Groupx+1

Zoe Sehlke, Agostina Campodonico, Martina Treggiari, Ben Parker, Imran Hussain, Muzlif Mulaffer

Our problem: With the polymorphic traits occurring in //Cepaea nemoralis// it is difficult to ascertain whether the range of traits is a random effect of genetic drift or a direct effect of selection. Our solution: Sample different habitats for //C. nemoralis// and observe the frequencies to determine if there is a correlation between habitat and characteristics.

Alternative hypothesis: If there is a significant difference in polymorphic characteristics frequencies between certain locations, then it is suggested that selection is acting on these characteristics (along with genetic drift).

Null hypothesis: If there is no difference in polymorphic characteristics frequencies between certain locations then selection is not acting on these characteristics (just genetic drift).

Allele-A version of a gene. Fitness-A property of the genotype. The expectation of the number of descendant genes in the next generation at the same stage of the life cycle. Often given relative to the fittest genotype. Gene flow-The exchange of genes between distinct populations. Genetic drift- Describes the process by which allele frequencies change over time due to the effects of random sampling. It is stronger in a small population and only present in finite populations. Genotype-A description of the alleles of an individual. Phenotype-The physical characteristics of the individual. A combination of the genes and their interaction with the environment. Polymorphism- Genetic variation in the form of multiple alleles of many genes in a population, resulting in multiple phenotypes. Population-A collection of interbreeding individuals of the same species in a certain, defined area. Sampling error-Error as a result of sampling a subset of a population. Also occurs because of the way a sample is collected. Selection-Acts on the phenotype. This is where there are certain genotypes that are more likely to increase in frequency.

Sampling Methods: 1) Deep Woods -//C. nemoralis// mainly or only in the woods

2) Disturbed area between Woods and Grass Land -//C. nemoralis// in both wood and grass land able to migrate to either habitat

3) Isolated Grassland -//C. nemoralis// exclusively in the grass area

4) Grass/ Hedgerow -//C. nemoralis// in the area near both hedgerow and grassland able to migrate to either habitat

5) Hedgerow -//C. nemoralis// in hedgerow

6) Opposite wood* -this option is to either sample on the opposite side which may have better conditions for //C. nemoralis (//e.g. cooler, damper) or if the woods are composed of significantly different tree species in regards to color we will take samples from two separate tree areas. This will allow for some flexibility in our sampling method.

We will attempt to sample at a consistent height as we do not think height is a significant factor in our experiments goal because of limitations in our sampling. We realize height is a factor, however for this experiment we will be focusing on habitat difference as opposed to height (this decision will be discussed further in our final write up). Each strip represents 24 meters squared (not shown to scale) collecting all the snails (dead and alive) in this area. We will record the snails shell color and number of bands (as much as visible). We will record predated upon snails in an additional data set to observe if there is correlation between predation and characteristic.

Sampling method revised

1 woods 2grass 3hedgerow 4hedgerow 5grass 6woods

We changed our sampling to prefer replication over habitat change.

- our samples are moved farther apart to minimize gene flow between populations. - we also want to avoid pseudo replication - we still maintaining height

