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Replication, pseudoreplication and model experiment in the study of population genetics

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The paper is dedicated to the problem of true and pseudoreplication of a biological experiment, in particular in the educational process. It was found that this issue is relatively new and actual for the methodology of biological experiments in general. Its solution in science ensures the veracity of the results obtained and the relevancy of the formulated conclusions. In biology teaching methods at school, the problem of true and pseudoreplication of the experiment was not reflected. The author covers an issue of true replication teaching when setting up a model experiment to study genetic-evolutionary processes in populations. The paper discloses the experience in evolution of a model experiment and its development aimed at formation of ideas about technical and biological replication by the example of study of the genetic structure of an ideal population in generations. For this purpose, there was developed a web page that allows to automatically implement technical and biological experiment replication. There was described an experience of approbation of the proposed variant of the experiment, and its difficulties and advantages were revealed.
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Replication, pseudoreplication and model experiment in the study of
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Journal of Physics: Conference Series 1840 (2021) 012010
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doi:10.1088/1742-6596/1840/1/012010
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Replication, pseudoreplication and model experiment in the
study of population genetics
E V Komarova
Immanuel Kant Baltic Federal University, 14 Aleksandra Nevskogo Str., Kaliningrad,
236041, Russia
E-mail: komarova1978@mail.ru
Abstract. The paper is dedicated to the problem of true and pseudoreplication of a
biological experiment, in particular in the educational process. It was found that this
issue is relatively new and actual for the methodology of biological experiments in
general. Its solution in science ensures the veracity of the results obtained and the
relevancy of the formulated conclusions. In biology teaching methods at school, the
problem of true and pseudoreplication of the experiment was not reflected. The author
covers an issue of true replication teaching when setting up a model experiment to study
genetic-evolutionary processes in populations. The paper discloses the experience in
evolution of a model experiment and its development aimed at formation of ideas about
technical and biological replication by the example of study of the genetic structure of
an ideal population in generations. For this purpose, there was developed a web page
that allows to automatically implement technical and biological experiment replication.
There was described an experience of approbation of the proposed variant of the
experiment, and its difficulties and advantages were revealed.
1. Introduction
The educational subject “Biology” is a didactically adapted system of scientific biological knowledge.
As came about from Aristotle times, the natural sciences, and the biology is undoubtedly one of them,
have an experiment as one of the main research methods. It is this that allows, on the basis of the various
factual material obtained, to make wide generalizations, to proceed to the establishment of connections,
patterns that allow deeper penetration into the essence of the phenomena under study. A lot has already
been said about the experiment in biological science, about its types, methods, requirements for
organization, limitations and difficulties of application. A huge number of scientific works are dedicated
to the issues of experimental method history in biology. We, however, were interested and are interested,
at this point in time, in the experimental method from the point of view of the possibilities of its use in
biological education. In addition, having narrowed the subject field we are interested in, it is worth
pointing out that a model experiment occupies a special place in high school. It allows to create models
of real objects and to prototype the processes occurring with them in reality. In previous works, the
author highlighted some aspects of this issue [16].
In the process of studying the topic of the display of experimental method at the level of school
biological education, transformation of ideas about how it is possible to implement the experimentation
with complex biological systems, including those inaccessible to the student for direct study, we initially
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started out from the following. An educational biological experiment should maximally meet the
requirements that are put forward for scientific biological experimentation. These, in particular, are the
reliability in essence, the rule of single difference, replication, mass nature. From the mid-20th century,
a lot of attention has been paid to the organization of a school biological experiment, moreover to its
various types, differing both in the object of research (botanical, zoological, physiological tests,
functional tests, etc.), and in the form of carrying out under the conditions of school laboratory, class
(demonstrational, laboratorial, mental) [3], [4], [5], [6], [8], [11], [24], [25], [29]. Regardless of the type
and form of carrying out, all various types of educational biological experiment must meet the
abovementioned requirements in order that the results obtained were maximum consistent.
The biggest difficulties in the educational process are caused by the observance of such requirements
as replication and mass nature. In other words, to ensure the veracity of results, the educational
experiment should be conducted several times using a sufficiently large number of objects.
