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
bDepartment of Life Sciences, National Taiwan Normal University, 88 Ting-Chow Rd, Sec 4, Taipei 116, Taiwan
cInstitute of Zoology and Department of Life Science, National Taiwan University, 1 Roosevelt Road, Section 4, Taipei 106, Taiwan
a r t i c l e
i n f o
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
a b s t r a c t
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 trutinaTsai et al. (2003)and Metaphire tahanmontaChang 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 bununaTsai et al. (2000b), Metaphire bununa glareosa Tsai et al. (2000b), Metaphire taiwanensis Tsai et al. (2004), Metaphire feijaniChang and Chen (2004), Metaphire hengchunensis (James et al., 2005), and Metaphire nanaoensisChang 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).
Contents lists available atScienceDirect
Molecular Phylogenetics and Evolution
formosae and Pheretima yuhsi, respectively (Michaelsen, 1922; Tsai, 1964). The two species were re-assigned to Amynthas bySims and Easton (1972) when the authors revised the systematics of the Pheretima complex. After that, re-inspection of specimens ledChang and Chen (2005b)to re-assign the two species to Metaphire due to the presence of copulatory pouches in the male pores. In addition, M. yuhsii was once regarded as a synonym of M. formosae (Tsai et al., 2000a), but was later resurrected as a valid species (Chang and Chen, 2005b) by the distinctive differences in the distances be-tween the paired spermathecal pores and divergences of the mito-chondrial cytochrome c oxidase subunit I (COI) gene, the former being the only morphological difference between the two species.
The other 10 taxa of this species group were described recently (Chang and Chen, 2004, 2005a; James et al., 2005; Tsai et al., 2000b, 2003, 2004). Metaphire trutina was described as a sexthecate spe-cies (spespe-cies bearing only three pairs of spermathecae) (Tsai et al., 2003), but as the octothecate Metaphire yuanpowa Chang and Chen (2005a)was regarded as a synonym of M. trutina ( Blake-more et al., 2006), this species was proved to be an originally oct-othecate earthworm. M. hengchunensis was described as a member of the genus Amynthas (James et al., 2005). However, the assign-ment of this species to Amynthas was due to different criteria regarding the presence and absence of copulatory pouches (James et al., 2005), which is the only diagnostic character between Meta-phire and Amynthas (for detailed discussion on this dispute, see James, 2005a,bandJames et al., 2005). By using the same criteria as those used in M. trutina, M. tahanmonta and M. taiwanensis, we tentatively re-assigned this species to Metaphire. Considering the blooming of new taxa and names proposed in recent years, and the confusion that previous studies may lead to, a comprehensive taxonomic revision of this species group using molecular phyloge-netic approaches is necessary for future systematic and biodiver-sity studies.
The morphological similarity among these earthworms sug-gests that they comprise a group of closely related species. Re-cently,Chang and Chen (2005a) hypothesized the monophyly of M. nanaoensis, M. tahanmonta, M. formosae, M. paiwanna paiwanna, M. paiwanna liliumfordi, M. bununa bununa, M. bununa glareosa, M. taiwanensis, and M. trutina by comparing their morphology. Subsequently, the monophyly of M. formosae, M. yuhsii, M. paiwan-na paiwanpaiwan-na, M. bunupaiwan-na bunupaiwan-na, M. trutipaiwan-na, and M. tahanmonta was supported by molecular studies using the COI gene (Chang and Chen, 2005b). However, the relationships among these species were not unraveled due to insufficient sequence lengths analyzed. Using a molecular phylogenetic approach, we attempted to clarify the relationships.
The molecular phylogenetic analyses of these earthworms in a geographical context can also provide opportunities to test specia-tion hypotheses of these species. The M. formosae species group has been suggested to be derived from their mainland siblings in Southeast Asia or southeastern China (Chang and Chen, 2005a). Most of these species show an allopatric distribution in Taiwan (Chang and Chen, 2004, 2005a,b). This allopatric distribution sug-gests possible causal relationships between the speciation of these species and the geological history of this island. Recently, DNA bar-coding has been used to evaluate morphologically similar earth-worm species (Chang and Chen, 2005b; Chang et al., 2007; Pérez-Losada et al., 2005), and systematic revisions of some earth-worm groups were also conducted using molecular phylogenetic analyses (Heethoff et al., 2004; James, 2005b; Jamieson et al., 2002; Pop et al., 2003, 2007) Nevertheless, no phylogeographical hypothesis concerning speciation of earthworms was inferred. In this study, we demonstrate the first case to use molecular phy-logenetic approaches on the evolutionary and phylogeographical study of closely related earthworm species. We used mitochondrial DNA sequences to study the systematics and evolution of the
M. formosae species group. We revised the taxonomy of these spe-cies using the COI sequences, the DNA barcode as proposed by He-bert et al. (2003a,b), and evaluated the validity of the M. formosae species group proposed herein. We then hypothesized the phylog-eny of the M. formosae species group and made phylogeographical inferences.
