #1][ worksheet PNG #2][ answers PNG #2]

Methods to Study Virus Structures

[ worksheet Google Slide ][ worksheet PDF ][ worksheet PNG ][ answers PNG ]

Icosahedral Virus Capsids

Human DNA Viruses

Human RNA Viruses

This is selection of worksheets relating to DNA, RNA, transcription, translation, genetic crosses, plasmid mapping, etc. See the full collection of genetics worksheets if you’re don’t see what you need.

DNA Replication

Types of Mutations

Monohybrid Cross Worksheet #1

Monohybrid Cross Worksheet #2

Monohybrid Cross Worksheet #3

Monohybrid Cross #4 – Multiple Alleles

• Monohybrid Cross Worksheet #5: Multiple Alleles [ worksheet Google Apps ][ worksheet PDF ][ worksheet PNG ][ answers PNG ]

Monohybrid Cross #6 – Sex-Linked Inheritance

Monohybrid Cross #7 – Sex-Linked Inheritance

Dihybrid Cross Worksheet #1

Dihybrid Cross Worksheet #2

Dihybrid Cross Worksheet #3

Dihybrid Cross Worksheet #4

Dihybrid Cross #5 – Epistasis

Dihybrid Cross #6 – Epistasis

Incomplete Dominance Worksheet #1

Incomplete Dominance Worksheet #2

Natural Selection Worksheet

Convergent vs Divergent Evolution Worksheet

Intro to Pedigrees Worksheet #1

Pedigrees/Genealogy Worksheet #2

Pedigrees/Genealogy Worksheet #3 – X-Linked Dominant Traits

Pedigrees/Genealogy Worksheet #4 – X-Linked Recessive Traits

Pedigrees/Genealogy Worksheet #5 – Autosomal Dominant Traits

Pedigrees/Genealogy Worksheet #6 – Autosomal Recessive Traits

Pedigrees/Genealogy Worksheet #7 – Comprehension Skills

Pedigrees/Genealogy Worksheet #8 – Identifying Inheritance Patterns

Autosomal vs Sex-Linked Inheritance

Plasmid Mapping

• Genotype vs Phenotype [ PNG ]
• Genetic Codon Chart [ PNG ][ PDF ]
• RNA vs DNA [ JPG ]

Use labelled diagrams as study guides.

Ecosystems Worksheet

Levels of Organization (Ecosystem)

Primary Succession

Secondary Succession

Label the Insect

Label the Bee

Chicken Life Cycle (Basic)

Bird Life Cycle (Basic)

Butterfly Life Cycle

• Biotic and Abiotic Factors [ worksheet PDF ][ worksheet Google Apps ][ worksheet PNG ][ answers PNG ]
• Kingdoms of Life Graphic [ PNG ]
• Autotroph vs Heterotroph Graphic [ PNG ]
• Commensalism Definition and Examples [ PNG ]
• Difference Between Butterflies and Moths [ JPG ]
• Difference Between Bugs and Insects [ PNG ]

Parasitology

Schistosome Life Cycle

Schistosome Anatomy

• Giardia Life Cycle [ worksheet Google Apps ][ worksheet PDF ][ worksheet PNG ][ answers PNG ]

Physical Science for Biology

There is also an entire section devoted to physical science worksheets and study guides.

• Adhesion vs Cohesion Graphic [ PNG ]
• What Is Entropy? [ PNG ]
• Freezing Point of Water [ PNG ]

Biology Labs

• How to Extract DNA From a Banana [ PNG ]

Biology Word Search Puzzles

• DNA Replication Word Search
• Gel Electrophoresis Word Search
• Citrus Fruits
• General Biology Word Search Puzzle [ PNG ]
• Life Science Word Search [ PNG ][ PDF ]
• Cell Biology Word Search [ PNG ][ PDF ]
• Amino Acid Word Search [ PNG ][ PDF ]
• Biome Word Search [ PNG ][ PDF ]
• Environmental Science Word Search [ PNG ][ PDF ]
• Photosynthesis Word Search [ PNG ]
• Human Skeleton Bones Word Search [ PNG ]
• Dinosaur Word Search [ PNG ][ PDF ]
• Different Dinosaur Word Search [ PNG ][ PDF ]
• Wild Cats Word Search [ PNG ][ PDF ]
• Shark Word Search [ PNG ]
• Trees Word Search [ PNG ][ PDF ]
• Flowers Word Search [ PNG ][ PDF ]
• Butterfly Word Search [ PNG ][ PDF ]
• Genetics Word Search [ PNG ][ PDF ]

Miscellaneous Biology Notes and Resouces

• How Long Can Germs Live on Surfaces? [ PNG ]
• 10 Radioactive Foods [ PNG ]
• Examples of Organic Compounds [ PNG ]

Biology Notes Terms of Use

You are welcome to print these resources for personal or classroom use. They may be used as handouts or posters. They may  not  be posted elsewhere online, sold, or used on products for sale.

This page doesn’t include all of the assets on the Science Notes site. If there’s a table or worksheet you need but don’t see, just let us know.

Related Posts

The Best Biology Review: Topic Summaries, Examples, and Free Practice

Welcome to Albert’s collection of science topic reviews for teaching and reviewing Biology. Teachers and students can explore our easy-to-follow guides below for use at home or in the classroom .

Introduction to Biology

What is the experimental design process how do scientists study feedback loops.

The review articles below explore the basic skills needed to be successful in a high school biology course. Review SI units, the nature of science, and experimental design.

What is the difference between mitosis and meiosis? How is genetic material created and pass on?

The review articles below explore many topics related to genetics. Review mitosis, meiosis, and the mechanism of genetic transfer with helpful examples and summaries.

Anatomy and Physiology

How do we explain the body what makes each system unique.

The review articles below explore the general ins and outs of the human body. From terminology to the inner-workings of the systems, many topics are examined below.

Advanced Biology Topics

Looking to take your biology mastery to the next level.

The review articles below dive deeper into the processes behind genetics and reproduction.

Interested in a school license?​

Choose Your Test

Sat / act prep online guides and tips, the complete ap biology review guide for 2023.

Advanced Placement (AP)

Reviewing for the AP Biology exam can seem daunting. There's so much material to cover, and much of it is highly complex. However, if you plan your time well and use appropriate study materials and strategies, you can expect a great score on the exam.

In this article, I'll give you an overview of what the AP Biology exam is like, what you need to know to ace it, and how you can use your study time effectively before the exam on Wednesday, May 10, 2023, at 12PM!

What's the Format of the AP Biology Exam?

The AP Biology exam is a long test — three hours long to be exact . Starting in 2020, the Bio test underwent some key structural changes in terms of questions and format, so it's important that you know what to expect and exactly how the test is structured.

Like other AP tests, AP Biology has two parts: a multiple-choice section and a free-response section, each of which is worth 50% of your overall score. These sections are then divided further into different types of questions.

The multiple-choice section is the first section. It consists of 60 multiple-choice questions and is one hour and 30 minutes long. Each question contains four answer choices.

Although you have one minute for each question, I would recommend keeping your pace at under a minute per question on your first pass through the section. This way you'll have some extra time at the end to go back and answer any tricky questions you skipped or guessed on.

There's no guessing penalty on the test, so you should answer every question, even if you have no idea which choice is correct (after you've tried to figure it out of course!).

The free-response section, which also lasts an hour and 30 minutes, is made up of six questions: four short-answer questions and two long questions. The short-answer questions are worth 4 points each, whereas the long questions are worth 8-10 points each.

You'll need to pace yourself wisely on this section. Divided up evenly, this would mean you'd get 15 minutes per question. However, try to spend no more than 10 minutes on each short answer. I recommend doing the short answers first to get yourself warmed up.

Then, if you manage your time well, you should have at least 20-25 minutes for each long free-response questions.

Here's a chart showing the current format of the AP Biology exam:

What Do Questions Look Like on the AP Biology Exam?

You now know the general format of the AP Biology test, but what do questions actually look like on it? And what kinds of topics do they test? Let's take a look.

Multiple-Choice Questions

As a reminder, there are 60 multiple-choice questions on the AP Bio exam. These can be discrete (meaning they are stand-alone questions) or they can come in sets with other questions.

Here's an example of a multiple-choice question you might see on the exam:

You don't necessarily need lots of in-depth biology knowledge to answer this. The answer is A because the total volume of gas wouldn't change (and oxygen consumption would be immeasurable) unless the carbon dioxide produced by the organisms were removed from the environment.

You can see this from the information contained in the question. This question is part of a group of three questions that pertain to the experiment and data chart. You'll see many question clusters like this in the multiple-choice section.

Short-Answer Questions

On the second section of the AP Biology exam, you'll get four short-answer questions (in addition to two long questions). These questions focus on the following topics:

• Scientific Investigation
• Conceptual Analysis
• Analysis of Model or Visual Representation
• Analysis of Data

Here's an example of a short free-response question from the 2013 exam:

This question requires an understanding of how evolution shapes the formation of new species.

To get the correct answer, you'll need to know the facts about evolution—but you'll also need to be able to apply that knowledge to make inferences about this specific scenario.

This is why a deeper understanding of the main topics in AP Biology is so critical: the difference between knowing the facts about something and comprehending how it works can be surprisingly large.

Long Questions

In addition to the four short-answer questions you'll get on the second part of the AP Bio exam, you'll get two long questions. Both of these focus on "interpreting and evaluating experimental results," with one requiring graphing (per the College Board description ).

Here's an example of a long question:

This question is heavy on analysis and isn't just testing your straight-up biology knowledge. Here, you need to be able to read and understand the graphs and table so you can use them to inform your answer to the question.

Once again, an understanding of evolution and the ability to apply that knowledge to a specific scenario is critical.

What Topics Does the AP Biology Exam Cover? 

According to the College Board's Course Description , AP Bio has shifted its focus from the content-heavy, memorization-based curriculum that defined the course and exam in the past to become a more concept-driven test.

The goal is for students to gain a deeper conceptual understanding of topics in biology. Reasoning skills and knowledge of the process of scientific inquiry are more important on the current version of the AP Biology test than they have ever been before.

The College Board has tried to structure the exam so that content knowledge and reasoning skills are intertwined. This can be both good and bad. The good is that you won't necessarily have to memorize as many little tidbits of information; the bad is that it can be harder to study for a test like this that covers more abstract forms of knowledge. (More on how to manage this in the "How to Review" section!)

The AP Bio exam and curriculum as a whole are centered around eight major units. Here they are:

Unit 1: Chemistry of Life Unit 2: Cell Structure and Function Unit 3: Cellular Energetics Unit 4: Cell Communication and Cell Cycle Unit 5: Heredity Unit 6: Gene Expression and Regulation Unit 7: Natural Selection Unit 8: Ecology

The Importance of Labs

Apart from background knowledge of all this content, it's important to understand your labs and the basic underlying principles that govern scientific experiments. If you know the ins and outs of experimental design, you'll earn a lot of points on the AP Bio exam.

