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 (available at, 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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