2. Material and methods
2.1. Sample collection and preservation
Earthworms of the M. formosae species group were collected throughout Taiwan during 2000–2004. In our analyses (see below), M. hengchunensis was regarded as a subspecies of M. paiwanna, M. bununa glareosa was elevated to specific status, namely M. glareosa, and a cryptic species, Metaphire sp. was revealed. Therefore, 11 species in total of the M. formosae species group were used, includ-ing M. formosae, M. yuhsii, M. paiwanna, M. bununa, M. taiwanensis, M. trutina, M. feijani, M. nanaoensis, M. tahanmonta, M. glareosa and Metaphire sp.. Moreover, M. paiwanna is composed of three subspe-cies, namely M. paiwanna paiwanna, M. paiwanna liliumfordi, and M. paiwanna hengchunensis, and all three subspecies were included in the analysis. Samples were anesthetized in a 10% ethanol solution; some muscle tissues were isolated and preserved in a 70% or 95% ethanol solution for DNA extraction; the residual earthworm sam-ples were fixed in 10% formalin and then preserved in a 70% etha-nol solution. Some other pheretimoid earthworms collected in Taiwan were treated using the same procedures for the phyloge-netic analysis (Table 1).
2.2. DNA extraction, polymerase chain reaction (PCR), and DNA sequencing
Muscle tissues were washed with distilled water, homogenized in liquid nitrogen, and digested in digestion buffer (10 mM Tris– HCl, 2 mM dihydrate EDTA, 10 mM NaCl, 10 mg/ml DTT, 1% SDS, and 0.4 mg/ml protease K) at 50 °C for 1530 min. Total DNA was extracted from the digested tissue-buffer solution with a stan-dard phenol/chloroform extraction method followed by ethanol precipitation (Palumbi et al., 1991). The ethanol-precipitated DNA was dissolved in distilled water, checked with 1.0% agarose gel electrophoresis, and stored at 20 °C.
Several DNA fragments from the earthworm mitochondrial gen-ome were amplified by PCR, including COI, 16S ribosomal (r)RNA, and NADH dehydrogenase subunit 1 (ND1). All primer sequences were listed inTable 2. For COI, two partially overlapping fragments were amplified by LCO1490 and HCO2198 (Folmer et al., 1994) for the 50 fragment and by COIF0622 and COIR1117 for the 30
frag-ment. When the second primer pair failed to work, instead of COIR1117, COIR1294 was used for M. trutina and Metaphire sch-mardae, and COIR1102 was used for M. feijani. For 16S rRNA, the universal primers 16Sar and 16Sbr (Hillis and Moritz, 1990) were used. For ND1, the primers LeuND1F and IleND1R designed in this study were used. The amplifications were carried out in a 50-
l
l to-tal volume using one cycle at 94 °C for 1 min, followed by 35 cycles of denaturation for 30 s at 94 °C, annealing for 30 s at 52 °C, and extension for 90 s at 72 °C, with a final cycle at 72 °C for 10 min.The PCR products were checked using 1.0% agarose gel electro-phoresis and sequenced in both directions using the same primers as for PCR. Sequencing was performed with a BigDye Terminator Cycle Sequencing Ready Reaction Kit, V3.1 (Applied Biosystems, CA, USA). Products were analyzed on an ABI 3730 XL DNA Analyzer (Applied Biosystems). The sequence of each sample was verified through a comparison of complementary light and heavy strands and double-checked by eye.
Table 1
Samples used in the phylogenetic study and the corresponding GenBank Accession Numbers
Species Locality Sample no. Voucher no. Accession no. of haplotypes
COI ND1 16S
Metaphire yuhsii Hsintien, Taipei County B0665 Afo-65 AY739309a
Wulai, Taipei County B0650 Afo-50 AY739310a
Shouyi, Taipei County B0604 Afo-4 AY739311a
Wanli, Taipei County B0601 Afo-1 AY960799 AY960786 AY960812 Tamsui, Taipei County B0612 Afo-12 AY739313a
Sanchih, Taipei County B0611 Afo-11 AY739314a
Tuchen, Taipei County B0663 Afo-63 AY739315a
Sanshia, Taipei County B0607 Afo-7 AY739316a
Mucha, Taipei City B0602 Afo-2 AY739317a
Nangang, Taipei City B0625 Afo-25 AY739318a