Important lab topics include the following:

• Artificial Selection
• Modeling Evolution
• Comparing DNA Sequences
• Diffusion and Osmosis
• Photosynthesis
• Cellular Respiration
• Mitosis and Meiosis
• Bacterial Transformation
• Restriction Enzyme Analysis of DNA
• Energy Dynamics
• Transpiration
• Animal Behavior
• Enzyme Catalysis

AP Biology Review Preview: 4 Key Tips to Keep in Mind

In this section, I'll give you some preliminary study tips that will help you get the most out of your AP Biology review time.

Tip 1: Plan Out Your Time

First of all, you should think about how much time you have left before the AP test. This will affect the structure of your study plan. If you're taking other AP classes or have a lot of personal commitments in general, you might want to start earlier depending on your confidence with the material.

Consider your schedule and the time you're willing to spend on AP Biology. Since there's so much content in this course, I think 20 hours of studying is a reasonable goal. However, if you find that you're already scoring at a high level (a high 4 or anywhere in the 5 range), you might aim for just 10 hours or so.

You should balance your time relatively evenly between studying the material and taking practice tests. In AP Bio, you might benefit from devoting a bit more time to practice testing.

Since the test is now more targeted toward assessing analytical skills, practicing real test questions might help you more than just memorizing content (although both are still important!). I'll give you more information about how to use practice tests and review materials effectively in a moment.

Tip 2: Use Appropriate Review Materials

The importance of using the right review materials can't be overstated, especially in the case of AP Biology. With the recent changes to the test, it's critical that you don't use old study materials and assume that they'll give you all the tools you need to succeed on the new format.

Some review books students have found the most useful include CliffsNotes' AP Biology for content review and Sterling's AP Biology Practice Questions for practice questions that give you a good sense of what the test is like.

Avoid using practice questions that come from exams before the 2013 test, when some of the more drastic changes were implemented. You might still be able to use older questions to refresh your memory on certain topics, but they won't really prepare you for the more analytical framework of questions on the current AP Biology test.

Furthermore, the College Board now offers a great online resource called AP Classroom , through which students can interact with teachers, complete homework and get feedback on assignments, and receive access to review materials for the AP Bio test, including real practice questions. You'll use your College Board student account login credentials to access AP Classroom, and once logged in, you can access a different section for each AP class you're taking.

Tip 3: Memorization Isn't Enough

Even though AP Biology still involves a fair amount of memorization, you can't focus exclusively on content knowledge and just assume you'll do great on the test.

AP Bio questions will test your critical-thinking skills and logical reasoning abilities, along with your general knowledge of biology. That's why it's so important that you spend a significant amount of time doing practice questions in addition to content review. Don't let the test surprise you!

Tip 4: Don't Forget About Labs

Revisiting old labs is not super fun (well, it wasn't for me), so you might be tempted to ignore them and just focus on studying content outside the lab context.

Try to avoid this temptation! Go through your labs, and make sure that you understand their methodologies and the reasoning behind the results. Understanding the scientific method and the components of a good experiment is key to acing the AP Bio exam.

The more lab review you do, the more comfortable you'll feel during the test.

How to Review for the AP Biology Exam: 5-Step Guide

As you study for the exam, follow the five steps below to ensure your AP Bio review is as thorough and effective as possible.

Step 1: Take a Diagnostic Test

The first step of your AP Biology review is to take a practice exam so you can see how much you'll need to study and which areas need the most work.

You should take your first complete practice test no later than the beginning of your second semester. You can use a practice exam from a review book or search online for a practice test. The review books I mentioned in the previous section have some useful materials.

When you take a practice test, make sure it's the newest 2020 version of the exam (or, if you can't find this, at least a version from 2013-2019). If you see practice tests that have 100 multiple-choice questions in the first section, you're looking at a very old version of the AP Bio exam! You won't be able to rely on your scores on this version to get a clear picture of where you fall on the new test.

Step 2: Calculate Your Score and Set a Goal

Once you've taken a diagnostic test, you can calculate your score on the 1-5 AP scale. According to the CliffsNotes review book mentioned above, you can estimate your score using the following method:

• Multiply the number of questions you answered correctly in Section 1 by 0.725
• Multiply the number of points you earned in Section 2 by 1.25
• Add those two numbers together to get your raw score

Then, convert the raw score to an AP score using the following chart:

For example, if you got 42 questions correct on the multiple-choice section and earned 25 points on the free-response section, your raw score would be (42 * .725) + (25 * 1.25) = 61.7 = just barely made it into the 5 category!

This is without taking the curve into account, which is different every year, but it should give you a rough idea of where you stand. Unless you're scoring a really high 5 (90+), you should still put in a bit of study time to make sure you're fully prepared.

If you score low (1 or 2), you might make it your goal to raise your score to 3. Just keep in mind that some schools don't accept 3s for college credit , so you might want to aim higher after you make it to this first milestone.

Most colleges consider 4 to be the standard cutoff for AP credit, so you should try for at least a 4 if you're hoping to get a head start in college. Once you're consistently scoring in the 3 range on this exam, you can set 4 or 5 as your goal.

Even if you're already at the 4 or 5 level on AP Biology, you probably still have some room to improve. It's nice to get in some extra practice so that you feel very comfortable on the real test.

Depending on how much you need to improve and on how long you want to spread out your prep, you might come up with different plans. To improve by 1 AP score point, you can get away with studying only two months or so in advance. On the other hand, if you're hoping to improve by 2 or more points, try to start midway through the school year to avoid cramming.

Step 3: Analyze Your Mistakes

This is the most critical part of the review process, and it's particularly important for AP Biology. There's a lot of material to learn, and you don't want to waste any time going over concepts you already have down.

Comb through your mistakes on the diagnostic test to see where the most errors happened and why. Did your mistakes center more around lack of knowledge of background information or difficulty analyzing the scenarios presented on the test (in other words, you knew the information but couldn't answer the question because it confused you)?

You'll most likely have a little of each type of mistake, but if one is more prevalent than the other, take that into account for your studying strategy. For example, it wouldn't be a good idea to keep drilling basic content knowledge if most of your mistakes were due to your misinterpreting complex questions or reading diagrams incorrectly. You'd want to devote less of your time to reviewing biological terms and more to doing realistic practice questions.

Even in those cases, you'll probably still have at least a few issues with content knowledge. As you go through your mistakes, keep a running list of the ideas you need to revisit in your notes or review book. If you're caught off guard by your unfamiliarity with a certain topic, you should pay special attention to that topic in your prep.

You might also notice mistakes due to carelessness or time pressure that aren't directly related to your knowledge of the material or understanding of the question. In this case, you'll need to think about revising your basic test-taking strategies. I'll go into more detail on this next.

Step 4: Fix Your Mistakes

There are a few things you can do to revise your strategies for taking the exam and effectively review concepts you didn't understand.

The obvious first step is to go back into your textbook, your notes, or a reliable AP Bio review book (or even all three!) and brush up on the information you forgot. Sometimes for biology, this can be a little overwhelming due to the complexity of the material.

If you're trying to understand systems or processes, I recommend testing yourself by drawing diagrams of how they work. This will allow you to make connections between dry facts presented in the text and the biological reality of what's happening in the system. It will help you not only in your content knowledge but also in your ability to analyze related scenarios on the test. You can use this strategy for many concepts in AP Biology, and it will make them much simpler to understand.

To correct your other mistakes that have more to do with question comprehension, you'll need to focus on doing similar practice questions. I suggest getting Sterling's AP Biology Practice Questions for some questions that are organized logically by topic area and well aligned with the new exam format.

More practice is also a good remedy for careless errors and time-management problems. You can learn how to better identify the key parts of each question and avoid distractions that might throw you off.

Underlining the most important parts of the question can be a good strategy if you're prone to careless errors. If time management is a problem, put some thought into why you might've run out of time. Did you linger for too long on difficult questions? Remember that it's a smart idea to skip questions that are giving you a lot of trouble (meaning, they're not answerable within a minute) and come back to them later once you've gotten through the whole section.

Step 5: Take Another Test and Repeat Previous Steps

Now that you've analyzed and fixed your mistakes on the diagnostic test and done more targeted studying, it's time to take another practice test.

Score this new test and then repeat steps 3 and 4. You should notice improvements as you continue to repeat this process and gain familiarity with the format and content of the AP Biology exam.

If you don't notice positive changes from one test to the next, it might be time to reevaluate your review techniques. Depending on how early you start studying for the AP Bio test and how much you want to improve, you might go through these steps once, twice, or seven times.

Continue the process until you achieve your score goals or run out of study time!

Conclusion: AP Biology Review Guide

The AP Biology test is a long exam, and it covers a wide range of material.

Recently, the test was updated to focus less on information recall and more on analytical thinking, which can be good and bad. On the one hand, you won't have to rely on memorization as much. On the other, your AP score will be highly dependent on your ability to think through complicated scenarios presented on the test.

In addition, the test underwent some structural changes in 2020. These key changes included going from 69 questions on the multiple-choice section to just 60 questions and reducing the number of short-answer questions from six to four. There will also no longer be any grid-in questions.

In your own AP Biology review, you should go over all the information you learned in the course. However, you should also devote a significant amount of your time to practice testing so that you can learn to think in the way the test wants you to think.

If you plan your study time wisely and learn how to solve the types of questions that are most difficult for you, you'll be on your way to a great AP Bio score!

What's Next?

Ready to jump into reviewing biology concepts? We have guides to help you review cell theory , enzymes , and homologous and analogous structures , as well as quick looks at parts of the cell ( cell membrane and endoplasmic reticulum ) and the photosynthesis equation .

If you're taking AP Biology, you probably have big plans for higher education. Find out how many AP classes you should take in high school if you're looking at highly selective colleges.

Considering an AP Calculus course? Read this article for some guidance on deciding whether you should take AP Calculus AB or BC .

These recommendations are based solely on our knowledge and experience. If you purchase an item through one of our links, PrepScholar may receive a commission.

Samantha is a blog content writer for PrepScholar. Her goal is to help students adopt a less stressful view of standardized testing and other academic challenges through her articles. Samantha is also passionate about art and graduated with honors from Dartmouth College as a Studio Art major in 2014. In high school, she earned a 2400 on the SAT, 5's on all seven of her AP tests, and was named a National Merit Scholar.

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• Review appropriate  Citation and Documentation Style  for your assignment and literature review

Common Questions

What is a literature review?

A literature review is a type of scholarly, researched writing that discusses the already published information on a narrow topic . 

What is the purpose of a writing literature review?

Writing a literature review improves your personal understanding of a topic, and demonstrates your knowledge and ability to make connections between concepts and ideas. The literature review is a service to your reader, summarizing past ideas about a topic, bringing them up to date on the latest research, and making sure they have all any background information they need to understand the topic.  

What is "the literature"?

