Biodiversity, Classification and Evolution — Diagnostic Tests

Unit Tests

UT-1: Hardy-Weinberg Equilibrium and Population Genetics

Question:

In a population of moths, wing colour is determined by a single gene with two alleles: $B$ (dark, dominant) and $b$ (light, recessive). A sample of 500 moths is collected: 455 are dark and 45 are light.

(a) Calculate the frequency of the $b$ allele and the $B$ allele in this population. Show all working.

(b) Calculate the expected number of heterozygous moths in a population of 500 assuming Hardy-Weinberg equilibrium.

(c) State the five conditions required for a population to be in Hardy-Weinberg equilibrium.

(d) The frequency of the $b$ allele was found to decrease from 0.30 to 0.20 over five generations. Explain whether this change is consistent with Hardy-Weinberg equilibrium, and suggest a possible evolutionary mechanism that could explain this change.

Solution:

(a) The frequency of the recessive phenotype (light moths, genotype $bb$) is: $q^2 = \frac{45}{500} = 0.09$

The frequency of the $b$ allele: $q = \sqrt{q^2} = \sqrt{0.09} = 0.30$

The frequency of the $B$ allele: $p = 1 - q = 1 - 0.30 = 0.70$

(b) The expected frequency of heterozygotes ($Bb$) is: $2pq = 2 \times 0.70 \times 0.30 = 0.42$

Expected number of heterozygous moths: $0.42 \times 500 = 210$

(c) The five conditions for Hardy-Weinberg equilibrium:

1. No mutations — the allele frequencies are not changing due to new mutations.
2. Random mating — individuals mate without regard to genotype; no sexual selection.
3. No natural selection — all genotypes have equal fitness; no selective advantage or disadvantage.
4. Extremely large population size — genetic drift is negligible (no random fluctuations in allele frequencies).
5. No gene flow (no migration) — no individuals entering or leaving the population (no immigration or emigration that would introduce or remove alleles).

(d) The change in allele frequency ($b$ decreasing from 0.30 to 0.20) is not consistent with Hardy-Weinberg equilibrium, which predicts that allele frequencies remain constant from generation to generation in the absence of evolutionary forces. A possible mechanism for this change is natural selection — if dark moths ($BB$ or $Bb$) have a selective advantage over light moths ($bb$) in the environment (e.g., better camouflage against dark tree bark, making them less visible to predators), then the $B$ allele would increase in frequency and the $b$ allele would decrease. Other possible mechanisms include genetic drift (if the population is small), gene flow (immigration of dark moths or emigration of light moths), or non-random mating (if dark moths preferentially mate with other dark moths).

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UT-2: Speciation — Allopatric vs Sympatric

Question:

The cichlid fish of Lake Victoria show remarkable species diversity, with over 500 species believed to have evolved from a common ancestor within the last 15,000 years.

(a) Distinguish between allopatric speciation and sympatric speciation.

(b) Explain how reproductive isolation can occur in sympatric speciation, using cichlid fish as an example.

(c) Explain the role of geographical isolation in allopatric speciation, and describe how the isolated populations may diverge over time.

(d) Polyploidy is a common mechanism of sympatric speciation in plants. Explain how polyploidy can lead to speciation, and explain why polyploidy is less common in animals than in plants.

Solution:

(a) Allopatric speciation occurs when populations of a species are separated by a geographical barrier (e.g., mountain range, river, ocean), preventing gene flow between them. The populations evolve independently and accumulate genetic differences until they can no longer interbreed to produce fertile offspring.

Sympatric speciation occurs without geographical separation — populations within the same geographic area become reproductively isolated and diverge into separate species. This is rarer and typically involves mechanisms such as polyploidy (in plants), behavioural isolation, ecological niche specialisation, or sexual selection.

