Conservation Genet Resour (2010) 2:159163 DOI 10.

1007/s12686-010-9295-1

TECHNICAL NOTE

Microsatellite markers for the proboscis monkey (Nasalis larvatus)

M. Salgado-Lynn D. W. G. Stanton R. Sakong J. Cable B. Goossens M. W. Bruford

Received: 21 July 2010 / Accepted: 21 July 2010 / Published online: 1 August 2010 Springer Science+Business Media B.V. 2010

Abstract We describe eight polymorphic microsatellite loci for the proboscis monkey (Nasalis larvatus). These markers were tested with 33 samples, collected from Sabah and exhibited a mean of 6.25 alleles per locus and a mean expected heterozygosity of 0.674. All but one locus were in HardyWeinberg equilibrium, and no evidence for linkage disequilibrium was detected between any loci. Another 30 loci were isolated but remain to be fully examined. These markers should be useful for the future study of population genetic diversity and genetic structure in this emblematic species. Keywords Proboscis monkey Microsatellite loci Population genetics Nasalis larvatus

Classied as endangered by IUCN (2010) and listed in Appendix I of CITES (UNEP-WCMC, 2010), the proboscis monkey (Nasalis larvatus van Wurmb 1787) is endemic to the island of Borneo. Its distribution is restricted to lowland coastal and riverine forests, mangrove, and peat swamp. With a declining population, major threats include hunting, re and, most importantly, anthropogenic habitat
M. Salgado-Lynn (&) D. W. G. Stanton J. Cable B. Goossens M. W. Bruford School of Biosciences, Cardiff University, Museum Avenue, Cardiff CF10 3AX, UK e-mail: salgado-lynnm@cf.ac.uk R. Sakong Red Ape Encounters, Kinabatangan Orangutan Conservation Project, PO Box 3109, 90734 Sandakan, Sabah, Malaysia B. Goossens Danau Girang Field Centre, c/o Sabah Wildlife Department, Wisma Muis, 88100 Kota Kinabalu, Sabah, Malaysia

loss and fragmentation (Meijaard and Nijman 2000; Sha et al. 2008). Despite its uniqueness and conservation status, limited research on genetic variation in proboscis monkeys has been carried out due to a lack of reliable genetic markers and challenging sample collection (Jalil 2007). Here, we describe the isolation and characterization of microsatellite markers which can be used for individual and population-level genetic analyses, suitable for both invasive and non-invasive samples, for the conservation of this species. Muscle samples were opportunistically collected from two deceased proboscis monkeys at Lok Kawi Wildlife Park, and from two road killed animals, in Sabah, Malaysia; the former were used for the isolation of microsatellite loci. Fecal samples from another 29 wild individuals, also from Sabah, were collected as part of a population study and were used for characterizing the markers along with the tissue samples. Stool samples were stored in 70% ethanol, and muscle samples in a 70C freezer. Faecal DNA was extracted via the DNA Stool Mini Kit (Qiagen GMBH, Germany) using a previously described protocol (Goossens et al. 2005). Tissue samples were extracted with DNeasy Blood & Tissue Kit (Qiagen GMBH, Germany) following the recommendations of the manufacturer, with minor modications during elution (namely, 5 min incubation at 70C with buffer AE, which was also preheated at the same temperature). To verify the existence of primate DNA from faecal extracts, a partial mitochondrial Control Region fragment was amplied using species specic primers (Jalil 2007) while DNA from the muscle samples was visualized in agarose gels (1.5%) and quantied by spectrouorometry TM (Invitrogens Quant-iT PicoGreen Kit microtiter assay, Molecular Devices SOFTmax Pro). Genomic libraries were constructed based on the protocol from Glenn and Schable (2005). DNA was digested

