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IUCN/SCC Otter Specialist Group Bulletin
©IUCN/SCC Otter Specialist Group

Volume 28A Proceedings Xth International Otter Colloquium, Hwacheon, South Korea

Citation: Trinca, C.S, Waldemarin, H.F., Rosas, F.C.W. and Eizirik, E. (2011) Characterization and Assessment of 12 Autosomal Microsatellite Loci Suitable for Population and Evolutionary Studies of the Neotropical Otter (Lontra longicaudis) . Proceedings of Xth International Otter Colloquium, IUCN Otter Spec. Group Bull. 28A: 162 - 166

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Characterization and Assessment of 12 Autosomal Microsatellite Loci Suitable for Population and Evolutionary Studies of the Neotropical Otter (Lontra longicaudis)

Cristine S. Trinca1,2 , Helen F. Waldemarin3 , Fernando C. W. Rosas4 and Eduardo Eiziri2,5

1Departamento de Genética, Instituto de Biociências, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves, 9500, Prédio 43323, CEP 91501-970, Porto Alegre, RS, Brazil.,
2Laboratório de Biologia Genômica e Molecular, Faculdade de Biociências, Pontifícia Universidade Católica do Rio Grande do Sul, Av. Ipiranga, 6681, Prédio 12C, CEP 90619-900, Porto Alegre, RS, Brazil
3Associação Ecológica Ecomarapendi / Projeto Ecolontras, Rua Paissandu, 362, Laranjeiras, CEP 22210-080, Rio de Janeiro, RJ, Brazil
4Laboratório de Mamíferos Aquáticos, Instituto Nacional de Pesquisas da Amazônia, Av. André Araújo,2936, Aleixo, CEP 69060-001, Manaus, AM, Brazil
5Instituto Pró-Carnívoros, Av. Horácio Neto, 1030, casa 10, Parque Edmundo Zanoni, CEP 12940-970, Atibaia, SP, Brazil

(received 23rd March 2011, accepted 10th April 2012)

Abstract:We tested 12 microsatellite markers previously isolated from two different otter species (Lutra lutra and Lontra canadensis) in the Neotropical otter aiming to help in the choice of markers that could be used even in noninvasive samples of this species. Thirty-one individuals from several localities of Brazil were genotyped. The mean expected heterozigosity values for the ten best loci ranged from 0.613 to 0.891 (mean He for all loci = 0.769). To investigate the reliability of individual identification with the selected markers, we calculated the maximum P(ID) using the ten loci, and also a subset of seven loci exhibiting the shortest product length (< 300 bp), which are more likely to be useful in noninvasive samples. The maximum estimated P(ID) was 1.68 x 10-11 and 3 x 10-8, respectively, indicating a good potential of both marker sets for secure individual identification. The results obtained in this study revealed that ten of the 12 loci analyzed are suitable for application in ecological and evolutionary studies of the Neotropical otters, even on the basis of noninvasive sampling.

Keywords: Lontra longicaudis; microsatellite loci; noninvasive sampling; population dynamic; phylogeographic studies

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INTRODUCTION

Strategies for species conservation must integrate genetic and ecological data to allow for adequate planning and implementation. For such conservation efforts to be successful, accurate estimates of genetic diversity, geographic structuring, density, and population abundance are essential.  In spite of their importance, these data are often difficult to obtain for most carnivore species, due to their elusive behavior.

In this context, microsatellite DNA has been extensively used as an informative type of molecular marker in population and evolutionary studies of carnivores ( Walker et al. 2001 ; Blundell et al. 2004 ; Pertoldi et al. 2006 ; Carmichael et al. 2007 ). Microsatellites are highly polymorphic regions of DNA widely scattered throughout eukaryotic genomes ( Schlötterer 1998 , 2004 ). This kind of molecular marker is a useful genetic tool that, in association with noninvasive sampling, increases the potential for collecting genetic, ecological and demographic data for elusive species, such as the Neotropical otter.

Recently, a variety of microsatellite loci has been isolated from mustelids (e.g. Dallas and Piertney 1998 ; Carpenter et al. 2003 ; Beheler et al. 2004 , 2005 ) and tested for cross-amplification in related species (e.g. Davis and Strobeck 1998 ; Beheler et al. 2004 , 2005 ). However, until now there is no published study that has attempted their use in the Neotropical otter (Lontra longicaudis). The characterization of polymorphic molecular markers in the Neotropical otter will allow the development of ecological and genetic studies on this species, expanding our knowledge on its biology and providing a suitable basis for planning adequate management and conservation strategies.

