Population genetic structure
Individual samples of M. myotis suitable for genetic analyses
were 120 for the microsatellite analysis and 112 samples for the
mitochondrial analysis, as detailed in Table 1. Data were obtained from
25-30 female bats sampled in May from four out of five colonies, while
no more than nine samples were collected from colony three due to its
small size and scarce accessibility. In order to secure the robustness
of genetic analyses, we then run an additional set of tests using a
reduced dataset including only nine individuals for each colony. These
analyses showed consistent results with the main tests (Supplementary
Methods).
Among the 15 microsatellites markers published for M. myotis(Castella & Ruedi, 2000), 12 were selected to carry out this study as
they i) could be successfully amplified in most samples, ii) showed
variability within the populations, iii) had no evidence for null allele
and iv) respected the Hardy Weinberg equilibrium (Table S1). Genotyping
of all individuals allowed for the detection of 109 distinct alleles,
with an overall mean of 9.5 alleles across loci, ranging from 6.83 to
10.58 within each of the investigated roost (Table 2). Genetic analyses
based on allele frequency for the 12 selected microsatellites supported
low genetic differentiation between different roosts
(FST = 0.030, SE 0.006). Indeed, pairwise
FST values between colonies ranged between 0.009 and
0.036 (mean 0.0193) and showed significant although little
differentiation only for the small colony three (p-value = 0.001) (Table
3). This same colony also showed the lowest mean allelic richness across
loci (Table 2).
Regarding genetic analyses on the mitochondrial DNA, we aligned
sequences of 413 bp, among which 20 sites were variable among the South
Tyrolean dataset, seven within the HVI and 13 within the HVII regions,
defining nine distinctive haplotypes included in two main clades (Figure
2, Table S4). Mean nucleotide distance was 0.12% within groups and
4.5% between groups. The most common haplotype was Hc_01, found in
66% of all individuals and in all colonies, with frequency ranging
between 41% and 75% in different locations. The overall frequency of
the other haplotypes varied between 0.9% and 17%, with five haplotypes
found in a single colony, one found in two colonies and two found in
four colonies (Table S4). In each colony, we found three to seven
distinct haplotypes, with colony two showing the highest variability
(n=7) and the highest number of private alleles (n=3) (Figure 3, Table
2, TableS4). Indeed, mean gene and nucleotide diversity confirmed to be
higher within this colony (Table 2). In turn, colony two showed the
highest mean nucleotide distance from other colonies based on sequences
from HVI+HVII (Table 3). Similarly to what was observed for
microsatellites, our data support a very limited geographical
structuring in the area, with FST values ranging between
-0.013 and 0.084 (mean 0.02), and showing significant although low
differentiation only between colonies 1 and 2 (FST:
0.084; p value: 0.019) (Table 3). The Mantel test detected a no sign for
isolation by distance based on either mitochondrial (R = 0.287,
p=0.052), or nuclear markers (R= 0.011, p=0.43) (Figure S3).