= = =**Final Method and Write Up**=

__Investigating polymorphism in //Cepaea nemoralis//__ All species evolve through random genetic mutations which give rise to new alleles [1] at a particular locus. Once in a population [2], the frequency of the new allele may fluctuate due to evolutionary processes such as genetic drift, selection [3] and gene flow [4] which may act in conjunction with each other, though the strength of each process may differ. The accumulation of mutations can create different variants of a certain trait, this is commonly referred to as polymorphism. Polymorphism is fundamental because it provides the variation upon which evolution can act. Many evolutionary processes, such as those listed above, can shape and maintain polymorphism at a locus. To investigate polymorphism without genetic analysis, it is necessary to find a population in which phenotypic polymorphisms are abundant and can be readily identified. The Grove snail (//Cepaea nemoralis//) is a suitable model organism for the study of polymorphism due to the distinctive variation in shell colour (different shades of yellow, pink or brown) and banding (may have 0 to 5 bands), for which a supergene is responsible (Cain et al. 1960) a. In addition to this, the genotype [5] of //C.nemoralis// can be easily identified from the phenotype [6], the snails have a relatively low dispersal rate (approximately 20m per generation) which limits gene flow, short generation times, and are found locally in Buckinghamshire. There has been much research into the maintenance of polymorphism in this species. In his review, Steve Jones discusses many possible factors which influence polymorphism in //C.nemoralis// and concludes that there is no single process responsible for the observed polymorphic patterns in the studied populations (Jones et. al, 1977) b. Jones suggests a frequency dependent form of selection and visual selection by predators as well as climatic selection for shell colour, amongst others. These processes may occur in addition to genetic drift which causes stochastic changes in allele frequencies over time This study aimed to establish whether there is a difference in the proportion of phenotypic polymorphism (shell banding number and colour) between grass and woodland habitats at Pulpitt Wood. 6 sample sites were selected with 3 samples in grassland habitat and 3 samples in woodland to ensure replication (Figure 1).

[1] Allele-A version of a gene.

[2]Population- A collection of interbreeding individuals of the same species in a certain, defined area.

[3]Selection- Acts on the phenotype. This is where there are certain genotypes that are more likely to increase in frequency. [4] Gene flow-The exchange of genes between distinct populations [5] Genotype-A description of the alleles of an individual [6] Phenotype-The physical characteristics of the individual. A combination of the genes and their interaction with the environment

References [a] Cain, A. J., King, J. M. B., Sheppard, P. M. 1960. New data on the genetics of polymorphism in the snail Cepaea nem- oralis L. Genetics 45:393-411 34 [b] Jones, J., Leith, B. and Rawlings, P. (1977). Polymorphism in Cepaea: A Problem with Too Many Solutions?. //Annu. Rev. Ecol. Syst.//, 8(1), pp.109-143

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Final Copy

** SBS 633 Evolutionary Genetics ** ** Professor Nichols ** Investigating Polymorphism in //Cepaea nemoralis// ** 9th December 2014 ** ** By Group Turbo: ** We hereby declare that this work is our own and any reference are cited. ||  Zoe Sehlke
 * 130404781  ||  ** ………………… **  ||
 * Agostina Campodonico  ||   130049098   ||  ** ………………… **  ||
 * Martina Treggiari  ||   130496384   ||  ** ………………… **  ||
 * Ben Parker  ||   130172592   ||  ** ………………… **  ||
 * Imran Hussain  ||   130007445   ||  ** ………………… **  ||
 * Muzlif Mulaffer  ||   130778291   ||  ** ………………… **  ||