This already published information- called the literature- can be from primary information sources such as speeches, interviews, and reports, or from secondary information sources such as peer-reviewed journal articles, dissertations, and books. These type of sources are probably familiar to you from previous research projects you’ve done in your classes.

Is a literature review it's own paper?

You can write a literature review as a standalone paper , or as part of a larger research paper . When a standalone paper, the literature review acts as a summary, or snapshot, of what has been said and done about a topic in the field so far. When part of the a larger paper, a literature review still acts as a snapshot, but the prior information it provides can also support the new information, research, or arguments presented later in the paper.

Does a literature review contain an argument?

No, a literature review does NOT present an argument or new information. The literature review is a foundation that summarizes and synthesizes the existing literature in order for you and your readers to understand what has already been said and done about your topic.

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Review Essays for the Biological sciences

A review essay for the biological sciences serves to discuss and synthesize key findings on a particular subject. Review papers are helpful to the writer and their colleagues in gaining critical awareness in specialized fields that may or may not be their own.

This guide explains what a review essay is and identifies several approaches to writing a review essay. Although much of the information is geared directly to the biological sciences, it is generally applicable to review essays in all fields.

What is a Review Essay?

A review essay is a synthesis of primary sources (mainly research papers presented in academic journals) on a given topic. A biological review essay demonstrates that the writer has thorough understanding of the literature and can formulate a useful analysis. While no new research is presented by the writer, the field benefits from the review by recieving a new perspective. There are several approaches one may take when writing a biological review:

A State of the art review

A state of the art review considers mainly the most current research in a given area. The review may offer new perspectives on an issue or point out an area in need of further research.

A Historical review

A historical review is a survey of the development of a particular field of study. It may examine the early stages of the field, key findings to present, key theoretical models and their evolution, etc.

A Comparison of perspectives review

A comparison of perspectives review contrasts various ways of looking at a certain topic. If in fact there is a debate over some process or idea, a comparison of perspectives review may illustrate the research that supports both sides. A comparison of perspectives review may introduce a new perspective by way of comparing it to another.

A Synthesis of two fields review

Many times researchers in different fields may be working on similar problems. A synthesis of two fields review provides insights into a given topic based on a review of the literature from two or more disciplines.

A Theoretical model building review

A theoretical model building review examines the literature within a given area with the intention of developing new theoretical assumptions.

Key Considerations for Writing a Biological Review Essay

This guide will inform you of certain things not to miss when writing a review essay. It will also give you some information about using and documenting your sources.

Keep your focus narrow.

When writing a review essay it is important to keep the scope of the topic narrow enough so that you can discuss it thoroughly. For example a topic such as air quality in factories could be narrowed significantly to something like carbon dioxide levels in auto manufacturing plants .

A good way to narrow your focus is to start with a broad topic that is of some interest to you, then read some of the literature in the field. Look for a thread of the discussion that points to a more specific topic.

Analyze, synthesize, and interpret.

A review essay is not a pure summary of the information you read for your review. You are required to analyze, synthesize, and interpret the information you read in some meaningful way.

It is not enough to simply present the material you have found, you must go beyond that and explain its relevance and significance to the topic at hand.

Establish a clear thesis from the onset of your writing and examine which pieces of your reading help you in developing and supporting the ideas in your thesis.

Use only academic sources.

A review essay reviews the academic body of literature—articles and research presented in academic journals. Lay periodicals such as, Discover , Scientific America , or Popular Science , are not adequate sources for an academic review essay.

If you are having trouble finding the academic journals in your field, ask one of your professors or a reference librarian.

Document your sources.

The material that you discuss in a review essay is obviously not your own, therefore it is crucial to document your sources properly. Proper documentation is crucial for two reasons: 1. It prevents the writer from being accused of plagiarism and 2. It gives the reader the opportunity to locate the sources the writer has reviewed because they may find them valuable in their own academic pursuits. Proper documentation depends on which style guide you are following.

Quote sparingly and properly.

No one wants to read a paper that is simply a string of quotes; reserve direct quotations for when you want to create a big impact. Often times the way a quote is written will not fit with the language or the style of your paper so paraphrase the authors words carefully and verbage as necessary to create a well formed paragraph.

Choose an informative title.

The title you choose for your review essay should give some indication of what lies ahead for the reader. You might consider the process you took in narrowing your topic to help you with your title—think of the title as something specific rather than a vague representation of your paper's topic. For example the title Wastewater Treatment might be more informative if rewritten as The Removal of Cloroform Bacteria as Practiced by California's Municipal Water Treatment Facilities .

Consider your audience.

More than likely your audience will be your academic peers, therefore you can make a couple assumptions and choose a writing style that suits the audience. Though your audience may lack the detailed knowledge you have about your topic, they do have similar background knowledge to you. You can assume that you audience understands much of the technical language you have to use to write about your topic and you do not have to go into great detail about background information.

Elements of a Review Essay

This guide explains each section of a review essay and gives specific information about what should be included in each.

On the title page include the title, your name, and the date. Your instructor may have additional requirements (such as the course number, etc.) so be sure to follow the guidelines on the assignment sheet. Professional journals may also have more specific requirements for the title page.

An abstract is a brief summary of your review. The abstract should include only the main points of your review. Think of the abstract as a chance for the reader to preview your paper and decide if they want to read on for the details.

Introduction

The introduction of your review should accomplish three things:

• It may sound redundant to "introduce" your topic in the introduction, but often times writer's fail to do so. Let the reader in on background information specific to the topic, define terms that may be unfamiliar to them, explain the scope of the discussion, and your purpose for writing the review.
• Think of your review essay as a statement in the larger conversation of your academic community. Your review is your way of entering into that conversation and it is important to briefly address why your review is relevant to the discussion. You may feel the relevance is obvious because you are so familiar with the topic, but your readers have not yet established that familiarity.
• The thesis is the main idea that you want to get across to your reader. your thesis should be a clear statement of what you intend to prove or illustrate by your review. By revealing your thesis in the introduction the reader knows what to expect in the rest of the paper.

The discussion section is the body of your paper. The discussion section contains information that develops and supports your thesis. While there is no particular form that a discussion section must take there are several considerations that a writer must follow when building a discussion.

• A review essay is not simply a summary of literature you have reviewed. Be careful not to leave out your own analysis of the ideas presented in the literature. Synthesize the material from all the works—what are the connections you see, or the connections you are trying to illustrate, among your readings.

A review essay is not a pure summary of the information you read for your review. You are required to analyze, synthesize, and interpret the information you read in some meaningful way. It is not enough to simply present the material you have found, you must go beyond that and explain its relevance and significance to the topic at hand. Establish a clear thesis from the onset of your writing and examine which pieces of your reading help you in developing and supporting the ideas in your thesis.

• Keep your discussion focused on your topic and more importantly your thesis. Don't let tangents or extraneous material get in the way of a concise, coherent discussion. A well focused paper is crucial in getting your message across to your reader.
• Keeping your points organized makes it easier for the reader to follow along and make sense of your review. Start each paragraph with a topic sentence that relates back to your thesis. The headings used for this guide give you some idea of how to organize the overall paper, but as far as the discussion section goes use meaningful subheadings that relate to your content to organize your points.
• Your thesis should illustrate your objectives in writing the review and your discussion should serve to accomplish your objectives. Make sure your keep your discussion related to the thesis in order to meet your objectives. If you find that your discussion does not relate so much to your thesis, don't panic, you might want to revise your thesis instead of reworking the discussion.

Conclusions

Because the conclusions section often gets left for last it is often the weakest part of a student review essay. It is as crucial a part of the paper as any and should be treated as such.

A good conclusion should illustrate the key connections between your major points and your thesis as well as they key connections between your thesis and the broader discussion—what is the significance of your paper in a larger context? Make some conclusions —where have you arrived as a result of writing this paper?

Be careful not to present any new information in the conclusion section.

Here you report all the works you have cited in your paper. The format for a references page varies by discipline as does how you should cite your references within the paper.

Bastek, Neal. (1999). Review Essays for the Biological Sciences. Writing@CSU . Colorado State University. https://writing.colostate.edu/guides/guide.cfm?guideid=79

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Ten Simple Rules for Writing a Literature Review

Marco pautasso.

1 Centre for Functional and Evolutionary Ecology (CEFE), CNRS, Montpellier, France

2 Centre for Biodiversity Synthesis and Analysis (CESAB), FRB, Aix-en-Provence, France

Literature reviews are in great demand in most scientific fields. Their need stems from the ever-increasing output of scientific publications [1] . For example, compared to 1991, in 2008 three, eight, and forty times more papers were indexed in Web of Science on malaria, obesity, and biodiversity, respectively [2] . Given such mountains of papers, scientists cannot be expected to examine in detail every single new paper relevant to their interests [3] . Thus, it is both advantageous and necessary to rely on regular summaries of the recent literature. Although recognition for scientists mainly comes from primary research, timely literature reviews can lead to new synthetic insights and are often widely read [4] . For such summaries to be useful, however, they need to be compiled in a professional way [5] .

When starting from scratch, reviewing the literature can require a titanic amount of work. That is why researchers who have spent their career working on a certain research issue are in a perfect position to review that literature. Some graduate schools are now offering courses in reviewing the literature, given that most research students start their project by producing an overview of what has already been done on their research issue [6] . However, it is likely that most scientists have not thought in detail about how to approach and carry out a literature review.

Reviewing the literature requires the ability to juggle multiple tasks, from finding and evaluating relevant material to synthesising information from various sources, from critical thinking to paraphrasing, evaluating, and citation skills [7] . In this contribution, I share ten simple rules I learned working on about 25 literature reviews as a PhD and postdoctoral student. Ideas and insights also come from discussions with coauthors and colleagues, as well as feedback from reviewers and editors.

Rule 1: Define a Topic and Audience

How to choose which topic to review? There are so many issues in contemporary science that you could spend a lifetime of attending conferences and reading the literature just pondering what to review. On the one hand, if you take several years to choose, several other people may have had the same idea in the meantime. On the other hand, only a well-considered topic is likely to lead to a brilliant literature review [8] . The topic must at least be:

• interesting to you (ideally, you should have come across a series of recent papers related to your line of work that call for a critical summary),
• an important aspect of the field (so that many readers will be interested in the review and there will be enough material to write it), and
• a well-defined issue (otherwise you could potentially include thousands of publications, which would make the review unhelpful).

Ideas for potential reviews may come from papers providing lists of key research questions to be answered [9] , but also from serendipitous moments during desultory reading and discussions. In addition to choosing your topic, you should also select a target audience. In many cases, the topic (e.g., web services in computational biology) will automatically define an audience (e.g., computational biologists), but that same topic may also be of interest to neighbouring fields (e.g., computer science, biology, etc.).