It is difficult to implement both the first and the second condition in the educational process due to
the following reasons:
– firstly, the temporal limitations of the educational process;
– secondly, due to the inaccessibility of objects for study in the required quantity;
– thirdly, in the principle of inaccessibility of some objects and processes for direct study, primarily
due to their objective specificity: either too small (organic molecules, cells, viral particles), or too large
(populations).
Let's turn our attention to these reasons, possible ways of their elimination.
Analysis of scientific literature in regards to the experimentation in biology showed that since the
end of the 1980s, one of the actively discussed problems became the problem of pseudoreplication in
ecological and biological research. In the classical variation, from the moment of publication of the first
paper on this topic, pseudoreplication was considered as a negative experimental practice [13]. Even
now, one of the criteria by which reviewers evaluate the submitted paper for a journal indexed in the
authoritative international scientometrical Scopus and Web of Science databases is the true and
pseudoreplication of the experiments conducted [7]. Please note that at the moment the scientific
community is still not so categorical in regards to pseudoreplication of experimental research.
Discussions are being conducted on the issue of reality and contrivedness of the problem [18], [22],
[23].
We proceed from the assumption that the biology teaching methods cannot stay on the sidelines of
the problems actively discussed in biological science. Moreover, this question lies in the plane of the
science methodology. The mastery of methodological knowledge and the ability to apply them is the
basis for the formation of a system of biological knowledge for senior high school students. The author's
early works were devoted to this question [15]. So, we consider the question of to what extent in school
experimentation in biology it is necessary to take into account the requirements that Stuart H. Hurlbert
identified as the problem of pseudoreplication of experimental research in science, as definitively
solvable in the direction of their observance. At the same time, given that the educational subject still
differs from the basic science in that it is a didactically adapted version of it, it is necessary to achieve a
double effect in organization of a school biological experiment. The first effect is that the results of the
educational experiment should be maximum consistent, obtained by true replication. The second effect
is that the use of true replication should be maximum ergonomical. Ergonomic in time, cost and
complexity.
The purpose of this paper is to demonstrate the capabilities of the school model experiment in study
of the genetic structure of populations in time while meeting the requirements of true technical and
biological replication of experimental objects. Or, in another way: to consider the possibilities of solving
the problem of pseudoreplication in the school biological experiment aimed at study of the genetic-
evolutionary processes in populations.
We consider it logical to state the essence of the declared problem in the sequence of answers to the
following questions: what is the essence of replication in biological research? What kind of experimental
replication occurs, what goals does it pursue? What is the essence of pseudoreplication in biological
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research, what is the history of the problem? Is pseudoreplication as scary as it might seem? How to
ensure true replication in a model experiment by studying complex ideal and real biological objects in
school biology (by the example of genetic structure of the population)?
The answers to the set of the abovementioned questions make sense if we answer one of the most
important questions: “Why should we conduct at school a model experiment in study of the genetic
structure of the population?” It can be concretized in the following way: what is the purpose of this
experiment, if it is possible manage without it, to replace it? What fundamental knowledge and skills do
pupils acquire when performing this experiment? Why the model experiment in study of population
genetics is considered by us as the most important instrument for the formation of not only genetic-
evolutionary concepts, but also of metasubject skills?
2. Technique and methods
2.1. Main issue. Why should we conduct at school a model experiment in study of the genetic
structure of the population?
Study of the issue of model experimentation with the genetic structure of the population during 2015-
2020 convinced us that its goal is the obtention by the pupils of a direct subject and mediated activity
result. Subject result – 1) mastery of the essence of the law of genetic balance and the conditions under
which it is consistent; 2) understanding of the mechanism of influence of evolutionary factors, such as
natural selection, gene drift, gene flow, mutation process on the genetic structure of the population;
understanding of the mechanisms forming the basis of micro- and macroevolutionary processes. The
fundamental significance of the law of genetic equilibrium (Hardy-Weinberg) is that it is the central law
of population genetics, it is based on the application of statistical methods in genetics [10].
Activity result – mastery by pupils of the method of the result achievement maximally approximate
to the consistent value. In other words, this refers to the formation of a metasubject ability to plan and
set up an experiment methodologically correctly, to collect data, to process them and to formulate
reasonable conclusions.
Before the development of methods of the model experiment in study of the genetic structure of the
population, we posed two questions:
1. Is it necessary to conduct a model experiment when studying the law of genetic equilibrium and
the conditions for its consistency?
