Molecular Phylogenetics and Evolution 41 (2006) 257–265 www.elsevier.com/locate/ympev Phylogeography of ground tit (Pseudopodoces humilis) based on mtDNA: Evidence of past fragmentation on the Tibetan Plateau Shu-Juan Yang a,b, Zuo-Hua Yin a, Xin-Ming Ma a,c, Fu-Min Lei a,¤ a Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, China b Graduate School of the Chinese Academy of Sciences, Beijing 100049, China c University of Connecticut, Farmington, CT 06030, USA Received 29 October 2005; revised 27 May 2006; accepted 1 June 2006 Available online 7 June 2006 Abstract Pseudopodoces humilis, a long misclassiWed terrestrial tit, is the only species of parid whose distribution is limited to treeless terrain and endemic to the Tibetan Plateau. We revealed the phylogeographic structure of the species by using mitochondrial control region, as well as comparing morphological characters. The distinct geographic distributions of two major clades suggest spatial and temporal separa- tions that coincide with important climatic and paleogeographic changes following the uplift of the Tibetan Plateau. Population expan- sion was inferred for the population at the platform of the Plateau 0.17 million years before present (Ma B.P.), and restricted gene Xow with isolation by distance was detected within this region, congruent with expansion occurring after the extensive glacial period. A signiW- cant decrease in body size with decreasing altitude was found, possibly indicating selection for larger-sized birds at higher altitude. © 2006 Elsevier Inc. All rights reserved. Keywords: Pseudopodoces humilis; Phylogeography; Population expansion; Past fragmentation; Tibetan Plateau 1. Introduction (e.g. past fragmentation, colonization, or range expansion events) (Hewitt, 1999, 2004; Avise, 2000). The uplift of the Many studies have focused on speciation events and Tibetan Plateau (Qinghai-Xizang Plateau) beginning in the genetic structure associated with the Pleistocene Refugia Pliocene and continuing through the Quaternary glacia- Hypothesis, wherein repeated glaciations followed by gla- tions has considerably inXuenced the evolution and distri- cial retreat and habitat recovery are proposed to account butions of many species (Zheng et al., 1981; Shi et al., 1998; for patterns of avian species diversity across Eurasia and Liu et al., 2002; Pang et al., 2003). Three stages of the uplift North America (Baker and Marshall, 1997; Klicka and have been hypothesized to potentially account for specia- Zink, 1997; Merilä et al., 1997; Avise, 2000; Mila et al., tion of some plant, Wsh and pika species (Liu et al., 2002; He 2000; Peck and Congdon, 2004). Phylogeography, the study et al., 2001; Yu et al., 2000). Much intraspeciWc diVerentia- of the distribution of genealogical lineages to understand tion occurred in mammals and birds during Quaternary factors involved in the origin and maintenance of present- (Avise et al., 1998; Klicka and Zink, 1997), therefore the day spatial genetic structure (Avise, 2000), has proved very phylogeographic studies within some species endemic to the useful in tests of this hypothesis. These factors include geo- Tibetan Plateau can help us to understand the inXuence of graphical barriers resulting in recurrent but restricted gene the topographic and environmental changes on the specia- Xow and historical events operating at the population level tion process. Nevertheless, it is poorly understood below species level since there are few intraspeciWc phylogeo- graphic studies about the species distributed on the Tibetan * Corresponding author. Fax: +86 10 62565689. Plateau due to inaccessibility of samples. Two recent stud- E-mail address: [email protected] (F.-M. Lei). ies of the phylogeography of the red-necked snow Wnch 1055-7903/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2006.06.003 258 S.-J. Yang et al. / Molecular Phylogenetics and Evolution 41 (2006) 257–265 (Pyrgilauda ruWcollis) and the white-rumped snow Wnch In this paper, we analyze variation in both mitochon- (Onychostruthus taczanowskii), both endemic to the Tibetan drial control region DNA sequence and morphology of P. Plateau, found no distinct phylogeographic structure. These humilis from seven populations covering most of its distri- two related species show some diVerence in overall nucleo- bution. We address three questions: (1) Is P. humilis geneti- tide diversity, but their phylogeographic patterns are con- cally structured over its distribution or does it exhibit the gruent, suggesting rapid range expansion following a weak structure evident in other sympatric species? (2) How population bottleneck and high gene Xow due to strong have potential geological barriers (e.g. mountains, deserts, mobility (Qu et al., 2005; Yang et al., 2006). habitats) aVected contemporary patterns of genetic varia- The Hume’s Ground Tit (Pseudopodoces humilis) is tion in this species? (3) What is the importance of historic- another species endemic to the Tibetan Plateau (Fig. 1). geographical versus contemporary processes in