(b) In cichlid fish, sympatric speciation can occur through several mechanisms:

• Sexual selection: female cichlids often choose mates based on male colouration. If a mutation arises that produces a different colour pattern, females with a preference for that colour will mate selectively with males displaying it. Over time, this assortative mating (preference for similar mates) leads to reproductive isolation between colour morphs.
• Ecological (dietary) specialisation: if some cichlids specialise on one food source (e.g., scraping algae from rocks) and others on a different source (e.g., eating insect larvae), they may occupy different ecological niches within the same lake. Differences in jaw morphology and feeding behaviour reduce interbreeding between the groups.
• Behavioural isolation: differences in courtship displays, mating rituals, or spawning times can prevent interbreeding between groups that live in the same geographic area.

Once reproductive isolation is established (pre-zygotic barriers prevent mating, or post-zygotic barriers produce infertile or inviable offspring), the two populations accumulate further genetic differences through natural selection, genetic drift, and mutation, eventually becoming distinct species.

(c) In allopatric speciation, a geographical barrier (e.g., a river forming, a mountain range rising, continental drift, or the formation of an island) physically separates a population into two or more isolated groups. This prevents gene flow between the groups (individuals cannot migrate between them to interbreed). Once isolated, each population experiences different environmental conditions, different selection pressures, and different mutations. Over many generations, natural selection favours different alleles in each population, and genetic drift causes random changes in allele frequencies. Mutations that arise in one population do not spread to the other. Eventually, the populations diverge so much genetically that if they were brought back together, they would not be able to interbreed to produce fertile offspring — they have become separate species. The key point is that geographical isolation leads to reproductive isolation as a consequence of independent evolution.

(d) Polyploidy is a condition in which an organism has more than two complete sets of chromosomes (e.g., tetraploid — 4n). Polyploidy can arise from errors in meiosis (e.g., failure of chromosomes to separate during anaphase, producing diploid gametes instead of haploid gametes). If a diploid gamete fuses with a normal haploid gamete, the resulting offspring is triploid (3n), which is usually sterile. However, if two diploid gametes fuse, the offspring is tetraploid (4n) and may be fertile. The tetraploid individual can reproduce with other tetraploids but cannot interbreed with the original diploid population because any offspring would be triploid (sterile). This instant reproductive isolation makes polyploidy a powerful mechanism of sympatric speciation in plants.

Polyploidy is less common in animals because:

1. Animals typically have chromosomal sex determination systems (e.g., XX/XY). Polyploidy disrupts the balance of sex chromosomes, leading to sterility.
2. Animal development is more sensitive to gene dosage imbalances caused by extra chromosome sets.
3. Animals generally have more complex developmental processes that are disrupted by polyploidy.
4. Many plants can self-fertilise (or reproduce asexually), allowing a polyploid individual to reproduce alone and establish a new population. Most animals require a mate of the same ploidy, which is unlikely to arise simultaneously.

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UT-3: Taxonomy, Classification, and Molecular Evidence

Question:

Traditionally, organisms were classified based on observable morphological characteristics. Modern classification increasingly uses molecular evidence such as DNA sequencing and protein electrophoresis.

(a) Describe the hierarchy of biological classification from domain to species, and explain why scientists use a standardised system.

(b) Explain the biological species concept and describe two limitations of this concept.

(c) Explain how comparisons of DNA base sequences and amino acid sequences in proteins can be used to determine the evolutionary relationships between species.

(d) Two species of insect look morphologically identical but have significantly different DNA sequences in a conserved gene. Explain what this suggests about their evolutionary relationship and why morphological classification alone can be misleading.

Solution:

(a) The hierarchy of classification (from broadest to most specific): Domain $\rightarrow$ Kingdom $\rightarrow$ Phylum $\rightarrow$ Class $\rightarrow$ Order $\rightarrow$ Family $\rightarrow$ Genus $\rightarrow$ Species

A standardised system (binomial nomenclature — two-part Latin name: genus + species, e.g., Homo sapiens) is used because: (1) it provides a universal language that all scientists worldwide can understand, avoiding confusion caused by common names that vary between languages and regions; (2) it allows clear and unambiguous communication about organisms; (3) it reflects evolutionary relationships (closely related species share the same genus).