2. Can a model experiment be replaced with other educational methods?
The answer to the first question is: no, not necessarily. It is possible to limit to the demonstration of
the multimedia presentation and video on this topic.
The answer to the second question is: yes, it is possible. An alternative is familiarization with
theoretical material on a printed basis about the factors of change in the genetic structure of a population,
overlearning of Hardy-Weinberg equations, teaching of the solution of problems on determination of
the genetic structure of a population.
The answers to both questions demonstrate that in the alternative version, at the best, only one result
will be achieved – a subject one. Without performing experimental actions, it is extremely difficult to
form such elements of methodological knowledge as a variant of experience, replication, sampling. In
addition, it has to be considered that the law of genetic equilibrium is a law, the substantial part of which
consists of abstract categories not attached to a specific biological object (abstract homozygotes and
heterozygotes, dominant and recessive alleles, conditions for the veracity of the law). And the law itself
is applicable to some really non-existent ideal object, or, conversely, is not applicable to any really
existing object (real population).
The abovementioned reasons are the answer for us to the main question, namely: 1) model
experimentation contributes to the mastery by the pupils of abstract biological categories on concrete
material objects; 2) allows to visualize the processes in an ideal population non-existent in reality;
3) allows to simulate the changes taking place in real populations over several generations. Thus, for
educational purposes, the time frame of the actually occurring processes is condensed; 3) allows to vary
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the replications and variants of the experiment with minimal material costs; 4) allows to teach true
replicates (replications) of the experimental impact; 5) allows to artificially quickly change the
conditions (factors) affecting the population, including acting stochastically [20].
2.2. Issue No. 2. What is the essence of replication in biological research?
A person possessing a basic level of biological knowledge within the scope of the school curriculum of
complete secondary education, the term “replication” is known as a process related to the molecular
level of organization of a living being. In the English-language scientific biological literature, the term
“replication” is used not only in the meaning of the synthesis of new nucleotide sequences, but also in
the meaning of the replicate of experimental attempts. In other words, the principle of replication in
experiment is the well-known principle of replication. The last term is more widely used in domestic
scientific works.
Replication in biological research can be technical and biological [26].
Technical replicates give us these things:
1. They give us an accurate measurement they give this particular object.
2. If we want to tell more about this object or we do not want to generalize the data and transfer it to
the population – a technical experiment is what we need.
3. They will also tell us how accurately we performed the measurements.
4. “If we wanted to publish a paper about how awesome our new method is, we’d use technical
replicates” [26].
5. If the experimental technique is transformed, different samples are taken simultaneously from one
object, then technical replication will also take place, since they tell us about an individual.
In the biological replicates each measurement comes from different sample that comes from different
objects.
Biological replicates give us these things:
1. Biological replicates tell us about a trait that occurs in a group. In biological replicates, each
measurement comes from different samples or is obtained differently from one object.
2. You can mix biological and technical replicates, but the wisdom of doing this depends on the type
of the experiment. Sometimes you get more bang for your buck if you add more biological replicates
and ignore technical replicates.
So, the difference between technical and biological replication is as follows:
a) technical replicates are just repetition of the same experiment on the same person;
b) biological replicates use different biological sources of samples (i.e. different people, different
plants, and different cell lines) [26].
When choosing the type of replication of a biological experiment, it is necessary to proceed from the
purpose in view. If it is planned to describe a specific object, whether it be an individual, a population,
or to research a method, it is necessary to use technical replication. If the goal is to study a group of
objects, it is necessary to choose biological replication.
2.3. Issue No. 3. What is the history of the problem of pseudoreplication in biological research?
The problem of pseudoreplication was raised for the first time in 1984 by Stuart H. Hurlbert, who
published a critical analysis of 156 experimental scientific papers in English-language editions
published in 1960-1980. He came to the conclusion that in 27% of cases there was one of two variants:
1) the experimental influence was applied in one replication; 2) the experimental replications were not
statistically independent. Such errors were called pseudoreplication by Stuart H. Hurlbert. Mikhail V.
Kozlov notes that in Russian academic journals in 1998-2001 the part of papers based on
pseudoreplication turned out to be twice as high (47%) than in the English-language periodicals for
1960-1980, i.e. before the publication of Stuart H. Hurlbert’s paper. This situation was considered as
non-normal, at the same time it was pointed out that the reason for the pseudoreplication lies not only
in errors in experiment planning, but also in the incorrect application of statistical analysis to the results
of a well-planned experiment [17].