generating Previously known as Hume’s Ground Jay, it had long been intraspeciWc phylogeographic pattern? misclassiWed as a member of crow and jay family (Corvi- dae) according to its initial description (Hume, 1871) and 2. Materials and methods was considered to be close to the genus Podoces until 1978 because of several traits shared in common with it. A recent 2.1. Sampling and laboratory methods study (James et al., 2003) removed it from the Corvidae and suggested that it should be the most aberrant member of Samples of 67 P. humilis individuals from 17 localities the tits and chickadees (family Paridae) based on three (Fig. 1 and Table 1) covering almost the entire distribution, independent datasets drawn from comparative osteology, were collected at elevations from 3200 to 4550 m during the the nuclear c-myc gene, and the mitochondrial cytochrome years 2000–2003. b gene, although Pseudopodoces shows little superWcial DNA was extracted from muscle, feather and blood with resemblance to tits and chickadees (Chin, 2003; Lei et al., EZ Spin Column Genomic DNA Isolation Kit (BBI) fol- 2003). Pseudopodoces is the only species of parid with a dis- lowing the manufacturer’s protocols. Polymerase chain tribution limited to treeless terrain (3000 to perhaps reactions (PCRs) were performed with primers L16700 and 5480 m) and one of the few species of parid with weak H636 which cover the Wrst domain and part of the second Xight. This explanation is consistent with a hypothesis that domain of the mitochondrial control region as described in the uplift of the Tibetan Plateau provided a tectonically Kvist et al. (2002). DNA samples were isolated from toe active geomorphological region in which Pseudopodoces pads of three individuals in Northern Tibet and two in Cen- has a limit to its distribution, that a species of parid invaded tral Tibet collected from the years 1960 to 1980 following the emergent high steppes and became the ancestor of the protocols as described in Nishiguchi et al. (2002), and Pseudopodoces; Wnally, that morphological evolution in a were ampliWed in two fragments using primers: novel adaptive zone altered its appearance. (L16700 + HP328: 5Ј-CTGTGACATTATTCGTATTCG Fig. 1. Distributions and sampling localities of P. humilis. Filled circles represent distributions; unWlled circles represent sampling localities; sands repre- sent desert area. (Note: NT, Northern Tibet; CT, Central Tibet; ST, Southern Tibet; ET, Eastern Tibet; SQ, Southern Qinghai; CQ, Central Qinghai; NQ, Northern Qinghai.) S.-J. Yang et al. / Molecular Phylogenetics and Evolution 41 (2006) 257–265 259 Table 1 Polymorphic sites within mtDNA control region of P. humilis , nucleotide diversity; Hd, haplotype diversity; n, sample size. A-3Ј and LP288: 5Ј-AAACATCTTGCACTCGAATACG ences and haplotype frequencies (ExcoYer et al., 1992), A-3Ј + H636) modiWed from Kvist et al. (2003). measure of the extent of DNA divergence between popula- W W Y The ampli cation pro le was 94 °C for 5 min followed tions (FST; Exco er et al., 1992) was calculated, and the sig- by 35 cycles of 94 °C for 1 min, 53 °C for 1 min and 72 °C for niWcance was tested using 10,000 permutations with 1 min and a Wnal extension in 72 °C for 5 min. Sequencing Arlequin (version 2.0, Schneider et al., 2000). Mismatch dis- reactions were performed with the primers H636, HP328 tributions (SSD) and Harpending’s raggedness index (Hri; and HP451: 5Ј-AGTTTAAGTCCCTGAAGC-3Ј (modi- Harpending, 1994) were estimated to test whether the Wed from H450 in Kvist et al., 2002) with Big Dye Termina- sequence data deviate signiWcantly from the expectation of tor Cycle Sequencing Kit v.2.0 and run with ABI 377 a population expansion model. The parameters , tau () automatic sequencer. Sequences have been deposited in and Fu’s Fs were also calculated with Arlequin. The esti- GenBank under Accession Nos. DQ267830–DQ267895. mates ( 0 and 1) are the product of 2 N, where N is the eVective population size, and is the mutation rate; here, 2.2. Genetic data analysis we assumed the widely accepted average mutation rate of 2% Ma¡1 per site of mtDNA control region for passerine The DNA sequences were aligned using the program birds, despite the controversies concerning the 2% rule (Kli- ClustalX1.83. Nucleotide diversity (, Nei, 1987; Eq. 10.5), cka and Zink, 1997; Avise and Walker, 1998; Randi et al., and haplotype diversity (Hd; Nei, 1987; Eqs. 8.4 and 8.12) 2001; Lovette, 2004). Tau () is equal to 2kT, where k is were calculated with DnaSP v. 4.00 (Rozas et al., 2003) for the number of nucleotides assayed, and T is the population each of the seven groups (see Fig. 1). Haplotypes were con- expansion time (Rogers and Harpending, 1992; Gaggiotti nected in a network obtained using the 95% parsimony cri- and ExcoYer, 2000). Fu’s Fs tests whether mutations are terion implemented in the program TCS (Clement et al., neutral or under inXuence of selection, and signiWcantly 2000). We calculated long term eVective female population negative values indicate population expansion (Fu, 1997).