Rule 2: Search and Re-search the Literature

After having chosen your topic and audience, start by checking the literature and downloading relevant papers. Five pieces of advice here:

• keep track of the search items you use (so that your search can be replicated [10] ),
• keep a list of papers whose pdfs you cannot access immediately (so as to retrieve them later with alternative strategies),
• use a paper management system (e.g., Mendeley, Papers, Qiqqa, Sente),
• define early in the process some criteria for exclusion of irrelevant papers (these criteria can then be described in the review to help define its scope), and
• do not just look for research papers in the area you wish to review, but also seek previous reviews.

The chances are high that someone will already have published a literature review ( Figure 1 ), if not exactly on the issue you are planning to tackle, at least on a related topic. If there are already a few or several reviews of the literature on your issue, my advice is not to give up, but to carry on with your own literature review,

The bottom-right situation (many literature reviews but few research papers) is not just a theoretical situation; it applies, for example, to the study of the impacts of climate change on plant diseases, where there appear to be more literature reviews than research studies [33] .

• discussing in your review the approaches, limitations, and conclusions of past reviews,
• trying to find a new angle that has not been covered adequately in the previous reviews, and
• incorporating new material that has inevitably accumulated since their appearance.

When searching the literature for pertinent papers and reviews, the usual rules apply:

• be thorough,
• use different keywords and database sources (e.g., DBLP, Google Scholar, ISI Proceedings, JSTOR Search, Medline, Scopus, Web of Science), and
• look at who has cited past relevant papers and book chapters.

Rule 3: Take Notes While Reading

If you read the papers first, and only afterwards start writing the review, you will need a very good memory to remember who wrote what, and what your impressions and associations were while reading each single paper. My advice is, while reading, to start writing down interesting pieces of information, insights about how to organize the review, and thoughts on what to write. This way, by the time you have read the literature you selected, you will already have a rough draft of the review.

Of course, this draft will still need much rewriting, restructuring, and rethinking to obtain a text with a coherent argument [11] , but you will have avoided the danger posed by staring at a blank document. Be careful when taking notes to use quotation marks if you are provisionally copying verbatim from the literature. It is advisable then to reformulate such quotes with your own words in the final draft. It is important to be careful in noting the references already at this stage, so as to avoid misattributions. Using referencing software from the very beginning of your endeavour will save you time.

Rule 4: Choose the Type of Review You Wish to Write

After having taken notes while reading the literature, you will have a rough idea of the amount of material available for the review. This is probably a good time to decide whether to go for a mini- or a full review. Some journals are now favouring the publication of rather short reviews focusing on the last few years, with a limit on the number of words and citations. A mini-review is not necessarily a minor review: it may well attract more attention from busy readers, although it will inevitably simplify some issues and leave out some relevant material due to space limitations. A full review will have the advantage of more freedom to cover in detail the complexities of a particular scientific development, but may then be left in the pile of the very important papers “to be read” by readers with little time to spare for major monographs.

There is probably a continuum between mini- and full reviews. The same point applies to the dichotomy of descriptive vs. integrative reviews. While descriptive reviews focus on the methodology, findings, and interpretation of each reviewed study, integrative reviews attempt to find common ideas and concepts from the reviewed material [12] . A similar distinction exists between narrative and systematic reviews: while narrative reviews are qualitative, systematic reviews attempt to test a hypothesis based on the published evidence, which is gathered using a predefined protocol to reduce bias [13] , [14] . When systematic reviews analyse quantitative results in a quantitative way, they become meta-analyses. The choice between different review types will have to be made on a case-by-case basis, depending not just on the nature of the material found and the preferences of the target journal(s), but also on the time available to write the review and the number of coauthors [15] .

Rule 5: Keep the Review Focused, but Make It of Broad Interest

Whether your plan is to write a mini- or a full review, it is good advice to keep it focused 16 , 17 . Including material just for the sake of it can easily lead to reviews that are trying to do too many things at once. The need to keep a review focused can be problematic for interdisciplinary reviews, where the aim is to bridge the gap between fields [18] . If you are writing a review on, for example, how epidemiological approaches are used in modelling the spread of ideas, you may be inclined to include material from both parent fields, epidemiology and the study of cultural diffusion. This may be necessary to some extent, but in this case a focused review would only deal in detail with those studies at the interface between epidemiology and the spread of ideas.

While focus is an important feature of a successful review, this requirement has to be balanced with the need to make the review relevant to a broad audience. This square may be circled by discussing the wider implications of the reviewed topic for other disciplines.

Rule 6: Be Critical and Consistent

Reviewing the literature is not stamp collecting. A good review does not just summarize the literature, but discusses it critically, identifies methodological problems, and points out research gaps [19] . After having read a review of the literature, a reader should have a rough idea of:

• the major achievements in the reviewed field,
• the main areas of debate, and
• the outstanding research questions.

It is challenging to achieve a successful review on all these fronts. A solution can be to involve a set of complementary coauthors: some people are excellent at mapping what has been achieved, some others are very good at identifying dark clouds on the horizon, and some have instead a knack at predicting where solutions are going to come from. If your journal club has exactly this sort of team, then you should definitely write a review of the literature! In addition to critical thinking, a literature review needs consistency, for example in the choice of passive vs. active voice and present vs. past tense.

Rule 7: Find a Logical Structure

Like a well-baked cake, a good review has a number of telling features: it is worth the reader's time, timely, systematic, well written, focused, and critical. It also needs a good structure. With reviews, the usual subdivision of research papers into introduction, methods, results, and discussion does not work or is rarely used. However, a general introduction of the context and, toward the end, a recapitulation of the main points covered and take-home messages make sense also in the case of reviews. For systematic reviews, there is a trend towards including information about how the literature was searched (database, keywords, time limits) [20] .

How can you organize the flow of the main body of the review so that the reader will be drawn into and guided through it? It is generally helpful to draw a conceptual scheme of the review, e.g., with mind-mapping techniques. Such diagrams can help recognize a logical way to order and link the various sections of a review [21] . This is the case not just at the writing stage, but also for readers if the diagram is included in the review as a figure. A careful selection of diagrams and figures relevant to the reviewed topic can be very helpful to structure the text too [22] .

Rule 8: Make Use of Feedback

Reviews of the literature are normally peer-reviewed in the same way as research papers, and rightly so [23] . As a rule, incorporating feedback from reviewers greatly helps improve a review draft. Having read the review with a fresh mind, reviewers may spot inaccuracies, inconsistencies, and ambiguities that had not been noticed by the writers due to rereading the typescript too many times. It is however advisable to reread the draft one more time before submission, as a last-minute correction of typos, leaps, and muddled sentences may enable the reviewers to focus on providing advice on the content rather than the form.

Feedback is vital to writing a good review, and should be sought from a variety of colleagues, so as to obtain a diversity of views on the draft. This may lead in some cases to conflicting views on the merits of the paper, and on how to improve it, but such a situation is better than the absence of feedback. A diversity of feedback perspectives on a literature review can help identify where the consensus view stands in the landscape of the current scientific understanding of an issue [24] .

Rule 9: Include Your Own Relevant Research, but Be Objective

In many cases, reviewers of the literature will have published studies relevant to the review they are writing. This could create a conflict of interest: how can reviewers report objectively on their own work [25] ? Some scientists may be overly enthusiastic about what they have published, and thus risk giving too much importance to their own findings in the review. However, bias could also occur in the other direction: some scientists may be unduly dismissive of their own achievements, so that they will tend to downplay their contribution (if any) to a field when reviewing it.

In general, a review of the literature should neither be a public relations brochure nor an exercise in competitive self-denial. If a reviewer is up to the job of producing a well-organized and methodical review, which flows well and provides a service to the readership, then it should be possible to be objective in reviewing one's own relevant findings. In reviews written by multiple authors, this may be achieved by assigning the review of the results of a coauthor to different coauthors.

Rule 10: Be Up-to-Date, but Do Not Forget Older Studies

Given the progressive acceleration in the publication of scientific papers, today's reviews of the literature need awareness not just of the overall direction and achievements of a field of inquiry, but also of the latest studies, so as not to become out-of-date before they have been published. Ideally, a literature review should not identify as a major research gap an issue that has just been addressed in a series of papers in press (the same applies, of course, to older, overlooked studies (“sleeping beauties” [26] )). This implies that literature reviewers would do well to keep an eye on electronic lists of papers in press, given that it can take months before these appear in scientific databases. Some reviews declare that they have scanned the literature up to a certain point in time, but given that peer review can be a rather lengthy process, a full search for newly appeared literature at the revision stage may be worthwhile. Assessing the contribution of papers that have just appeared is particularly challenging, because there is little perspective with which to gauge their significance and impact on further research and society.

Inevitably, new papers on the reviewed topic (including independently written literature reviews) will appear from all quarters after the review has been published, so that there may soon be the need for an updated review. But this is the nature of science [27] – [32] . I wish everybody good luck with writing a review of the literature.

Acknowledgments

Many thanks to M. Barbosa, K. Dehnen-Schmutz, T. Döring, D. Fontaneto, M. Garbelotto, O. Holdenrieder, M. Jeger, D. Lonsdale, A. MacLeod, P. Mills, M. Moslonka-Lefebvre, G. Stancanelli, P. Weisberg, and X. Xu for insights and discussions, and to P. Bourne, T. Matoni, and D. Smith for helpful comments on a previous draft.

Funding Statement

This work was funded by the French Foundation for Research on Biodiversity (FRB) through its Centre for Synthesis and Analysis of Biodiversity data (CESAB), as part of the NETSEED research project. The funders had no role in the preparation of the manuscript.

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Assignment 2

Primary article vs. review article.

After a scientist conducts an experiment, it is common for them to analyze the results and create an article about their findings. This type of article is called a primary article, an article that reports on newly found information, including experiment results or an analysis of a combination of information for several studies. An example is when a college professor finalizes a study about how blue light affects several aspect of life for college students. That professor than constructs a primary article about the results of the study as well as using information they used from pervious studies done withs similar parameters. Since primary articles are developed from the individuals that carried out the studies, primary article are considered to be a more reliable source of information.

After primary articles are constructed, there are some people that have different perspectives on the studies performed and want to express their ideas about the primary article. This type of article is called a review article. In its name, a review article is a review of a primary article. An example could be a graduate student that, was not in the study, but still experiences the results found in the study done about effect of blue light on college students. This student will add their ideas and insight on how blue light affected them, while also citing back to the primary article as to why they are adding their ideas. Since the review article is not created by someone that physically performed the study or experiment, a review article is not as reliable as a primary article.

In order for articles to be published to be published to a journal, they must go through the peer review process. This process includes having an authors peers review the article they created to deem it acceptable for submission to the journal. An example is when the professor that created the primary article about blue light effect on college students, the article is then sent to other professors that work in the same discipline or have conducted a similar study. These professors than can make edits to the article for it to be revised later or deem it worthy of being in a journal. The process denies any faulty or misleading studies to be submitted to a journal and accepted for others to see.

