Chromosomal Evolution in Tenrecs (Microgale and Oryzorictes, Tenrecidae) from the Central Highlands of Madagascar
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Chromosome Research (2007) 15:1075–1091 # Springer 2007 DOI: 10.1007/s10577-007-1182-6 Chromosomal evolution in tenrecs (Microgale and Oryzorictes, Tenrecidae) from the Central Highlands of Madagascar C. Gilbert1, S. M. Goodman2,3, V. Soarimalala3,4, L. E. Olson5,P.C.M.O_Brien6, F. F. B. Elder7, F. Yang8, M. A. Ferguson-Smith6 & T. J. Robinson1* 1Evolutionary Genomics Group, Department of Botany and Zoology, University of Stellenbosch, Stellenbosch, South Africa; Tel: +27-21-8083955; Fax: +27-21-8082405; E-mail: [email protected]; 2Department of Zoology, Field Museum of Natural History, Lake Shore Drive, Chicago, IL, USA; 3Vahatra, BP 738, Antananarivo (101), Madagascar; 4De´partement de Biologie Animale, Universite´ d_Antananarivo, BP 906, Antananarivo (101), Madagascar; 5University of Alaska Museum, University of Alaska Fairbanks, Fairbanks, AK, USA; 6Centre for Veterinary Science, University of Cambridge, Cambridge, UK; 7Department of Pathology, Cytogenetics Laboratory, UT Southwestern Medical Center, Dallas, TX, USA; 8The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, UK *Correspondence Received 13 August 2007. Received in revised form and accepted for publication by Pat Heslop-Harrison 2 October 2007 Key words: Afrotheria, cytogenetics, evolution, speciation, Tenrecidae Abstract Tenrecs (Tenrecidae) are a widely diversified assemblage of small eutherian mammals that occur in Madagascar and Western and Central Africa. With the exception of a few early karyotypic descriptions based on conventional staining, nothing is known about the chromosomal evolution of this family. We present a detailed analysis of G-banded and molecularly defined chromosomes based on fluorescence in situ hybridization (FISH) that allows a comprehensive comparison between the karyotypes of 11 species of two closely related Malagasy genera, Microgale (10 species) and Oryzorictes (one species), of the subfamily Oryzorictinae. The karyotypes of Microgale taiva and M. parvula (2n = 32) were found to be identical to that of O. hova (2n = 32) most likely reflecting the ancestral karyotypes of both genera, as well as that of the Oryzorictinae. Parsimony analysis of chromosomal rearrangements that could have arisen following Whole Arm Reciprocal Translocations (WARTs) showed, however, that these are more likely to be the result of Robertsonian translocations. A single most parsimonious tree was obtained that provides strong support for three species associations within Microgale, all of which are consistent with previous molecular and morphological investigations. By expanding on a recently published molecular clock for the Tenrecidae we were able to place our findings in a temporal framework that shows strong chromosomal rate heterogeneity within the Oryzorictinae. We use these data to critically examine the possible role of chromosomal rearrangements in speciation within Microgale. Introduction Mozambique Channel (probably by rafting from East Africa between 42 and 25 million years ago; Poux One of the intriguing events in the evolutionary history et al. 2005), the ancestors of the Malagasy tenrecs of Afrotheria is the colonization of Madagascar by gave rise to an assemblage comprising eight genera the afrosoricid family, Tenrecidae. After crossing the that is virtually unique among eutherians in its 1076 C. Gilbert et al. morphological diversity, and range of ecological distribution, with 11 new or resurrected species adaptations (Olson & Goodman 2003). Recent recognized in newer treatments (e.g., Jenkins 1993, phylogenies based on molecular and morphological Jenkins et al. 1997, Jenkins & Goodman 1999, characters strongly support two main clades within Goodman & Soarimalala 2004, Olson et al. 2004, Malagasy tenrecs. The first corresponds to the sub- Goodman et al. 2006). Several other aspects of the family Tenrecinae and includes four genera of spiny, biology of Microgale have received attention in the hedgehog-like tenrecs (Echinops, Hemicentetes, relatively recent past including physiology and Tenrec and Setifer). The second clade groups reproduction (Stephenson & Racey, 1995 and refer- the semi-aquatic Limnogale, the shrew-like Micro- ences therein), as well as the morphology of its hind gale, and the mole-like Oryzorictes in the subfamily limb musculature (Endo et al. 2006), cranium (Asher Oryzorictinae (Poux et al. 2005, Asher & Hofreiter 2001), placenta (Enders et al. 2006), male reproduc- 2006). The placement of the mouse-like Geogale is, tive tract (Bedford et al. 2004) and spermatozoa however, uncertain, being either sister to all Mala- (Bedford 2004). However, apart from karyotypic data gasy tenrecs as a separate subfamily, the Geogalinae