Molecular systematics and phylogeography of the gigantic earthworms of the Metaphire formosae species group (Clitellata, Megascolecidae) Chih-Han Chang a , Si-Min Lin b , Jiun-Hong Chen c, * a Institute of Zoology, National Taiwan University, 1 Roosevelt Road, Section 4, Taipei 106, Taiwan b Department of Life Sciences, National Taiwan Normal University, 88 Ting-Chow Rd, Sec 4, Taipei 116, Taiwan c Institute of Zoology and Department of Life Science, National Taiwan University, 1 Roosevelt Road, Section 4, Taipei 106, Taiwan article info Article history: Received 13 June 2008 Revised 29 August 2008 Accepted 31 August 2008 Available online 10 September 2008 Keywords: Phylogeography Systematics Metaphire formosae species group Megascolecidae Pheretima complex abstract The earthworms of the Metaphire formosae species group distributed in Taiwan are members of the Phere- tima complex within the Megascolecidae. In this study, the systematics and phylogeography of this spe- cies group were investigated using DNA sequences of mitochondrial cytochrome c oxidase subunit I (COI), 16S ribosomal (r)RNA, and NADH dehydrogenase subunit 1 (ND1). The results indicated that the 13 taxa of the M. formosae species group form a clade, including a cryptic species discovered in this study. In addi- tion, Metaphire hengchunensis (James et al., 2005) should be regarded as a subspecies of Metaphire pai- wanna Tsai et al., 2000, and Metaphire bununa glareosa Tsai et al., 2000 should be elevated to specific status. Phylogeographical inferences showed that allopatric speciation occurred in this species group dur- ing the rapid uplift of the main island of Taiwan between 5.0 and 2.5 million years ago. Our analysis exposes non-monophyly within each of the genera Amynthas and Metaphire, and more generally within the Pheretima complex. Further revisions of this speciose complex are urgently needed. Ó 2008 Published by Elsevier Inc. 1. Introduction The Asiatic earthworm genus Metaphire is a member of the Pheretima complex, a speciose group of more than 800 described species within 12 genera belonging to the Megascolecidae (Blake- more, 2002; Easton, 1979, 1982; James, 2005a,b; Sims and Easton, 1972). This genus is widely distributed in East and Southeast Asia with more than 160 species belonging to 25 species groups (Blake- more, 2004; Sims and Easton, 1972). In pheretimoid earthworms, the number and position of the testes are considered to be impor- tant taxonomic characters. Generally, most species are holandric (two pairs of testes, one each in segments 10 and 11), but occasion- ally proandry (one pair of testes in segment 10) or metandry (one pair of testes in segment 11) occurs (Sims and Easton, 1972). These differences in testis condition are crucial in within-genus group- ings as well as in species identification. In Metaphire, octothecate species (species bearing four pairs of spermathecae) without secondary copulatory pouches and precli- tellar genital markings are further divided into two species groups: the ignobilis species group (holandric) and the stephensoni species group (proandric) (Tsai et al., 2004; Sims and Easton, 1972). How- ever, it has been remarked in many studies that 12 taxa belonging to these two groups share a number of morphological character states (Chang and Chen, 2004, 2005a; James et al., 2005; Tsai et al., 2000b, 2004). These taxa are Metaphire trutina Tsai et al. (2003) and Metaphire tahanmonta Chang and Chen (2005a) of the former species group, and Metaphire formosae (Michaelsen, 1922), Metaphire yuhsii (Tsai, 1964), Metaphire paiwanna paiwanna Tsai et al. (2000b), Metaphire paiwanna liliumfordi Tsai et al. (2000b), Metaphire bununa bununa Tsai et al. (2000b), Metaphire bununa glareosa Tsai et al. (2000b), Metaphire taiwanensis Tsai et al. (2004), Metaphire feijani Chang and Chen (2004), Metaphire hengchunensis (James et al., 2005), and Metaphire nanaoensis Chang and Chen (2005a) of the latter. These species all have large body sizes exceeding 30 cm in length and 10 mm in width, bluish-gray body coloration, male pores within copulatory pouches with one or two oval pads and four pairs of spermathecae in segments 6– 9. Their burrowing behaviors and casts are also very similar. Mor- phologically, these species differ only in the condition of the testes, the distance between the paired spermathecal pores, and the struc- ture of the male pores. Accordingly, considering the morphological similarity of the 12 taxa noted above, we herein included these taxa in a newly proposed Metaphire formosae species group, and defined this group as large octothecate Metaphire species without secondary copulatory pouches and preclitellar genital markings, but with oval pads in the male pores. The M. formosae species group is endemic to Taiwan, an island between the Ryukyu Archipelago and the Philippines, and locally nicknamed ‘‘snake earthworms” for their large body size. M. for- moae and M. yuhsii were described in 1922 and 1964 as Pheretima 1055-7903/$ - see front matter Ó 2008 Published by Elsevier Inc. doi:10.1016/j.ympev.2008.08.025 * Corresponding author. E-mail addresses: r91225025@ntu.edu.tw (C.-H. Chang), fishdna@ms31.hinet.net (S.-M. Lin), chenjh@ntu.edu.tw (J.-H. Chen). Molecular Phylogenetics and Evolution 49 (2008) 958–968 Contents lists available at ScienceDirect Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev