Results
Specimen Collection. For initial microsatellite PCR
amplification and gel electrophoresis assessments, we sampled 48 adult
specimens from populations of O. lignaria collected in California
(El Dorado County [n = 16], Fresno County [n =
11], Placer County [n = 13]) and Idaho (Franklin County
[n = 8]). Using other wild-caught specimens, we conducted a
population genetic analysis of 35 O. lignaria female specimens
from Birch Creek and 41 O. lignaria female specimens from Cub
River.
Microsatellite mining and assessment. Based on the filtering
criteria we defined for our microsatellite query with the O.
lignaria genome assembly, we identified 48,816 imperfect
microsatellites, 10,553 perfect microsatellites, and 263 compound
microsatellites. Average imperfect microsatellite product size is 153
nts ± 0.14 SE (maximum = 500, minimum = 100); average perfect
microsatellite size is 148 nts ± 0.27 SE (maximum = 241, minimum = 100);
and average compound microsatellite size is 171 nts ± 2.35 SE (maximum =
353, minimum = 100). We provide the filtered imperfect, perfect, and
compound microsatellite primer sets in Supplementary Tables 1, 2, and 3,
respectively.
From the queried loci, we haphazardly selected 22 microsatellites for
testing, 13 of which were perfect microsatellites and nine of which were
imperfect microsatellites (Table 1). We did not include compound
microsatellites in our study due to their small sample size of 263 and
product size overlap with perfect microsatellites. In the 76 O.
lignaria specimens tested from Cub River and Birch Creek, average
amplification success of the 22 markers was 95% ± 1% SE (maximum =
100%, minimum = 80%) (Table 1). Oli61901 demonstrated the least
amplification success with 20% of specimens not amplifying at this
locus. Oli064 demonstrated 100% amplification success in all tested
specimens, whereas Oli160, Oli84207, Oli119, Oli131, Oli216, Oli127,
Oli076, Oli053, Oli76146, and Oli03483 demonstrated 99% amplification
success in all tested specimens. Average genotyping error rate was 11%
± 3% SE (Median = 3%; maximum = 42%, minimum = 0%). Eight markers
had a genotyping error rate of 0% (i.e., sampled specimens were
genotyped consistently at these loci), and included Oli156, Oli105252,
Oli104572, Oli963, Oli144609, Oli76146, Oli141, Oli103483. Markers with
a genotyping error rate > 30% included Oli053, Oli076,
Oli107, and Oli127 (Table 1). Furthermore, a Wilcoxon Rank-sum test
found that perfect microsatellites (M = 0.11, n = 13) had
significantly larger genotype error rates than imperfect microsatellites
(M = 0, n = 9) (W = 20.5, P = 0.01).
The 22 novel microsatellite loci tested in our study were distributed
across 16 scaffolds of the O. lignaria genome assembly
(Supplementary Table 4). Oli127 and Oli84207 were placed in scaffold
NW_023009259.1; Oli107, Oli119, Oli131, and Oli216 were placed in
scaffold NW_023009260.1; and Oli104572, Oli105252, and Oli156 were
placed in scaffold NW_023009264.1. Based on the start position of each
locus, mean pairwise nucleotide distances between loci co-located on a
scaffold exceed a nucleotide distance of 500,000 nts:
NW_023009259.1DIST = 559,304 nts (n = 1),
NW_023009260.1DIST = 2,369,751 nts ± 514,195 SE
(n = 6), and NW_023009264.1DIST = 6,599,034 nts
± 3,062,327 SE (n = 3). Comparison of the loci with the annotatedO. lignaria genome assembly suggests that four of the tested
microsatellite loci are in an intergenic space, whereas 18 of the loci
are in an intragenic space of a predicted gene. The average estimated
distance of the intergenic loci to the nearest gene was 8,803 nts.
Additional information, including gene identity, of the 22 novel
microsatellites examined in this study are available in Supplementary
Table 4.
Sibship identity and population genetic analysis. Of the 35O. lignaria individuals surveyed in Birch Creek, 21 full-sibling
families were identified. Family size of the surveyed specimens in Birch
Creek range from one to three individuals (mean = 1.67 ± 0.16 SE). Of
the 41 O. lignaria individuals surveyed in Cub River, 24
full-sibling families were identified. Family size of the surveyed
specimens in Cub River ranged from one to five individuals (mean = 1.70
± 0.19 SE) (Table 2).
Seven loci, Oli160, Oli053, Oli064, Oli076, Oli127, Oli61901, and
Oli84207, were not in HWE after Bonferroni correction (Table 1). Of the
markers tested for LD and not in HWE, Oli053 was in LD with Oli076.