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After the publication of Stuart H. Hurlbert 's paper in 1984 during the period from 1987 to 2001,
according to Mikhail V. Kozlov: 1) the term “pseudoreplication” firmly came into the ecological
scientific lexicon of foreign authors, the problem of pseudoreplication in foreign ecological studies is
actively discussed; 2) the number of foreign publications based on pseudoreplication began to decrease.
Back in 2003, Mikhail V. Kozlov paid attention to the fact that the concept of pseudoreplication is
completely unknown to the overwhelming majority of Russian ecologists. In addition, the author
emphasized that Stuart H. Hurlbert's work was never cited in Russian-language periodicals, against the
background of more than 2000 references (2015 references as of 2001) in English-language publications.
Mikhail V. Kozlov repeatedly published his works on standing up for the position that the problem of
pseudoreplication is a problem of the world scientific community, which should be treated with all
possible seriousness [17], [18].
The English term “pseudoreplication” does not have a direct analogue in Russian, since it primarily
denotes a process – an erroneous choice of replicates for assessment of intragroup variability in
statistical analysis [13], [18]. In this regard, direct translation of terminology is difficult enough; the
authors provide English equivalents of key concepts. “In medical experiments, where they are
designated to as “spurious replication”, “trial inflation”, or “the unit of analysis problem or error” [1],
[2], [30]. Although the concept of “pseudoreplication”, which is most adequately translated as
“statistical analysis based on pseudoreplication”, is not found in all works listed above, and we do not
agree with all the conclusions of the indicated authors, all the cited studies are united by a serious
approach to the problem” [18].
2.4. Issue No. 4. Is pseudoreplication as scary as it might seem?
In Russian-language sources, the attitude to the problem specified by Stuart H. Hurlbert and supported
by Mikhail V. Kozlov can be characterized as far-fetched and already well-known and studied (Vasilii
V. Nalimov, Alexander A. Lyubishchev, Aleksandr I. Bakanov, Nikolai A. Plokhinskii, Tatiana I.
Golikova). The Russian-speaking authors agree that there are two indisputable theses in the ideas of
Stuart H. Hurlbert:
1) “it is not always correctly to extend the conclusions, obtained in the study of private samplings, to
the entire general population;
2) assessment of the degree of factor influence may turn out to be erroneous if the studied effect is
not properly localized, and the compared data are taken from insufficiently randomized sources” [22].
The conducted analysis of literary sources [21], [9], [27], [28], [14], [19], [12] on the problem
allowed us to single out the “pros” and “cons” of the consideration of the problem of pseudoreplication
as significant for biological research. The analysis results are presented in table 1.
2.5. Issue No. 5. How to ensure true replication in a model experiment in school biology?
In previously published materials [15], [16], we described the method of model experimentation
developed for senior high school students to study the supraorganismal levels of life organization,
namely, population-specific.
The development of a model experiment methodology aimed at the study of the essence of genetic-
evolutionary changes in the population by pupils, and its improvement during 2015-2020, was carried
out by us in a staged manner. This was dictated by the objective and subjective difficulties of
implementation of a model experiment into teaching practice.
At the first stage, we used only material models of gene alleles, created models of genotypes in a
manual way, and, respectively, models of parental and daughter populations in generations.
Mathematical calculations were performed without the use of a computer, the participants in the
experiment manually calculated the frequencies of genotypes and alleles in populations, and presented
the results obtained in the graphical representation.
At the second stage, we combined material modelling and use of the computer. Work with material
models consisted of carrying out of the experiment itself, creation of a model of the parental population
in manual way, and combination of the gene alleles at random (this is how panmixia was simulated).
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The participants entered the results of the experiments into a table on the developed web pages. With
the help of a computer, the obtained frequencies of alleles and genotypes were automatically calculated.
In automatic mode, the results of the experiment were optionally presented in the graphical
representation.
Table 1. Pseudoreplication – a real problem in biological research.
Pro
arguments
arguments
1. Each object in the sampling is a functional part of the
whole, and not a separate element of a set. In a number of
studies, the results and conclusions obtained for discrete
objects apply to the entire population, which does not
correspond to one of the requirements for biological
experimentation – consistency in essence.