Keelung City B0624 Afo-24 AY739319a
Taoyuan, Taoyuan County B0660 Afo-60 AY739320a
Lungtan, Taoyuan County B0619 Afo-19 AY739321a
Fushing, Taoyuan County B0657 Afo-57 AY739322a
Chudong, Hsinchu County B0628 Afo-28 AY739323a
Guanhsi, Hsinchu County B0659 Afo-59 AY739324a
Jianshi, Hsinchu County B0637 Afo-37 AY739325a
M. formosae Yamgmei, Taoyuan County B1801 AfoTII-1 AY960807 AY960794 AY960820 Baushan, Hsinchu County B1808 AfoTII-8 AY739327a
Sanwan, Miaoli County B1811 AfoTII-11 AY739328a
Sanyi, Miaoli County B1847 AfoTII-47 AY739329a
Nanchuang, Miaoli County B1855 AfoTII-55 AY739330a
Heping, Taichung County B1875 AfoTII-75 AY739331a
Guoshing, Nantou County B1867 AfoTII-67 AY739332a
Shinyi, Nantou County B18105 AfoTII-105 AY739333a
Mayshan, Chiayi County B1899 AfoTII-99 AY739334a
M. tahanmonta Taoyuan, Kaohsiung County B0830 sp40-2 AY739335a
Taoyuan, Kaohsiung County B0831 sp40-3 AY960800 AY960787 AY960813 Chunri, Pingtung County B0807 sp40-1 AY962115
Taoyuan, Kaohsiung County B0874 sp40-4 AY962116 M. paiwanna paiwanna Liouguei, Kaohsiung County B0827 Mpa-45 AY739336a
Sandimen, Pingtung County B1906 Mpa-6 AY962117 Majia, Pingtung County B1911 Mpa-11 AY962118 Taiwu, Pingtung County B1920 Mpa-20 AY962119 Taiwu, Pingtung County B1930 Mpa-30 AY962120 Chunri, Pingtung County B1941 Mpa-41 AY962121 Taimali, Taitung County B08156 Mpa-55 AY962133 Taimali, Taitung County B08157 Mpa-56 AY962135 Dazen, Taitung County B08159 Mpa-57 AY962136 Dazen, Taitung County B08174 Mpa-59 AY962137 Dawu, Taitung County B08163 Mpa-58 AY962138 M. paiwanna hengchunensis (M. hengchunensis) Nanjenshan, Pingtung County B1901 Mph-1 AY962122 Nanjenshan, Pingtung County B08179 Mph-6 AY962123 Nanjenshan, Pingtung County B08180 Mph-7 AY962124 M. paiwanna liliumfordi Shulin, Hualien County B0816 Mpl-3 AY962125 Guangfu, Hualien County B0812 Mpl-2 AY962126 Juoshi, Hualien County B0847 Mpl-18 AY962127 Juoshi, Hualien County B0845 Mpl-16 AY962128 Yuli, Hualien County B0857 Mpl-26 AY962129 Fengbin, Hualien County B0814 Mpl-24 AY962130 Fengbin, Hualien County B0853 Mpl-25 AY962131
Beinan, Taitung County B08101 Mpl-27 AY960802 AY960789 AY960815 Beinan, Taitung County B0810 Mpl-1 AY962132
Taimali, Taitung County B08154 Mpl-28 AY962134 M. bununa Heping, Taichung County B08145 Mbu-9 AY739337a
Datong, Ilan County B0815 Mbu-3 AY962139 Renai, Nantou County B0802 Mbu-1 AY962140
Alishan, Chiayi County B08143 Mbu-7 AY960804 AY960791 AY960817 M. trutina Wulai, Taipei County B0729 sp21-29 AY739338a
Pinglin, Taipei County B0704 sp21-4 AY962144 Neihu, Taipei City B0701 sp21-1 AY962145 Hsiaochaochi, Ilan County B0803 sp21-13 AY962146 Jianshi, Hsinchu County B08110 sp21-34 AY962147
Wufeng, Hsinchu County B0714 sp21-14 AY960808 AY960795 AY960821 Nanchuang, Miaoli County B0726 sp21-26 AY962148
M. nanaoensis Shulin, Hualien County B1315 sp62-2 AY962149 Shulin, Hualien County B1323 sp62-5 AY962150
Shulin, Hualien County B1314 sp62-1 AY960805 AY960792 AY960818 Nanao, Ilan County B1322 sp39-22 AY962151
Nanao, Ilan County B1307 sp39-7 AY962152 Nanao, Ilan County B1306 sp39-6 AY962153 Nanao, Ilan County B1320 sp39-20 AY962154 M. taiwanensis Renai, Nantou County B1504 Mta-4 AY962155 Renai, Nantou County B1505 Mta-5 AY962156
2.3. Sequence alignment, phylogenetic analysis, and topological tests The sequences obtained were checked by aligning the sequenc-ing results with the correspondsequenc-ing sequences of Lumbricus terres-tris in GenBank using the default settings of Clustal X 1.81 (Thompson et al., 1997) and then submitted to GenBank (Table 1). All ingroup sequences used inChang and Chen (2005b)and se-quences of L. terrestris and some other megascolecid earthworms were retrieved from GenBank (Table 1) and used in the phyloge-netic analysis. Alignments were performed using the default set-tings of Clustal X 1.81 and then manually adjusted using BioEdit