from three species based on standard stained prepa- (Olson & Goodman 2003), or occupying an unre- rations (Borgaonkar & Gould 1968, 1969), virtually solved position within Oryzorictinae (Asher & nothing is known about the chromosomal evolution Hofreiter 2006). Within Oryzorictinae, Olson & of Microgale. This observation can in fact extend to Goodman (2003) found that Limnogale nested within other Tenrecidae since the only information available Microgale, a position that was supported by several on the karyotypes of the other genera is restricted to non-ambiguous molecular characters. Moreover, diploid chromosome numbers and/or the occasional although subsequent studies included only one presentation of unbanded karyotypes (i.e., Micro- species of Microgale, and could therefore not test potamogale, 2n = 40; Tenrec and Hemicentetes,2n= the monophyly of this genus, strong support was 38; Echinops and Setifer, 2n = 40; Borgaonkar 1967, found for an (Oryzorictes,(Limnogale + Microgale)) Borgaonkar & Gould 1965, Bernischke 1969). topology by Poux et al. (2005) and Asher & Moreover, there is no well-supported phylogenetic Hofreiter (2006). hypothesis detailing interspecific relationships within With its 34 species (including three African Microgale. Olson & Goodman_s (2003) cladistic species), the Tenrecidae is the most diversified analysis of the Tenrecidae is the most comprehensive family within Afrotheria, an endemic African clade in terms of taxonomic sampling, including a large of mammals that includes the Paenungulata (ele- number of species of shrew tenrecs. Importantly, phants, sirenids and hyraxes), and the Afroinsecti- however, relationships within Microgale were not phillia comprising elephant shrews, tenrecs, golden discussed in this study. Finally, although two recent moles and the aardvark (see Springer et al. 1997, studies have utilized both molecular and morpho- Murphy et al. 2001, reviewed in Robinson & Seiffert metric characters to define species limits in selected 2004). This high diversity within the Tenrecidae can shrew tenrec taxa (Olson et al. 2004, Goodman et al. be attributed to the spectacular radiation of Micro- 2006), neither was intended to produce a compre- gale, the most speciose terrestrial mammal genus on hensive phylogeny for the genus. Madagascar (Goodman et al. 2006). Most of these Here, we use conventional banding as well as small, shrew-like tenrecs are found in the eastern chromosome painting by fluorescence in-situ hybrid- humid forests of the island where they generally have ization (FISH) to describe and compare the karyo- a broad distribution, with many species occurring types of 10 Microgale species and that of the closely sympatrically (Jenkins 2003). The taxonomy of the related genus Oryzorictes, represented by O. hova. genus has undergone extensive revision since its We interpret the observed rearrangements in a original description by Thomas (1882). For example, cladistic framework and examine the outcomes with MacPhee (1987) retains only 10 of the 22 species respect to two hypotheses of chromosomal evolu- described during the preceding century. During the tionVone involving Whole Arm Reciprocal Trans- past 20 years, however, extensive field surveys locations (WARTs), and the other only Robertsonian coupled with comprehensive morphometric and/or translocations. We place our findings in a temporal molecular investigations resulted in a considerable framework by expanding the molecular clock analy- refinement of their taxonomy and patterns of sis of Poux et al. (2005) and show that extreme rate Chromosomal evolution in tenrecs of Madagascar 1077 differences exist in the chromosomal evolution of Fluorescence in-situ hybridization (FISH) Microgale spp. Using these data, we critically examine the role for chromosomal rearrangements Chromosome specific painting probes were generated in speciation within the genus. from flow-sorted suspensions of Hoechst 33258 and chromomycin A-3 stained chromosomes that were separated on size and AT:GC ratio, and labelled with Material and methods DOP-PCR (Telenius et al. 1992). FISH followed the protocol described in Gilbert et al. (2006) with the Tissue samples, cell culture and chromosome exception that chromosome preparations were dena- preparation tured for 10 s in 70% formamide/0.6% SSC solution at 65-C rather than 30Y45 s at the temperature of Tissue samples were collected during three inventory 70-C originally described. Biotin-labelled probes surveys of study sites situated in the rainforests of the were detected by Cy3-avidin. Slides were mounted Central Highlands of Madagascar (Table 1). Fibro- in antifade (Vectashield: Burlingame, California, USA) blast cell lines were established using DMEM or and images were captured using Genus 3.7 software Amniomax (Gibco: Paisley, Scotland, UK) culture (Applied Imaging).