Computational protocol: Morphological and genetic divergence in Swedish postglacial stickleback (Pungitius pungitius) populations

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Protocol publication

[…] Total genomic DNA was extracted from finclips of all individuals in each sampled location using a DNeasy ® Blood & Tissue Kit (product #69581, Quigen, Valencia, USA). Nine polymorphic microsatellite markers (Pbbe1125, Stn19, Stn49, Stn96, Stn148, Stn163, Stn173, Stn196, Stn198) previously used to characterize P. pungitius [] were individually labelled with florescent primers. Microsatellite markers were amplified using polymerase chain reaction (PCR) in a 10 μl reaction using a multiplex PCR kit (Qiagen Inc.). PCR conditions for each reaction using the multiplex kit were 5 μl of Qiagen Master Mix (containing HotStarTaq DNA polymerase, dNTPs and 3 mM MgCl2), 0.625 μM of each primer, and 3 μl of genomic DNA. Temperature profile for thermal cycling: 96°C for 15 min followed by 35 cycles of 94°C for 30 sec, 60°C for 90 sec, 72°C for 90 sec, and a final 20 min extension at 72°C. Fragment lengths of PCR products were analyzed using CEQ™ 800 Genetic Analysis System (Beckman Coulter Inc., Brea, USA).Microsatellite data were analyzed with ARLEQUIN v. 3.11 [] to test for Hardy-Weinberg equilibrium (Fisher's exact test) and for genotypic disequilibrium for pairs of loci within populations (Fisher's exact test). Loci were checked the scoring errors and the presence of null alleles using MICROCHECKER v 2.2.3 []. We also tested all loci for neutrality using the program LOSITAN using a multi-step mutation model and 10,000 replicates of data collection [,].A single microsatellite locus, Stn49 showed a mutation consisting of a one base pair insertion. This mutation is common in eastern European lineages of P. pungitius [] and it was included in all subsequent analyses except for analyses conducted with MICROCHECKER, LOSITAN, and BOTTLENECK because this locus does not follow a typical dinucleotide stepwise mutation pattern [].Nei's genetic distance (DA) [] was used to construct a phylogeny based on microsatellite data for the 18 nine-spined stickleback populations using the program POPULATIONS v. 1.2.30 []. Levels of bootstrap support were obtained from 10,000 replicates and the resulting neighbour-joining tree was drawn with POPULATIONS.Genetic differentiation, as measured by FST [], was calculated between each population pair with ARLEQUIN. Significance of global pairwise FST values was estimated with ARLEQUIN using 10,000 permutations. In order to compare measures of genetic differentiation corrected for differences between populations with different levels of allelic richness and heterozygosity, we calculated standardized FST values with GENODIVE v. 2.0b18 [].We tested for differences in within-group genetic variation (Baltic coast, coastal lakes, inland lakes with or without predators) and between coastal (pooled Baltic coast, coastal lakes) and inland lakes (pooled predators, no predators). Our estimates of genetic variation included observed heterozygosity (Ho), expected heterozygosity (He) allelic richness (A), and global FST values with FSTAT v [] using the "comparison among groups of samples" subfunction. Significance was ascertained for these parameters over 10,000 permutations.To investigate whether populations bear the signature of a recent fluctuation in population size, we investigated heterozygosity deficiency and heterozygosity excess with respect to gene diversity using the program BOTTLENECK v. 1.2.02 with 1,000 replications of a two-phase mutation model []. The two-phase model of mutation consisted of mostly of one-step changes with a low percentage of multistep changes (9:1), recommended by Di Rienzo et al. [] for microsatellites. […]

Pipeline specifications

Software tools Arlequin, Genodive
Applications Phylogenetics, Population genetic analysis
Organisms Pungitius pungitius, Hemisus marmoratus