Using the information provided earlier, there are two different articles that must be determined either a primary article or review article. After close analysis, the article titled “Rapid and Sensitive Detection of SARS-CoV-2 Using Clustered Regularly Interspaced Short Palindromic Repeats” by J.H. Tsou et. al, is a primary article. “We obtained NR-52286 and NR52349 samples from the BEI Resources” (Tsou 2019). This quote like many others in the article talks in the first person in the point of view of the ones carrying out the studies. Moving onto the second article, “Development of clustered regularly interspaced short palindromic repeats/CRISPR-associated technology for potential clinical applications” by Y. Y. Haung et. al, is a review article. “In CRISPR/Cas systems, researchers have discovered various Cas proteins with different characteristics” (Haung 2022). This quote speaks in the third person, as if it was viewing the researcher and discussing about how those researchers conducted the study. It is also easy to differentiate the two because the second article also has a date when the peer review article was started.

Reference Huang, Y.-Y., Zhang, X.-Y., Zhu, P., & Ji, L. (2022). Development of clustered regularly interspaced short palindromic repeats/CRISPR-associated technology for potential clinical applications. World Journal of Clinical Cases, 10(18), 5934–5945. https://doi.org/10.12998/wjcc.v10.i18.5934 Tsou, J.-H. Liu, H. Stass, S.A.; Jiang, F. Rapid and Sensitive Detection of SARS-CoV-2 Using Clustered Regularly Interspaced Short Palindromic Repeats. Biomedicines 2021, 9, 239. https://doi.org/10.3390/biomedicines9030239

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• Published: 13 May 2024

Integrating population genetics, stem cell biology and cellular genomics to study complex human diseases

• Nona Farbehi   ORCID: orcid.org/0000-0001-8461-236X 1 , 2 , 3   na1 ,
• Drew R. Neavin   ORCID: orcid.org/0000-0002-1783-6491 1   na1 ,
• Anna S. E. Cuomo 1 , 4 ,
• Lorenz Studer   ORCID: orcid.org/0000-0003-0741-7987 3 , 5 ,
• Daniel G. MacArthur 4 , 6 &
• Joseph E. Powell   ORCID: orcid.org/0000-0002-5070-4124 1 , 3 , 7  

Nature Genetics volume  56 ,  pages 758–766 ( 2024 ) Cite this article

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• Population genetics
• Transcriptomics

Human pluripotent stem (hPS) cells can, in theory, be differentiated into any cell type, making them a powerful in vitro model for human biology. Recent technological advances have facilitated large-scale hPS cell studies that allow investigation of the genetic regulation of molecular phenotypes and their contribution to high-order phenotypes such as human disease. Integrating hPS cells with single-cell sequencing makes identifying context-dependent genetic effects during cell development or upon experimental manipulation possible. Here we discuss how the intersection of stem cell biology, population genetics and cellular genomics can help resolve the functional consequences of human genetic variation. We examine the critical challenges of integrating these fields and approaches to scaling them cost-effectively and practically. We highlight two areas of human biology that can particularly benefit from population-scale hPS cell studies, elucidating mechanisms underlying complex disease risk loci and evaluating relationships between common genetic variation and pharmacotherapeutic phenotypes.

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Acknowledgements

Figures were generated with BioRender.com and further developed by A. Garcia, a scientific illustrator from Bio-Graphics. This research was supported by a National Health and Medical Research Council (NHMRC) Investigator grant (J.E.P., 1175781), research grants from the Australian Research Council (ARC) Special Research Initiative in Stem Cell Science, an ARC Discovery Project (190100825), an EMBO Postdoctoral Fellowship (A.S.E.C.) and an Aligning Science Across Parkinson’s Grant (J.E.P., N.F., D.R.N. and L.S.). J.E.P. is supported by a Fok Family Fellowship.

Author information

These authors contributed equally: Nona Farbehi, Drew R. Neavin.

Authors and Affiliations

Garvan Weizmann Center for Cellular Genomics, Garvan Institute of Medical Research, Sydney, New South Wales, Australia

Nona Farbehi, Drew R. Neavin, Anna S. E. Cuomo & Joseph E. Powell

Graduate School of Biomedical Engineering, University of New South Wales, Sydney, New South Wales, Australia

Nona Farbehi

Aligning Science Across Parkinson’s Collaborative Research Network, Chevy Chase, MD, USA

Nona Farbehi, Lorenz Studer & Joseph E. Powell

Centre for Population Genomics, Garvan Institute of Medical Research, University of New South Wales, Sydney, New South Wales, Australia

Anna S. E. Cuomo & Daniel G. MacArthur

The Center for Stem Cell Biology and Developmental Biology Program, Sloan-Kettering Institute for Cancer Research, New York, NY, USA

Lorenz Studer

Centre for Population Genomics, Murdoch Children’s Research Institute, Melbourne, Victoria, Australia

Daniel G. MacArthur

UNSW Cellular Genomics Futures Institute, University of New South Wales, Sydney, New South Wales, Australia

Joseph E. Powell

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All authors conceived the topic and wrote and revised the manuscript.

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Correspondence to Joseph E. Powell .

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D.G.M. is a founder with equity in Goldfinch Bio, is a paid advisor to GSK, Insitro, Third Rock Ventures and Foresite Labs, and has received research support from AbbVie, Astellas, Biogen, BioMarin, Eisai, Merck, Pfizer and Sanofi-Genzyme; none of these activities is related to the work presented here. J.E.P. is a founder with equity in Celltellus Laboratory and has received research support from Illumina. The other authors declare no conflict of interest.

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Farbehi, N., Neavin, D.R., Cuomo, A.S.E. et al. Integrating population genetics, stem cell biology and cellular genomics to study complex human diseases. Nat Genet 56 , 758–766 (2024). https://doi.org/10.1038/s41588-024-01731-9

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DOI : https://doi.org/10.1038/s41588-024-01731-9

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• Published: 27 May 2024

Which contributes more to the relict flora distribution pattern in East Asia, geographical processes or climate change? New evidence from the phylogeography of Rehderodendron kwangtungense

• Jiehao Jin 1   na1 ,
• Wanyi Zhao 1   na1 ,
• Sufang Chen 1 ,
• Chao Gu 2 ,
• Zhihui Chen 1 ,
• Zhongcheng Liu 1 ,
• Wenbo Liao 1 &
• Qiang Fan 1  

BMC Plant Biology volume  24 , Article number:  459 ( 2024 ) Cite this article

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Relict species are important for enhancing the understanding of modern biogeographic distribution patterns. Although both geological and climatic changes since the Cenozoic have affected the relict flora in East Asia, the contributions of geographical processes remain unclear. In this study, we employed restriction-site associated DNA sequencing (RAD-seq) and shallow genome sequencing data, in conjunction with ecological niche modeling (ENM), to investigate the spatial genetic patterns and population differentiation history of the relict species Rehderodendron kwangtungense Chun.

A total of 138 individuals from 16 populations were collected, largely covering the natural distribution of R. kwangtungense . The genetic diversity within the R. kwangtungense populations was extremely low ( H O = 0.048 ± 0.019; H E = 0.033 ± 0.011). Mantel tests revealed isolation-by-distance pattern (R 2  = 0.38, P  < 0.001), and AMOVA analysis showed that the genetic variation of R. kwangtungense occurs mainly between populations (86.88%, K = 7). Between 23 and 21 Ma, R. kwangtungense underwent a period of rapid differentiation that coincided with the rise of the Himalayas and the establishment of the East Asian monsoon. According to ENM and population demographic history, the suitable area and effective population size of R. kwangtungense decreased sharply during the glacial period and expanded after the last glacial maximum (LGM).

Our study shows that the distribution pattern of southern China mountain relict flora may have developed during the panplain stage between the middle Oligocene and the early Miocene. Then, the flora later fragmented under the force of orogenesis, including intermittent uplift during the Cenozoic Himalayan orogeny and the formation of abundant rainfall associated with the East Asian monsoon. The findings emphasized the predominant role of geographical processes in shaping relict plant distribution patterns.

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Introduction

Relict species are considered fascinating ‘living fossils’ or remnants of ancient clades and biotas and can be divided into geographical relicts and phylogenetic relicts [ 1 , 2 ]. These organisms are often viewed as providing compelling evidence for the conservation of ancestral character states in terms of morphology, ecology [ 3 , 4 ], and spatial distribution [ 5 , 6 ]. Relict species are often confined to ecological refugia and have fragmented distributions, and they are commonly high priority targets for biodiversity conservation [ 7 ]. They must adjust their physiological adaptability [ 8 ] and purge the accumulation of deleterious mutations within the population to quickly respond to the pressure of environmental changes [ 9 ]. Thus, relict species serve as a “window to the past”, allowing us to understand the conditions that enabled them to survive for so long [ 1 , 10 , 11 ].

East Asia contains the most abundant Tertiary relict flora in the Northern Hemisphere [ 12 , 13 , 14 ]. Southeast China is an important host of such relict flora and is considered a floristic museum characterised by phylogenetic and distribution centres of ancient relict species [ 15 , 16 ]. In general, climatic fluctuations during the Cenozoic prompted the southward migration of Northern Hemisphere flora [ 17 ]. High topographic heterogeneity and climate stability has allowed East Asia to serve as a long-term stable habitat, and many species remained in situ during the Pleistocene glaciation [ 18 , 19 , 20 , 21 , 22 ]. Many phylogeographic studies have indicated that plants can survive the climatic fluctuations associated with an ice age and take refuge in mountains in situ [ 23 , 24 , 25 , 26 ]. It is clear that the establishment of relict flora in East Asia occurred much earlier than the Pleistocene. Previous studies have shown that past geographic events and climate changes have jointly influenced the lineage differentiation of relict plants in East Asia [ 13 , 27 , 28 , 29 ]. However, the distribution ranges of relict species prior to fragmentation have not been explored, and the independent effects of climate and geography on relict patterns still need further study.

In fact, a topographic reversal has occurred in South China during the Cenozoic era, and the implications on biota of this phenomenon may have been previously overlooked. Before the late Eocene, the terrain of South China was high along the eastern coast of Cathaysia and low in the western Yangtze interior [ 30 , 31 , 32 ]. Several lacustrine sedimentary basin, such as the Danxia Basin, Nanxiong Basin, Heyuan Basin, and Guiping Basin, formed in the interior under hot and arid climates [ 26 , 33 ]. During the early Oligocene, the uplift of the SE Tibetan Plateau triggered a topographic reversal in the South China Block [ 34 , 35 , 36 , 37 , 38 ]. This period was also the key period for the transformation of the palaeo-Pearl River, which flowed from east to west, to the present Pearl River, which flows from west to east [ 39 , 40 ]. Although, the exact time frame during which the modern Pearl River formed, such as during the early Miocene [ 41 ], late Oligocene [ 39 ], or ∼  30 Ma [ 42 ], is debated. There is no doubt that a peneplain existed between the Cathaysian coast (current coastal mountains of South China) and the Yangtze interior between the Oligocene and Miocene [ 43 ]. This peneplain can still be observed as the present mountain planation surface in the Nanling Mountains, specifically at elevations of approximately 1000–1350 m in northern Guangdong [ 44 , 45 ] and 1416–1780 m in northern Guangxi and southern Hunan [ 46 ]. A large number of relict plants appeared in the high altitudes of the Nanling Mountains, e.g. Fokienia hodginsii [ 22 ], Bretschneidera sinensis [ 25 ]. The occurrence of this continuous relict flora is highly improbable to be attributed to random chance. Thus, we suspect that the peneplain stage between the Oligocene and Miocene in South China was important for establishing continuous warm East Asian relict flora.

