Print version ISSN 1415-4757
Genet. Mol. Biol. vol.34 no.1 São Paulo 2011 Epub Dec 22, 2010
Milene Moura Martins; Pedro Manoel Galetti Junior
Laboratório de Biodiversidade Molecular e Conservação, Departamento de Genética e Evolução, Universidade Federal de São Carlos, São Carlos, SP, Brazil
Leontopithecus caissara is a critically endangered primate species from the Brazilian Atlantic Forest. Nineteen microsatellite loci, previously developed for congeneric species, were tested with 34 L. caissara individuals from Superagüi Island. Of the 19 loci, 17 (89.4%) produced robust alleles, nine (47.4%) of these proved to be polymorphic, with a total of 23 alleles and an average of 2.56 alleles per locus. Expected and observed heterozygosity averaged 0.483 and 0.561, respectively. The exclusion power for identifying the first parent of an arbitrary offspring was 0.315 over all loci. The results thus indicate both the usefulness and limitations of these nine microsatellite loci in the genetic analysis of L. caissara, as well as their potentiality for genetic investigation in other congeneric species.
Key words: lion tamarins, endangered species, genetic diversity, New World primate, SSR transferability.
The black-faced lion tamarin (Leontopithecus caissara), whose specific status has recently received support from molecular data (Perez-Sweeney et al., 2008), is a critically endangered Neotropical primate (Kierulff et al., 2008). Its distribution range lies in lowland swampy forests of southeastern Brazil (Lorini and Persson, 1994), with a population currently estimated at less than 500 individuals (A. Nascimento, pers comm). This has generated apprehension when considering the impact on such a small population, of barriers hindering gene flow between main populations, to the point of genetic evaluation being considered top priority in any conservation plan involving this primate (Holst et al., 2006).
Microsatellites are useful for investigating behavioral ecology (Di Fiore, 2009) and shedding light on questions concerning biological conservation (Selkoe and Toonen, 2006). They are considered expedient, notably in antecipation of management decisions beneficial to wildlife conservation. Microsatellites present relatively high rates of transferability among mammals (Barbará et al., 2007), which is advantageous, since their development can be time-consuming (Squirrell et al., 2003; Sarre and Georges, 2009). Thus, exploiting microsatellite available for one or more species could be a plausible alternative in the genetic investigation of congeners. Here we investigated feasibility of employing microsatellites previously isolated in other Leontopithecus species in L. caissara.
Blood samples were taken from 34 free-ranging black-faced lion tamarins from Superagüi Island, state of Paraná, Brazil. DNA was extracted according to a modified phenol-chloroform method (Sambrook et al., 1999). Nineteen microsatellites, previously developed for Leontopithecus rosalia (Grativol et al., 2001), L. chrysopygus (Perez-Sweeney et al., 2005) and L. chrysomelas (Galbusera and Gillemot, 2008), were tested. A primer for each locus was constructed with an M13 tail. A fluorescently-labeled M13 primer was also used in a three primer-PCR (polymerase chain reaction), following an established protocol (Schuelke, 2000). Microsatellite loci were amplified in a 10 μL reaction volume containing 20 ng of template DNA, 1 μL of each primer, 0.2 mM of dNTP, 1.5 mM of MgCl2, and 1 U Taq polymerase (Fermentas). After annealing-temperature optimization, amplifications were carried out in either a Perkin Elmer 2400 thermal cycler or an Eppendorf Gradient Mastercycler, under the following conditions: 5 min at 94 °C, 30 cycles of 30 s denaturation at 94 °C, annealing at 51-61 °C for 45 s, extension for 45 s at 72 °C, and finally 10 cycles of 30 s denaturation at 94 °C, annealing at 53 °C for 45 s, extension for 45 s at 72 °C, followed by a final extension step of 10 min at 72 °C. Amplified fragments were checked on 2% agarose gels. PCR products were analyzed on a MegaBace automatic sequencer, and allele sizes scored using the FRAGMENT PROFILER version 1.2 program (Applied Biosystem®). The GENEPOP version 4.0 program (Raymond and Rousset, 1995) was used to test for departures from Hardy-Weinberg equilibrium (HWE) and linkage disequilibrium (LD). Genetic-diversity parameters and the non-exclusion probability from parentage were estimated using the CERVUS version 3.0.3 software (Marshall et al., 1998).
From the 19 microsatellite loci tested, 17 (89.4%) produced robust alleles, of which nine (47.4%) were polymorphic and eight (42.1%) monomorphic. The remaining two (10.5%) failed to amplify fragments under all the tested conditions. Analysis using the nine polymorphic microsatellites and the 34 black-faced lion tamarins revealed a total of 23 alleles. The number of alleles per locus ranged from two to three (Table 1), with an average of 2.56 alleles per locus. Among loci, expected heterozygosity varied from 0.327 to 0.644, with an average of 0.483, whereas observed heterozygosity ranged from 0.382 to 0.794, with an average of 0.561. The total exclusion power for identifying an unrelated candidate parent of an arbitrary offspring [Pr(Ex1)] when neither parent was known, was estimated to be 0.315 over all loci. This value indicates that these loci may not be suitable for paternity testing. Only the Leon15c85 locus deviated significantly (p = 0.015 for corrected p = 0.017) from HWE (Table 1). Analysis using MICRO-CHECKER version 2.2.3 software (Van Oosterhout et al., 2004), failed to indicate the presence of null alleles (p = 0.05) at this locus. Four loci pairs (Leon21c75 - LrP2BH6, Leon21c75 - Lchμ04, Leon3c20 - Lchμ04 and LrP2BH6 - Lchμ04) displayed significant LD after Benjamini and Yekutieli (2001) correction.