2. During the experiment, there is a multiple
determination of reaction of the same organism in the
course of sampled counts. As an alternative, the same
sampling is studied in different time intervals. In this case,
living objects (their populations) are pseudoreplications.
3. Two main problems of pseudoreplication are an
insufficient mass nature of experimental objects and their
initial incomparability with each other. In the first case,
the researcher receives insufficient data for the consistent
statistical result. In the second case, the problem has an
objective causality due to the initial uniqueness of living
objects.
1. Each object in the sampling is discrete and
individual.
2. Factors acting independently on the sampling,
act on a set of separate biological objects, and not
on an integral object. The specificity of a biological
experiment lies in the uniqueness of the objects
and, in certain cases, in the impossibility of
repeating the experiment in an accurate manner.
3. Living objects react to the actions of factors
independently on a physical level, and thus they are
statistically independent. In a majority of research
variants, living objects are true replications.
4. The specificity of living objects in their
uniqueness and originality. Some ecological
research involves study of the reactions of
individuals or parts to the impact. In a number of
studies, it is not possible to repeat a unique
biological object, whether it be an individual or a
population.
5. The problem of pseudoreplication is artificial,
since technical and biological replication is
distinguished in biology. The attempt to apply the
goals and requirements of technical replication to
biological is a prime cause of the issue of
pseudoreplication in biological experiments.
6. According to one of the points of view, the
attention of English-speaking authors to the
problem of pseudoreplication is explained by
several reasons:
– the desire to join the campaign of criticism and
to incriminate colleagues in pseudoreplication;
– the attempt to divert the stigma of
pseudoreplication from their work and the work of
colleagues;
– as a warning signal to the reviewer that the author
is acquainted with the work of Stuart H. Hurlbert,
therefore there should be no comments on the
paper
[6]
.
But both variants did not allow to work with a large number of experimental objects. That is, it was
impossible to comply with the condition of mass nature. The reasons are as follows:
1. It is physically impossible in the course of the educational process to explore a large number of
material model objects – homozygous dominant, homozygous recessive and heterozygous individuals.
The work was accompanied by the enormous time spent on manual modelling and counting of randomly
formed pairs of alleles. Such a calculation had to be carried out both within one generation, and in
several replications. Note that in this variant we are talking about the difficulties with the technical
replication of the experiment.
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2. The use of material models was limited to elementary material costs for the manufacture of model
elements. The maximum number of individuals whose genotype models were used in the experiment
was equal to 50. In the case of diallelic inheritance of a trait (as the simplest variant of inheritance), the
number of alleles was equal to 100. Let's point to the fact that in the classrooms there were carried out
parallel experiments on the study of influence of different factors of the dynamics of the population
genetic structure, the work was carried out in small groups, each of which worked with a separate set of
elements for modelling. There were 5 such groups. The first group studied the genetic structure of an
ideal population in generations. The second group studied the effects of gene drift. The third group
studied the essence of the gene flow phenomenon. The fourth group studied the influence of natural
selection on the genetic structure of the population. The fifth group studied the role of the mutational
process in the dynamics of the genetic structure of the population. In total, at least a set of 500 material
elements was needed for modelling.
We place the emphasis on the fact that even with 50 simulated members of the population, we
obtained results that allowed to illustrate the essence of genetic transformations in populations in the
absence of any factors and in their presence.
In work on the improvement of the experimental methodology, we tried to: 1) get closer in school
modelling of genetic-evolutionary processes to the real process taking a course in populations; 2) take
into account significant differences and commonality between scientific and educational experiment.
Particularly, this was expressed in the fact that it was necessary to:
1. Cover by the experiment the maximally large number of individuals. It has been assumed that the
hundreds and thousands of individuals could be the experimental objects.
2. Reduce the amount of routine work for pupils on the calculation of the resulting genotypes and
alleles in one generation.
3. Simulate a larger number of replications (replicates) of the experiment, which would increase the
veracity of results and their closeness to the mathematical formula of Hardy-Weinberg. We also set the
task to provide the possibility for technical and biological replication of an experiment on one topic.
3. Results
Taking into account the abovementioned tasks, we have developed a web page
http://mybio.education/mod/exp6/en/index.html# (Model experiment 1. Study of the genetic structure
of the ideal population (third variant).