Rehderodendron Hu (Styracaceae) is a typical relict genus, whose fossil fruits and pollen are widely known in Europe from the early Eocene to the Pliocene [ 47 , 48 , 49 , 50 , 51 ]. However, the modern species of Rehderodendron are restricted mostly to warm tropical and subtropical climates in the mountains of East Asia [ 52 ]. Our previous phylogenetic evidence indicated that R. kwangtungense Chun is the most ancient species within Rehderodendron (unpublished data). This fact is further demonstrated by the similarities between modern pollen and pollen discovered in the lower Eocene strata of England [ 48 ]. R. kwangtungense is sporadically distributed in fog-dependent forests in the subtropical mountains of southern China, exhibiting an island-like (near-mountaintop, mountain planation surface) distribution pattern (Fig.  1 a). This island-like distribution pattern is widely observed in East Asian relict species but remains poorly studied. R. kwangtungense is an ancient species with low dispersal capacity, making it an ideal species for tracing the spatiotemporal dynamic history of relict flora in East Asia.

Here, we examined the genetic diversity and population divergence of R. kwangtungense by employing RAD-seq data [ 53 ]. Then, we used ENM to identify the climatic niche of this species under current environmental conditions and to predict its potential distribution during the LGM and mid-Holocene. We combined this information to assess the formation of the modern distribution pattern of R. kwangtungense . Our specific objectives were to: (1) whether the distribution patterns of R. kwangtungense formed in the panplain stage in the Oligocene, (2) reveal the population structure and population demographic history, and (3) conservation genetics research on R. kwangtungense . This study is expected to provide new understanding of East Asian relict flora.

The studied species, sample collection and DNA extraction

Rehderodendron kwangtungense is a 5–15 m tall arbor with a diameter up to 30 cm at breast height. Its corolla is white, bell-shaped and fragrant (Fig.  1 b), and its fruits are cylindrical ellipsoid in shape (Fig.  1 c). The woody exocarp is approximately 1 mm thick, the fibrous corky mesocarp is 8–12 mm thick, and the woody endocarp is hard and radiates into the mesocarp (Fig.  1 d). The tillering ability of R. kwangtungense is relatively strong. On the one hand, when the trunk of a tree is broken, the base can branch and grow into a larger tree; On the other hand, after the main trunk falls down and touches the soil, it is easy to take root and clone many trees. During field investigation, we found two major distribution areas of R. kwangtungense with obvious phenological differences, one on the eastern margin of the Yunnan-Guizhou Plateau (northwestern populations, P1-7; Fig.  1 a) and the other in the Nanling and southern mountains (southeastern populations, P8-16; Fig.  1 a). In March and April, when the southeastern populations are blooming, the northwestern populations are still in the early bud stage.

Fresh leaves were collected from a total of 138 individuals (16 populations) of R. kwangtungense , covering most of its geographic range (Table  1 ; Fig.  1 a). The geographical information of the populations was recorded using a Garmin GPS unit (GPSMAP 62sc, Shanghai). Voucher specimens were deposited at the Herbarium of Sun Yat-sen University (SYS). The fresh leaves were dried and deposited with silica gel in sealed bags. Genomic DNA was isolated using the modified cetyl trimethylammonium bromide (CTAB) method [ 54 ].

Geographic distribution and morphology of R. kwangtungense . ( a ) The white dots indicate the 16 sampling sites in this study. ( b ) Flowering branch. ( c ) Fruiting branch. ( d ) Transverse section of a fruit

RAD-seq data processing

RAD-seq library preparation and Illumina sequencing were performed by Novogene Bioinformatics Technology Co. Ltd. (Tianjin, China). The standard protocol was followed. Stacks was developed to work with RAD-seq for the purpose of building genetic maps and conducting population genomics and phylogeographic analyses. Therefore, we processed the RAD-Seq data using Stacks v2.55 [ 55 ]. The program “process_radtags” was used to decomplex reads for each sample, and “denovo_map” was used to determine the optimum values for M (number of mismatches allowed between stacks within individuals) and n (number of mismatches allowed between stacks among individuals) with subset data containing 12 samples. Then, “denovo_map” was reused to process all 138 samples with the optimum M and n values (M =  n  = 4). Finally, the results were filtered using the program “populations”, in which polymorphic RAD loci that were present in at least 40% of the individuals across populations, had a minor allele frequency > 0.05 and had an observed heterozygosity < 0.7 were retained (-R 0.4 --min-maf 0.05 --max-obs-het 0.7). And for each locus, unlinked SNP were retained using the command “--write-single-snp”. To detect non-neutral loci under selection, Tajima’s D was calculated using VCFtools [ 56 ], in which the window size was set as 3,000 bp. Loci with Tajima’s D value < − 1.771 or > 2.086 were deemed as non-neutral [ 57 ]. The produced vcf files (Supplementary Material S1 ) were used for downstream analyses, and converted to other formats using VCFtools.

Genetic diversity and population structure analysis

The program “populations” of Stacks was used to calculate genetic diversity parameters ( H O , H E , π and F IS ) and pairwise fixation index ( F ST ) [ 55 ]. Population structure was examined using Admixture v1.3.0 [ 58 ]. The value of the hypothetical ancestral population (K) was set from 1 to 10, the optimal K was determined by the smallest cross-validation (CV) error value, and Q estimates were used as a proxy for ancestry fractions. Principal component analysis (PCA) was performed using Plink v1.90b to evaluate population genetic variation [ 59 ]. Analysis of molecular variance (AMOVA) was performed to estimate standardized genetic differentiation using Arlequin v3.5 [ 60 ], in which the 16 populations of R. kwangtungense were divided into two regions (northwestern and southeastern regions according to variation in flowering time), four regions according to PCA, and seven regions according to Admixture analysis. The python script “vcf2phylip.py” ( https://github.com/edgardomortiz/vcf2phylip ) was used to convert the vcf file to the phylip format, and the maximum likelihood phylogenetic tree was produced with IQ-TREE v2.2 [ 61 ] and plotted with iTOL v6 [ 62 ].

Population historical dynamics inference

The site frequency spectra (SFS) were generated using easySFS ( https://github.com/isaacovercast/easySFS ), and the value for projecting down samples was set to 112 to maximize the number of segregating sites. Stairway Plot v2.1.1 [ 63 ] was subsequently used to infer demographic history based on the folded SFS. Stairway Plot is based on SFS, which does not require whole-genome sequence data or a reference genome to infer recent population size changes [ 64 ]. The mutation rate of R. kwangtungense was set to 2.29 e-9 per site per year according to the estimate for walnut by Luo et al. [ 65 ]. The generation time was set to ten years, which was observed in the field investigation.

Shallow genome sequencing and plastid genome, ribosomal cistron assembly

For each population, we randomly selected one individual for shallow genome sequencing. In addition, 5 other species of Rehderodendron have undergone shallow sequencing and were utilized in the construction of the phylogenetic tree. The extracted DNA was sent to JieRui BioScience Co. Ltd. (Guangzhou, China) for library construction and Illumina sequencing on the Illumina 2000 platform. The program Novoplasty v2.7.2 [ 66 ] was used to assemble the chloroplast genome, using the R. macrocarpum chloroplast genome (GenBank accession MG719844) as a reference and its chloroplast rbcl gene sequence as a seed. Meanwhile, GetOrganellle v1.7.7 [ 67 ] was used to assemble the ribosomal cistron, using the Melliodendron xylocarpum ribosomal cistron (GenBank accession MF171073) as a seed.

Phylogenetic tree construction and divergence time estimation

According to Yan et al. [ 68 ], the plastid genomes of 6 related genera were downloaded from the NCBI nucleotide database (Supplementary Table 1 ). Together with the 16 newly assembled plastid genomes of R. kwangtungense and 5 other species of Rehderodendron in this study, 31 plastid genome sequences were aligned using MAFFT v7.508 [ 69 ]. Gaps and ambiguous bases (N) were removed using MEGA 7 [ 70 ]. A similar method was used to standardize ribosomal DNA data (Supplementary Table 2 ). The processed sequences were subsequently used to construct a phylogenetic tree, and the divergence times were inferred via Beast v1.8.4 [ 71 ]. The tree was calibrated with three fossils, Rehderodendron stonei (52 − 49 Ma) [ 49 ], Pterostyrax coronatus (33.9–28.1 Ma) [ 72 ] and Halesia reticulata (37.2–33.9 Ma) [ 73 ]. The lognormal prior distributions were enforced with a mean = 1.5, a Stdev = 2.0 and an offset set so that 95% of the distribution would fit the age of the upper (youngest) stratum from which each fossil was described. A GTR + I + F + G4 substitution model and lognormal relaxed-clock model were used to allow rate variation among branches. Additionally, a constant size was chosen as the tree prior. The analysis was run for 400 million Markov Chain Monte Carlo (MCMC) steps. MCMC samples were imported into TRACER v1.5 (available from http://beast.bio.ed.ac.uk/Tracer ) to inspect the sampling adequacy and convergence of the chains to a stationary distribution. The final plot was visualized using Figtree v1.4.4.

Ecological niche modelling

The ecological niche model (ENM) was used to predict the potential distribution of R. kwangtungense . In addition to our sample locations, geographic distributions of the species were also obtained from the Chinese virtual herbarium portal (CVH, https://www.cvh.ac.cn/ ). After filtering duplicate records and invalid records without a specific location, a total of 51 records were used in the following step. Climate layer information for 19 bioclimatic variables was downloaded from WorldClim [ 74 , 75 ] at a resolution of 2.5 arc-minutes. Based on geographical distribution data from R. kwangtungense and 19 climatic factors, a MaxEnt model was initially established to evaluate the contributions of different variables to the distribution of climate data [ 76 ]. To avoid overfitting of the simulation results due to the mutual influence between highly correlated climatic variables, Pearson correlation coefficient analysis was performed among the 19 climatic variables using SPSS v26.0. A pair of climatic variables with correlation coefficients greater than |0.8| was considered non-independent. Finally, for each pair of significantly correlated variables, only one variable with a large contribution was used for projection. After the selection procedure, 7 variables were retained for modelling the distribution of R. kwangtungense : the mean diurnal range (Bio2), isothermality (Bio3), minimum temperature of coldest month (Bio6), temperature annual range (Bio7), annual precipitation (Bio12), precipitation of driest quarter (Bio17), and precipitation of coldest quarter (Bio19). We employed MaxEnt 3.4.4 to simulate and predict the evolution of potentially suitable areas of R. kwangtungense in different periods, using default settings and specific parameters: random test (25%), training (75%), regularization multiplier (1), maximum iterations (5000), convergence threshold (0.00001), maximum background points (10,000), and 10 bootstrap replications. Model accuracy was gauged using the AUC of receiver operating characteristic (ROC). The output file was imported into ArcGIS 10.8, and the suitability was manually divided at 0.05 intervals, and areas with suitability values less than 0.1 were considered unsuitable areas.