(b) The biological species concept defines a species as a group of similar organisms that can interbreed to produce fertile offspring, and are reproductively isolated from other such groups.

Limitations:

1. Asexual organisms: the concept cannot be applied to organisms that reproduce asexually (e.g., bacteria, many plants), as they do not interbreed.
2. Fossils: the concept cannot be applied to extinct organisms preserved in the fossil record, as their reproductive behaviour cannot be observed.
3. Hybridisation: some closely related species can interbreed to produce fertile offspring (e.g., some species of Canis), blurring the species boundary.
4. Geographically isolated populations: it may be impractical to test whether allopatric populations can interbreed.

(c) DNA base sequence comparison: Scientists extract DNA from two species and compare the sequence of bases in a specific gene. The greater the similarity in the base sequence, the more closely related the two species are (they shared a more recent common ancestor). Differences accumulate over time through mutations (substitutions, insertions, deletions), so species that diverged longer ago have more differences. Molecular clocks use the rate of mutation in specific genes to estimate the time since two species diverged from a common ancestor.

Amino acid sequence comparison: Similarly, the sequence of amino acids in a protein (e.g., cytochrome c, haemoglobin) can be compared between species. Because the amino acid sequence is determined by the DNA sequence (via transcription and translation), the same principle applies — more similar amino acid sequences indicate closer evolutionary relationships. Protein comparison has the advantage that some proteins (e.g., cytochrome c) are highly conserved across many species, allowing comparisons across very distantly related organisms.

(d) If two insect species are morphologically identical but have significantly different DNA sequences in a conserved gene (a gene that changes very slowly over evolutionary time), this suggests that they are not closely related despite their similar appearance. They likely evolved similar morphologies independently through convergent evolution — similar environmental pressures led to the evolution of similar adaptations (analogous structures) in unrelated lineages. This demonstrates why morphological classification alone can be misleading: similar appearances can result from convergent evolution rather than shared ancestry (homology). Molecular evidence (DNA and protein sequences) provides a more objective and reliable measure of evolutionary relationships because it reflects the actual genetic changes that have accumulated over time, independent of the selective pressures that shape morphology.

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Integration Tests

IT-1: Natural Selection and Antibiotic Resistance (with Genetics and DNA)

Question:

The evolution of antibiotic resistance in bacteria is a significant public health concern. MRSA (methicillin-resistant Staphylococcus aureus) is resistant to beta-lactam antibiotics including methicillin.

(a) Describe how natural selection leads to the increase in frequency of antibiotic-resistant bacteria in a population exposed to antibiotics.

(b) The mecA gene confers methicillin resistance in MRSA. This gene is carried on a section of DNA called SCCmec, which can be transferred between bacterial cells. Explain how horizontal gene transfer contributes to the rapid spread of antibiotic resistance compared with vertical gene transfer alone.

(c) Explain why the overuse and misuse of antibiotics (e.g., not completing a prescribed course, using antibiotics for viral infections) increases the rate at which antibiotic resistance evolves.

(d) Using the Hardy-Weinberg principle, explain why the frequency of antibiotic resistance genes in a bacterial population does not increase in the absence of antibiotics, assuming the resistance gene carries a small metabolic cost.

Solution:

(a) In a population of S. aureus, random mutations arise naturally. Occasionally, a mutation (or acquisition of the mecA gene) confers resistance to methicillin. Before antibiotic exposure, resistant bacteria may be at a slight disadvantage (see part d). When methicillin is applied: (1) susceptible bacteria are killed or inhibited; (2) resistant bacteria survive and reproduce; (3) the resistant bacteria pass the resistance gene to their offspring (vertical gene transfer); (4) the proportion of resistant bacteria in the population increases over successive generations. This is natural selection in action: the antibiotic acts as a selective pressure, and resistant bacteria have a selective advantage (higher fitness) in the presence of the antibiotic. Over time, the population evolves to become predominantly resistant.