(Hall, 1999). Gaps and ambiguously aligned regions in the DNA se-quences were eliminated in all of the following analyses. Metaphire tschiliensis tschiliensis, Dichogaster samjamesi or L. terrestris was used as the outgroup in the analyses. The 50fragment of COI
ampli-fied using the primer pair, LCO1490 and HCO2198, was first used to reexamine the taxon status; then, one specimen for each species identified in the COI analysis was chosen and used in the following analyses. The 16S rRNA gene sequences were used to test the hypothesis of a monophyletic M. formosae species group. The 16S rRNA gene was chosen instead of COI because of the availability of sequences in GenBank and the slower evolutionary rate of this
Table 1 (continued)
Species Locality Sample no. Voucher no. Accession no. of haplotypes
COI ND1 16S
Renai, Nantou County B1502 Mta-2 AY960806 AY960793 AY960819 Yuanshan, Ilan County B1605 sp22-5 AY962157
Yuanshan, Ilan County B1607 sp22-7 AY962158
M. feijani Majia, Pingtung County B2401 sp92-1 AY960809 AY960796 AY960822 Majia, Pingtung County B2402 sp92-2 AY962159
Majia, Pingtung County B2403 sp92-3 AY962160 Wutai, Pingtung County B2404 sp92-4 AY962161 Wutai, Pingtung County B2405 sp92-5 AY962162 Metaphire sp. (morphologically identified
as M. paiwanna paiwanna)
Taoyuan, Kaohsiung County B0873 sp90-2 AY962163 Taoyuan, Kaohsiung County B08147 sp90-5 AY962164
Taoyuan, Kaohsiung County B0833 sp90-1 AY960801 AY960788 AY960814 Taoyuan, Kaohsiung County B0832 sp90-3 AY962165
Taoyuan, Kaohsiung County B0834 sp90-4 AY962166 M. glareosa (M. bununa glareosa) Shitzi, Pingtung County B08150 Mam-18 AY962167 Shitzi, Pingtung County B08149 Mam-17 AY962168 Dazen, Taitung County B08169 Mam-21 AY962169 Ruisui, Hualien County B0813 Mam-2 AY962170 Ruisui, Hualien County B0856 Mam-8 AY962171 Yuli, Hualien County B0861 Mam-3 AY962172 Yuli, Hualien County B0863 Mam-6 AY962173 Fuli, Hualien County B08148 Mam-16 AY962174 Dunghe, Taitung County B0872 Mam-4 AY962175 Luyee, Taitung County B0875 Mam-9 AY962176 Beinan, Taitung County B0879 Mam-10 AY962177 Taimali, Taitung County B08108 Mam-12 AY962178 Taimali, Taitung County B08151 Mam-19 AY962179
Taimali, Taitung County B08106 Mam-11 AY960803 AY960790 AY960816 Chupun, Taitung County B08113 Mam-15 AY962180
Chupun, Taitung County B08112 Mam-14 AY962181 Chupun, Taitung County B08111 Mam-13 AY962182 Chupun, Taitung County B08166 Mam-20 AY962183
M. californica Taipei City B0106 Mca-6 AY960810 AY960797 AY960823 M. schmardae Taipei City B2502 Msc-2 AY960811 AY960798 AY960824 M. posthuma Hsintien, Taipei County B0203 Mpo-3 AY960825 Amynthas binoculatus Baushan, Hsinchu County B2111 sp61-11 AY962184 AY968683 A. carnosus Chaochi, Ilan County B1201 sp34-1 AY960830
Wulai, Taipei County B1205 sp34-5 AY962185
A. aspergillum Guting, Taipei City B0301 Aas-1 AY960826 A. incongruus Wanli, Taipei County B0503 Ain-3 AY960827 A. robustus Yuanshan, Ilan County B1101 Aro-1 AY960829 A. gracilis Hsintien, Taipei County B1005 Agr-5 AY960828
Begemius queenslandicus AF406578a
Pontodrilus litoralis AF003256a
AF406586a
Fletcherodrilus sigillatus AF406588a
Spenceriella cormieri AF406589a
Spenceriella sp. AF406572a
Diporochaeta sp. AF406574a
Perionychella kershawi AF406567a
Digaster lingi AF406583a
Dichogaster saliens AF406573a
Dic. samjamesi AF406571a
Terrisswalkerius grandis AF406566a
T. moritzi AF406560a
T. millamilla AF406565a
Perionyx excavatus AF406582a
Didymogaster sylvaticus AF406575a
Lumbricus terrestris U24570a
U24570a
U24570a
a
gene. The combined sequence set of 16S rRNA, COI, and ND1 was then used to reconstruct the phylogeny of the M. formosae species group. Before combining the nucleotide sequences of the three genes, the incongruence length difference test (ILD; Farris et al., 1994) was conducted to check whether all of the sequences were suitable for combination. Since the result of the ILD test was not significant (P = 0.38), the three genes were combined.
