Microsatellite Selection
We tested three sample types: tissue, ‘high’, and ‘low’ quality museum
specimens to evaluate differential amplification across microsatellites
of varying lengths. Degradation of samples is thought to occur quickly,
but age alone is a poor predictor of the overall quality (Campana et
al., 2012; Hawkins, Hofman, et al., 2016; McDonough et al., 2018).
Previously published microsatellites from the northern flying squirrel
(G. sabrinus ) - the sister taxa of the Humboldt’s flying squirrel
- were used in this study (Loci names: GS-2, GS-4; Zittlau, Davis, &
Strobeck, 2000, and GLSA-12, GLSA-22, and GLSA-52; Kiesow, Wallace, &
Britten, 2011, Table 2).
Microsatellites were selected by length and type of repeat motif. Two
microsatellites were selected in our ‘short’ size range (GS-2 and GS-4),
which was considered any marker under 150 bp in length according to
published allele sizes, two ‘medium’ (GLSA-12 and GLSA-22, 150-200 bp in
length) and one ‘long’ microsatellite (GLSA-52, >200 bp in
length). The repeat motif was also considered, and of these
microsatellites, one was considered a ‘complex’ motif (GLSA-12) while
all others were considered ‘simple’. The specific motif composition can
be found in Table 2. The rationale for testing microsatellites with both
types of motifs was to examine homoplasy. Direct sequencing can reveal
if microsatellites with complex motifs accrew variants in different
regions of the repeat complex that result in the same genotype in a
different individual with polymorphisms in another region.