(b) Horizontal gene transfer (HGT) allows bacteria to acquire resistance genes from other bacteria, even from different species, without being their descendant. This is much faster than waiting for a resistance mutation to arise de novo in each lineage. In the context of MRSA:

• Conjugation: a donor bacterium transfers a copy of the SCCmec element (carrying mecA) to a recipient through a pilus.
• Transformation: bacteria take up free DNA from dead bacteria in the environment, potentially acquiring resistance genes.
• Transduction: bacteriophages (viruses) transfer resistance genes between bacteria.

HGT allows a single resistance gene to spread through a diverse population of bacteria in a single generation, whereas vertical gene transfer requires the gene to be passed from parent to offspring over many generations. HGT is the primary reason antibiotic resistance can spread so rapidly in hospitals and communities.

(c) Not completing a prescribed course: stopping antibiotic treatment early means that the most susceptible bacteria are killed, but some less susceptible (but not yet resistant) bacteria survive. These surviving bacteria have been exposed to sub-lethal concentrations of the antibiotic, which provides a strong selective pressure for the development of resistance (resistant mutants survive and reproduce). Completing the full course ensures that all susceptible bacteria are eliminated, reducing the chance of resistant mutants surviving.

Using antibiotics for viral infections: viruses are not affected by antibiotics (antibiotics target bacterial structures such as cell walls, ribosomes, and DNA replication machinery). Using antibiotics for viral infections provides no therapeutic benefit but does expose the normal bacterial flora (commensal bacteria) to the antibiotic. This creates a selective pressure for resistance in these bacteria, which can then transfer resistance genes to pathogenic bacteria via HGT. The unnecessary use of antibiotics is one of the strongest drivers of resistance evolution.

(d) According to the Hardy-Weinberg principle, allele frequencies remain constant in the absence of evolutionary forces. In the absence of antibiotics, the resistance gene may carry a metabolic cost — the bacteria must expend energy to produce the resistance mechanism (e.g., an altered penicillin-binding protein encoded by mecA) that provides no benefit without the antibiotic. This means resistant bacteria are at a slight selective disadvantage compared to susceptible bacteria — they grow more slowly or are outcompeted. Natural selection will therefore decrease the frequency of the resistance gene in the population (susceptible bacteria have higher fitness without the antibiotic). In Hardy-Weinberg terms, the resistance allele has a lower fitness coefficient, and selection acts against it, reducing its frequency until it may be lost from the population entirely (or maintained at a very low frequency by mutation-selection balance). This is why prudent antibiotic use (minimising unnecessary exposure) helps preserve the effectiveness of existing antibiotics.

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IT-2: Classification Using Molecular Evidence and Immunology (with Cells and Biological Molecules)

Question:

Scientists studying the evolutionary relationships between five species of primate compare the amino acid sequence of cytochrome c in each species. They also test the immunological response by injecting serum containing antibodies from human cytochrome c into blood samples from each species and measuring the degree of antibody-antigen binding.

(a) Explain why cytochrome c is a suitable protein for comparing evolutionary relationships between distantly related species.

(b) The amino acid sequence comparison reveals that Species A differs from humans by 2 amino acids, Species B by 5, Species C by 12, Species D by 0, and Species E by 25. Construct a likely evolutionary tree showing the relationships between these species and humans, and explain your reasoning.

(c) Explain the principle behind using antibody-antigen reactions (immunological comparison) to determine evolutionary relationships. Why would a stronger antibody-antigen reaction indicate a closer evolutionary relationship?

(d) Explain how both DNA hybridisation and DNA sequencing can be used to compare the genetic similarity between species, and state which method provides more detailed information.