All species evolve through random genetic mutations which give rise to new alleles[A] at a particular locus. Once in a population[B], the frequency of the new allele may fluctuate due to evolutionary processes such as genetic drift, selection[C] and gene flow[D] which may act in conjunction with each other, though the strength of each process may differ. The accumulation of mutations can create different variants of a certain trait, this is commonly referred to as polymorphism. Polymorphism is fundamental because it provides the variation upon which evolution can act. Many evolutionary processes, such as those listed above, can shape and maintain polymorphism at a locus. To investigate polymorphism without genetic analysis, it is necessary to find a population in which phenotypic polymorphisms are abundant and can be readily identified. The Grove snail (//Cepaea nemoralis//) is a suitable model organism for the study of polymorphism due to the distinctive variation in shell colour (different shades of yellow, pink or brown) and banding (may have 0 to 5 bands), for which a supergene is responsible (Cain et al. 1960)1. Cain utilised breeding experiments between parents with different phenotypes and proved that variation in phenotype[E] was under the control of a single gene, the “supergene”. Linkage was also apparent for genes affecting band shading and colour. In addition to this, the genotype[F] of //C.nemoralis// can be easily identified from the phenotype, the snails have a relatively low dispersal rate (approximately 20m per generation) which limits gene flow, short generation times, and are found locally in Buckinghamshire. There has been much research into the maintenance of polymorphism in this species. In his review, Steve Jones discusses many possible factors which influence polymorphism in //C.nemoralis// and concludes that there is no single process responsible for the observed polymorphic patterns in the studied populations (Jones et. al) 1977)2. Jones suggests a frequency dependent form of selection and visual selection by predators as well as climatic selection for shell colour, amongst others. These processes may occur in addition to genetic drift which causes stochastic changes in allele frequencies over time   Based on previous work (Jones et al, 1977)2 it is assumed that there is phenotypic variation in snail populations between habitats. The study aimed to establish which process most strongly affects this variation: genetic drift, gene flow or selection. In order to investigate this assumption samples were taken from 6 sites with 3 sites in grassland habitat and 3 in woodland to ensure replication (Figure 1). By comparing relative phenotype frequencies between habitats and populations within habitats a statistical analysis could reveal if differences were significant. Sites were chosen as a deliberate attempt to try and minimise the effects of gene flow between sub populations as well as reduce the risk of pseudoreplication[G]. If some phenotypes were consistently more/less frequent in certain habitats this would suggest a form of selection; however if there was variation within the same habitat between sampling sites this may suggest genetic drift as the predominant process acting upon the population.
 * __ Introduction: __**

Circles represent sampling locations for study. Colour key; green=bushes, brown=woodland blue=grass
 * __ Figure 1. Sampling site locations at Monk’s Risborough __**

The total collected data for the study is shown in Table 1 with categories merged for live/dead and also adult/subadults. This was done to produce categories with values high enough to run through statistical tests. For the analysis brown snails were discarded from the results due to low frequencies. For the same reason, the number of bands for yellow and pink snails were merged to form a 0-2 or 3-5 band category (Table 2 and Table 3). For the Chi squared banding analysis in grass sampling sites χ2=4.09, P=0.6645 and for woodland sites χ2=6.81, P=0.3388. This suggested there is no significant difference in the frequency of number of bands within grass and woodland habitats. The proportion of 4 and 5 banded snails, for both yellow and pink shells, was much greater in every sample site (Table 1). In the second set of tables (Tables 4, 5, and 6) only the frequency of each colour was analysed for each location. For grass habitat χ2=0, P=1 and χ2=2.82 ,P=0.2441 for woodland. Once again this suggested no significant difference, in this case, in the frequency of pink and yellow shelled snails within the same habitat. The Chi squared analysis comparing the total number of pink and yellow shelled snails between habitats produced a χ2 value of 9.22, P=0.0024 (Pearson’s uncorrected) suggesting a significant difference. As the Chi squared value (9.22) was greater than the critical value for χa=0.005 (7.879), the null hypothesis was rejected at the 0.5% level; this result demonstrated a significant difference in shell phenotype frequency between different habitats (grass and woodland). B=brown shell; Y=yellow shell; P=pink shell; 0-5=number of bands
 * __ Results: __**
 * __ Table 1. Site sample data: __**
 * Site ||  B0   ||   B1/B2   ||   B3   ||   B4/B5   ||   P0   ||   P1/P2   ||   P3   ||   P4/P5   ||   Y0   ||   Y1/Y2   ||   Y3   ||   Y4/Y5   ||   TOTALS   ||
 * Grass1 ||  -   ||   1   ||   1   ||   -   ||   1   ||   1   ||   2   ||   4   ||   -   ||   1   ||   4   ||   21   ||   36   ||
 * Grass2 ||  -   ||   -   ||   1   ||   3   ||   3   ||   -   ||   -   ||   12   ||   -   ||   9   ||   7   ||   32   ||   67   ||
 * Grass3 ||  -   ||   3   ||   3   ||   2   ||   2   ||   2   ||   -   ||   7   ||   -   ||   6   ||   5   ||   25   ||   55   ||
 * Wood1 ||  -   ||   1   ||   1   ||   1   ||   5   ||   1   ||   1   ||   20   ||   1   ||   -   ||   2   ||   31   ||   64   ||
 * Wood2 ||  1   ||   1   ||   -   ||   -   ||   4   ||   2   ||   1   ||   14   ||   -   ||   4   ||   1   ||   41   ||   69   ||
 * Wood3 ||  1   ||   -   ||   -   ||   -   ||   8   ||   3   ||   2   ||   13   ||   -   ||   2   ||   -   ||   32   ||   61   ||
 * ||  ||   ||   ||   ||   ||   ||   ||   ||   ||   ||   ||   Total:   ||   352   ||
 * __ Table 2. Contingency table for pink and yellow banding categories within grass sites (G1-G3): __**
 * || Pink (0-2) || Pink (3-5) || Yellow (0-2) || Yellow (3-5) || Total ||
 * Grass1 ||  2   ||   6   ||   1   ||   25   ||   34   ||
 * Grass2 ||  3   ||   12   ||   9   ||   39   ||   63   ||
 * Grass3 ||  4   ||   7   ||   6   ||   30   ||   47   ||
 * Total ||  9   ||   25   ||   16   ||   94   ||   144   ||

