Computational protocol: Populations of a cyprinid fish are self-sustaining despite widespread feminization of males

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[…] To understand the extent to which roach populations are restricted to various stretches of river, several approaches were used to investigate population genetic structure. We analysed microsatellite loci variation in 1,769 fish sampled between 1995 and 2011. Each fish was genotyped at between 14 to 19 microsatellite loci. Microsatellite genotypes are provided in Additional file . Protocols for DNA extraction and details of amplification of the microsatellite loci are illustrated in Additional file . Data for 14 microsatellite loci were used to calculate three measures of genetic diversity: observed heterozygosity (HO) and expected heterozygosity (He) using GenAlEx 6 []; allelic richness (AR) was calculated using Fstat v2.9.3 [] – see Additional file for full details. The programme BOTTLENECK [,] was used to test for recent genetic bottlenecks. This programme tests for a relative excess in heterozygosity that is apparent for a few generations after a bottleneck and develops because allelic diversity declines faster than heterozygosity, due to loss of rare alleles. Pairwise genetic differentiation between the sampled sites was estimated using FST, calculated using Arlequin 3.5 [] and Jost’s D, Dest[], calculated using SMOGD []. The significance of the FST estimates was assessed based on 10,000 permutations. AMOVA was performed using Arlequin. In order to test whether fish are more likely to produce offspring with local mates, compared to mates in geographically distant locations within the Thames catchment, isolation by distance analysis was performed using the Mantel test [] in GenAlEx 6 []. Genetic similarity between populations was investigated using population based trees, calculated in POPULATIONS, v1.2.30beta [], PCA in GenAlEx 6 [] and a Bayesian clustering approach in STRUCTURE []. Finally, the program IMA2 [,] was used to estimate migration rates between adjacent populations within high effluent stretches of the Lee, giving relatively high pairwise FST values (LeeHyd, LeeWhe, and LeeSta). See Additional file for further details. To investigate the influence of restocking, genetic assignment of fish from the Wandle was undertaken using the ‘leave one out test’ in the computer program, ONCOR [], based on their microsatellite genotypes. The reporting regions comprised: (1) the Wandle, (2) Lee/Stort, (3) rest of the Thames, (4) Trent, (5) Nene, (6) Arun, (7) Chelmer and (8) Anglian Blackwater. All animals used in this research were treated humanely and with regard for the alleviation of suffering; all procedures were subject to approval by the local ethical review process as required under the U.K. Animals (Scientific Procedures) Act (1986). [...] To test whether WWTW effluents substantially reduce the size of breeding populations, effective population sizes (Ne), which relate to the number of breeding fish and skews in breeding success, were estimated using the microsatellite genotypes. We compared Ne from sites ranging from little/no upstream WWTW effluent inputs to those where the majority of the flow can comprise WWTW effluent. Two single sample (generation) methods, that use different aspects of the microsatellite data, were used to estimate Ne for each population; the Approximate Bayesian Computation (ABC) method using ONeSAMP 1.2 [], hereafter referred to as Ne(ABC); and the sibling assignment method (SA), Ne(SA)[]. Temporal estimates for Ne, which are calculated from the change in allele frequencies between generations, were also estimated for sites where fish had been sampled more than once using TempoFs [] and NeEstimator []. For further details see Additional file . […]

Pipeline specifications

Software tools GenAlEx, Arlequin, NeEstimator
Application Population genetic analysis
Chemicals Estrogens