The white-collared lemur, Eulemur cinereiceps (=albocollaris; Johnson et al. 2008), is a medium sized (1.6–2.7 kg), cathemeral, frugivorous lemur that is sexually dichromatic (Tattersall 1982; Johnson et al. 2005). Males are grey–brown with distinctive white cheeks and beard, while females are not bearded, have grey faces, and are more rufous in color. Eulemur cinereiceps live in rain forests between the Andringitra Massif and the Mananara River in southeastern Madagascar (Petter and Petter-Rousseaux 1979; Johnson and Wyner 2000). It is estimated that there is approximately only 700 km2 of habitat remaining within this species range (Irwin et al. 2005). As a result of habitat loss and hunting, E. cinereiceps is one of the 25 most endangered primates in the world (Mittermeier et al. 2007). We report here the isolation and characterization of 18 polymorphic markers for E. cinereiceps.

Genomic DNA was isolated from tissue from a free-ranging E. cinereiceps collected from Mahabo Forest. Procedures for construction of the genomic DNA library, identification of plasmids containing (GT) n inserts as well as plasmid preparation and sequencing were carried out as generally described by Hillis et al. (1996). Isolated genomic DNA was digested using Sau3A restriction enzyme. The digested DNA was sized using Clontec© chromaspin columns to remove fragments under 400 bp. Sized DNA was ligated to primers forming blunt-ended DNA pieces. Ligated DNA was enriched using a PCR-based method by Moraga-Amador et al. (2001), a modification of Kandpal et al. (1994). Enriched DNA was denatured and a biotinilated probe annealed to the DNA. The biotinilated DNA was captured using Vector Laboratory (Burlingame, CA) Vectrex Avidin D© and non-annealed DNA was washed away. After releasing captured DNA from the Vectrex Avidin D©, a second round of PCR enrichment was performed. An Invitrogen (Carlsbad, CA) TOPO A© plasmid ligation was performed following this PCR. Following transformation, cells were plated onto LB agar plates including ampicillin and X-gal. Plates were picked for positive white colonies that were placed on Pall (East Hill, NY) Biodyne B nylon membranes. A Southern blot of the colonies was done using DIG-labeled oligonucleotide. Plasmid preps of positive colonies from the Southern blot were sequenced and primers were designed from the two regions flanking the microsatellite repeat motif. Of 1,878 clones screened, 268 were positive for a microsatellite insert and the first 18 were designed and polymorphic are being reported here.

Tissue samples were collected and stored in ambient temperature storage buffer (Longmire et al. 1992) from 21 white-collared brown lemurs in multiple locations in Vevembe Forest, Madagascar, in 2000 and 2007. Genomic DNA was isolated using standard protocols (Sambrook et al. 1989). PCR amplification was carried out in a 25 μl reaction volume using a MBS Satellite 0.2G thermal cycler (Thermo Electron Corporation; Gormley, ON) with approximately 50 ng of genomic DNA as template. Final amplification concentrations consisted of 12.5 pmol unlabelled reverse primer, 12.5 pmol fluorescently labeled forward primer, 1.5 mM MgCl2, 200 μM each dNTP, and 0.5 units of Taq DNA polymerase (Promega; Madison, WI). The thermal profile for PCR amplification was 95°C for 5 min, followed by 35 cycles of 95°C for 30 s, a primer-specific annealing temperature for 30 s (Table 1), 72°C for 30 s, ending with a single 10 min extension of 72°C. Allele sizes were determined by separation of PCR products via POP 4 capillary buffer electrophoresed in an ABI 3100 DNA Analyzer (Applied Biosystems, Inc; Foster City, CA). Fragment length genotypes were assigned by GeneScan (Applied Biosystems, Inc.) using GeneScan-500 [Tamra] size standard. Loci characterizations are presented in Table 1.

Table 1 Primer sequences with fluorescent dye label used, repeat motif, optimized annealing temperature, number of alleles (k), allele size range and GenBank accession numbers of 18 Eulemur cinereiceps—specific microsatellite loci
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The data set was analyzed for errors using MICRO-CHECKER (Van Oosterhaut et al. 2004) and Microsatellite Analyzer (MSA; Dieringer and Schlötterer 2003). Null alleles and polymorphic information content (PIC) were estimated using CERVUS v.2.0 (Marshall et al. 1998; Slate et al. 2000). Marker independence was verified following Bonferroni correction for multiple tests in FSTAT (Goudet 1995, 2001) before population genetic parameters were estimated in FSTAT and web-based Genepop (Raymond and Rousset 1995).

Global population genetic parameter estimates for the marker suite are presented in Table 2. Two loci deviated significantly from Hardy Weinberg Equilibrium and two others were estimated to have excessive null allele frequencies (>0.10). The average observed heterozygosity for the data was 0.577 with an expected heterozygosity of 0.631. Polymorphic information content for the loci averaged 0.568 (0.214 < PIC < 0.719). This novel marker suite will be useful to further the understanding of population dynamics in Eulemur cinereiceps.

Table 2 Hardy–Weinberg exact tests P-values with standard errors, within population fixation indices (F IS), observed (H O) and expected (H E) heterozygosities, and polymorphic information content (PIC) among 18 Eulemur cinereiceps microsatellite loci screened across 21 white-collared lemurs from Vevembe Forest
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