Chi square: 4.09 P value: 0.6645 Df: 6
 * __ Table 3. Contingency table for pink and yellow banding categories within woodland sites (W1-W3): __**
 * || Pink (0-2) || Pink (3-5) || Yellow (0-2) || Yellow (3-5) || Total ||
 * Wood1 ||  6   ||   21   ||   1   ||   33   ||   61   ||
 * Wood2 ||  6   ||   15   ||   4   ||   42   ||   67   ||
 * Wood3 ||  11   ||   15   ||   2   ||   32   ||   60   ||
 * Total ||  23   ||   51   ||   7   ||   107   ||   188   ||

Chi square: 6.81 P value: 0.3388 Df: 6
 * __ Table 4. Contingency table for pink and yellow snails in grass samples (G1-G3): __**
 * || P || Y || Total ||
 * Grass1 ||  8   ||   26   ||   34   ||
 * Grass2 ||  15   ||   48   ||   63   ||
 * Grass3 ||  11   ||   36   ||   47   ||
 * Total ||  34   ||   110   ||   144   ||

Chi square: 0 P value: 1 Df: 2
 * __ Table 5.Contingency table for pink and yellow snails in woodland samples (W1-W3): __**
 * || P || Y || Total ||
 * Wood1 ||  27   ||   34   ||   61   ||
 * Wood2 ||  21   ||   46   ||   67   ||
 * Wood3 ||  26   ||   34   ||   60   ||
 * Total ||  74   ||   114   ||   188   ||

Chi square: 2.82 P value: 0.2441 Df: 2
 * __ Table 6. Contingency table for pink and yellow snails (woods vs grass): __**
 * || Grass || Woods || TOTAL ||
 * Pink ||  34   ||   74   ||   108   ||
 * Yellow ||  110   ||   114   ||   224   ||
 * TOTAL ||  144   ||   188   ||   332   ||

Chi square: 9.22 (Pearson’s uncorrected) P value: 0.0024 (Pearson’s uncorrected) Df: 1