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160 Table 1 Primer and motif sequences of isolated microsatellite loci for the proboscis monkey Locus NlB5a NlD10 NlD5 NlE10 NlF1 NlG8a NlP1A6 NlP1C3 NlP1C5 NlP1C8a NlP1E9a NlP1F2a NlP1F5 NlP1G7a NlP2B8a NlP2B9 NlP2C12 NlP2C5a NlP2D6 NlP2E2a NlP2F3 NlP2F7a NlP3A12a NlP3B2 Primers (F-forward; R-reverse) F-CCCATCACCTCATGTAGTTACC R-CCTGAAATTTGCTAAGGGAGT F-TGTCCTTCTCCACTGCCTCT R-TGCAATTTCATTACACCAATGAT F-TGATTTTGCTCTCACCCTTG R-CCGATTCTCTGTTGGAGGAA F-CCATCACACCTGGCTGCTTA R-ATGCCTTGTTGGGAAGACAG F-GCCAATGTTGTAAACTCTATACCC R-TTTATCAACCTGGCCTTTGA F-GGAAATCCAAAGCCTACTGC R-CAGGAAATGTGAAATGGAGGA F-TCTCACTGGTAAAGAAATGTGGA R-CGGACTCTCTGGCTTTTCAG F-CGACCCTCCAGGTTTAAGTG R-ACGCTTGTAATCCCACCTTG F-AGGCCACTGAAGGCTGTCTA R-TGAGTCTAGCTTGGGCAACA F-CCAAATGGTTATTTTGCGAGA R-TTTTGGAAACACCAAAAATGG F-GCTGGCCTGCATACTCAAAT R-CAGACCAGTAGGGGGAGACA F-TGCAGTGAACCTAAACCTGCT R-CTCTGACTTGTGCCAGTGGA F-CCTATCACTTCAAGGGCATAAAA R-TGGCTTGGAGATGCATTTATT F-GGAGCTGGTGCTTCTACAGG R-GGCACCATAGCTTTCTATTCAA F-GAGGTGGTCAGCTGGTCATAA R-GTGCACTGGCTCACTCATGT F-CGATTGAGTTCAGGTATCTTTTG R-TTCAATAATGATGGAAGAATACCG F-CCACAAAACACCATCTCCAA R-TGCTTCATGTCAAGGGATTG F-TCCTTTTGAATTGCCAAGTTTT R-AAGGCACCATGGTCTCAAAG F-AGGGGAAAACACATTTGCAG R-TTTTCCACTCCTCGTTTTGG F-TTGAGGCCTACCTGGTCAAC R-GCACTGAATTGCATCCAGAA F-CAGAACATTTTGCCCAACAG R-GTGGGCAGAAAAGAGAATCG F-CATTCAGACTCACTGGATTAAAAA R-AGATAGAGCCAGAACCTTTCCA F-CTGTGGCCAAACAGTTCATC R-CAGCAGTGGTTTTATTCATTTTTG F-GCAATTTTGCTGAATTTGCTC R-GGCATCGAATTGAAAAGGAA (GT)23 (GT)17 (AC)21 (AG)17 (CT)20 (GT)17 (AC)21 (AC)16 (GT)17 (GTT)9 (GT)20 (GT)16 (AG)17 (GT)19 (GT)20 (GT)15 (GAT)10 (GT)14 (AAC)7 (AC)10AT(AC)TC(AC)8 (GT)16 (CA)6AA(CA)6TG(CA)6 (CT)10 Motif (GT)10

Conservation Genet Resour (2010) 2:159163

Ta (C) 57 54 54 58 52 54 54 54 54 60 54 58 52 52 56 52 52 58 54 60 58 56 50 58

Size (bp) 107 209 171 169 176 226 158 229 204 221 218 240 234 160 234 209 191 184 160 247 191 150 246 165

GenBank ID HM588998 HM588999 HM589000 HM589001 HM589002 HM589003 HM589004 HM589005 HM589006 HM589007 HM589008 HM589009 HM589010 HM589011 HM589012 HM589013 HM589014 HM589015 HM589016 HM589017 HM589018 HM589019 HM589020 HM589021

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Conservation Genet Resour (2010) 2:159163 Table 1 continued Locus NlP3B4a NlP3B6a NlP3C11a NlP3E1a NlP3E7 NlP3E8a NlP3G2 NlP3H5a NlP4B1 NlP4B2 NlP4C11 NlP4C6 NlP4E10a NlP4E6
a

