Groupx+22

__**The Snailers**__ Adil Aslam Taran Dhillon Oliver Farrell Anna Forizs Cameron Mills Isabel Robson

__**Board Meetings:**__ __01/10/2014__ Attendees: Adil Aslam, Taran Dhillon, Oliver Farrell, Anna Forizs, Cameron Mills, Isabel Robson Discussion: Adil suggested using a line transect, Cameron added 'stratified'. Anna suggested using quadrats and Oliver pointed out we may not find enough //Cepaea nemoralis// in a one metre squared quadrat. Isabel suggested using a ten metre squared area to collect. Anna brought up whether both dead and alive //C. nemoralis// should be collected, Cameron said collecting both may be useful to see what polymorphic traits are successful (e.g. small dead //C. nemoralis// would be deemed unsuccessful, large dead //C. nemoralis// would be deemed successful since size indicates how long they have lived). Taran suggested collecting just dead //C. nemoralis// so we avoid aggravating the //C. nemoralis// of Buckinghamshire, the rest of the group agreed to collect only dead //C. nemoralis//. The whole group vowed not to kill live //C. nemoralis// in order to count them in our 'dead //C. nemoralis//'. The whole group came to the conclusion that we should do collecting over all three habitats (grass, shrubbery and forest) twice so we have a repeat to make our results more reliable. Oliver noticed, that for this to work, we have to do both sets of the three at the same elevation to be able to compare them fairly. But, in a dramatic twist of events, Taran pointed out the shrubbery in the top right hand corner of the map and suggested we do one set of three at low elevation and the other at high elevation - this means we can compare elevation as well as habitat.

__08/10/2014__ Attendees: Adil Aslam, Taran Dhillon, Oliver Farrell, Anna Forizs, Cameron Mills, Isabel Robson Discussion: Adil and Isabel discuss revision of the hypotheses after an enlightening lecture. Adil shortened 'polymorphic traits' to 'polymorphism' and they discussed how in the experiment, the effects of selection, gene flow, genetic drift and mutation have to be considered but what we were focusing on was selection therefore agreed on the null/alternative hypotheses being 'Polymorphism in //C. nemoralis// is not/is due to selection' respectively. It would then be explained in the introduction that we are aware of the effects of selection, gene flow, genetic drift and mutation on polymorphism. Our hypotheses indicate what will or will not be mainly responsible for polymorphism. Anna asked the group why we are studying //C. nemoralis// rather than another species. The group referred to the lecture where we previously discussed this - Anna remembered that when she watched //C. nemoralis// in her childhood, they moved very slowly. Taran expanded on this saying this creates distinct polymorphic habitats that would be easy for us to view and Oliver rounded it off by summarising that this created a small geographical scale that is manageable to experiment on. Isabel compared this to //Homo sapiens// where there is a lot (thousands of kilometres by aeroplane) of travel between almost all populations allowing for a lot of gene flow. Anna remembered that snails 'wear their genes on their shells' making it easy to determine genotype from phenotype. Anna remembered that we are studying //C. nemoralis// since it is the most polymorphic UK land snail meaning that it has the largest/clearest variations in phenotype. Cameron asked whether elevation should be taken into account and the group voted for and against including elevation in the experiment respectively 2 (Oliver and Anna) - 4 (Adil, Taran, Cameron and Isabel). Isabel stressed the importance of sampling all three habitats (grass, shrubbery and forest) at the same elevation largely resembling a line transect. Oliver brought up the different distances between the shubbery on the left and the forest compared with the shrubbery on the right and the forest. The group recognised this as an important consideration for our write up but acknowledged that little could be done to resolve this problem without varying elevation. Adil advised against a half-and-half habitat sample (e.g. half forest, half grass) and the group concurred. Cameron suggested whether we would want to reconsider collecting only dead //C. nemoralis// and suggested collecting both dead and alive recording which individuals were and alive and which alive on our record - the group agreed.