The results suggest that selection is the predominant process acting upon the phenotype for the colours pink and yellow (P value: 0.0024). Selection is further supported by the fact that such large difference between the two habitats is unlikely to be due to sampling variation[H]. Furthermore, the P values (Table 4 and Table 5) also support a selection mechanism whereas if genetic drift was operating without selection more variation would be expected between sampling sites within the same habitat. There were several consistent trends in the data such as both greater phenotypic variation and a greater density of snails in woodland habitat. Also a higher frequency of yellow snails than pink was recorded in both habitats. Sampled brown snail frequency was extremely low and, for this reason, they were excluded from the Chi squared test. This could have biased the results as processes acting upon brown snails were overlooked in this study. A lower frequency of brown snails could be due to the effects of selection, genetic drift and gene flow in which case our sample could be representative of the actual snail population. The lack of brown snails in the collection could also be due to a form of sampling variation, possibly “search image”[I]. In snail populations, banding patterns 2 and 4 are rare and were therefore collated with 1 and 5 banded snails respectively (Table 1). Banding categories were further merged for the Chi squared tests in Tables 2 and 3, but the results were insignificant for this analysis. An inconclusive result could be due to an insufficient amount of data or because banding is mostly affected by the effects of drift, with very little or even no selection acting upon this trait. Further reductions to the reliability of the data are caused by disruptions in snail distribution such as: anvil stones[J] and previous students’ work. Variation in factors such as temperature and sunlight of sampling sites, which could not be controlled in the study, could have affected the reliability of the results. Previous work (Emberton and Bradbury, 1963)3 has uncovered a negative correlation between sunlight absorbance and band number in the snails. Emberton found snails with more bands absorbed heat from the sun more efficiently allowing activity at lower temperatures. Absorbance rate in shell sections of different colours and banding numbers were also measured using a photodetector to record the amount of light (and hence heat) absorbed by each shell type. These results would predict a greater banded proportion in woodland habitats where there is less sunlight present. This study’s results support previous works done on the topic and the results consistently integrate with current hypotheses. Future study recommendations would be to control height, as it is a possible key factor pertaining to banding and to only collect living snails to minimize generational mixing in the sample. Finally it would be recommended to maximize replication both within and between habitats in order to study the effects of a broader range of selective pressures.
 * __ Discussion: __**
 * __ Key terms: __**
 * 1) ** A. ** ** Allele: ** A version of a gene.
 * 2) ** B. ** ** Population ** : A collection of interbreeding individuals of the same species in a certain, defined area.
 * 3) ** C. ** ** Selection ** : Acts on the phenotype. This is where there are certain genotypes that are more likely to increase in frequency.
 * 4) ** D. ** ** Gene flow ** : The exchange of genes between distinct populations
 * 5) ** E. ** ** Phenotype ** : The physical characteristics of the individual. A combination of the genes and their interaction with the environment
 * 6) ** F. ** ** Genotype ** : A description of the alleles of an individual
 * 7) ** G. ** ** Pseudoreplication: ** Where samples are thought to be independent but are actually related as a subset of the whole population (possibly due to gene flow).
 * 8) ** H. ** ** Sampling variation: ** Where the sample collected is not representative of the population as a whole.
 * 9) ** I. ** ** Search image: ** Where there is a bias or preference for a certain phenotype. This can be bias by the experimenter or a predator.
 * 10) ** J. ** ** Anvil stones: ** Sites where snail predating birds bring the snails to break their shells open. This can translocate snails from their original location and leads to an accumulation of snail shells at these sites.
 * __ References: __**
 * [1] ** Cain, A. J., King, J. M. B., Sheppard, P. M. 1960. New data on the genetics of polymorphism in the snail //Cepaea nemoralis// L. Genetics 45:393-411 34
 * [2] ** Jones, J., Leith, B. and Rawlings, P. (1977). Polymorphism in //Cepaea//: A Problem with Too Many Solutions?. //Annu. Rev. Ecol. Syst.//, 8(1), pp.109-143
 * [3] ** Emberton, l. (1963). Relationships between pigmentation of shell and of mantle in the snails //Cepaea nemoralis// (l.) And //Cepaea hortensis// (mull.). //Proceedings of the Zoological Society of London//, 140(2), pp.273-293.


 * __ Word Count: __**
 * o ** Introduction ** : 498 words
 * o ** Results ** : 261 words
 * o ** Discussion ** : 496 words