Computational protocol: Genetic differentiation between cave and surface-dwelling populations of Garra barreimiae (Cyprinidae) in Oman

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[…] Sequences were edited in BioEdit version 5.0.9 []. The alignment of sequences from the genus Garra was straightforward because there were very few insertions or deletions. Besides G. rufa, the following sequences from GenBank were used as outgroup: Cyprinus carpio (X61010), Carassius gibelio (GQ985484), Labeo senegalensis (AB238968), and Barbus barbus (AB238965). The alignment including sequences from other genera was performed with t-coffee [] and adjusted manually (no regions of the alignment were deleted prior to the analyses). Average p-distances (pairwise exclusion of gaps) were calculated using MEGA version 4 [], which was also used to calculate NJ trees []. Nodal support was evaluated with nonparametric bootstrapping based on 1000 replicates. BI analyses were performed using MrBayes 3.1.2 [] applying the following models of sequence evolution for nucleotide sequences according to the Akaike information criterion (AIC) as implemented in the program MrModeltest v2.2 []: HKY+G (for the CR1 data set) and TrN+G (for the combined: CR1+CR2 data set). Runs were started with random trees and performed for 2 million generations each with four Markov chains and a sampling frequency of every 100th generation. Those trees generated prior to stationarity were discarded as burn-in and were not included in the calculation of the consensus trees. An unrooted parsimony haplotype network (95% probability level) of clade 3 was constructed by the method of [] with the software TCS version 1.21 [], treating gaps as 5th character state. A full median-joining network [] of the complete G. barreimiae CR1 data set was constructed with network 4.6.0.0 (available at http://www.fluxus-engineering.com), putting equal weight on each site. The numbers of haplotypes and the haplotype diversities were calculated with ARLEQUIN 3.11 []. Past demographic fluctuations were assessed by calculating the mismatch distribution [] as implemented in DnaSP v5 [] to fit the observed data with models of population expansion. As a statistical test for population growth, we calculated the R2 statistic [] and Fu's Fs test statistic []. In both cases small values suggest recent demographic expansion. Significance (α = 0.05) of both the R2 statistic [] and Fu's Fs test statistic were evaluated by comparison to a distribution generated from 10,000 random simulations of the data in DnaSP v5.Analyzing demographic fluctuations requires having a reasonable assumption about what a population actually is and to have an appropriate sampling. We chose Al Hoota Cave (25 specimens) and the group of localities found within clade 3 (except Al Hoota Cave; 97 individuals) as the only two populations for which a mismatch analysis is suitable. For the other genetically isolated clades, a mismatch analysis is not meaningful: The sample size of clade 2 is too small, and clade 3 is composed of two geographically disjunct populations (see results).The sequences determined in this study are deposited at GenBank under the accession numbers: CR1: JF709013 - JF709129; CR1+CR2: JF709135 - JF709162; complete CR: JF709130 - JF709134. […]

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