Cryptic Speciation and Chromosomal Repatterning in the South African Climbing Mice Dendromus (Rodentia, Nesomyidae) Emanuela Solano1*, Peter J. Taylor2,3, Anita Rautenbach3, Anne Ropiquet4, Riccardo Castiglia1 1 Department of Biology and Biotechnologies ‘‘Charles Darwin’’, Sapienza University of Rome, Rome, Italy, 2 Department of Ecology & Resource Management, School of Environmental Sciences, Thohoyandou, South Africa, 3 School of Life Sciences, University of KwaZulu-Natal, Durban, South Africa, 4 Imperial College London Silwood Park Campus, Ascot, Berks, United Kingdom Abstract We evaluate the intra- and interspecific diversity in the four South African rodent species of the genus Dendromus. The molecular phylogenetic analysis on twenty-three individuals have been conducted on a combined dataset of nuclear and mitochondrial markers. Moreover, the extent and processes underlying chromosomal variation, have been investigated on three species by mean of G-, C-bands, NORs and Zoo-FISH analysis. The molecular analysis shows the presence of six monophyletic lineages corresponding to D. mesomelas, D. mystacalis and four lineages within D. cfr. melanotis with high divergence values (ranges: 10.6% – 18.3%) that raises the question of the possible presence of cryptic species. The first description of the karyotype for D. mesomelas and D. mystacalis and C- and G- banding for one lineage of D. cfr. melanotis are reported highlighting an extended karyotype reorganization in the genus. Furthermore, the G-banding and Zoo-FISH evidenced an autosome-sex chromosome translocation characterizing all the species and our timing estimates this mutation date back 7.4 mya (Late Miocene). Finally, the molecular clock suggests that cladogenesis took place since the end of Miocene to Plio-Pleistocene, probably due to ecological factors, isolation in refugia followed by differential adaptation to the mesic or dry habitat. Citation: Solano E, Taylor PJ, Rautenbach A, Ropiquet A, Castiglia R (2014) Cryptic Speciation and Chromosomal Repatterning in the South African Climbing Mice Dendromus (Rodentia, Nesomyidae). PLoS ONE 9(2): e88799. doi:10.1371/journal.pone.0088799 Editor: Roscoe Stanyon, University of Florence, Italy Received August 14, 2013; Accepted November 9, 2013; Published February 13, 2014 Copyright: ß 2014 Solano et al. 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 author and source are credited. Funding: Part of this study was carried out during the post-doctoral fellowship of E. Solano at the University of Stellenbosch and the work was supported by the South African National Research Foundation and the University of Stellenbosch. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] Introduction The four species cannot be all easily distinguished morpholog- ically since some diagnostic character may be equivocal. However, Rodentia are the most diversified mammals, with more than even if sympatric, the species show a certain degree of ecological two thousand species [1]. This number is ever increasing, as new separation. Dendromus melanotis, the grey climbing mouse, is a species are continually described through molecular phylogenetics morphologically variable species with a wide distribution in South and DNA barcoding studies, revealing frequently the presence of Africa and lives in drier areas with short grassland. Dendromus cryptic species [2]. Besides these molecular approaches, the study mesomelas, Brants’s climbing mouse, overlaps with D. melanotis of the karyotype in rodents contributed in delimiting species [3] except for the northern part of the country where it is absent and the chromosomal rearrangements are suspected to have a role (Fig. 1). D. mesomelas is markedly different from the sympatric in the speciation itself [4–6]. species in pelage colour and other morphological traits. Dendromus This study focuses on the South African species of the climbing mystacalis, the chestnut climbing mouse, occurs predominantly in mice, genus Dendromus Smith, 1829 (Rodentia, Nesomyidae), that the eastern parts of South Africa, is smaller than D. mesomelas but belong to the Dendromurinae, currently restricted to Africa. In very similar in morphology and they both prefer wet zones and this region, several rodents lineages have been molded by complex long grass. Finally, Dendromus nyikae, the Nyika climbing mouse, is events during Miocene and Plio-Pleistocene, yet not completely dark-furred and large-bodied. It has a distribution mostly outside understood [7]. While the phylogenetic positions of Dendromus and of South Africa; it fringes into the Limpopo