__15/10/2014__ Attendees: Adil Aslam, Anna Forizs, Cameron Mills, Isabel Robson Discussion: *Any numbers in brackets refer to the designs as seen below, names are given beside to indicate the person responsible for the idea* After another awe-inspiring lecture, Isabel recommended having fewer variables to be able to draw more reliable conclusions. This also means more experimental designs available so the team started on making new designs. Anna suggests three samples in the forest far away from each other with three samples in the grass adjacent to each of them (1). Isabel and Adil say we won't be able conclude selection if gene flow is prominent (i.e. samples are close together) then Anna says this depends on how similar our individuals (in phenotype) are. Adil suggests doing all six samples in grass (6). Isabel suggests rather than ten metre squared samples, picking a point then collecting thirty individuals nearest to that point because it is unclear whether we are collecting all individuals in a sample or only thirty. This is suggested to make sure there are no groups with zero frequency to make chi squared test more reliable. Adil suggests sticking to ten metre squared sample and collecting thrity individuals randomly. Anna suggests (2), Cameron says we need fewer variables and Anna agrees. Cameron likes (5) since samples are all fairly close. Adil reminds the group to consider what is practical when it comes to designing our experiment and stresses that shrubbery may be an awkward place to collect. Anna likes (8) but Cameron thinks we could afford to include more variables. Adil and Cameron democratically say that all designs have their merits.



__29/10/2014__ Attendees: Adil Aslam, Taran Dhillon, Oliver Farrell, Anna Forizs, Cameron Mills, Isabel Robson Discussion: This meeting mainly consisted of The Snailers agreeing on one final design for the collection day and outlining the reasons for the chosen design. After agreeing which design we should follow (see below) Isabel suggested going around the group giving each person the opportunity to say one point about why they thought this idea was the most suitable. Adil said that we are sampling two different habitats and therefore two different selection pressures so we should see polymorphism in accordance with these two selection pressure and therefore habitats. Anna mentioned the importance of the replication (two habitats, repeated twice) and how this will lead to our results being more reliable. Cameron noted that all samples are taken at very similar elevation which would decrease the effects that elevation may have on //C. nemoralis// phenotype. Ollie mentioned the replication of samples in order to reduce sampling variation. Isabel brought up that they are 'isolated' populations since one snail on average in its lifetime will travel twenty metres so gene flow between  be reduced significantly. Isabel said that gene flow between 'A's and 'B's was a good thing if there is phenotypic variation between them and different selection pressures since this will support our hypothesis in the face of possible gene flow. The group agrees on collecting both dead and alive snails, noting which are dead and which are alive. 'A's and 'B's should be 20m apart to allow some gene flow so 'A's and 'B's can be considered as connected populations rather than individual ones. 1, 2 and 3 will be at least 60m apart so they can be considered individual populations. Isabel reminds the group that snails are able to travel one metre per hour but this is not often in a straight line, snails move on average twenty metres in their lifetime and have been shown to be somewhat loyal to certain areas. There shall be a ten metre squared sampling area for all samples.

Introduction of the Research Design

The aim of this research is to try to establish the relative importance of the underlying processes (mutation, selection, drift and gene flow) that maintain polymorphism of the species //Cepaea nemoralis//. It is suggested by Jones et al. that rather than trying to identify the one single process that explains polymorphism in //C. nemoralis//, all populations require a unique explanation. The focus here is on the processes maintaining polymorphism in the samples collected from the Costwolds National Park. //C. nemoralis// has distinct
 * “The polymorphism of //C. nemoralis// is well understood since the genes controlling major polymorphism for shell phenotypes are borne together as a Mendelian supergene (Jones et al. 2009). This implies that genotype can be inferred from the obvious variations of phenotype and can be easily quantified (discrete phenotypic classes)
 * The shells of dead snails can be studied

“The shell of //C. nemoralis// is polymorphic for colour and for the presence, number, and appearance of up to five dark bands” (Jones et al. 2009). This research only distinguishes between the tree major shell colour types (brown, pink and yellow) and the presence and number of bands regardless of shading of colours and appearance of bands.

__Technical Terms__

Gene: defined set of amino acids that code for one or more traits

Allele: version of a gene

Fitness: average contribution of a genotype to the gene pool of the next generation

Genotype: alleles of a specific gene

Phenotype: physical traits determined by genotype

Polymorphism: different phenotypes in the same species

Habitat: area inhabited by a species

Elevation: height above a given level

Selection: acts on individuals and their fitness in a specific environment

Inbreeding:

Hybrid zone:

Heterozygote:

Homozygote:

Supergene: is a group of neighbouring genes on a chromosome which are inherited together because of close genetic linkage and are functionally related in an evolutionary sense (Wikipedia definition).

