Computational protocol: Mature habitats associated with genetic divergence despite strong dispersal ability in an arthropod

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[…] Total genomic DNA was extracted by using QuickExtract (Epicentre). Samples were homogenized in 30–50 μL of the QuickExtract solution, incubated at 65°C for 2 h and 98°C for 10 min, and stored at -20°C. A c. 1000 bp fragment of the mitochondrial protein-coding NADH-2 (ND2) gene was amplified using the primers MetF3 (5'-GTT CAT GCC CCA TTT ATA GGT TA-3') and TrpR (5'-GAA GGT TTT TAG TTT AGT TAA CTT AAA ATT CT-3') []. A c. 760 bp fragment of the nuclear protein-coding HSP90 gene was amplified using the primers (5'-TTA CGA GTC CAG ATG GGC TT-3') and (5'-ATC CGT TAT GAA TCC CTG ACT GA-3') []. Each 50 μL PCR reaction consisted of 5 μL of extracted DNA, 10× PCR buffer [50 mM KCl, 1.5 mg MgCl2, 10 mM Tris-HCl pH 8.3, 0.01% (w/v) gelatin], 2 mM of each dNTP, 1 μM of each primer and 1 unit of Taq DNA polymerase. The PCR temperature profile for the mitochondrial ND2 gene was as follows: 40 cycles of 94°C for 30 s, 48°C for 30 s and 72°C for 1 min, and final extension at 72°C for 5 min. The PCR temperature profile for the nuclear HSP90 gene was as follows: 40 cycles of 94°C for 30 s, 50°C for 30 s and 72°C for 1 min, and final extension at 72°C for 5 min. Because ND2 PCR products from two Japanese populations had multiple peaks in their sequences, cloning was performed for the products using the TOPO TA Cloning Kit (Invitrogen). Then, three cultured colonies were used for sequencing. For HSP90, all PCR products were cloned using the TOPO TA Cloning Kit, and then four or five cultured colonies were sequenced. Sequences of ND2 and HSP90 were obtained in both directions by Genaissance pharmaceuticals (Connecticut, USA) or Roswell Park Cancer Institute (New York, USA). Sequences were assembled, edited with Sequencher 4.2 (Gene Code Corporation), and aligned manually with Se-Al 2.0 []. [...] For the mitochondrial phylogeny of Holarctic D. rosea s.l., we used a total of 403 individuals of D. rosea s.l from 84 Holarctic locations: 212 individuals from 30 non-Beringia North American populations (in North America except Alaska and Yukon), 43 individuals from nine Beringia North American populations (in Alaska, USA and Yukon, Canada), 97 individuals from 19 Japanese populations, 25 individuals from six Siberian populations, and 26 individuals from 20 European populations [see Additional file ]. The best-fit maximum likelihood (ML) model was selected by hierarchical likelihood ratio tests in the program Modeltest 3.7 []. The ML distance was based on the best fit model and calculated using PAUP v4.0b10 []. An Neighbor-joining (NJ) tree was constructed with the ML distance and 1000 bootstrap replicates using PAUP.Population level information was available for the mtDNA of specimens from North America and Japan. We calculated FST with the estimator of Hudson et al. [] and the nearest-neighbor statistic (Snn) of Hudson [] as implemented in DnaSP 4.10 []. With Hudson et al.'s estimator of FST, each polymorphic site in an alignment is treated as a locus and FST = 1-Hw/Hb. Hw is the mean number of sequence differences among different haplotypes sampled from the same subpopulation and Hb is the mean number of sequence differences among samples from different subpopulations []. This estimator is equivalent to the NST estimator of Lynch & Crease [] without a Jukes-Cantor correction for multiple substitutions. Snn is a measure of how often the most closely related sequences (nearest-neighbors) occur in the same locality (water bodies in the present case). The number of private haplotypes (which occur in only one population) was counted. Haplotype diversities and nucleotide diversities were calculated using DnaSP [].We further analyzed phylogeography of the North American and Japanese populations using mitochondrial and nuclear data. For mitochondrial analysis, we used the same data of ND2 sequences that was used in construction of the Holarctic D. rosea s.l. phylogeny. For nuclear analysis, we used 36 individuals of the D. rosea s.l: 13 individuals from 13 non-Beringia North American populations, six individuals from six Beringia North America, and 17 individuals from 17 Japanese populations. Intron boundaries of the HSP90 sequences were identified by comparing arthropod HSP90 mRNA sequences (e.g. AY528900, AY423488) and by examining the intron-splicing signature sequences. Two introns were identified and aligned using clustalW. For mitochondrial and nuclear data of North American and Japanese D. rosea s.l., the best-fit ML model was selected by hierarchical likelihood ratio tests using Modeltest. ML distance was calculated using PAUP. NJ phylogeny was constructed with ML distance and 1000 bootstrap replicates using PAUP. The Mantel test [] was used to determine the relationship between genetic distance (pairwise FST from Arlequin 3.01 [], and pairwise ML divergence) and geographic distance to test an isolation-by-distance model. The geographic distance was calculated using the great-circle distance formula. The Mantel test was implemented in IBD 1.52 [] using 1000 permutations. The computer program TCS 1.21 [] was used to estimate the mitochondrial haplotype network that illustrates all connections that have 95% probability of being the most parsimonious. Analysis of molecular variance (AMOVA) was carried out for Japanese regional groups (Jp1-8 in Fig. ) using the TrN + G (Tamura-Nei distance with gamma distribution) by Arlequin with 1000 permutations.To address the population expansion of major North American clades in mitochondrial and nuclear phylogenies, we performed the neutrality tests of Fu's FS [] and Tajima's D [] and revealed the frequency distribution of the number of pairwise sequence differences (i.e. the mismatch distribution). The values of Fu's FS and Tajima's D are expected to have significantly negative values for population expansion (other explanations are background selection and hitch-hiking associated with selective sweeps). The significance of Fu's FS and Tajima's D were tested by random permutation using 1000 replicates in ARLEQUIN 3.01 []. The mismatch distributions were calculated using ARLEQUIN. The observed distributions were compared with the simulated distributions under the models of pure demographic expansion and spatial expansion. The model of pure demographic expansion assumes that non-subdivided populations suddenly expand in population size and increase the total number of individuals []. The model of spatial expansion assumes that subdivided populations expand the distribution range and increase the total number of individuals [,]. Both models estimate Tau = 2ut where u = mTμ. t is the estimated time of expansion, mT is the number of nucleotide sequences under study, and μ is the mutation rate per time. A least square procedure and bootstrap approach (1000 replications) provided the probability that the sum of squared deviations is lower in the observed distribution than in the simulated distributions, and calculated the 95% confidence intervals of the expected distribution and Tau. […]

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