Here, we report the characterization of a set of 12 polymorphic markers suitable for studies in L. longicaudis, derived from cross-species testing of previously developed microsatellite loci of Lutra lutra and Lontra canadensis.

MATERIAL AND METHODS

The aim of this work was to identify a set of loci that: (i) show good amplification efficiency and (ii) are polymorphic in L. longicaudis; and (iii) present short amplification products (≤ 300 bp), facilitating their use in DNA recovered from noninvasive samples (e.g. hairs and scats). We tested 12 primer sets chosen on the basis on the following features reported for the original species (from which the markers were isolated) and, in some cases, additional mustelids also typed for the same loci: (i) number of observed alleles; (ii) heterozygosity; and (iii) product length.

The primers were tested in genomic DNA from thirty-one individuals of the Neotropical otter sampled from wild populations and kept in captivity. The samples were obtained from several localities in Brazil, including the states of Rio Grande do Sul, Santa Catarina, Paraná, Mato Grosso do Sul, Rio de Janeiro, São Paulo, Goiás, Minas Gerais a Amazonas. Tissue and blood samples were obtained opportunistically by collaborators from road-killed and captive individuals, respectively. Tissue samples were stored in ethanol 96% and blood samples were preserved in a salt saturated solution (100mM Tris, 100mM EDTA, 2% SDS). Both tissue and blood samples were stored at -20°C DNA prior the DNA extraction. Genomic DNA was extracted using a standard phenol-chloroform-isoamyl alcohol protocol following digestion of the tissue with Proteinase K ( Sambrook et al. 1989 ).

The markers selected for this study were developed by Dallas & Piertney (1998) for Lutra lutra and by Beheler et al. (2004 , 2005 ) for Lontra canadensis. We initially tested all 12 loci for 14 individuals, and selected the ten best markers (based on amplification performance, product size, and level of polymorphism) for genotyping in the remaining individuals.

Every forward primer was 5’-tailed with an M13 sequence (Boutin-Ganache et al. 2001), and used in combination with an M13 primer that had the same sequence but was dye-labeled on its 5’ end. The PCR reactions were performed in 10 μl reactions containing 0.5 – 1.5 μl of empirically diluted DNA, 1x PCR Buffer (Invitrogen), 1.5 - 2 mM MgCl2, 200 μM dNTPs, 0.2 μM of the reverse and M13-fluorescent primers, 0.013 μM of the M13-tailed forward primer, and 0.5 unit of Taq DNA Polymerase. The conditions were kept constant and general for all loci, to maximize standardization. The PCR cycles profile consisted of 10 cycles (touchdown) of 94 °C for 45s, 60-51 °C for 45s, 72 °C for 1.5 min; this was followed by 30 cycles of 94 °C for 45s, 50 °C for 45s, 72 °C for 1.5 min and final extension of 72 °C for 30min. PCR reactions were carried out for each locus separately, and products from 1 to 3 loci were diluted and pooled together based on yield, size range and fluorescent dye, and then analyzed in a MegaBACE1000 automated sequencer (GE Healthcare). Allele sizes of PCR products were determined using the software GENETIC PROFILER 2.2 (GE Healthcare) and an internal size standard (ET Rox-400 or ET Rox-550, GE Healthcare).

We tested the selected loci for linkage disequilibrium and for Hardy-Weinberg disequilibrium using the software ARLEQUIN 3.01 ( Excoffier et al. 2005 ) with the allowed percentage of missing values set to 0.1. We also estimated the maximum P(ID) using the panel of ten markers and also with a subset containing the shortest-product loci using the software CERVUS 3.0 ( Marshall et al. 1998 ). Both marker sets were tested since the DNA recovered from noninvasive samples tends to be degraded, and therefore the reliable amplification and genotyping of large products may be compromised. 

RESULTS AND DISCUSSION

The initial tests indicated that all loci were amplifiable in the Neotropical otter. However, two of them (LUT818 and RIO17) presented low amplification efficiency. The ten remaining loci presented the desirable features of good efficiency, short product and allelic diversity.

