Genetic differences between the two remaining wild populations of the endangered Indian rhinoceros (Rhinoceros unicornis) Samuel Zschokke a,⇑ , Georg F.J. Armbruster a,b , Sylvain Ursenbacher a , Bruno Baur a a Department of Environmental Sciences, Section of Conservation Biology, University of Basel, St. Johanns-Vorstadt 10, CH-4056 Basel, Switzerland b Department of Environmental Sciences, Institute of Botany, Section of Plant Ecology, University of Basel, Schönbeinstrasse 6, CH-4056 Basel, Switzerland article info Article history: Received 12 March 2011 Received in revised form 18 July 2011 Accepted 27 July 2011 Available online 25 August 2011 Keywords: Bottleneck Microsatellite markers Mitochondrial D-loop Population genetics Population management abstract The management of rare and endangered species in the wild and in captivity requires an understanding of the characterization of the genetic units within each species and their relationships to each other. The Indian rhinoceros (Rhinoceros unicornis) is an endangered species with a current population size of c. 2800 individuals. We analyzed 26 individuals of known origin kept in captivity and 21 wild ranging individuals of the two remnant large wild populations in Assam (India) and Nepal employing mitochondrial and microsatellite markers to determine whether the two geographically isolated populations show distinct patterns of genetic diversity, and whether the genetic diversity of the populations is influenced by past demographic bottlenecks. We identified 10 different mitochondrial D-loop haplotypes, of which 4 were specific to the Assam population (10 sequences examined) and 6 specific to the Nepal population (19 sequences). Similarly, the microsatellite analysis demonstrated a strong genetic differentiation between the Assam and Nepal populations and allowed to assign each individual to its origin with high confidence. Furthermore, our analyses revealed the occurrence of a bottleneck in the Assam population long before the reported bottleneck in 1908, and it revealed that the Nepal population is a recent (probably post-gla- cial) colonization. In summary, the extent of genetic divergence between the two remnant R. unicornis populations suggests separate conservation programs (even for captive individuals) as long as the persis- tence of the entire species is not severely threatened. The microsatellite markers can also be used to determine the origin of confiscated material such as horns. Ó 2011 Elsevier Ltd. All rights reserved. 1. Introduction The fourth largest extant terrestrial mammal, the Indian (or greater one-horned) rhinoceros (Rhinoceros unicornis), is threa- tened according to the World Conservation Union (IUCN) red list (Talukdar et al., 2008). Recent estimates revealed a total number of c. 2800 rhinoceros living in isolated populations in India and Nepal (Talukdar, 2009; van Strien and Talukdar, 2007). In historic times (c. 1400 AD), the Indian rhinoceros occurred along the flood plains from north-western Myanmar across the Gangetic plain to the Indus River Valley in northern Pakistan with a minimal total population of more than 450,000 individuals (Blanford, 1891; Dinerstein and McCracken, 1990; Laurie, 1979). Since the 19th cen- tury, land alterations significantly reduced and fragmented the habitat suitable for the rhinoceros. Hunting and later poaching further reduced the populations. Today, natural populations of the Indian rhinoceros only occur in the states of Uttar Pradesh, West Bengal and Assam in Eastern India and the Terai of Nepal (Foose and van Strien, 1997). The population in Assam was estimated to contain less than 20 individuals in the area of today’s Kaziranga National Park when hunting was banned in 1908 (Laurie et al., 1983; Ryhiner, 1961; Ullrich, 1972). This population recovered to c. 2300 individuals in 2009 and has expanded into neighboring areas, including the Laokhowa, Pabitora and Orang wildlife sanctuaries (Merenlender et al., 1989; Molur et al., 1995; Ryhiner, 1961; van Strien and Talukdar, 2007). In the Chitwan valley in Nepal, a population of more than 1000 individuals persisted until 1950 when poaching and land alterations began to reduce the number of rhinoceros to 60–80 individuals in 1962 (Laurie, 1979). This population also recovered from its bottleneck, attaining an estimated size of 544 individuals in 2000, with a subsequent decrease to 372 individuals due to renewed poaching (Poudyal et al., 2009) and a slow increase to 408 individuals in 2008 (Talukdar, 2009). A distance of 700 km separates the two main populations in Assam and Nepal and no individuals were translocated between the two populations. Further 180 individuals live in other areas in India and Nepal 0006-3207/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocon.2011.07.031 ⇑ Corresponding author. Tel.: +41 61 267 08 54; fax: +41 61 267 08 32. E-mail addresses: samuel.zschokke@unibas.ch (S. Zschokke), g.armbruster@ unibas.ch (G.F.J. Armbruster), s.ursenbacher@unibas.ch (S. Ursenbacher), bruno. baur@unibas.ch (B. Baur). Biological Conservation 144 (2011) 2702–2709 Contents lists available at SciVerse ScienceDirect Biological Conservation journal homepage: www.elsevier.com/locate/biocon