In the phylogenetic analyses, the most-appropriate model of DNA substitution was chosen using hierarchical likelihood ratio tests with PAUP 4.0b10 (Swofford, 2000) and Modeltest 3.0 (Posada and Crandall, 1998). For the 50 fragment of COI, the TVM model
(Rodríguez et al., 1990) with invariable sites of 0.5907 and a gamma shape parameter of 0.9298 (TVM + I + G) was chosen (Base frequen-cies: A, 0.3744; C, 0.2120; G, 0.1451; and T, 0.2684. Substitution rates: A–C, 1.2390; A–G, 11.5090; A–T, 0.6402; C–G, 0.2470; C–T, 11.5090; and G–T, 1.0000). For 16S rRNA, the general-time revers-ible model (Tavar´e, 1986) with invariable sites of 0.3930 and a gam-ma shape parameter of 0.4588 (GTR + I + G) was chosen (Base frequencies: A, 0.4365; C, 0.1458; G, 0.1492; and T, 0.2684. Substi-tution rates: A–C, 1094.7264; A–G, 2152.7852; A–T, 1349.1843; C– G, 285.2955; C–T, 6401.8551; and G–T, 1.0000). The maximum likelihood (ML) analysis was performed using PAUP 4.0b10 with heuristic searches, starting trees obtained by neighbor joining (NJ), and TBR branch swapping. Three different approaches were used to evaluate the reliability of the inferred phylogenetic tree. First, Bayesian analysis was applied to generate a posterior proba-bility distribution using the Metropolis-coupled Markov Chain Monte Carlo (MCMC) with MrBayes 3.0b4 (Huelsenbeck and Ron-quist, 2001; Ronquist and Huelsenbeck, 2003). The search was run for 1 106generations, and every 500th tree was sampled after
a burn-in of 105generations. Posterior probabilities for each branch
were calculated from the sampled trees. Second, an NJ analysis with 1000 bootstrap replicates was conducted using PAUP 4.0b10. Third, an unweighed maximum parsimony (MP) analysis with 1000
bootstrap replicates was performed using PAUP 4.0b10 with heuris-tic searches, random starting trees, 10 random additions of se-quences, and TBR branch swapping. Because of the extensive computational time, the maximum number of trees saved was lim-ited to 1000 in the COI dataset in this procedure.
For the combined dataset of COI, ND1, and 16S rRNA, Tamura– Nei’s model (Tamura and Nei, 1993) with invariable sites of 0.5446 and a gamma shape parameter of 0.9102 (TrN + I + G) was the most-appropriate model of DNA substitution. Parameters were set to unequal base frequencies (A, 0.3473; C, 0.2200; G, 0.1294; and T, 0.3033), unequal transition rates (A–G, 9.4422; and C–T, 11.2124), and equal transversion rates (1.0000). The ML analyses were performed using random starting trees and 100 random addi-tions of sequences with the other settings the same as those used in the COI and 16S rRNA analyses. In addition, the same three ap-proaches used in the 16S rRNA analysis were employed to evaluate the reliability of the inferred phylogenetic tree except 100 instead of 10 random additions of sequences were used in the unweighed MP analysis.
The ML tree with the highest ln(L) score was compared to alter-native tree hypotheses. The Shimodaira–Hasegawa (SH) test ( Shi-modaira and Hasegawa, 1999) was performed using PAUP 4.0b10 with 1000 bootstrap replications.
3. Results
3.1. Sequence characteristics
For the 50 fragment of COI, 94 haplotypes were observed from
104 individuals of the M. formosae species group (Table 1). All hap-lotypes were 535 bp in length, without insertions or deletions. Within the M. formosae species group, the mean interspecific se-quence divergences ranged from 12.9% (M. bununa vs. M. feijani) to 27.7% (M. trutina vs. M. yuhsii) (Table 3).
Table 3
Mean interspecific sequence divergences of the Metaphire formosae species group calculated using the most-appropriate model of DNA substitution based on the COI sequences (lower left) and the combined sequences of COI, ND1, and 16S rRNA (upper right)
MSP FEI PAI BUN TAH TRU TAI GLA NAN YUH FOR
MSP 0.086 0.087 0.095 0.102 0.105 0.109 0.110 0.114 0.109 0.125 FEI 0.151 0.086 0.088 0.095 0.110 0.107 0.107 0.111 0.116 0.120 PAI 0.164 0.147 0.080 0.086 0.091 0.096 0.100 0.104 0.105 0.115 BUN 0.192 0.129 0.156 0.086 0.095 0.097 0.100 0.099 0.110 0.115 TAH 0.201 0.155 0.156 0.152 0.093 0.105 0.102 0.095 0.112 0.121 TRU 0.241 0.241 0.190 0.184 0.174 0.097 0.109 0.098 0.113 0.125 TAI 0.256 0.230 0.225 0.206 0.217 0.241 0.105 0.099 0.110 0.122 GLA 0.224 0.226 0.197 0.204 0.216 0.222 0.253 0.095 0.108 0.116 NAN 0.232 0.178 0.234 0.199 0.192 0.232 0.228 0.200 0.109 0.117 YUH 0.216 0.205 0.223 0.193 0.197 0.277 0.275 0.222 0.224 0.122 FOR 0.226 0.222 0.193 0.207 0.215 0.249 0.233 0.242 0.239 0.228
Abbreviations: MSP, Metaphire sp.; FEI, M. feijani; PAI, M. paiwanna; BUN, M. bununa; TAH, M. tahanmonta; TRU, M. trutina; TAI, M. taiwanensis; GLA, M. glareosa; NAN, M. nanaoensis; YUH, M. yuhsii; FOR, M. formosae.