Solution:

(a) Cytochrome c is a suitable protein because:

1. It is ubiquitous — it is found in almost all aerobic organisms, from bacteria to humans, allowing comparisons across a wide range of species.
2. It is functionally conserved — it plays a critical role in the electron transport chain (carrying electrons between complex III and complex IV), so its function has been conserved over billions of years. This means the protein has changed very slowly over evolutionary time, and differences between species represent ancient divergences.
3. It is of appropriate length — approximately 104 amino acids in mammals, long enough to provide statistically meaningful comparisons but short enough to sequence easily.
4. Its slow rate of change means it is useful for comparing deeply divergent lineages (e.g., between kingdoms), whereas faster-evolving proteins (e.g., fibrinopeptides) are better for comparing closely related species.

(b) The number of amino acid differences from human cytochrome c indicates evolutionary distance (fewer differences $=$ more closely related):

Species	Differences from human
D	0
A	2
B	5
C	12
E	25

Likely evolutionary tree (branching pattern, closest to most distant):

Human --- D (0 differences — possibly same species or extremely close relative)
  |
  |-- A (2 differences — very close)
  |
  |-- B (5 differences — moderately close)
  |
  |-- C (12 differences — more distant)
  |
  |-- E (25 differences — most distant)

Species D is most closely related to humans (identical sequence), followed by A, then B, C, and E. The increasing number of differences reflects increasing time since divergence from a common ancestor. (Note: in a more sophisticated tree, A and D might share a common ancestor that diverged from the human lineage, but with only 0 and 2 differences from human, they are extremely closely related.)

(c) Immunological comparison works on the principle that antibodies are highly specific to the antigens (proteins) that stimulated their production. If antibodies are raised against human cytochrome c (injected into a rabbit, for example), they will be specific to the epitopes (antigenic determinants) on human cytochrome c. When these antibodies are mixed with serum from another species, they will bind to that species' cytochrome c if and only if it has similar epitopes. A stronger antibody-antigen reaction (more precipitate formed, stronger agglutination, or stronger signal in an ELISA) indicates greater similarity between the two cytochrome c proteins, because more antibodies are able to bind. Greater protein similarity indicates closer evolutionary relationship — the two species shared a more recent common ancestor, so their cytochrome c has had less time to accumulate mutational differences.

(d) DNA hybridisation: DNA from two species is extracted, heated to separate the strands (denaturation), and then mixed. Complementary strands from the two species will anneal (hybridise) to form hybrid DNA. The mixture is then gradually heated again, and the temperature at which the hybrid DNA separates (melting temperature, $T_m$) is measured. The more similar the DNA sequences, the more hydrogen bonds form between complementary bases, and the higher the melting temperature. A higher $T_m$ indicates greater genetic similarity and closer evolutionary relationship.

DNA sequencing: The actual sequence of nucleotide bases in a gene is determined for each species and directly compared using computer analysis. The percentage similarity in base sequence is calculated, and the number and position of mutations (substitutions, insertions, deletions) are identified.

DNA sequencing provides more detailed information because it reveals the exact nature, position, and number of differences between sequences. DNA hybridisation gives only a general measure of overall similarity (the $T_m$) without revealing specific differences. Sequencing can identify specific mutational changes, detect insertions and deletions, and be used to construct detailed phylogenetic trees with molecular clock dating. DNA hybridisation is a coarser, less precise method.

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IT-3: Biodiversity Measurement and Conservation (with Ecology)

Question:

A conservation organisation wants to compare the biodiversity of two nature reserves (Reserve X and Reserve Y) to determine which should receive priority for funding. Reserve X is a managed meadow, and Reserve Y is an ancient woodland.

(a) Explain the difference between species richness and species evenness, and explain why both are needed to give a complete picture of biodiversity.

(b) Describe how Simpson's Diversity Index is calculated, and explain the advantage of using an index rather than simply counting the number of species.

(c) The following data were collected using quadrats in each reserve:

Reserve X: Species A (50 individuals), Species B (30), Species C (10), Species D (5), Species E (5) — 100 individuals total, 5 species. Reserve Y: Species F (20), Species G (20), Species H (20), Species I (20), Species J (20) — 100 individuals total, 5 species.