Conversely, the following LD marker comparisons had at least one marker
that was in HWE: Oli216 (in HWE) was in LD with Oli053 (not in HWE);
Oli131 (in HWE) was in LD with Oli076 (not in HWE); Oli101 (in HWE) was
in LD with Oli156 (in HWE) and Oli119 (in HWE); and Oli119 (in HWE) was
in LD with Oli127 (in HWE) (Table 1). For the final analysis, we elected
to remove all seven loci that were not in HWE and removed Oli101 as it
was in LD with two other markers (Oli156 and Oli119). The loci either in
HWE or demonstrating LD also demonstrated high genotype error rates
(Supplementary Tables 5, 6). These loci are unlikely to be reliable
markers for O. lignaria population genetic analysis due to
phenomena like null alleles or primers binding to multiple annealing
sites. Removal of these eight loci resulted in 14 loci suitable for
further population genetic analysis: Oli021, Oli107, Oli119, Oli131,
Oli141, Oli144609, Oli156, Oli216, Oli963, Oli74400, Oli76146,
Oli103483, Oli104572, Oli105252.
We found no significant difference between meanHO and Nei’s HE in the
Birch Creek population (mean HE = 0.63 ± 0.05 SE,
mean HO = 0.60 ± 0.07 SE; t = 0.32,df = 26, P = 0.75) or Cub River population (meanHE = 0.60 ± 0.06 SE, meanHO = 0.53 ± 0.06 SE; t = 0.71, df =
26, P = 0.48). Furthermore, we found no significant difference in
average HE between the Birch Creek or Cub River
populations (t = 0.38, df = 26, P = 0.71). In
addition to heterozygosity comparisons across populations, we found no
significant difference between the Birch Creek and Cub River populations
in Simpson’s Index of Diversity (1 - D) (Birch Creek mean [1-D] =
0.61 ± 0.05 SE; Cub River mean [1-D] = 0.58 ± 0.06 SE; t =
-0.35, df = 28, P = 0.73), allelic richness (Birch Creek
mean AR = 0.40 ± 0.06 SE; Cub River mean AR = 0.33 ± 0.07 SE; t =
0.79, df = 26, P = 0.44), the number of alleles (NA)
(Birch Creek mean NA = 6.86 ± 0.94 SE; Cub River mean NA = 7.36 ± 1.51
SE; t = -0.26, df = 26, P = 0.79), or private
alleles (PA) (Birch Creek M PA = 2; Cub River M PA = 1.5; Wilcoxon
rank-sum test, W = 112.5, P = 0.51). All population genetic
diversity and richness statistics and indices are described in Table 3.
AMOVA found evidence for genetic differentiation between the two
Intermountain North America populations (Table 4). Specifically, 1.87%
of the genetic variance was partitioned between populations, 4.83% of
the genetic variance was partitioned between individuals within a
population, and 93.3% of the genetic variance was partitioned within
individuals across populations (Table 4). While high variance across
individuals in both populations was observed, we found that
ΦST is highest in variance comparisons between
populations (ΦST = 0.07) as opposed to between
individuals within populations (ΦST = 0.04) (Table 4).
Furthermore, based on 999 permutations of the data, we present
statistical inference to support the conclusion that these populations
are differentiated at all population stratifications (Table 4). While
population structure estimates are low, estimates of ΦSTbetween the two populations is further supported by analogs of this
index in FST (1.66 X 10-2,P = 0.01), GST (8.39 X
10-3, P = 0.01) and Jost’s D (1.18 X
10-2, P = 0.05). Finally, an assessment of the
inbreeding with FIS implicates no evidence of
inbreeding across populations (FIS = 4.20 x
10-3, P = 0.99).
Osmia subspecies, species, and clade marker
amplification. In total, eight loci consistently amplified in all four
clades: Oli101, Oli84207, Oli131, Oli216, Oli127, Oli076, Oli156, and
Oli105252. Nine loci amplified in three species/subspecies ofapicata , 11 loci amplified in seven species/subspecies ofemarginata , and all 22 loci amplified 11 species/subspecies ofbicornis and two species/subspecies of ribifloris (Table
5, Supplementary Table 7). Average marker amplification rate across
specimens was 21% ± 6% (n = 3), 51% ± 5% (n = 27),
22% ± 6% (n = 7), and 66% ± 7% (n = 11) in the cladesapicata , bicornis , emarginata , andribifloris, respectively.