Revised Proof Journal of Genetics © Indian Academy of Sciences DOI 10.1007/s12041-017-0823-6 RESEARCH ARTICLE Genetic variability and structure of an isolated population of Ambystoma altamirani, a mole salamander that lives in the mountains of one of the largest urban areas in the world ROSA-LAURA HEREDIA-BOBADILLA1, OCTAVIO MONROY-VILCHIS1∗, MARTHA M. ZARCO-GONZÁLEZ1, DANIEL MARTÍNEZ-GÓMEZ2, GERMÁN DAVID MENDOZA-MARTÍNEZ2 and ARMANDO SUNNY1 1Centro de Investigación en Ciencias Biológicas Aplicadas, Universidad Autónoma del Estado de México, Instituto literario # 100, Colonia Centro, CP 50000 Toluca, Estado de México, México 2Departamento de Producción Agrícola y Animal, Universidad Autónoma Metropolitana Xochimilco, Calz. Del Hueso 1100, Col. Villa Quietud, 04960 México, México ∗ For correspondence. E-mail: [email protected]; [email protected]. Received 17 October 2016; revised 8 December 2016; accepted 6 January 2017 Abstract. Amphibians are globally threatened by habitat loss and fragmentation; species within the order Ambystoma are not the exception, as there are 18 species of mole salamanders in México, of which 16 are endemic and all species are under some national or international status of protection. The mole salamander, Ambystoma altamirani is a microendemic species, which is distributed in central México, within the trans-Mexican volcanic belt, and is one of the most threatened species due to habitat destruction and the introduction of exotic species. Nine microsatellite markers were used to determine the genetic structure, genetic variability, effective population size, presence of bottlenecks and inbreeding coefficient of one population of A. altamirani to generate information which might help to protect and conserve this threatened species. We found two genetic subpopulations with significant level of genetic structure (FST = 0.005) and high levels of genetic variability (Ho = 0.883; He = 0.621); we also found a small population size (Ne = 8.8), the presence of historical (M = 0.486) and recent bottlenecks under IAM and TPM models, with a low, but significant coefficient of inbreeding (FIS =−0.451). This information will help us to raise conservation strategies of this microendemic mole salamander species. Keywords. population genetics; conservation genetics; microsatellites; microendemic species; threatened species. Introduction the surrounding area of Mexico city, where this species live, are severely changed, leading to a severely degraded Amystoma altamirani is an endemic mountain mole sala- habitat fragmented by agriculture and urban settlements mander that lives in small, permanent streams which flows (Lemos-Espinal et al. 1999; Griffiths et al. 2004; Frías- in the high mountains in central Mexico in Pinus and Álvarez et al. 2008; Contreras et al. 2009; Lemos-Espinal Abies religiosa forests. This species occur in isolated pop- et al. 2016; Sunny et al. 2017) and the wooded areas are ulations of the valley of Mexico, in the trans-Mexican subjected to illegal logging (FAO 2006; Ellis and Porter- volcanic belt (TMVB), at altitudes from 2700 to 3450 masl Bolland 2008; Becker et al. 2016), stream pollution and (meters above sea level) (Lemos-Espinal 2003; Shaffer sedimentation (Shaffer et al. 2008). Also, local people tend et al. 2008). The Abies–Pinus forest and the streams in to eat molesalamanders (Casas-Andreu et al. 2004)and Electronic supplementary material: The online version of this article (doi:10.1007/s12041-017-0823-6) contains supplementary material, which is available to authorized users. Revised Proof Rosa-Laura Heredia-Bobadilla et al. introduce trout, carp and tilapia farms that predate and Materials and methods compete with mole salamanders, even in protected natu- ral areas (Lemos-Espinal et al. 1999, 2016; Griffiths et al. Study site and population sampling 2004; Frías-Álvarez et al. 2008; Shaffer et al. 2008; Con- treras et al. 2009; Farías-Álvarez et al. 2010). This has Population samplings were done from January to June eliminated the species from many streams, and many pop- 2014 in a small stream (±1km2) surrounded by induced ulations showed a severe decline of >50% over the last and alpine grassland near a Pinus hartwegii and Abies three generations (Shaffer et al. 2008). The environmen- religiosa forest and some rural houses in Tlazala, state ◦ ◦ tal vulnerability score of A. altamirani is 13, in a range of of México (19 31 31 N, 99 26 09.52 W with an altitude 3–19; this score positions it between medium and high vul- of 3185 masl; figure 1). This site is a private land, where nerabilities, primarily because of its restricted geographic ecotourism activities are carried out, along with trout and ecological distributions (Wilson et al. 2013; Lemos- farming, subsistence