Mutation: origin of all genetic differences thus polymorphism, introduces new alleles to a population but it has very small effect on allele frequency.

Selection: increases or decreases allele frequency in a population depending on allele fitness except when selection favours heterozygotes, or in hybrid zones.

Gene Flow: introduces new alleles or reintroduces lost alleles to populations, decreases differences in allele frequency between population and increases frequency variety within a population.

Genetic Drift: always in operation (unless allele is already fixed or lost), increases the differences between populations, leads to the fixation or loss of an allele.

__Hypotheses __ Null hypothesis: There is no difference in relative frequencies of phenotypes in the populations of //C. nemoralis// Alternative hypothesis: There is a difference in relative frequencies of phenotypes in the populations of //C. nemoralis//

If the null hypothesis is accepted, phenotypes will have the same frequency in all samples. If the alternative hypothesis is accepted, phenotypes will have varying frequencies in samples. Mutation is the origin of any polymorphism, selection and genetic drift create loss within a population and gene flow creates movement from one population to another.

This method only focuses on two habitat types (shrub and grass). The samples are collected at the same elevation in a 10m X 10m area. The team will collect the samples together to reduce sampling variation. Samples 1,2 and 3 (and A, B and C) will have a distance of at least 40 m apart but preferably more to minimise effects of gene flow and to collect independent samples. That will allow to analyse relationships between selection and drift in the absence of gene flow. On the other hand, sample pairs 1-A, 2-B, 3-C, will be kept close (no more than 20 m apart), where if any differences in phenotype frequencies are detected will be present despite the effects of gene flow trying to decrease differences between populations.

Jones, J. S., Leith, B. H. and Rawlings, P. 1977. Polymorphism in Cepaea: A Problem with Too Many Solutions? //Annual Review of Ecology and Systematics,// **8**, 109-143. __ Method (Old) __ The black line across the map represents the transect line to be followed while the black squares shown on the map represent the areas in which samples of 30 individuals (this amount will allow for an accurate representation of the area in which the indiviuals were sampled) of the //C. nemoralis// species of snail will be collected, both dead and live snails will be sampled to provide a an accurate representation of not only the currently total population of snails within the habitat (using the live snails sampled at each point along the transect) but also the historical populations of snails within the habitat (by sampling the dead ones). This method was chosen as it allows the maximum possible repeats for sampling locations (two samples at each different habitat (woodland, grassland, bushes)) all at the same hight. This was chosen over the previous method mentioned earlier that not only sampled each location twice, but sampled each location at two different heights, however it was decided that this would not give enough repeating power to the results due to the added variable of height reducing the repeating power significantly, and therefore is less accurate than the method now chosen.

This Method has been Revised, see below.

__ Method (New) __

The new method simplifies the research design further by eliminating one habitat type and focusing only on woodland and grass habitat. The samples are collected at the same elevation in a 10m X 10m area. Sample size is aimed to be 30 snails in one location. The team will collect the samples together to reduce sampling variation. This method increases repeating power by having an additional sample in the same habitat type. Samples 1,2 and 3 (and A, B and C) will have a distance of at least 40 m apart but preferably more to minimise effects of gene flow and to collect independent samples. That will allow to analyse relationships between selection and drift in the absence of gene flow. On the other hand, sample pairs 1-A, 2-B, 3-C, will be kept close (no more than 20 m apart), where if any differences in phenotype frequencies are detected will be present despite the effects of gene flow trying to decrease differences between populations.

__**Revised method**__ Due to physical barriers in the field location it is not possible to sample the woodland habitat. Therefore our new sampling strategy will require 3 samples to be taken in open grass habitats and the remaining 3 in shrub land habitat. By having 3 repeats of each habitat the reliability of the results will be strengthened and any pattern seen amongst all three samples is more likely to be significant. Sampling sites will be no less than 40m apart to ensure that the samples being collected are completely independent to reduce the effects of pseudo replication. At each sampling site snails will be collected by 6 individuals for 10 minutes over a 10m X 10m area. This should provide adequate time gain a thorough representation of the organisms present. By spreading out our sample sites we hope to reduce the effects of gene flow and identify phenotypic differences between the two habitat types due to selection and genetic drift.



In Field method