Mitochondrial Phylogeographic Structure of the White-Browed Piculet (Sasia Ochracea): Cryptic Genetic Differentiation and Endemi
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Journal of Biogeography (J. Biogeogr.) (2008) 35, 565–575 ORIGINAL Mitochondrial phylogeographic structure ARTICLE of the white-browed piculet (Sasia ochracea): cryptic genetic differentiation and endemism in Indochina Je´roˆme Fuchs1,2,3,4*, Per G.P. Ericson5 and Eric Pasquet1,2 1UMR5202, ‘Origine, Structure et Evolution de ABSTRACT la Biodiversite´’, De´partement Syste´matique et Aim Our understanding of the geographic patterns of gene flow between Evolution, Muse´um National d’Histoire Naturelle, 55 rue Buffon, 75005 Paris, France, populations of birds in the Indo-Malayan faunal region is surprisingly poor 2Service Commun de Syste´matique compared with that in other parts of the world. A thorough knowledge of general Mole´culaire, IFR CNRS 101, Muse´um patterns of phylogeographic structure is, however, of utmost importance for National d’Histoire Naturelle, 43 rue Cuvier, conservation purposes. Species with poor dispersal capabilities could serve as 75005 Paris, France, 3DST-NRF Centre of indicators of endemism and genetic isolation in the Indochinese subregion. From Excellence at the Percy FitzPatrick Institute, their morphology (tiny size, short tail, short and rounded wings), piculets of the University of Cape Town, Rondebosch 7701, genus Sasia are inferred to have poor dispersal capabilities, and thus form a Cape Town, South Africa, 4Museum of suitable focal species. This study analysed the pattern of genetic variation within Vertebrate Zoology and Department of the White-browed Piculet (Sasia ochracea). Integrative Biology, 3101 Valley Life Science Location Southeast Asia, north of the Isthmus of Kra. Building, University of California, Berkeley, CA 94720-3160, USA and 5Department of Methods We sampled 43 individuals throughout the breeding range of Vertebrate Zoology and Molecular Systematics S. ochracea. DNA was extracted both from fresh tissues (n = 15) and from toe Laboratory, Swedish Museum of Natural pads from ancient museum skins (n = 28). We amplified a 801-bp fragment of History, PO Box 50007, SE-104 05 Stockholm, the mitochondrial ND2 gene to reconstruct the phylogeographic history of the Sweden White-browed Piculet. The sequence data were analysed using Bayesian inference, statistical parsimony, and population genetics methods (analysis of molecular variance, mismatch distributions). We estimated the amount of ongoing gene flow between populations using the coalescent-based method implemented in Mdiv. Results The analysis of molecular variance indicated that the current taxonomy does not adequately reflect the amount of genetic variation within S. ochracea,as the great majority of genetic variation was nested within the nominal subspecies, which is distributed from Nepal to southern Vietnam. Bayesian inference analyses and haplotype networks suggested the occurrence of five main lineages that are strongly correlated with geography. Our coalescent-based analyses indicated a very limited amount of ongoing gene flow between these five lineages. Our dating analyses suggested that the genetic structuring probably occurred during the last 400,000 years. Main conclusions Our analyses revealed that S. ochracea is composed of at least five lineages: south Vietnam (South Annam and ‘Cochinchina’), India and Nepal, Myanmar and India, the remainder of Indochina, and probably southern Myanmar (Tenasserim). We strongly recommend that studies aiming to understand the phylogeographic structure within Indo-Malayan species sample *Correspondence: Je´roˆme Fuchs, Museum of Vertebrate Zoology and Department of these areas. Integrative Biology, 3101 Valley Life Science Keywords Building, University of California, Berkeley, CA 94720-3160, USA. Gene flow, haplotype network, phylogeography, Sasia ochracea, Southeast Asia, E-mail: [email protected] systematics. ª 2007 The Authors www.blackwellpublishing.com/jbi 565 Journal compilation ª 2007 Blackwell Publishing Ltd doi:10.1111/j.1365-2699.2007.01811.x J. Fuchs, P. G. P. Ericson and E. Pasquet subcontinent. From their morphology (tiny size, short tail, INTRODUCTION short and rounded wings), piculets of the genus Sasia are The Indo-Malayan faunal region is one of the most species-rich inferred to have poor dispersal capabilities. Indeed, one areas of the world. An impressive number of 1169 bird species individual of Sasia abnormis was captured only 800 m from (> 10% of all the species in the world) live there, of which 70% its initial capture location several years earlier (Winkler & are endemic to the region (Newton, 2003). However, heavy Christie, 2002). Species with poor dispersal capabilities, such as exploitation of natural resources for wood and the pet trade has S. ochracea, could serve