Computational protocol: Ongoing niche differentiation under high gene flow in a polymorphic brackish water threespine stickleback (Gasterosteus aculeatus) population

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Protocol publication

[…] Two ecto-parasites were additionally used as ecological markers to reflect long-term habitat- and diet, reflecting niche occupation. The choice of parasite species to be analyzed was based on their easy visual recognition and also their robustness with regard to sticklebacks being removed from storage in EtOH. Thus, we did not analyse Gyrodactylus spp. as the fish had been dried several times. First, the number of the crustacean copepod Thersitina gasterostei (family Ergasilidae) [Pagenstecher, 1861] was counted on the inner side of the operculum and on the muscle tissue close to the branchial arch on both sides of the fish. The sum of the copepods on both sides of the fish was used in analyses. T. gasterostei is a parasite on Holarctic euryhaline fishes [], often on gasterosteids [–], and particularly on threespine stickleback [].The second parasite was an unidentified metacercaria of Trematoda spp. encysted only on the pectoral, dorsal, anal and caudal fins. The number of Trematoda spp. metacercaria cysts were counted on both pectoral fins, the dorsal, anal and caudal fins and then summed. To clarify the identity we used three primer-pairs to amplify a 1410 bp region (combined into one sequence) of the rDNA ITS gene [] partly covering the ribosomal gene clusters 18S, ITS1, 5.8S, ITS2, 28S. PCR was performed using PuReTaq ready-to-go PCR beads (GE Healthcare), 1 mMol− 1 of each primer, and 5 ml of the extracted DNA in a 50-ml reaction volume. The thermal cycling was: 94 °C for 3 min; 35 cycles of 94 °C for 30 s, 55 °C for 47 s, and 72 °C for 1 min; and a final extension at 72 °C for 5 min. PCR products were purified by 10× diluted exoSAP-IT (USB). Cycle sequencing, using the same primers as in the PCR, was performed in 10-ml reactions using 2-ml BigDye terminator cycle sequencing ready kit (Applied Biosystems), 2 ml 5× sequencing buffer, 10 pmol primer, and 3-ml cleaned PCR product. One cyst from each of eight sticklebacks was sequenced. Data analyses were done in Sequencher 5.0 (Gene Codes Corporation, Ann Arbor, Michigan, USA) and aligned with the Crustal W algorithm in MEGA 6.0 [] using default settings. Sequences were compared in BLAST (blast.ncbi.nlm.nih.gov/Blast.cgi) using a reduced set of 871 bp to increase number of comparisons to four sequences. Evolutionary analyses were conducted in MEGA6 with evolutionary history inferred using the Minimum Evolution (ME) method [] with 1000 bootstraps []. Evolutionary distances were computed using the Maximum Composite Likelihood method [] and are in units of number of base substitutions per site. The ME tree was searched using the Close-Neighbor-Interchange (CNI) algorithm [] at a search level of 1. The Neighbor-joining algorithm [] was used to generate the initial tree. Codon positions included were 1st + 2nd + 3rd + Noncoding. All positions with gaps and missing data were eliminated. [...] DNA was extracted from pectoral fins using standard proteinase K phenol chloroform protocol []. A set of 25 microsatellites, where 12 loci were a priori suggested to be neutral and 13 loci to be QTLs, were analyzed. Specific information about all the primers, PRC run conditions, binning and applied laboratory methods are reported in Le Rouzic et al. [] and Klepaker et al. []. Two of the markers, Stn381 and Stn382, are situated in two introns of the Ectodysplasin gene (Eda) that is a major determinant of lateral plate morphs partitioning the completely plated, partially plated and the low plated morph [, ]. We analyzed individuals in the upper and lower part of Lake Engervann in spring 2007 (Table ) as this should be sufficient for testing for potential population genetic structure. Genetic analyses were performed on all genotyped stickleback in Lake Engervann together assuming that all plate morphs belonged to a single population.Microsatellites were first screened in MICRO-CHECKER 2.2.3 [] to evaluate presence of stutter, allelic drop-out, homozygote excess and null-alleles. Four loci (Stn152, Stn180, Stn211, Stn271) showed homozygote excess/null alleles in two or three comparisons and were removed from all the further analyses.To test if microsatellites were neutral or candidates for either directional or balancing selection, all 21 loci were run in LOSITAN [, ] under the stepwise mutation model (SMM) and the infinite alleles model (IAM). Here, we used 100,000 simulation with the “Neutral mean Fst” and “Force mean Fst” options when analyzing data separately for IAM and SMM models. For all the simulations, the two microsatellites Stn381 and Stn382 emerged as candidates for directional selection and was thus removed before analyzing the rest of the markers collectively. None of the remaining 19 microsatellites showed any signals of selection and were therefore inferred and used as neutral markers in population genetic structure analysis.Genotypic linkage disequilibrium and deviations from Hardy-Weinberg equilibrium (HWE) were analyzed by the exact (probability) test estimated in GENEPOP 4.0.10 []. The results showed that a total of 4 loci differed from HWE, and only 3 after Bonferroni corrections. To be conservative, we thus removed the three loci (Stn178, Stn180 and Gac2111) from further population genetic analysis.To test for population genetic structure, we used Bayesian population clustering in STRUCTURE 2.3.3. [] with the 17 neutral microsatellites. We used an admixture model, correlated gene frequencies, 500,000 burn-in steps and 700,000 MCMC iterations, with K set to vary between 1 and 3 clusters with 5 replicates for each K. In addition, we conducted similar STRUCTURE analyses with the same burn-in steps and MCMC iterations, but now using the locprior option comparing (i) upper versus lower lake section regardless of morph (K:1–3, 5 replicates), (ii) three plate morphs (K:1–5, 5 replicates), (iii) or contrasting the morphs x location (K:1–6), 5 replicates. These additional analyses were performed with all loci (including loci deviating from HWE), but excluding Stn381 and Stn382 (being Eda linked loci), and null allele loci. The results were evaluated based on Evanno et al. [] and Pritchard et al. [] also using STRUCTURE-HARVESTER v0.6.93 []. We also ran a DAPC analysis (a principal component analyses) using adegent (http://adegenet.r-forge.r-project.org/). […]

Pipeline specifications

Software tools MUSCLE, Sequencher, MEGA, Genepop, adegenet
Applications Population genetic analysis, Nucleotide sequence alignment
Organisms Gasterosteus aculeatus, Hemisus marmoratus, Toxoplasma gondii
Chemicals Ectodysplasins