Table 2
Primers used in the phylogenetic analyses
Primer Sequence Reference
LCO1490 50-GGT CAA CAA ATC ATA AAG ATA TTG G-30 Folmer et al., 1994
HCO2198 50-TAA ACT TCA GGG TGA CCA AAA AAT CA-30 Folmer et al., 1994
COIF0622 50-ACA GAT CGA AAC CTA AAT AC-30 This study
COIR1117 50-ATT CTC AAC ACG TAG TGG AAG TG-30 This study
COIR1294 50-TCA GAA TAT CGC CGA GGT ATA CC-30 This study
COIR1102 50-TGA AAA TGT GCT ACN ACA TAG TA-30 This study
16Sar 50-CGC CTG TTT ATC AAA AAC AT-30 Hillis and Moritz, 1990
16Sbr 50-CCG GTC TGA ACT CAG ATC ACG T-30 Hillis and Moritz, 1990
LeuND1F 50-CAA GAT GGC AGA GTG CCA-30 This study
The lengths of the M. formosae species group 16S rRNA gene haplotypes ranged from 385 to 388 bp. The lengths of the phere-timoid earthworm 16S rRNA gene haplotypes ranged from 381 to 388 bp. The total aligned sequences were 395 bp, with insertions or deletions of 12 bp.
The lengths of the combined sequences ranged from 2435 to 2442 bp, including 456464 bp from the 16S rRNA gene, 1,056 bp from the COI gene, and 922 or 925 bp from the ND1 gene. The aligned sequences were 2453 bp, with insertions or deletions of 14 bp due to a 3-bp insertion of the L. terrestris ND1 gene (corre-sponding to an amino acid) and variations in the 16S rRNA gene frag-ments. Within the M. formosae species group, interspecific sequence
divergences ranged from 8% (M. paiwanna vs. M. bununa) to 12.5% (M. formosae vs. M. trutina and M. formosae vs. Metaphire sp.) (Table 3). 3.2. Phylogenetic analyses
In the analyses using the 50fragment of COI, the ML analysis
re-sulted in two trees with the highest ln(L) score of 6202.47. The two trees differed only slightly in arrangements of specimens within M. paiwanna liliumfordi, and hence only one tree is shown (Fig. 1). The posterior probabilities from the Bayesian analysis and the bootstrap values from the MP and NJ analyses were plotted on the ML tree (Fig. 1). The phylogenetic analysis based on the 50
Fig. 1. One of the two maximum likelihood trees of the Metaphire formosae species group based on the cytochrome c oxidase subunit I (COI) gene. The two trees differed only slightly in arrangements of specimens within M. paiwanna liliumfordi, and hence only one tree is shown. Nodes for each species identified are emphasized with black dots. Intraspecific clades with allopatric distributions are identified in M. paiwanna (A1A3), M. glareosa (B1B3), M. nanaoensis (C1 and C2), M. formosae (D1D3), M. taiwanensis (E1 and E2), and M. yuhsii (F1 and F2), and their supporting values are presented.
fragment of COI revealed that there are 11 monophyletic groups, corresponding to 11 species, within the M. formosae species group (Fig. 1). These monophyletic groups were largely congruent with morphological species previously identified but with a few excep-tions. A cryptic species morphologically identified as M. paiwanna paiwanna was discovered, namely Metaphire sp.. M. bununa glare-osa was elevated to a specific status, namely M. glareglare-osa (see Dis-cussion). In addition, intraspecific clades for M. paiwanna, M. taiwanensis, M. nanaoensis, M. formosae, M. yuhsii, and M. glareosa were defined (Fig. 1), and their genetic divergences were estimated (Table 4) for further discussion. However, although the COI
fragment proved to be useful in species clustering, it showed poor resolution for interpreting the interspecific relationships.
In the analyses using 16S rRNA, the ML analysis resulted in a tree with the highest ln(L) score of 3657.68 (Fig. 2). The posterior probabilities from the Bayesian analysis and bootstrap values from the MP and NJ analyses were plotted on the ML tree (Fig. 2). The analysis of the 16S rRNA gene sequence supported the M. formosae species group being monophyletic within the Pheretima complex. Furthermore, the monophyly of the Pheretima complex within the Megascolecidae was also supported (Fig. 2).
In the combined analyses using COI, ND1, and 16S rRNA, the ML analysis resulted in a tree with the highest ln(L) score of 13389.88 (Fig. 3). The posterior probabilities from the Bayesian analysis and the bootstrap values from the MP and NJ analyses were plotted on the ML tree (Fig. 3). In addition, three groups with-in the M. formosae species group were defined for further discus-sion: at the basal part of the inferred phylogenetic tree is the western hill group, including M. formosae and M. yuhsii; the mono-phyletic eastern mountain group includes M. nanaoensis and M. glareosa; the monophyletic western mountain group includes M. paiwanna, M. bununa, M. trutina, M. taiwanensis, M. tahanmonta, M. feijani and Metaphire sp. (Fig. 3).