as models with which to detect areas of rendered the Indo-Malayan region among the most threatened local endemism as a result of genetic isolation. Here, we aim to on Earth (Sodhi et al., 2004, 2006). The severe threats have address the phylogeographic structure within S. ochracea in sparked intensive work to protect the fauna and flora, including order to: (1) propose new hypotheses about patterns of genetic the creation of conservation programs all over the region. variation within widespread Southeast Asian species, and (2) Among many other things, such work requires a thorough highlight areas where high genetic distinctiveness occurs in understanding of the patterns of genetic diversity among and order to inform conservation practices. within species (Kahindo et al., 2007). Furthermore, widespread species may exhibit zones of molecular endemism, suggesting MATERIALS AND METHODS incomplete understanding of intraspecific taxonomy and the existence of morphologically cryptic species. In birds, such Laboratory procedures information is essentially lacking in all parts of the Indo- Malayan region, whereas it has received considerably more We sampled 43 individuals of S. ochracea, and used two attention in other areas of the world, such as the Palaearctic individuals of S. abnormis as outgroups. Sampling localities (e.g. Pavlova et al., 2005; Zink et al., 2006), Afrotropics (e.g. encompassed the distribution of S. ochracea (Fig. 1, Appendix Bowie et al., 2004, 2006), the Nearctic (e.g. Barrowclough et al., S1). Several ancient collecting localities were retrieved using 2004; Burns & Barhoum, 2006), and the Neotropics (e.g. Marks Hennache & Dickinson (2000) and Lozupone et al. (2004). DNA et al., 2002; Bates et al., 2004). The only two studies from the was extracted from fresh tissues (blood, liver, muscle) (n = 15) Indo-Malayan region are of the White-crowned Forktail and from toe-pads from museum skins collected during the (Enicurus leschenaulti, Moyle et al., 2005) and Grey-cheeked period 1910–1970 (n = 28) using a Cethyl Trimethyl Ammo- Fulvetta (Alcippe morrisonia, Zou et al., 2007). These studies nium Bromide (CTAB)-based protocol (Winnepenninckx et al., highlight regions of high genetic differentiation within Borneo 1993). Two further sequences (one White-browed and one (Moyle et al., 2005) and China (Zou et al., 2007). These two Rufous Piculet) were retrieved from GenBank (http:// studies do not, however, involve a comprehensive geographic www.ncbi.nlm.nih.gov). We amplified an 801-bp fragment of sampling across the breeding range of the study species, so a the ND2 gene using primer pairs L5219–H6313, L5219–SaH650 thorough understanding of the patterns of genetic diversity and SaL450–H6313 for fresh samples, and L5219–Sa200H, within an Indo-Malayan species has not yet been gained. SaL150–SaH350, SaL300–SaH500, SaL450–SaH650, SaL600– In our previous survey of the phylogenetic relationships and SaH800, SaH750–H6313, 750F–950R for museum samples biogeographical history of the piculets (Fuchs et al., 2006), we (Appendix S2). The amplification protocol was standard sampled four white-browed piculets (Sasia ochracea Hodgson, (2 min at 94°C, followed by 36 cycles of 94°C for 40 s, 54°Cfor 1836) and discovered substantial genetic differentiation among 45 s, 72°C for 40 s, and a final extension at 72°C for 5 min). the four individuals. Here, we aim to describe and understand, Three-microlitre samples of the amplification products were using a more thorough sampling of the breeding range, the electrophoresed on a 1.5% agarose gel and examined under UV geographic structure of the genetic variation within the White- light with ethidium bromide to check for the correct fragment browed Piculet, a woodpecker species endemic to Southeast size, control for the specificity of the amplifications, and rule out Asia. Sasia ochracea has a widespread distribution throughout contaminations (positive blank). The polymerase chain reaction the Indochinese subregion, and three subspecies are currently (PCR) products were purified using a QiaQuick PCR purification recognized (Winkler & Christie, 2002): S. o. ochracea Hodgson, kit (Qiagen, Holden, Germany). Cycle-sequencing reactions were 1836 is present from northern India to southern and central performed using a CEQ Dye terminator cycle sequencing kit Vietnam; S. o. kinneari Stresemann, 1929 can be found in (Beckman Coulter, Inc., Fullerton, CA, USA) or a Big Dye northern Vietnam (‘Tonkin’) and South China (Yunnan and (Applied Biosystems, Inc., Foster City, CA, USA) terminator Guangxi); and S. o. reichenowi Hesse, 1911 is restricted to chemistries kit using the same primers as for PCR amplifications. south Myanmar (Tenasserim) and south-west Thailand south DNA strands were sequenced on an automated CEQ2000 DNA to the Isthmus of Kra, where the species is replaced by its analysis system