The number of observed alleles per locus ranged from six to 13 (mean number of alleles per locus = 8.6) and the mean expected heterozygosity values for the ten best loci ranged from 0.613 to 0.891 (mean He for all loci = 0.769) ( Table 1 ).


Table 1: Characterization of polymorphic microsatellite loci in Lontra longicaudis.
NI =number  of analyzed individuals; NA =  number of alelles; He = expected and Ho =observed heterozygosities;
HWE = Hardy-Weinberg equilibrium test P value and PIC =  polymorphic information content.
Sample size = 31 individuals

Locus Name Isolated from NI NA Size range (bp) He Ho HWE PIC Dye Reference
Lut453 Lutra lutra 31 6 136-146 0.749 0.677 -- 0.693 FAM Dallas & Piertney 1998
Lut701 Lutra lutra 29 8 172-198 0.702 0.793 -- 0.643 FAM Dallas & Piertney 1998
Lut733 Lutra lutra 31 11 152-178 0.810 0.677 -- 0.770 NED Dallas & Piertney 1998
Lut782 Lutra lutra 31 6 178-198 0.613 0.516 -- 0.552 NED Dallas & Piertney 1998
RIO06 Lontra canadensis 30 7 261-293 0.718 0.600 -- 0.660 FAM Beheler et al. 2004
RIO07 Lontra canadensis 31 7 176-192 0.788 0.290 * 0.743 FAM Beheler et al. 2004
RIO11 Lontra canadensis 31 10 168-186 0.891 0.903 -- 0.864 NED Beheler et al. 2005
RIO18 Lontra canadensis 30 13 142-174 0.834 0.500 -- 0.801 NED Beheler et al. 2005
RIO19 Lontra canadensis 31 9 286-306 0.744 0.548 * 0.718 NED Beheler et al. 2005
RIO20 Lontra canadensis 30 9 257-273 0.844 0.333 * 0.809 HEX Beheler et al. 2005

* significant after Bonferroni correction (α = 0.005)

Furthermore, no locus was in linkage disequilibrium (LD) after Bonferroni correction (α = 0.001) ( Rice 1989 ). On the other hand, three loci (RIO07, RIO18 and RIO20) were out of Hardy-Weinberg equilibrium (HWE) even after Bonferroni correction, but this result can be biased by the sampling design, since we employed samples collected from a large geographic area whose genetic structuring is presently unknown. To further investigate this possibility, we repeated this analysis with samples collected from a more restricted area (Rio Grande do Sul state in southern Brazil; n = 11), and only one locus remained out of HWE (RIO20), supporting the hypothesis that population subdivision may account for the departure from equilibrium observed in the broader sample.

The maximum P(ID) obtained for the ten loci were 1.68 x 10-11, and for the seven shortest-length microsatellites it was 3 x 10-8, indicating a high probability of accurate individual identification using either ten or seven loci. This result indicates that this set of markers should be effective in individual-based studies of L. longicaudis employing noninvasive samples.

The set of markers tested here demonstrated to have a great potential for application in population-genetic, ecological and evolutionary studies of the Neotropical otter, allowing their use even in studies based on noninvasive sampling. The characterization of these microsatellite loci in L. longicaudis opens a wide range of opportunities for studies addressing population structure and dynamics, kinship and mating system, as well as phylogeographic and historical investigations focusing on this so-far-ignored species. The data obtained from these studies will likely be of significant utility for the development of adequate conservation strategies for L. longicaudis, not only in Brazil but throughout its geographic distribution.

ACKNOWLEDGMENTS -We would like to thank all people and institutions that helped with the collection of biological samples used in this study. This work was supported by Centro Nacional de Pesquisas para a Conservação de Predadores Naturais – CENAP/IBAMA, Instituto Pró-Carnívoros and CNPq.