To test if M. paiwanna and Metaphire sp. comprise a monophy-letic group, the SH test was applied. In addition, the monophyly of species with dorsally positioned spermathecal pores, M. formo-sae and M. yuhsii, was also tested using the SH test. The constrained topologies inferred from these monophyly hypotheses were com-pared with the topology of the ML tree derived from the combined
Table 4
Mean intraspecific sequence divergences between clades within Metaphire paiwanna, M. glareosa, M. nanaoensis, M. formosae, M. taiwanensis and M. yuhsii calculated using the most-appropriate model of DNA substitution based on the COI sequences Species Clade pair Sequence divergence M. paiwanna A1 and A2 0.083 A1 and A3 0.095 A2 and A3 0.094 M. glareosa B1 and B2 0.081 B1 and B3 0.118 B2 and B3 0.135 M. nanaoensis C1 and C2 0.090 M. formosae D1 and D2 0.093 D1 and D3 0.118 D2 and D3 0.106 M. taiwanensis E1 and E2 0.057 M. yuhsii F1 and F2 0.120
Fig. 2. Maximum likelihood tree of Metaphire, Amynthas, and Begemius of the Pheretima complex and some other megascolecid earthworms based on the 16S rRNA gene. The Pheretima complex forms a monophyletic group within the Megascolecidae, and the monophyly of the M. formosae species group is also supported. When three numbers are shown around the nodes, the numbers above the branches are Bayesian posterior probabilities followed by neighbor joining bootstrap values, and the numbers below branches are maximum parsimony bootstrap values; values <50 are marked as ‘-’. When only one number is shown above the branch, this number is the Bayesian posterior probability, and the neighbor joining and maximum parsimony bootstrap values were both <50. For some nodes, the three values were all <50, and no numbers are presented.
analyses. The result indicated that all of the constrained topologies were significantly worse than those of the ML tree (Table 5). 4. Discussion
4.1. Systematics of the M. formosae species group and the Pheretima complex
According to the phylogenetic analyses, the M. formosae species group is composed of at least 11 species, including ten described species and a cryptic species previously identified as M. paiwanna paiwanna. In addition, the monophyly of the 11 species was strongly supported. Among them, M. formosae and M. yuhsii have spermathecal pores near the central dorsal lines, a rare feature in the Pheretima complex (Chang and Chen, 2005b). This unique fea-ture, together with other similarities in morphology, resulted in our hypothesizing the monophyly of these two species. However, this hypothesis was rejected by the SH test. Similarly, although Metaphire sp. is a cryptic species morphologically similar to M. p. paiwanna, the monophyly of the two species was also rejected by the SH test.
Metaphire sp. is morphologically similar to M. p. paiwanna. However, this species has a smaller body size, less apparent hori-zontal ridges in the male pore areas, and more regularly coiled spermathecal diverticulum stalks. In addition, the two species re-quire different habitats: M. p. paiwanna lives in evergreen broad-leaf forests, while Metaphire sp. lives in deciduous broadbroad-leaf forests at higher elevations where M. p. paiwanna has never been found (Fig. 4).
In the original description, M. bununa glareosa was described based on four specimens, but these specimens were destroyed in a strong earthquake on 21 September 1999 that devastated central Taiwan. Our phylogenetic analyses do not support the monophyly of M. b. bununa and M. b. glareosa. On the contrary, two indepen-dent monophyletic groups corresponding to M. b. bununa and M. b. glareosa are supported (Figs. 1 and 3). Moreover, the average ge-netic distances between the two taxa are equivalent to those among species (Table 3). Therefore, M. b. glareosa should be ele-vated to specific status, namely M. glareosa.
According to the phylogenetic analysis, M. hengchunensis, M. p. paiwanna and M. p. liliumfordi, which correspond to clades A1, A2 and A3 inFig. 1, respectively, are supported to form a monophy-letic group (Fig. 1). The genetic distances between M. hengchunensis and each of M. p. paiwanna and M. p. liliumfordi (9.5% and 9.4%, respectively), are almost equivalent to that between M. p. paiwanna and M. p. liliumfordi (8.3%), and are obviously lower than interspe-cific distances within the M. formosae species group (Table 3). Mor-phologically, the type specimens of M. hengchunensis are almost indistinguishable from specimens of M. p. paiwanna examined in this study, except that the seminal grooves and oval pads in the male pore areas of M. hengchunensis are slightly degenerated. In addition, the three taxa show an allopatric distribution. Altogether, we strongly suggest that M. hengchunensis should be regarded as one of the subspecies of M. paiwanna, namely M. paiwanna hengchunensis.
In the M. formosae species group, M. trutina and M. tahanmonta are holandric (with two pairs of testes, one each in segments 10 and 11), while other species are proandric (with only one pair of testes in segment 10). There are two hypotheses with two steps of changes that may explain this character evolution. In one hypothesis, M. tahanmonta and M. trutina independently acquired the second pair of testes (Fig. 5, left). Alternatively, in the other equally parsimonious hypothesis, the common ancestor of M. tahanmonta and M. trutina acquired the second pair of testes, and then the common ancestor of M. bunina, M. paiwanna, M. feijani and Metaphire sp. lost it (Fig. 5, right). Although the predominance of holandry in pheretimoid earthworms implies that this character state may be plesiomorphic, a hypothesis generally accepted
Fig. 3. Maximum likelihood tree of the Metaphire formosae species group based on the combined sequences of cytochrome c oxidase subunit I (COI), NADH dehydrogenase subunit 1 (ND1), and 16S rRNA. Numbers above the branches are Bayesian posterior probabilities, neighbor joining bootstrap values, and maximum parsimony bootstrap values. Values <50 are marked as ‘-’. Three groups were defined for further discussion: WM, western mountain group; EM, eastern mountain group; WH, western hill group.