Province in the north- of Dendromurinae in the family has been defined [8–9], the eastern part of South Africa. D. nyikae is considered closely related evolutionary relationships among the constituent species are to D. melanotis and sometimes they can be morphologically unresolved and the species limits require an extensive revision. confused since the differences mainly concern the size and Musser and Carleton [10] recognized 12 species, but recently, sometimes the pelage colour pattern. an additional species was described from Guinea [11]. Four Cytogenetic data for Dendromus are sparse and are mainly species, with partially overlapping ranges, occur in South Africa limited to standard stained karyotypes [13–15]. Matthey [13], [12] (Fig. 1): D. melanotis Smith, 1829, D. mesomelas Brants, 1827, D. comparing diploid numbers, shape of chromosomes and funda- mystacalis Heuglin, 1863 and D. nyikae Wroughton, 1909. mental number in related genera argued that complex rearrange- PLOS ONE | www.plosone.org 1 February 2014 | Volume 9 | Issue 2 | e88799 Genetic Diversity in South African Dendromus Figure 1. Dendromus species distribution in South Africa. The scale of grey areas (from light to dark tone) indicate D. melanotis, D. mesomelas and D. mystacalis respectively. The numbers indicate the sample localities listed in Table S1. The symbols represent the species sampled in each locality - Black triangles: D. melanotis; White circles: D. mesomelas; White squares: D. mystacalis; White triangles: Dendromus sp. doi:10.1371/journal.pone.0088799.g001 ments occurred in the speciation of genus. Furthermore, to the Animal Protection Act (1962) and the National Environ- Dippenaar et al. [15] recorded a 2n = 52 (NFA = 62) for a D. mental Management: Protected Areas Act 57 (2003). Animal melanotis from the Kalahari region of South Africa. Only recently, a handling procedures were approved by the animal ethics sub- C- and G-banded karyotype was studied in D. lachaisei from committee of the University of Kwazulu-Natal. The capture and Guinea [11]. These data suggest that considerable chromosomal euthanasia of animals were performed in accordance with variability exists among the recognized species, with variation prescribed guidelines of the American Society of Mammalogists recorded from 2n = 30 to 2n = 52. for the use of wild mammals in research, outlined in Sikes and The aim of this investigation was to evaluate the intra- and Grannon (2011). Each animal was anesthetized and euthanized at interspecific genetic diversity in species of Dendromus and to the capture site by overdose of inhalant anesthetic according to the estimate their phylogenetic relationships. We also provide the first ‘‘Guideline to the care and use of animals in research and comparative cytogenetics study for the genus including the first teaching’’ of the Animal Ethics Committee of the University of description of the karyotype for D. mesomelas and D. mystacalis and KwaZulu-Natal. All other samples were sourced from existing the first C- and G-banding patterns for a single lineage of D. cfr. tissue collections associated with museum collection. Specimens melanotis. The extent and processes underlying temporal chromo- were deposited in the Durban Natural Science Museum, South somal variation was estimated by a molecular phylogenetic Africa. Voucher numbers and grid references to the collection analysis. localities are reported in Table S1. Materials and Methods Samples Twenty-three Dendromus specimens were analysed from nine localities in South Africa (Table 1, Fig. S1). The specimens, Table 1. Pairwise genetic distances. identified using external traits and skull morphology (see [16]), were assigned to D. melanotis (N = 9), D. mesomelas (N = 2) and D. mystacalis (N = 7). However, for five specimens morphologically L1 L2 L3 L4 C2 C3 similar to D. melanotis (indicated herein as Dendromus sp.), the identification using existing taxonomic keys was equivocal. L1------ L2 0,183 - 0,034 0,041 0,061 0,064 Ethic statement L3 0,141 0,159 - 0,024 0,063 0,067 No protected species were used in this study. The three animals L4 0,166 0,171 0,106 - 0,068 0,072 used for the cytogenetic procedures were live trapped with C2 0,205 0,212 0,200 0,196 - 0,006 Sherman traps placed in transects on the ground. The bait was C3 0,193 0,190 0,170 0,163 0,105 - a mixture of peanut butter, apples and oatmeal. Traps were set in the late afternoon and inspected in the early morning. The The pairwise genetic divergence (net between-group mean distances) among sampling was conducted in accordance with regulations of the four lineages