Genetic structure of M. myotis from South Tyrol
DNA was extracted from wing biopsies using the DNeasy blood and tissue commercial kit (QIAGEN) according to the manufacturer’s instructions. The partial cytochrome b (cytb) was amplified as described elsewhere (Irwin, Kocher, & Wilson, 1991), in order to confirm the identification based on morphological parameters. Only samples genetically confirmed asM. myotis (n=166) were included in the following analyses.
The genetic structure of the target population was determined combining analyses based on nuclear DNA (bi-parental inheritance) and mitochondrial DNA (maternal inheritance), in order to account for sex-biased gene flow. In addition, we used only data from females sampled from each of the five colonies during the same week in May (Table 1), thus avoiding possible issues related to animal movements during the reproductive season. On the other hand, we considered the sampling of adult males within maternity colonies as occasional and excluded these individuals (n=11 sampled in May) from analyses.
Nuclear genetic structure was investigated using microsatellites available for this species in the literature (Castella, Ruedi, & Excoffier, 2001) (Table S1). Forward primers were labelled with florescent dyes (MWG-Operon) and PCR amplification was carried out in three multiplex mixtures containing 10-20 ng of template DNA, using the Qiagen multiplex kit (QIAGEN) (Table S1). The amplification protocol was as follows: initial denaturation step at 95°C for 15 minutes; 35 cycles of denaturation for 30 seconds, annealing at 60°C, 55°C or 50°C for 90 seconds for mix 1, 2 and 3 respectively, and extension at 72°C for 1 minute; final extension step at 72°C for 10 minutes. PCR products diluted 1:100 were analyzed on an ABI PRISM® 3130xl automatic sequencer (Applied Biosystem, Foster City, CA). The molecular size of microsatellite alleles was evaluated by using GeneMapper4.0. The reliability of all microsatellites was tested for each locus in terms of the presence of null alleles, the coefficient of inbreeding and the deviation from Hardy-Weinberg equilibrium (HWE) using a Chi-square test implemented in Micro-checker 2.2.3.
Two hypervariable domains of the mitochondrial control region (CR) were used to investigate the genetic structure at the mitochondrial level. In detail, the full control region of the mitochondrion was PCR-amplified in two overlapping fragments of about 1200 bp, using the Platinum TAQ kit (Invitrogen), according to the manufacturer’s instructions. Primers were developed using reference sequences from the mitochondrion ofM. myotis (Table S2). The amplification protocol was as follows: initial denaturation step at 94°C for 2 minutes; 40 cycles of denaturation for 30 seconds, annealing at 51°C or 56°C for 30 seconds respectively for PCR1 and 2, and extension at 72°C for 2 minutes; final extension step at 72°C for 5 minutes. PCR products were sequenced on an ABI PRISM® 3130xl genetic analyzer. Sequences were trimmed to obtain two fragments of the hypervariable HVI and HVII domains. Haplotypes for the concatenated HVI and HVII were inferred in MEGA 6.0 (Tamura, Stecher, Peterson, Filipski, & Kumar, 2013).
Genetic information obtained for the nuclear and mitochondrial DNA were used to investigate the genetic population structure of M. myotiswithin South Tyrol. Analyses were carried out assuming each bat belonged to a distinct population based on its roost of sampling. We first determined the genetic variability existing within each separate roost. In particular, we calculated allelic richness across loci (A) and expected heterozygosity (HE) from microsatellites data using GeneAlex, while the number of haplotypes (N), gene (h) and nucleotide (π) diversity of mitochondrial sequences were calculated in Arlequin 2.0 (Castella et al., 2001; Ruedi et al., 2008). In addition, we used MEGA 6 to determine mean nucleotidic distance between individuals from different roosts based on the concatenated HVI and HVII. We then assessed the genetic differentiation between populations at both the nuclear and mitochondrial level, as expressed by the fixation index (FST) calculated using Genealex and Arlequin 2.0 respectively. We considered little difference for FST values ranging from 0 to 0.05, moderate from 0.05 to 0.15 and great for values above 0.15 (Balloux & Lugon-Moulin, 2002). We tested the hypothesis of genetic isolation by distance (IBD) by plotting linearized FST values=FST/(1-FS) (Slatkin, 1995) against the ln(geographical distance) using a Mantel test implemented in GeneAlex (Rousset, 1997). In particular, we used geographical 2D distances calculated along the valleys’ bottom rather than Euclidean distances, as they are likely closer to real distances covered by bats when flying.
For mitochondrial data only, we also investigated the phylogenetic relationship between haplotypes through Maximum Likelihood (ML) phylogenetic analyses implemented in PhyML 3.0 software using the general time-reversible (GTR) model of nucleotide substitution with gamma-distributed rate variation among sites (with four rate categories, G4) and a heuristic SPR branch-swapping search (Dereeper et al., 2008). One thousand bootstrap replications were performed to assess the robustness of individual nodes. Mean nucleotide distance between and within clades thus identified was calculated in MEGA 6 (Tamura et al., 2013).