Correlation of genetic structure with geographic isolation and environment factor

The isolation-by‐distance (IBD) reveals that genetic differentiation among populations increases with geographic distance, while the isolation‐by‐environment (IBE) assumes a linear relationship between genetic variation and environmental differences among populations [ 77 ]. To explore the existence of IBD and IBE, we applied Mantel test to assess the relationships between genetic distance and geographic or environmental distance with R v4.3.2 ( https://www.r-project.org/ ). The pairwise genetic distance among populations was calculated by F ST /(1 −  F ST ), the geographic distance matrix was transformed by the geographic coordinates of populations, and 7 climatic variables used in ENM were matrix transformed using PCA to obtain the environmental distance. A partial mantel test was conducted to control the influence of geographical and environmental distance, respectively.

RAD data processing and genetic diversity

A total of 11,190,163 loci were genotyped by the “denovo_map” program, and the mean, minimum and maximum values of effective per-sample coverage were 17.1×, 10.1× and 23.7×, respectively. After data filtration, a total of 1,943 SNPs remained for all subsequent analyses. The Tajima’s D values of all 1943 variant sites ranged from − 0.394 to 2.029, all sites are neutral. The observed heterozygosity ( H O ) for the 16 populations ranged from 0.022 to 0.078, with an average value of 0.048 ± 0.019; the expected heterozygosity ( H E ) ranged from 0.019 to 0.052, with an average value of 0.033 ± 0.011; and the inbreeding coefficient ( F IS ) was close to 0 (-0.0009 to 0.0005) (Table  1 ).

Population structure

Based on the results of the Admixture program, the CV error decreases and becomes stable at K = 7 (Supplementary Fig.  1 ). When K = 2, the populations of R. kwangtungense were divided into northwestern group (P1-7) and southeastern group (P8-16) (Fig.  2 a), which was consistent with phenological differences. When K = 7, the populations were subdivided gradually, with the northwestern cluster divided into four groups (G1-4) and the southeastern cluster divided into three groups (G5-7) (Fig.  2 b).

Delimitation of 16 populations of R. kwangtungense based on Admixture analysis. ( a ) Genetic structure of R. kwangtungense based on K  = 2. ( b ) Genetic structure of R. kwangtungense based on K  = 7. Each individual (indicated as columns along the X-axis) is probabilistically assigned (probability of assignment q on the Y-axis) to one of the inferred genetic clusters

PCA showed that the contribution rates of the first and second principal components were 21.57% and 16.64%, respectively. The two-dimensional plot of PC1 and PC2 divided all the 138 individuals into four large clusters (Fig.  3 a): the seven populations located in the northwestern area were ascribed to one cluster, including the four groups G1-G4 according to Admixture analysis (Fig.  2 b), while the nine populations in the southeastern area were divided into three clusters, corresponding to the three groups G5-G7 according to Admixture analysis (Fig.  2 b). The unrooted phylogenetic tree based on population data also confirmed that individuals of most group (G1-G6) clustered into a monophyletic group with a high support value (Fig.  3 b). All terminal branches are very short in length (Fig.  3 b), indicating little genetic variation within the population.

Genetic and phylogenetic analysis of R. kwangtungense . ( a ) Two-dimensional clustering of individuals obtained via principal component analysis. ( b ) Phylogenetic tree constructed from RAD-seq data

AMOVA showed that the genetic variation among regions increased from 27.41 to 59.72% and that the variation among populations within groups decreased from 60.41 to 27.16% as the K value increased from 2 to 7, and the variation within populations remained stable (12.18-13.12%, Table  2 ).

IBD and IBE

The mantel test showed that genetic distance [ F ST /(1– F ST )] was significantly correlated with geographical distance and conformed to an isolation-by-distance pattern (R 2  = 0.38, P  < 0.001, Fig.  4 a). Meanwhile, environmental factors also affected the genetic variation (R 2  = 0.21, P  < 0.001, Fig.  4 b). The partial mantel test showed that after controlling the influence of environment matrix, geographical distance and genetic distance were still related (R 2  = 0.24, P  < 0.001). However, after controlling for the influence of geographical matrix, there was no correlation between environmental factors and genetic distance (R 2  = 0.018, P  = 0.881). Therefore, geographical isolation rather than environmental factors was the main factor that caused the genetic differences of R. kwangtungense populations.

Isolation-by-distance (IBD) and Isolation-by-environment (IBE) patterns for populations of Rehderodendron kwangtungense . ( a ) Mantel test result of genetic distance and geographical. ( b ) Mantel test result of genetic distance and environment distance. Each dot represents a pair of populations

Population demographic history

A demographic history investigation showed that the effective population size of R. kwangtungense increased quickly from approximately 16,000 years ago (YA) to 10,000 YA and then remained stable above 2000 until 8,00 YA. It subsequently gradually declined to the very low level observed at present (approximately 200) (Fig.  5 ).

Estimates of the effective population size of R. kwangtungense

Divergence history of populations inferred by phylogenetic tree

The plastid and ribosomal cistron phylogenetic tree yielded similar topologies and intra-genus divergence time. However, ribosomal phylogenetic tree showed lower posterior probability and larger time estimation range (Supplementary Fig.  2 ), and ribosomal DNA has limited credibility as one of the nuclear genes. Thus, we mainly referred to the plastid phylogenetic tree. Furthermore, the inclusion of other species within Rehderodendron genus exerts minimal impact on the estimation of populations divergence time, given that R. kwangtungense represents the most ancestral clade (Supplementary Fig.  3 ). Therefore, only the phylogenetic tree constructed from plastid genomes excluding other Rehderodendron species was presented (Fig.  6 a and b), which showed that existing R. kwangtungense populations differentiated at ca. 26.25 Ma. In addition, populations P14, P15, P16 and P8 were placed on the root of the tree; these populations diverged from the remaining populations at ca. 22–23 Ma. The populations P1-P7 formed a monophyletic clade, and the crown age was estimated to be ca. 12.92 Ma. Additionally, populations P9-P13 formed another monophyletic clade that diverged only recently (ca. 1.73 Ma).

( a ) The presumed migration path of R. kwangtungense . ( b ) Phylogenetic tree and divergent time estimation (below the bars) of R. kwangtungense based on the plastid genome. The fossil points are marked with triangles. Maximum likelihood standard bootstrap support values (BS) and Bayesian posterior probabilities (PP) are shown above the bars (*: BS ≥ 95% or PP ≥ 0.99). The median ages of the nodes are shown below the branches (those younger than 1 Ma are not displayed), with blue bars indicating the 95% highest posterior density intervals

Suitable distribution based on ENM

Among the 7 bioclimatic variables selected for ENM simulation, the bioclimatic variables with greater contribution percent are Bio17, Bio7, Bio6; the bioclimatic variables with greater permutation importance are Bio7, Bio2, Bio6; and the bioclimatic variables with greater regularized training gain of the jackknife test are Bio2, Bio17, Bio19 (Supplementary Tables 3 & Supplementary Fig.  4 ). In conclusion, the bioclimatic variables that dominate the potential geographical distribution of R. kwangtungense are mean diurnal range (bio2), min temperature of coldest month (Bio6), temperature annual range (Bio7) and precipitation of driest quarter (Bio17). The suitable areas for R. kwangtungense were few and scattered in southern China during the LGM (5.01 km 2 , Fig.  7 a), increased obviously at ca. 6000 YA (26.10 km 2 , Fig.  7 b), and reached a maximum at present (43.51 km 2 , Fig.  7 c). The predicted areas at ca. 6000 YA and at present are larger than the actual distribution area of the species, mostly in southeastern China.

Presence records (dark circles) and predicted distribution probability according to climatic conditions during the ( a ) last glacial maximum (22 kya), ( b ) mid-Holocene (6 kya), and ( c ) current (0 kya)

Origin and early dispersal of R. kwangtungense

Although there have been some phylogenetic studies of family Styracaceae, our study is the first to infer the internal divergence time of family Styracaceae, so there is no intra-family divergence time reference. Based on large-scale phylogenetic tree, Rose et al. [ 78 ] showed that the divergence time between the family Styracaceae and the family Diapensiaceae was about 93.2 Ma, and Li et al. [ 79 ] showed that the formation time of Ericales was between 103 and 107 Ma, which is close to our estimated time. Rehderodendron is suggested to be a member of “boreotropical flora” [ 48 ] because of the existence of a continuous zone of maritime influenced vegetation along the Tethys during the Cenozoic [ 80 ]. Our phylogenetic results showed that populations P14 and P15, which are located in the coastal mountains of Southeast China, harbor the most ancient plastid genomes (Fig.  6 b). These findings suggest that the ancestor species of R. kwangtungense may have initially spread to the Cathaysian coast through the lowland region along the Tethys Ocean during the early Eocene, when the Tibetan Plateau had not yet fully uplifted [ 34 ]. This early diffusion event is supported by the similarity of R. kwangtungense pollen and pollen from the early Eocene [ 48 ].

Most of the eastern populations (P8, P14, P15, P16) and early clades (P1-P7, P9-P11) of R. kwangtungense quickly diverged between 26.25 Ma and 21.48 Ma (Fig.  6 b). This result implies that the modern distribution area of R. kwangtungense was already established before the early Miocene.

Field observation revealed that the fruits of R. kwangtungense are large and cylindrical with thick spongy mesocarps (Fig.  1 c-d), and long-distance transport by animals is difficult (only rodents eat them). Thus, water and gravity are the main forces driving lateral fruit distribution [ 48 ]. The Mantel test also confirmed the weak dispersal ability of R. kwangtungense (Fig.  4 ). Therefore, the fruits of R. kwangtungense do not have the ability to spread between mountains in different river basins. The current island-like distribution pattern most likely formed before mountain uplift and erosion. Palynological evidence also suggests that the genus Rehderodendron seems to be more adapted to braided river system transmission [ 80 ]. The existence of a panplain stage between the Oligocene and Miocene with many nondirectional water systems [ 39 ], seems to be the most plausible explanation for the early dispersal of R. kwangtungense .