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Primers (F-forward; R-reverse) F-TTCCAGCTATCAAAATAGTGGCTA R-GAAGTGGCTTGCCTTACAGC F-ATCATTTCTGGGCCTGTTTT R-CCTGCGGAACAAGAGTGAA F-TCCATCCCCTTTTTATGATACTT R-AGGTATGCAGCCAAGCAAAG F-ACTGGGCATCAGAGTCATCC R-TCCATGCAATGGCACATAGT F-GGAGAGGTGGCCTTTGAACT R-TGTTCAGCAAACAATATAGAGCTAA F-CAAATGAAAAATGCCTCTAACAGA R-CAGAGCATGCAAGAAAGAGAGA F-TCCCATGTTTATTGCAGCAC R-TCAGTGCCTGGCTGATTTTA F-CATTGTGAGAAAACTTGCTTCTG R-CCCATCAACTTCAGAATACACA F-TTCCATGGTTTCCAGAGTCC R-AGAAATGGATGGGGCAGAG F-TCAGGTGAATTGCTGGCATA R-GCATCCAAACTGGAAAGGAA F-CTCCACAGTCCTGTGACCAA R-TGCAGAAAGCCAAAAGGATT F-TGTTGAAAATTCTTGCATTTGTG R-TCTTCCCCAAAACTGAGGAT F-CAAACCTGCATGTTCTCCAC R-GCTGGGAAACAATCAGTCCT F-CAAGGAAGAATTGTGCCAAGA R-GGCATTCCCAAACCTCATAA

Motif (AC)18 (AC)22 (AC)20 (GAT)10 (GAT)5 (ATCT)12 (AC)23 (AG)16 (AC)16 (GT)15 (GT)23 (GTT)7 (AC)20 (GT)19

Ta (C) 54 58 54 54 52 56 58 50 58 54 54 52 56 54

Size (bp) 185 237 199 208 151 223 250 167 158 199 248 160 218 234

GenBank ID HM589022 HM589023 HM589024 HM589025 HM589026 HM589027 HM589028 HM589029 HM589030 HM589031 HM589032 HM589033 HM589034 HM589035

Poor amplication in faecal DNA extracts

Sizes are based on the sequenced allele

overnight with RsaI (New England BioLabs) and the products were subsequently ligated to Super SNX24 linkers. Linker-ligated DNA was electrophoresed in a 1.5% agarose gel and fragments between 300 and 800 bp were electroeluted, precipitated with 3 M NaOAcethanol and resuspended in TE Buffer (Bruford et al. 1992). Fragments containing microsatellites were captured using biotinylated oligonucleotides (see mix 2 in Glenn and Schable 2005), and the biotinylated probeDNA complex was enriched by hybridization to streptavidin-coated magnetic beads (Dynabeads M-280, Invitrogen). Nonspecic DNA was removed by subsequent washes with SSCSDS buffers as described in the same protocol, and recovery was performed by PCR using the forward SuperSNX-24 primer. Enriched libraries were constructed using a TA Cloning Kit according to the manufacturers protocol (Invitrogen) and positive colonies were amplied using universal M13 forward and reverse primers (M13F: 50 -GTAAAACGACGGCCAG-30 ; M13R:

50 -CAGGAAACA- GCTATGAC-30 ). Fragments between 500 and 1200 bp were sequenced using the BigDye terminator kit v1.1 (Applied Biosystems. Sequences were assembled and edited in Mega 4.0 (Tamura et al. 2007) and visually checked for microsatellite repeats. Two libraries were constructed and a mean of 33% of sequenced clones yielded microsatellite motifs. Unique sequences with sufcient anking DNA and at least ve (trinucleotide) or ten (dinucleotide) repeat units were selected for primer design using Primer 3 in msatcommander (Faircloth 2008). Ninety-one primer pairs were designed with melting temperatures between 50 and 66C and length of PCR products between 100 and 300 bp. The tissue samples were used to optimize the PCR conditions for 46 unlabelled primer pairs. PCR reactions consisted of 19 Master Mix (QIAGEN, Multiplex Kit), 0.4 lg/ll BSA (New England BioLabs), 0.2 lM of each primer, and 200 pg of template DNA to a total volume of

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162 Table 2 Characteristics of 20 proboscis monkey microsatellite loci suitable for invasive and non-invasive genetic studies Locus Label A Range (bp) HO HE

