Many forest taxa, including squirrels, are becoming increasingly endangered due to the effects of forest loss and fragmentation (Sodhi et al. 2010). This is particularly true in Southeast Asia that has the highest rates of forest loss worldwide (Sodhi et al. 2004). In Southeast Asia, squirrel species vary in IUCN status from Critically Endangered (e.g. Biswamoyopterus biswasi (the Namdapha Flying Squirrel) in northeast India) to Least Concern (http://www.iucnredlist.org/details/2816/0). However the risks are certainly underestimated as true species diversity remains unknown and many taxa are classified by the IUCN as Data Deficient. It is important to note that even though some species are listed as Least Concern they actually face significant threats. For example, two of our study species: Hylopetes lepidus (the Grey-cheeked Flying Squirrel); and Hylopetes phayrei (Phayre’s Flying Squirrel), are hunted extensively and sold in food markets throughout Thailand (SJ personal observation), despite being officially protected under the Preservation and Protection of Wildlife Act of B.E. 2535 (1992) (http://www.forest.go.th). Here we developed conserved microsatellite markers to facilitate conservation genetics studies in a broad range of squirrel taxa but particularly flying squirrels of the genus Hylopetes from Indochina.

Genomic DNA was extracted from ear clips using a phenol–chloroform extraction method. Microsatellite-enriched genomic libraries were constructed separately following Armour et al. (1994) using one adult female from each of three species: the flying squirrels: H. phayrei and H. lepidus; and the Indochinese Ground Squirrel, Menetes berdmorei, all sampled in Thailand. From these we generated 89, 85 and 91 unique microsatellite sequences (EMBL accession numbers LN650709–650973), respectively. Microsatellite sequences were available from GenBank for five other squirrel species: Glaucomys sabrinus, G. volans, Sciurus lis, S. niger and S. vulgaris.

We followed the approach of Dawson et al. (2010) to identify conserved microsatellite sequences and create markers of high cross-species utility. We created a consensus sequence by aligning the newly isolated squirrel microsatellite sequences and/or other online squirrel sequences (Hylopetes/Menetes/Glaucomys/Sciurus) against their homologue in the thirteen-lined ground squirrel genome (Spermophilus tridecemlineatus; http://www.ensembl.org/Spermophilus_tridecemlineatus/index.html). Primer sets were designed for 40 microsatellite loci based on these consensus sequences using PRIMER3 v0.4.0 (avoiding bases mismatching between species, when possible). Each primer set matched S. tridecemlineatus and one of the other eight squirrel species cited above (the “source” species) with a maximum of three degenerate bases per primer (three per primer set) and a maximum of one base mismatching S. tridecemlineatus. The optimal difference between the forward and reverse primer melting temperatures was set to 0.5 °C (maximum 4 °C; Table 1).

Table 1 Characterization of eleven conserved microsatellite markers in three squirrel species
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The 40 conserved microsatellite markers were tested in our primary study species H. phayrei, H. lepidus and M. berdmorei (using four individuals per species). Twenty-six markers were variable in H. phayrei, 25 markers in H. lepidus and 25 markers in M. berdmorei (Supplementary Table 2). Fourteen loci amplified and were polymorphic in all three species (Supplementary Table 2). Primer sets designed from comparisons of the thirteen-lined ground squirrel with the flying squirrels (Hylopetes) amplified in more species than those designed from comparisons between the two ground squirrel species, M. berdmorei and S. tridecemlineatus (Supplementary Tables 1 and 2). This is likely due to the greater phylogenetic distance between Hylopetes/Glaucomys/Sciurus genera and S. tridecemlineatus than between M. berdmorei and S. tridecemlineatus (Mercer and Roth 2003), resulting in more highly conserved primers for the former. The 14 microsatellite loci were evaluated in a greater number of individuals: H. phayrei (28 individuals from Mae Rim, Thailand); H. lepidus (eight individuals from Phu Huay Sing, Thailand); and M. berdmorei (28 individuals from Wapipathum, Thailand). PCR reactions were carried out in a DNA Engine thermal cycler (MJ Research) in 2 µl volumes containing 10 ng genomic DNA, 1 µl Qiagen Multiplex PCR Master Mix (Qiagen Inc.) and primers (0.2 µM). Initial denaturation for 15 min at 95 ○C was followed by 34 cycles of 30 s at 94 °C, 90 s at the optimal annealing temperature (Table 1) and 60 s at 72 °C with a final extension step of 30 min at 60 °C. PCR products were run on a 48-capillary ABI3730 DNA Analyzer using prism set D and a ROX size standard and the alleles sized with GENEMAPPER ver. 3.7 (Applied Biosystems). Observed heterozygosity (H O ), expected heterozygosity (H E ) and estimated null allele frequencies were calculated using CERVUS v3.0.3. Deviations from Hardy–Weinberg equilibrium and tests for linkage disequilibrium were calculated using GENEPOP v.4.2 (http://genepop.curtin.edu.au/).

Three loci were excluded due to low variability in M. berdmorei, one of the three test species (Hph14, Hph54 and Hlep05; Supplementary Table 1). For the remaining 11 loci, the number of alleles per locus across the three species ranged from three to 19 (Table 1). Heterozygotes were observed in males and females for each species suggesting none of the 11 loci were sex-linked. Observed and expected heterozygosities ranged from 0.21 to 0.93 and from 0.23 to 0.96, respectively (Table 1). Two loci deviated from Hardy–Weinberg equilibrium in some species (p < 0.01, GLSA22 and Hph46, Table 1), which may be due to null alleles. There was no evidence of linkage disequilibrium between any groups of loci in any species.

The majority of the 11 markers could be amplified and were polymorphic in other squirrel species; ten loci in the flying squirrels Petaurista petaurista and Hylopetes spadiceus and seven loci in the tree squirrels Callosciurus caniceps and C. finlaysoni (Table 2). These markers will therefore be useful for conservation studies in a wide range of squirrel taxa.

Table 2 Cross-species utility of conserved squirrel microsatellite loci in four additional squirrel species (two flying squirrels and two Callosciurus tree squirrels)
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