Differentiation of R. kwangtungense since the establishment of the east Asian monsoon

The results of population structure analysis suggest that R. kwangtungense populations should be divided into seven groups (Fig.  2 ). Group 1–4 are located on the eastern side of the Yunnan-Guizhou Plateau on the second level of the terrain ladder in China, and the flowering period of these populations is late. Group 5–7 are located in the Nanling Mountains and the southern region on the third level of the terrain ladder, and the flowering period of these populations is early. The phylogenetic tree shows that the crown age of extant R. kwangtungense was estimated to be 26.25 Ma, and this species underwent a period of rapid populations divergence at 23 − 21 Ma (Fig.  6 b), coinciding with the rapid rise of the Himalayas and subsequent establishment of the East Asian monsoon. Ding et al. proposed that the Himalayas experienced a rapid increase in elevation to 4000 m from 23 to 19 Ma [ 81 ], while the Yunnan-Guizhou Plateau was uplifted at a much greater rate than the Nanling Mountains, thus forming the three-step ladder of China’s topography and restructuring the monsoon circulation system. Sun & Wang also suggested that the topographic inversion that occurred at ca. 25 Ma, resulted in the formation of the East Asian monsoon, which had a large impact on the dry and wet patterns in China [ 31 ]. The geographical isolation caused by geological movements hindered gene flow, and monsoonal rains intensified mountain erosion, which together promoted the differentiation of R. kwangtungense into two groups from 26 − 21 Ma. Moreover, a differentiated population structure between the Yunnan-Guizhou Plateau and South China is common among relict species, such as Bretschneidera sinensis [ 25 ], Eomecon chionantha [ 82 ] and Eurycorymbus cavaleriei [ 24 ].

Subsequently, continuous geographical processes have led to the formation of more complex mountain systems, and several phases of intensification in the Asian monsoon have led to climate change [ 83 ]. The R. kwangtungense in the Yunnan-Guizhou Plateau has been continuously differentiated since 12.69 Ma. Similar divergence processes were also observed in Cyclocarya paliurus and Eriobotrya during this period [ 84 , 85 ].

Effects of Quaternary glaciation on the R. kwangtungense and implications for conservation

Southern China, which has numerous mountains and valleys, provides long-term stable habitats for many species, allowing them to maintain in situ, and harbours a great abundance of Tertiary relict flora [ 18 , 19 ]. Under the influence of repeated ice ages in the Quaternary, the population of R. kwangtungense contracted dramatically and took refuge in mountainous areas. The geographic isolation caused by population contraction restricted gene flow, which combined with a changing climate, facilitated the second phase of rapid divergence in P1-2, P4-6 and P9-13. Similarly, the lineage differentiation has also occurred in Cercdiphyllum japonicum , Kalopanax septemlobus and other species [ 86 , 87 ].

Based on ENM and historical dynamic analysis, when the LGM occurred, the area suitable for R. kwangtungense decreased sharply, and the effective population size reached an extremely low level, indicating that R. kwangtungense was not adapted to the cold climate and that a large number of populations died out, with only a small minority taking refuge in the mountains. As the climate became warm and humid after the LGM, the suitable habitat area for R. kwangtungense continued to expand, and the historical dynamic simulation confirmed that the population size increased simultaneously, reaching a peak at approximately 10,000 YA (Figs.  5 and 7 ). However, since the modern landscape had been basically formed at this point, the dispersal of R. kwangtungense was limited, and the species only expanded in situ, forming an island-like distribution pattern. AMOVA indicated that genetic variation within populations was minimal (Table  2 ), as was that of H E and H O in various groups (Table  1 ), and the phylogenetic tree constructed from RAD data showed a very short branch length for all individuals (Fig.  3 b). This evidence points to the limited genetic diversity of R. kwangtungense populations, and a population may be the result of the cloning of a few individuals. Thus, we surmised that the dispersal type of fruits and pollination mode determined whether population expansion occurred after the ice age. Similarly, Cathaya argyrophylla did not experience long-distance dispersal or population expansion after the glacial period [ 23 ]. In contrast, Cyclocarya paliurus with samara experienced substantial spatial expansion [ 85 ].

As temperatures and precipitation become more favorable, the effective population size has shrunk over the past 1,000 years. The main reason for the inconsistency may be the weak diffusion ability and competition ability of R. kwangtungense . According to the field observation, the genus Rehderodendron can quickly grow many small trees after the forest gap period, while in the original forest with good vegetation, the seedlings are basically invisible (because the fruit will soon become mildew if it is not dried in time). In other words, we assume that the suitable habitat for R. kwangtungense is the plain, with rivers and flat land, and the current mountain habitat is not suitable for the diffusion and competition of R. kwangtungense. The ENM result is mainly based on temperature and precipitation, but in reality, temperature and precipitation are not the only limiting factors.

Another reason for the reduction in effective population size could be human activities. To our knowledge, many populations are currently located in natural reserves where the species may be well protected (P8, P10 and P13), although in some regions, such as P5, the species are susceptible to the impacts of nearby villages. The fragile genetic diversity of the populations suggests that all populations should be protected, and priority should be given to representative populations in each group. Field observations have shown that seedlings are rare, possibly due to failure to compete with other species, while manual cultivation has shown that successful germination of seeds can be as high as 80% [ 88 ]. Therefore, artificial breeding and other measures can be adopted for protection.

The understanding of the origin of the contemporary distribution pattern of relict plants is crucial for the scientific conservation of rare and endangered plant species. In this study, we revealed the population differentiation history of the relict species Rehderodendron kwangtungense Chun during the Cenozoic. The modern distribution area of R. kwangtungense was already established during the Oligocene-Miocene transition period, and subsequently underwent fragmentation in Miocene as a result of allometric plate uplift and mountain erosion. During the Quaternary glacial period, R. kwangtungense experienced a significant population decline and sought refuge in situ. The findings of our study emphasize the predominant role of geographical processes in shaping relict plant distribution patterns, while the impact of climate fluctuations primarily manifests through their influence on local population size. This means that scenario of population expansion and contraction under climate oscillations may be overestimated, especially for those species with low dispersal capacity. Furthermore, we propose a perspective on the existence of a panplain in south China during the Oligocene to Miocene epochs, which has significant implications for understanding the formation process of relict flora in East Asia.

Data availability

The RAD-seq data has been uploaded to NCBI ( https://www.ncbi.nlm.nih.gov/ ) Sequence Read Archive under the BioProject PRJNA1056300. The assembled chloroplast genomes data has been uploaded to NCBI GenBank under the accessions PP048903-PP048918, PP761266-PP761270. And the ribosomal DNA data has been uploaded to NCBI GenBank under the accessions PP765768-PP765788.

Abbreviations

Restriction-site associated DNA sequencing

Last glacial maximum

Observed heterozygosity

Expected heterozygosity

Nucleotide diversity

Inbreeding coefficient

Fixation index

Hypothetical ancestral population

Cross-validation

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Acknowledgements

We thank Fan Ye, Su-Juan Wei for their help in the field work.

This work was supported by National Natural Science Foundation of China (32300172), Natural Science Foundation of Guangdong Province (2021A1515110425), Guangdong Special Fund for Natural Resources Management and Ecological Forestry Construction (2021GJGY001), Guangdong Province Ecological Forestry Development Project, and the project of the Research on Coevolution of Vegetation and Geological Environment of Shenzhen Dapeng Peninsula Geopark (2020F36).

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Jiehao Jin and Wanyi Zhao contributed equally to this work.

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State Key Laboratory of Biocontrol and Guangdong Provincial Key Laboratory of Plant Resources, School of Life Sciences, Sun Yat-sen University, Guangzhou, 510275, China

Jiehao Jin, Wanyi Zhao, Sufang Chen, Zhihui Chen, Zhongcheng Liu, Wenbo Liao & Qiang Fan

Shenzhen Dapeng Peninsula National Geopark, Shenzhen, 518121, China

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Jiehao Jin: study design, fieldwork, data analysis, writing; Wanyi Zhao: study design, fieldwork, writing; Sufang Chen: data analysis, writing; Chao Gu, Zhihui Chen, Zhongcheng Liu: fieldwork; Wenbo Liao, Qiang Fan: study design.All authors read and approved the final manuscript.

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This study did not involve any human tissue materials or animal tissue materials. It did not require ethical approval. We declare that all plant materials used in this study were collected and sequenced in accordance with relevant institutional, national, and international guidelines and legislation. Relevant permits, permissions, and licenses were obtained prior to the collection and use of plant materials. All steps in the study were performed in accordance with the relevant guidelines and regulations. W.Z. undertook the formal identification of the plant material used in this study. Voucher specimens were deposited at the Herbarium of Sun Yat-sen University (SYS) under the voucher numbers ZWY-1728 (population NKS), ZWY-1762 (population DYS), ZWY-1831 (population SSP), ZWY-1850 (population SD), ZWY-1868 (population SHS) and ZWY-1900 (population TJS).

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Jin, J., Zhao, W., Chen, S. et al. Which contributes more to the relict flora distribution pattern in East Asia, geographical processes or climate change? New evidence from the phylogeography of Rehderodendron kwangtungense . BMC Plant Biol 24 , 459 (2024). https://doi.org/10.1186/s12870-024-05181-7

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With Donald Trump’s “hush money” trial heading into final arguments, it’s the perfect time to have Katelyn Polantz , CNN’s Senior Reporter for Crime and Justice, give a full review of the many trials of the former president.

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Middle school biology - NGSS

Welcome to middle school biology, unit 1: cells and organisms, unit 2: organism growth and reproduction, unit 3: matter and energy in organisms, unit 4: interactions in ecosystems, unit 5: matter and energy in ecosystems, unit 6: ecosystems and biodiversity, unit 7: inheritance and variation, unit 8: evolution, unit 9: natural and artificial selection, unit 10: hands-on biology activities.

RSC Chemical Biology

Protac chemical probes for histone deacetylase enzymes.

* Corresponding authors

a Leicester Institute of Structural and Chemical Biology, School of Chemistry, University of Leicester, Leicester, UK E-mail: [email protected]

Over the past three decades, we have witnessed the progression of small molecule chemical probes designed to inhibit the catalytic active site of histone deacetylase (HDAC) enzymes into FDA approved drugs. However, it is only in the past five years we have witnessed the emergence of proteolysis targeting chimeras (PROTACs) capable of promoting the proteasome mediated degradation of HDACs. This is a field still in its infancy, however given the current progress of PROTACs in clinical trials and the fact that FDA approved HDAC drugs are already in the clinic, there is significant potential in developing PROTACs to target HDACs as therapeutics. Beyond therapeutics, PROTACs also serve important applications as chemical probes to interrogate fundamental biology related to HDACs via their unique degradation mode of action. In this review, we highlight some of the key findings to date in the discovery of PROTACs targeting HDACs by HDAC class and HDAC isoenzyme, current gaps in PROTACs to target HDACs and future outlooks.

• This article is part of the themed collection: Medicinal Chemistry Small Molecule Probes

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U. Patel, J. P. Smalley and J. T. Hodgkinson, RSC Chem. Biol. , 2023,  4 , 623 DOI: 10.1039/D3CB00105A

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