Using the tools of this web page, we can conduct an experiment on the modelling of a structure of
an ideal population in the absence of such factors as natural selection, gene flow, gene drift, mutations.
Note that this is the third variant for conduction of a model experiment on the stated topic. The first two
are displayed on the following pages: http://mybio.education/mod/exp1/en/index.html and
http://mybio.education/mod/exp2/en/index.html.
What is the difference between the proposed third variant?
First. In two early variants, the number of individuals was limited by the physical ability to manually
count the resulting pairs of alleles and the number of material elements for modelling. The studied
population in the proposed variant can be very large – several hundreds, thousands, millions of
individuals. This contributes to the implementation of the first of the tasks pursued by us an increase
in the number of objects used in the model experiment. And in this case, it can be considered as a step
towards the increase in the veracity of the experimental results. And thus, the maximum convergence
with the actually occurring genetic and evolutionary transformations in the population. For example, for
an experiment, you can take several tens of thousands of individuals and several million (figures 1, 2).
Second. The user (student) can independently enter the initial allele frequencies in the graph for the
parental population. In the first two variants, the allele frequencies were calculated automatically after
data entering by the manual calculation of the randomly obtained genotypes. This function opens up an
opportunity to demonstrate the essence of biological replication of experiments. This function is
especially remarkable in the lesson during the simultaneous work of several groups of students with
different populations in number and frequency of alleles occurrence (figures 1, 3).
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Figure 1. Results of a model experiment with a number of 20000 individuals, allele frequencies
p (0, 7) and q (0, 3).
Figure 2. Results of a model experiment with a number of 2000000 individuals, allele frequencies
p (0, 7) and q (0, 3).
Third. The number of generations of the population has been increased. In the proposed variant, it is
equal to 5. I.e. together with the parental population, the total number of replications of the experiment
is equal to 6. In previous variants of the experiment, the number of replications was equal to 3 (one
parental generation and two daughter generations). In addition, note that it is technically possible to
increase the number of replications by times. This will offer an opportunity, first of all, to quickly get a
picture of the genetic structure of the population, without bothering students with mechanical work on
mixing and distribution of genotypes, since there is an automatic distribution of genotype frequencies
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within the limits of the ideal population. Secondly, it contributes to the implementation of one of the
tasks pursued by us – an increase in the number of replications of the experiment within the limit of one
sample (population). This function opens an opportunity to conduct the technical replication of
experiments.
Figure 3. Results of a model experiment with a number of 200000 individuals, allele frequencies
p (0, 2) and q (0, 8).
The visualized replication results are displayed on the user's screen by clicking the “Show graphs
and diagrams” button. Note that in one session the user can only see the results of technical replication,
i.e. distribution of alleles and genotypes in generations with initially specified parameters (number of
individuals and allele frequencies). The generations of the population will act as technical replications.
In order to simulate biological replication, it is necessary to load the page once again without closing
the previous one and enter other initial data (the number of individuals, allele frequencies). Within each
session, generations of a population in relation to each other will act as technical replications, but in
relation to the first population and its generations – biological replications.
In 2019/2020 academic year, the developed web page was tested with the participation of 6 students
of the 3rd year of the Institute of Living Systems of Immanuel Kant Baltic Federal University, in specialty
“Biology” and 12 students of the 11th form of the Municipal Budgetary General Education Institution
General Secondary School “School of the Future” of Guryevsky district of Kaliningrad Region (Russian
Federation). The approbation took place within the framework of carrying out by Municipal Budgetary
General Education Institution General Secondary School “School of the Future” together with the
National Research University Higher School of Economics of the conference “Effective High School”
(January 23-25). Within the framework of the conference, there were organized practical classes for
pupils of 11th forms on the topic “Modelling of the genetic evolutionary processes in the population”.
One of the proposed experiments for carrying out was a model experiment “Study of the genetic
structure of an ideal population” according to the methodology updated by us without using material
objects.