Conservation Genet Resour (2010) 2:159163

Multiplex 1 (Ta 54C) NlD10 NlP4C11 NlP1C3 NlD5 NlP2D6 NlP1A6 NlP1C5 NlP4B2 NlP4E6 NlE10 NlP2F3 NlP3B2 NlP4B1 NlP3G2 NlP3E7 NlP2B9 NlP4C6 NlP1F5 NlP2C12 NlF1 HEX HEX FAM FAM HEX NED FAM HEX FAM NED FAM FAM HEX HEX NED HEX HEX FAM FAM HEX 6 6 1 2 5 4 7 2 3 8 5 4 3 3 1 3 2 2 4 3 177189 241259 230 163169 146160 146158 177205 185201 237241 153205 175187 158172 152156 200204 152 212218 156162 235237 186194 171175 0.848 0.757 0.636 0.575 0.696 0.484 0.575 0.727 0.581 0.786 0.655 0.581 0.685 0.651 0.662 0.793

Multiplex 2 (Ta 54C)

Multiplex 3 (Ta 58C)

Multiplex 4 (Ta 52C)

Genetic diversity of eight microsatellite loci is based on genotypes of 33 individuals A-, Number of alleles per locus; , markers tested only with four tissue samples

samples under the conditions previously described. For genotyping, PCR products were electrophoresed along with GeneScan ROX 350, or GS-400 HD LIZ, in a Prism 3700 Genetic Analyzer (Applied Biosystems) and fragment lengths were scored using GeneMapper ID 3.2 (Applied Biosystems). Allele diversity and size ranges of all 20 markers are included in Table 2, along with other details for the eight loci. Exact HardyWeinberg probabilities were assessed, and linkage disequilibrium was tested using GENEPOP version 4.0.10 (Raymond and Rousset 1995; Rousset 2008). Signicance levels were adjusted using Bonferroni corrections for multiple testing (P \ 0.006 in our dataset). All loci were in HardyWeinberg equilibrium except NlD10 (P \ 0.001), and no evidence was found for linkage disequilibrium between any pair of loci. Observed and expected heterozygosities were calculated using ARLEQUIN version 3.5.1.2 (Excofer and Lischer 2010). The mean expected heterozygosity was 0.674 (range 0.5810.793) and mean observed heterozygosity was 0.662 (range 0.4840.848). The mean number of alleles was 6.25 (range 49) for the eight fecal-tested markers and 2.41 (range 14) for the other 12 loci. Deviation from Hardy Weinberg equilibrium for some loci may be due to samples coming from three distant sites and the wildlife park individuals whose origin was unknown. Large-scale testing on individuals from a single population is needed for locus NlD10. The eight polymorphic microsatellite loci will be useful for the future study of individual identication, population genetic diversity and genetic structure in the proboscis monkey.
Acknowledgments This work was supported by CONACYT (Consejo Nacional de Ciencia y Tecnologa- Mexico), Rufford Small Grants for Nature Conservation, and North of England Zoological Society. We thank Dr. XiangJiang Zhan, Dr. Patricia J. Faria and Dr. Eddie Brede for their valuable suggestions on the laboratory work and colleagues at Danau Girang Field Centre and the Kinabatangan Orangutan Conservation Project for their help in the eld. The authors are grateful to Lok Kawi Wildlife Park, Sepilok Rehabilitation Centre and Dr Henry Bernard, of the Unit for Primate Studies at the Institute of Tropical Biology and Conservation of Universiti Malaysia Sabah, for providing some of the samples used in this study.

10 ll and were performed in a GeneAmp PCR System 9700 (Applied Biosystems). The amplication conditions were as follows: 95C for 15 min, 45 cycles at 94C for 30 s, 4863C for 90 s, 72C for 90 s and a nal extension at 72C for 30 min. Thirty-eight primers, which produced a single target band, were further optimized by amplifying 29 dung samples with a multi-tube approach (Taberlet et al. 1996) using the above PCR protocol but with 2 ll of DNA template and the optimized annealing temperatures (Ta) shown in Table 1. Twenty primers which successfully (C80%) amplied in faecal samples were uorescently labeled (50 - FAM, HEX, or NED) and assembled in four multiplexes. Although amplication of the tissue samples with the multiplex systems was successful (100%), consistent amplication of the dung samples with the multiplex approach was not possible. The limited volume of the faecal DNA extracts allowed thorough characterization of only eight markers (singleplex) with the 29 dung samples and the four tissue

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