Preliminary data on the genetic structure of puma (Puma concolor) populations in Argentina

GALLO, ORLANDO; CASTILLO, DIEGO; GODINHO, RAQUEL; GUERISOLI, MARIA DE LAS MERCEDES; LUCHERINI, MAURO; CASANAVE, EMMA

The Wild Felid Monitor

Wild Felid Research and Management Association

Lugar: Hillsboro; Año: 2017 vol. 10 p. 19 - 20

2167-3861

Genetic isolation due to habitat fragmentation and the consequent loss of landscape connectivity is considered a major threat to wildlife conservation, especially for wide-ranging carnivores, such as puma. Intensive agriculture expansion and ranching, and the resulting habitat fragmentation process, as well as human persecution, are the main threats to puma conservation in Argentina (Novaro and Walker 2005; De Angelo et al. 2011). Although the puma is protected by an Argentinian National Law (N°22.421), the species is under high hunting pressure by livestock ranchers and hunters. Furthermore, some provincial governments permit puma hunting and reward livestock ranchers for each individual killed, without information about the population status as well as no control on the number of individuals harvested.In this work we investigated puma genetic diversity and population structure using 40 puma samples, including tissue, bone, skin and fecal samples opportunistically collected from three Argentinian provinces (Buenos Aires [N=24], Chubut [N=13] and Jujuy [N=3]; Figure 1) during 2013-2016. Samples were preserved in ethanol 96% and stored at -20°C until DNA extraction, except for bone samples that were stored at room temperature and without alcohol.EasySpin Kit (Citomed) was used to extract DNA from tissue samples following manufacture instructions. DNA extractions from fecal material followed the Guanidine Tiocyanate Protocol (Boom et al. 1990) and from bone and skin samples followed the Dabney et al. (2013) method. To be sure that each scat belonged to pumas we amplified a fragment of the mtDNA cytochrome b using primers GludG and CB2R. Sequences were compared with GenBank entries using BLAST (National Center for Biotechnology Information, Bethesda, MD).A total of 30 Puma concolor microsatellites (9 dinucleotide and 21 tetranucleotide; Kurushima et al. 2000 and Rodzen et al. 2007) were amplified in three multiplexes. Parameters for thermal cycling were as follows: 95°C for 15 min; followed by 13 cycles at 95°C for 30 s, an annealing temperature of 64°C for 1.30 min [Multiplex1 and 3] or of 62°C for 1 min [Multiplex2] with a decreasing value of 0.5°C/cycle, and 72°C for 45 s; 32 [Multiplex 1 and 3] or 27 [Multiplex 2] more cycles at 95°C for 30 s, one step in which the temperature was kept constant at 58°C [Multiplex 1 and 3] or 56°C [Multiplex 2], and 45 s at 72°C; finally a 30 min extension at 60°C followed by 10°C until further use. Microsatellites were scored using GeneMapper (Applied Biosystems) and sequences were analyzed using BioEdit (Hall 1999).Population genetics data analyses (observed [Ho] and expected [He] heterozygosity and Hardy-Weinberg equilibrium) were carried out using Arlequin 3.5.2 (Exoffier and Lischer 2010). To assess the existence of population structure, we used a Bayesian model based clustering method implemented in STRUCTURE 2.3.4 (Pritchard et al. 2000). We conducted five independent runs for each number of clusters (k) between 1 and 5, using the admixture model, no prior population information, 100,000 burnin iterations and 106 replications.Although we obtained DNA from 33 samples (9 muscles, 9 skins, 8 bones, and 7 faeces), we could only have a complete microsatellite profile for22 individuals. The four puma fecal DNA and six tissue samples had not enough quality for microsatellite amplification.All loci were polymorphic and the number of alleles per locus ranged from 3 to 13 with an average of 5.83. Across all samples and 29 loci (missing value<5%) the average Ho was 0.61 and the average He was 0.70. The 30 polymorphic loci did not deviate from Hardy-Weinberg equilibrium.Population structure analysis revealed two distinct genetic subdivisions in central-southern Argentina corresponding to Buenos Aires and Chubut Provinces (Figure 1 and 2), respectively. This preliminary result suggests the potential presence of a genetically structured population between two areas only 343 km apart.Maintaining landscape connectivity or any current corridor among regions may be necessary for the conservation of viable puma populations, mostly for a species living in a fragmented landscape and under heavy hunting pressure. In this context, our future work will include the analyses of a comprehensive dataset throughout almost the entire country combining recent and historical samples to confirm the observed population structure and estimate genetic diversity and gene flow across populations. Moreover, it will allow us to make inferences on possible demographic events of puma populations in Argentina and on the presence of barriers to puma dispersion.