In approbation, the participants were divided into 2 groups (3 students and 6 pupils). One group was
asked to start with an experiment at http://mybio.education/mod/exp1/en/index.html#, and then at
http://mybio.education/mod/exp6/en/index.html#. Another group was asked to click a link to the web
page http://mybio.education/mod/exp6/en/index.html# (Model experiment 1. Study of the genetic
structure of the ideal population (third variant) and simulate the genetic structure of a population of any
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number more than a thousand with an arbitrarily given combination of allele frequencies. It was
proposed three times to the participants to carry out model experiment in the third variant with different
initial data (number of individuals of the population, allele frequencies). Each of the participants of the
approbation both in the first and second groups in carrying out of the third variant of the experiment
worked separately. The participants were asked to use the “Show graphs and diagrams” function, and
also to formulate conclusions at the end of the experiment.
The goals pursued by us were as follows:
1. To find out the availability of understanding by users of the tasks and results of experiments.
2. To find out the main difficulties faced by users when working with a web page
http://mybio.education/mod/exp6/en/index.html#.
During the oral survey of the participants in the experiment, it was found:
1. Participants of the first group, when conducting an experiment with material objects at the
beginning of work, hardly understood the essence of the performed similar actions. Only after data
entering into the table, calculation of the frequencies of alleles and genotypes, the understanding of the
meaning of the uniformity of actions came.
2. Participants of the first group complained about the routine of the performed actions, increased
fatigue during their performance. Participants sought to complete the experiment more quickly, which
increased the error rate in calculation of the absolute number of genotypes. The latter was displayed at
the frequency of genotypes calculated by the program. Thus, the obtained results in several cases were
erroneous, the experimental actions had to be performed over again.
3. Participants of the first group, after passage to the second experiment, which, in fact, duplicated
the first variant, but did not require manual counting, expressed great approval of the possibility to
operate only with numbers.
4. Participants of the second group completed the assigned task more quickly. However, in both
groups, there arose questions about the purpose of three-time replicate of the experiment (with different
number of population and allele frequencies). Note that practically no questions arose in both groups
regarding the advisability of repeating the experiment in generations of the same population. It follows
that the essence and necessity of technical replication is recognized and accepted by the participants.
With biological replication, the situation is different. Its objectives were not clear to the participants,
most likely due to a lack of methodological awareness of this type of replication.
5. Before the performance of the experiment, we deliberately did not focus the participants' attention
on the goals of repeated replicate of experimental actions. This was done in order to find out whether
the participants understood the conditions for the veracity of the results of the biological experiment.
Since among the examinees there were both students of a biological specialty and pupils of graduating
profile chemical and biological classes. We assumed that the participants already possess the necessary
methodological tools for planning, conduction of biological experiments and interpretation of the
results. The results of approbation showed that teaching the methodology of a biological experiment
should be started with distinguishing between technical and biological replication of experimental
effects. We can only assume that a lack of understanding of the differences between them (for purposes,
methodology) could initiate the spread of the problem of pseudoreplication in biological research in
principle. We believe that in order to confirm this assumption, it is necessary to conduct additional
studies aimed at a retrospective analysis of biological scientific literature, primarily of scientific papers,
conference materials containing a description of the methods and results of experiments. The question
is, is it worth doing? Or to accept the fact that even if we consider the problem of pseudoreplication as
far-fetched, then the issue of distinguishing between technical and biological replications and teaching
this in the secondary school and in higher educational establishment definitely deserves further study.
4. Conclusions
As a result of work on the topic of true replication by means of a school biological experiment, we came
to the following conclusions:
1. The question of the artificiality and reality of the problem of true replications in biological science
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remains open.
2. One of the reasons for the artificiality of the problem may be an implicit distinction between the
purpose and methods of technical and biological replication.
3. Model experiment on the study of genetic-evolutionary processes in populations by means of
computer modelling is ideal for demonstration of the essence of technical and biological replication.
4. Computer modelling of genetic-evolutionary processes in populations allows to take into account
the requirement of mass nature of experimental impact, which is one of the necessary for obtention of
consistent results.
5. In the educational model experiment, it is impossible to take into account all the requirements for
a scientific biological experiment, therefore, it is necessary to rely only on its essential features:
replicativity, mass nature, principle of single difference, veracity in essence.
5. Outlook
Continuation of approbation of the effectiveness of the proposed method for studying the law of genetic
equilibrium and the essence of technical and biological replication of the experiment is scheduled to be
conducted in 2020/2021 academic year. Elective courses “Population Biology” and “Fundamentals of
Theoretical Biology” for students of 3rd and 4th academic years of the Institute of Living Systems of
Immanuel Kant Baltic Federal University will be used as an experimental site, as well as the course
“Olympiad Biology” for pupils of 10-11 forms of the School of the Future of Guryevsky district of
Kaliningrad region.