Calculate Simpson's Diversity Index ($D$) for each reserve using $D = 1 - \sum\left(\frac{n}{N}\right)^2$ and determine which reserve has higher biodiversity.

(d) Explain why maintaining genetic diversity within a species is important for conservation, and describe how inbreeding can threaten the survival of small, isolated populations.

Solution:

(a) Species richness is the number of different species present in a habitat (a simple count). Species evenness is a measure of how evenly individuals are distributed among the species — whether the community is dominated by one or a few species, or whether all species have similar abundances.

Both are needed because two habitats can have the same species richness but very different biodiversity. For example, a habitat with 5 species where one species makes up 95% of individuals has lower biodiversity than a habitat with 5 species where each makes up approximately 20%. Species richness alone does not capture this difference — evenness provides the additional information needed.

(b) Simpson's Diversity Index: $D = 1 - \sum\left(\frac{n}{N}\right)^2$, where $n$ is the number of individuals of a particular species and $N$ is the total number of individuals of all species. $D$ ranges from 0 (no diversity — only one species present) to approaching 1 (maximum diversity — many species, all equally abundant).

The advantage of using an index rather than simply counting species (species richness) is that the index accounts for both species richness and species evenness. A single number provides a more comprehensive measure of biodiversity, allowing easy comparison between different habitats or the same habitat at different times.

(c) Reserve X:

Species	$n$	$\frac{n}{N}$	$\left(\frac{n}{N}\right)^2$
A	50	0.50	0.2500
B	30	0.30	0.0900
C	10	0.10	0.0100
D	5	0.05	0.0025
E	5	0.05	0.0025

$\sum\left(\frac{n}{N}\right)^2 = 0.2500 + 0.0900 + 0.0100 + 0.0025 + 0.0025 = 0.3550$

$D_X = 1 - 0.3550 = 0.645$

Reserve Y:

Species	$n$	$\frac{n}{N}$	$\left(\frac{n}{N}\right)^2$
F	20	0.20	0.0400
G	20	0.20	0.0400
H	20	0.20	0.0400
I	20	0.20	0.0400
J	20	0.20	0.0400

$\sum\left(\frac{n}{N}\right)^2 = 5 \times 0.0400 = 0.2000$

$D_Y = 1 - 0.2000 = 0.800$

Reserve Y ($D = 0.800$) has higher biodiversity than Reserve X ($D = 0.645$), despite both having 5 species and 100 individuals. Reserve Y has greater evenness (all species equally abundant), which results in a higher Simpson's Index.

(d) Genetic diversity within a species is the variety of alleles present in the gene pool. It is important for conservation because:

1. Resistance to disease: a genetically diverse population is more likely to contain individuals with alleles that confer resistance to new diseases or pathogens. A genetically uniform population could be wiped out by a single disease.
2. Adaptation to environmental change: genetic diversity provides the raw material for natural selection. If the environment changes (e.g., climate change, habitat alteration), a genetically diverse population is more likely to contain individuals with alleles that confer adaptation to the new conditions.
3. Reduced inbreeding depression: genetic diversity means individuals are less likely to be closely related, reducing the expression of harmful recessive alleles.

Inbreeding occurs when individuals in a small, isolated population mate with close relatives. Inbreeding increases the probability that offspring will be homozygous for harmful recessive alleles (which are normally masked in heterozygous individuals). This leads to inbreeding depression: reduced fitness, lower survival rates, reduced fertility, and increased susceptibility to disease. In small, isolated populations (e.g., on habitat islands, in fragmented habitats), the gene pool is small, and inbreeding is unavoidable. Over time, the loss of genetic diversity through genetic drift and inbreeding reduces the population's ability to adapt and increases its risk of extinction. Conservation strategies such as creating wildlife corridors (to connect isolated populations and allow gene flow) and captive breeding programmes with careful management of mating pairs aim to maintain genetic diversity and reduce inbreeding.