Our results confirm the usefulness of the nine microsatellite loci in genetic analyses involving L. caissara. Lion tamarins have figured as flagship (Dietz et al., 1994) and umbrella species (Simberloff, 1998) favoring wildlife conservation at several sites in the Brazilian Atlantic Forest (Kleiman and Rylands, 2002). The wise management of lion tamarin populations could turn out to be a time saving procedure, in which the successful transferability of microsatellites between congeneric species will be of great assistance.
The authors thank the Instituto de Pesquisas Ecológicas (IPÊ) for donating the L. caissara blood samples. The present research received financial support from the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, grants 05/04346-2 and 07/07409-0), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Primate Action Fund from The Margot Marsh Biodiversity Foundation, and Rufford Small Grants for Conservation.
Barbará T, Palma-Silva C, Paggi GM, Bered F, Fay MF and Lexer C (2007) Cross-species transfer of nuclear microsatellite markers: Potential and limitations. Mol Ecol 16:3759-3767. [ Links ]
Benjamini Y and Yekutieli D (2001) The control of false discovery rate under dependency. Ann Stat 29:1165-1188. [ Links ]
Di Fiore A (2009) Genetic approaches to the study of dispersal and kinship in New World primates. In: Garber P, Estrada A, Bicca-Marques J, Heymann E and Strier K (eds) South American Primates, Developments in Primatology: Progress and Prospects. Springer, New York, pp 211-250. [ Links ]
Dietz J, Dietz L and Nagagata E (1994) The effective use of flagship species for conservation. In: Olney P, Mace G and Feistner A (eds) Creative Conservation. Chapman & Hall, London, pp 32-49. [ Links ]
Galbusera PHA and Gillemot S (2008) Polymorphic microsatellite markers for the endangered golden-headed lion tamarin, Leontopithecus chrysomelas (Callitrichidae). Conserv Genet 9:731-733. [ Links ]
Grativol A, Ballou J and Fleischer R (2001) Microsatellite variation within and among recently fragmented populations of the golden lion tamarin (Leontopithecus rosalia). Conserv Genet 2:1-9. [ Links ]
Holst B, Medici E, Marini-Filho O, Kleiman D, Leus K, Pissinatti A, Vivekananda G, Ballou JD, Traylor-Holzer K, Raboy B et al. (2006) Lion Tamarin Population and Habitat Viability Assessment Workshop 2005. Final Report. IUCN/SSC Conservation Breeding Specialist Group, Apple Valley, 208 pp. [ Links ]
Kleiman D and Rylands A (2002) Lion Tamarins: Biology and Conservation. Smithsonian Institution Press, Washington, 422 pp. [ Links ]
Lorini ML and Persson VG (1994) Status of field research on Leontopithecus caissara: The black-faced lion tamarin project. Neotrop Primates 2(Suppl):52-55. [ Links ]
Marshall TL, Slate J, Kruuk LEB and Pemberton JM (1998) Statistical confidence for likelihood-based paternity inference in natural populations. Mol Ecol 7:639-655. [ Links ]
Perez-Sweeney B, Valladares-Padua C, Burrell A, Di Fiore A, Satkoski J, Groot P, Boag P and Melnick D (2005) Dinucleotide microsatellite primers designed for a critically endangered primate, the black lion tamarin (Leontopithecus chrysopygus). Mol Ecol Notes 5:198-201. [ Links ]
Perez-Sweeney B, Valladares-Padua C, Martins C, Morales J and Melnick D (2008) Examination of taxonomy and diversification of Leontopithecus using the mitochondrial control region. Int J Primatol 29:245-263. [ Links ]
Raymond M and Rousset F (1995) Genepop v. 1.2: Population genetics software for exact tests and ecumenicism. J Hered 86:248-249. [ Links ]
Sambrook J, Fritsch E and Maniatis T (1999) Molecular Cloning: A Laboratory Manual. 2nd edition. Cold Spring Harbor Laboratory Press, New York. [ Links ]
Sarre SD and Georges A (2009) Genetics in conservation and wildlife management: A revolution since Caughley. Wildlife Res 36:70-80. [ Links ]
Schuelke M (2000) An economic method for the fluorescent labeling of PCR fragments. Nat Biotechnol 18:233-234. [ Links ]
Selkoe KA and Toonen RJ (2006) Microsatellites for ecologists: A practical guide to using and evaluating microsatellite markers. Ecol Lett 9:615-629. [ Links ]
Simberloff D (1998) Flagships, umbrellas, and keystones: Is single-species management passé in the landscape era? Biol Conserv 83:247-257. [ Links ]
Squirrell J, Hollingsworth PM, Woodhead M, Russel J, Lowe AJ and Gibby M (2003) How much effort is required to isolate microsatellites from plants? Mol Ecol 12:1339-1348. [ Links ]
Van Oosterhout C, Hutchinson WF, Willis DPM and Shipley P (2004) MICRO-CHECKER: Software for identifying and correcting genotyping errors in microsatellite data. Mol Ecol Notes 4:535-538. [ Links ]
Kierulff MCM, Rylands AB, Mendes SL and de Oliveira MM (2008) Leontopithecus caissara. In: IUCN 2010. IUCN Red List of Threatened Species, v. 2010.2. http://www.iucnredlist.org (May 23, 2010). [ Links ]
Send correspondence to:
Milene Moura Martins
Laboratório de Biodiversidade Molecular e Conservação
Departamento de Genética e Evolução
Universidade Federal de São Carlos
Rodovia Washington Luiz km 235
13565905 São Carlos, SP, Brazil
Received: May 25, 2010; Accepted: October 22, 2010.
Associate Editor: Louis Bernard Klaczko
License information: This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.