Further work on studying the possibilities of a model experiment in training of the pupils of 11 forms
and students-biologists in true replication, as well as the essence of technical and biological replication,
we can see in the following. It is necessary to develop and approbate web pages to model the structure
of a very large population under the influence on its numerous generations of such factors as natural
selection, gene flow, gene drift, mutations.
The modelling of the genetic structure should be fully automated. The initial platforms for
improvement of methodology will be the existing web pages
http://mybio.education/mod/exp3/en/index.html# (Model experiment 2. Study of the genetic structure
of the population under the influence of natural selection),
http://mybio.education/mod/exp4/en/index.html# (Model experiment 3. Modelling the effect of gene
flow on the genetic structure of the population), http://mybio.education/mod/exp5/en/index.html#
(Model experiment 4. Modelling the effect of random processes on the genetic structure of the
population, modelling the drift of genes*), providing one of the stages of work with material objects.
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... This article highlights further research by the authors, begun in [104][105][106]. The article "The learning-style-based approach and optimal use of e-resources in teaching ecological disciplines" [46] by Tetiana M. Derkach, Tetiana V. Starova (figure 58) and Alexander V. Krajnikov aims to optimise electronic resources used in teaching ecological chemistry following the educational preferences of students. ...
... The technique is currently developed to work only with very large ideal populations. This article highlights further research by the authors, begun in [104][105][106]. Third. ...
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Pseudoreplication is defined as the use of inferential statistics to test for treatment effects where treatments are not replicated and/or replicates are not statistically independent. It is a genuine but controversial issue in ecology particularly in the case of costly landscape-scale manipulations, behavioral studies where ethics or other concerns may limit sample sizes, ad hoc monitoring data, and the analysis of natural experiments where chance events occur at a single site. Here key publications on the topic are reviewed to illustrate the debate that exists about the conceptual validity of pseudoreplication. A survey of ecologists and case studies of experimental design and publication issues are used to explore the extent of the problem, ecologists' solutions, reviewers' attitudes , and the fate of submitted manuscripts. Scientists working across a range of ecological disciplines regularly come across the problem of pseudoreplication and build solutions into their designs and analyses. These include carefully defining hypotheses and the population of interest, acknowledging the limits of statistical inference and using statistical approaches including nesting and random effects. Many ecologists face considerable challenges getting their work published if accusations of pseudoreplication are made – even if the problem has been dealt with. Many reviewers reject papers for pseudoreplication, and this occurs more often if they haven't experienced the issue themselves. The concept of pseudoreplication is being applied too dogmatically and often leads to rejection during review. There is insufficient consideration of the associated philosophical issues and potential statistical solutions. By stopping the publication of ecological studies, reviewers are slowing the pace of ecological research and limiting the scope of management case studies, natural events studies, and valuable data available to form evidence-based solutions. Recommendations for fair and consistent treatment of pseudoreplication during writing and review are given for authors, reviewers, and editors.
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The use of differential statistics to test for treatment effect with data from experiments where either treatments were not replicated (though samples may be) or replicates are not statistically independent leads to serious methodological problem. This problem, discovered by Hurbert (1984), is called pseudoreplication. Due to unknown reasons, pseudoreplication issue was completely overlooked by the Russian ecologists, in spite of the fact that the international scientific community is aware of pseudoreplication for almost twenty years. As the result, up to 47% of the experimental ecological papers, published in six Russian academic journals (Botanicheskij zhurnal, Ekologia, Izvestija RAN Ser. Biol., Lesovedenie, Zhurnal Obshchei Biologii, Zooligicheskij zhurnal) in 1998-2001, are pseudoreplicated; this proportion is nearly twice as high as the proportion of pseudoreplicated studies in international journals during 1960-1980, e.g. before the problem was discovered by Hurlbert (1984). This situation is alarming, especially because a substantial part of pseudoreplication arise from incorrect use of statistics, not from incorrect designing of experiments. By using several examples from the recent papers of Russian ecologists I shortly review the situations where pseudoreplication may occur and discuss some aspects of the experimental design, which are critical for correct processing and interpretation of ecological data.
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