REFERENCES

Beheler, A.S., Fike, J.A., Murfitt, L.M., Rhodes, O.E. Jr., Serfass, T.S. (2004). Development of polymorphic microsatellite loci for North American river otters (Lontra canadensis) and amplification in related Mustelids. Molecular Ecology Notes, 4, 56-58.
Beheler, A.S., Fike, J.A., Murfitt, L.M., Dharmarajan G., Rhodes, O.E. Jr., Serfass, T.L. (2005). Ten new polymorphic microsatellite loci for North American river otters (Lontra canadensis) and their utility in related mustelids. Molecular Ecology Notes, 5, 602-604.
Blundell, G.M., Ben-David, M., Groves, P., Bowyer, R.T., Geffen, E. (2004). Kinship and sociality in coastal river otters: are they related? Behavioral Ecology, 5, 705–714.
Boutin-Ganache, I., Raposo, M., Raymond, M., Deschepper, C.F. (2001). M13-tailed primers improve the readability and usability of microsatellite analyses performed with two different allele-sizing methods. Biothecniques, 31, 25-28.
Carmichael, L.E., Krizan, J., Nagy, J.A., Fuglei, E., Dumond, M., Johnson, D., Veitch, A., Berteaux, D., Strobeck, C. (2007). Historical and ecological determinants of genetic structure in arctic canids. Molecular Ecology, 16, 3466–3483.
Carpenter, P.J., Dawson, D.A., Greig, C., Parham, A., Cheeseman, C.L., Burke, T. (2003). Isolation of 39 polymorphic microsatellite loci and the development of a fluorescently labeled marker set for the Eurasian badger (Meles meles) (Carnivora: Mustelidae). Molecular Ecology Notes, 3, 610-615.
Dallas, J.F., Piertney, S.B. (1998). Microsatellite primers for the Eurasian otter. Molecular Ecology, 7, 1248-1251.
Davis, C.S., Strobeck, C. (1998). Isolation, variability and cross-species amplification of polymorphic microsatellite loci in the family Mustelidae. Molecular Ecology, 7, 1776-1778.
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Résumé: Caracterisation et Evaluation de 12 Loci Microsatellitaires pour l‘Étude de l’Évolution des Populations de Loutre à Longue Queue (Lontra Longcaudis)
Nous avons testé sur la Loutre à longue queue, 12 marqueurs microsatellitaires précédemment isolés chez deux espèces de loutres (Lutra lutra et Lontra canadensis) ceci afin de choisir les marqueurs qui peuvent être utilisés dans le cadre d’un échantillonnage non invasif de cette espèce. Trente et un individus ont été génotypés à partir de plusieurs localités du Brésil. Les valeurs moyennes attendues d’hétérozygotie pour les dix meilleurs loci variaient de 0,613 à 0,891 (Moyenne He=0,769 pour tous les loci). Pour évaluer la fiabilité de l'identification individuelle à partir des marqueurs sélectionnés, nous avons calculé le P(ID) maximum (polymorphisme) en utilisant les dix loci mais aussi un sous-ensemble de sept loci présentant les plus courtes séquences (<200 pb). Les P(ID) maximum estimés étaient respectivement de 1,68x10-11 et 3x10-8, indiquant un bon potentiel des complexes de marqueurs pour une identification individuelle fiable. Les résultats obtenus dans cette étude ont révélé que dix des 12 loci analysés sont appropriés pour une application dans les études écologiques et évolutives des loutres à longue queue, même sur base d’un échantillonnage non invasif.
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Resumen: Caracterización y Evaluación de 12 Loci de Microsatélites Autosómicos Apropiados para Estudios Poblacionales y Evolutivos de la Nutria Neotropical (Lontra longicaudis)
Probamos 12 marcadores de microsatélites previamente aislados de dos diferentes especies de nutria (Lutra lutra y Lontra canadensis) en la nutria neotropical con el objetivo de ayudar en la elección de los marcadores que podrían ser utilizados incluso en muestras no invasivas de esta especie. Treinta y un individuos de varias localidades de Brasil fueron genotipados. La media de los valores esperados de heterocigosidad para los diez mejores loci variaron desde 0.613 hasta 0.891 (la media He para todos los loci = 0.769). Para investigar la fiabilidad de la identificación individual con los marcadores seleccionados, calculamos el P(ID) utilizando los diez loci, y también un subconjunto de siete locis exhibiendo la longitud más corta del producto (<200 pb), que probablemente sean de más utilidad en muestras no invasivas. La máxima estimada P(ID) fue de 1.68 x 10-11 y 3 x 10-8, respectivamente, indicando un buen potencial de ambos juegos de marcadores para asegurar la identificación individual. Los resultados obtenidos en este estudio revelaron que diez de los 12 loci analizados son apropiados para la aplicación en los estudios ecológicos y evolutivos de las nutrias neotropicales, incluso sobre la base del muestreo no invasivo.
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