logging, and cattle and sheep grazing. Espinal et al. 2016). Therefore, this species is protected by We obtained 96 tissue samples, the mole salamanders were Mexican law under the ‘Special Protection’ category and found in small ponds (transects) of ∼ 40 cm in depth and the IUCN categorized this species as endangered (Shaffer 1–2 m of area separated by ∼10−20 m. There were multi- et al. 2008; Semarnat 2010). The isolated and fragmented ple trapping locations along the rivers, so that the sampling populations by a matrix of agriculture and urbanization, areas were chosen for the differences in the microenviron- can be considered islands in terms of genetic variability, mental characteristics of the river; we found four types of gene flow and landscape connectivity (Kim et al. 1998; substrates (mud, gravel, bedrock and sand). To represent Sunny et al. 2014a). The fragmentation of the habitat all maturity stages of A. altamirani in the data, tissue was and the introduction of exotic species at the landscape obtained from both adults and larvae. Sampling of larvae scale may reduce connectivity among amphibian popula- was limited (≥10%) and efforts were made to avoid sam- tions leading to fragmentation and isolation, which could pling siblings. The individuals were collected with fishing reduce gene flow and increase the loss of genetic variability net and we sampled 2 mm2 of tail clips. This methodology through genetic drift and increase the chance of inbreeding is a low-impact method that does not affect the survival (Lande 1998; Frankham et al. 2002, 2005; Beebee and Grif- or growth of the mole salamanders (Arntzen et al. 1999; fiths 2005; Storfer et al. 2009; Hedrick 2011), bottlenecks Polich et al. 2013); tissue was preserved in 90% ethanol and ◦ and diseases (Frankham et al. 2005) and the probability then frozen at −20 C until processed. All mole salaman- of local extinctions (Newman and Tallmon 2001; Johans- ders were released immediately at the point of capture. Our son et al. 2006). Habitat change affects the populations of study received the approval of the ethics committee of Uni- amphibians and reptiles more than other vertebrate taxa versidad Autónoma del Estado de México (3047-2011E). (Jäggi and Baur 1999; Woinarski and Ash 2002; Anadón et al. 2006; Castellano and Valone 2006; Ribeiro et al. DNA extraction and microsatellite amplification 2009; Sunny et al. 2014a, 2015, 2017), owing to their small home ranges, high philopatry and low vagility (Huey Genomic DNA was extracted using a commercial kit 1982; Ribeiro et al. 2009). For these reasons, amphibians (Vivantis GF-1 tissue DNA extraction kit); we used nine are a high priority vertebrate group for which conser- microsatellite loci (At 52.2, At 52.10, At 52.143, At 60.3, vation strategies must be carried out (Cushman 2006; At 52.115, At 52.6, At 52.34, A5 52.20 and At 52.1) Storfer et al. 2009; Allentof and O’Brien 2010; Nori et al. specifically developed for Mexican Ambystomas and fol- 2015). Therefore, we studied the genetic variability and lowed published protocols (Parra-Olea et al. 2007)for structure, effective population size, inbreeding and genetic amplification. PCR reactions were performed in a Techne bottlenecks of a population of A. altamirani in a highly thermocycler, amplified products were multiplexed on an disturbed habitat. The study area is subjected to pres- ABI Prism3730xl and sized with a ROX-500 as an inter- sures, such as illegal logging introduction of exotic species nal size and then scored using PEAK SCANNER V1.0 such as trout, human settlements and pollution of the (Applied Biosystems, Foster City, USA) software; allele streams. Under this scenario, we expected that there will sizes were measured and rounded with the software TAN- be high genetic structure patterns induced by landscape DEM (Matschiner and Salzburger 2009). modification and because we studied a small population, isolated and subjected to anthropogenic pressures, this population would present low levels of genetic variability Microsatellite analysis and effective population size, and presence of inbreeding and genetic bottlenecks. This information will help us to Potential scoring errors: Typing errors and the presence raise conservation strategies of this microendemic mole of null alleles were determined using the Micro-Checker salamander species. software (Van Oosterhout et al. 2004). Revised Proof Population genetics of Ambystoma altamirani Figure 1. (a) Map of Mexico showing in gray, the TMVB and in dark gray, the State of Mexico. (b) State of Mexico showing in white, Isidro Fabela. (c) The sampling site in Tlazala, Isidro Fabela. Genetic structure: The Structure 2.3.4 software (Pritchard within populations based on FST values, using 999 per- et al. 2000) was used to