Table 5
Comparison of the ML tree (ln(L) score = 13389.88) with the constraint trees using the Shimodaira–Hasegawa test
Topological constraint ln L Diff. ln L p value Monophyly of M. paiwanna and Metaphire sp. 13406.37 16.49 0.02a
Monophyly of M. formosae and M. yuhsii 13398.76 8.88 0.039a
a
among earthworm taxonomists, the case of the M. formosae species group suggests that holandry can also be an apomorph. Moreover, the fact that testis condition changes even among closely related species indicates that this character is highly variable and cannot be used in grouping earthworm species within a genus. Therefore, the sub-grouping within a genus of the Pheretima complex, which is partly based on testis condition (Sims and Easton, 1972), may be inconsistent with the phylogeny. In addition, based on morpholog-ical phenetic analyses conducted about 30 years ago (Easton, 1979, 1982; Sims and Easton, 1972), the generic divisions in the Phereti-ma complex were recently challenged by overlapping diagnostic characters and serious homoplasy (Blakemore, 2002; James, 2005a). This opinion is further supported by the non-monophyly of Amynthas and Metaphire as revealed in the present study, as well as some other pheretimoid genera in previous DNA analyses (James, 2005b). All the evidence suggests the urgent need to revise the systematics of the Pheretima complex.
4.2. Phylogeography
Taiwan is a mountainous island about 170 km off the southeast-ern coast of China. The Central Mountain Range (CMR) runs north– south throughout the center of the island, with more than 200 mountain peaks exceeding 3000 m; along the southeast coast lies the Coastal Mountain Range, in which the majority of the moun-tain peaks are about 1000 m. The main island of Taiwan is the re-sult of a collision between the Luzon Volcanic Arc and the Eurasian Continental Margin between 5 and 2.5 million years ago (Ma), a geological event known as Penglai Orogeny (Huang et al., 1997, 2000; Teng, 1990;), and is still rising at present (Huang et al., 1997, 2000).
The mountains and the rivers in the CMR have long been considered as major factors resulting in intraspecific genetic dif-ferentiation of many terrestrial and freshwater animals in Tai-wan, such as mice (Hsu et al., 2001), lizards (Liu, 1995), frogs
Fig. 4. Location of Taiwan and the distribution of the Metaphire formosae species group on the island. Closed triangles indicate the general positions of the higher parts of the Central Mountain Range. Neighboring species not separated by a black line have a contact zone, which is not shown for clarity. The black lines indicate geographic barriers between species: A, Shuei-Shan Mountain Ridge; B, Launong River; C, Liwu River.
(Yang et al., 1994; Toda et al., 1997), spiders (Lin et al., 1999), crabs (Shih et al., 2006), and earthworms (Chang and Chen, 2005b). For instance, Chang and Chen (2005b) proposed that the formation of the ancient Tamsui River about 2.5 Ma in northern Taiwan caused the divergence between the two popu-lations of M. yuhsii (re-revealed as clades F1 and F2 in Fig. 1). Similarly, clades C1 and C2 of M. nanaoensis isolated by the Hep-ing River in northeastern Taiwan shows another case of river-driven genetic differentiation. Except the two cases of M. yuhsii and M. nanaoensis, intraspecific genetic differentiation has been observed among geographically isolated populations of four more species in the M. formosae species group, including M. pai-wanna (clades A1A3), M. glareosa (clades B1B3), M. formosae (clades D1D3), and M. taiwanensis (clades E1 and E2). This ge-netic structure is clear evidence that the formation of the moun-tains and the rivers in the CMR has resulted in intraspecific genetic differentiation in this species group.
In Taiwan, most species of the M. formosae species group are allopatrically distributed (Fig. 4). Furthermore, in the inferred phy-logenetic tree, species of the eastern mountain group are distrib-uted east of the CMR, those of the western mountain group are distributed west of the CMR, except M. paiwanna, and those of the western hill group are distributed in the western foothills to the west of the CMR (Figs. 3 and 4). This pattern suggests that allo-patric speciation may be the major mechanism driving the species diversity of this group. Using the estimated formation time of the ancient Tamsui River and the genetic divergence within M. yuhsii (Table 4), we can roughly estimate the evolutionary rate of earth-worm COI genes as 4.8% per million years, and consequently, the speciation events of the M. formosae species group are estimated to have occurred between 5.8 and 2.7 Ma. This inferred time range is congruent with the period of the Penglai Orogeny, during which the rapidly formed mountains and rivers were geographical barri-ers for many flightless invertebrates. Therefore, by combining the geological and phylogenetic evidence, we herein propose a vicari-ance hypothesis to explain the allopatric speciation events of the M. formosae species group. In this hypothesis, the ancestors of these species arrived in Taiwan before the rapid uplift of this is-land, probably during the late Miocene, and then dispersed throughout this island. During the period of rapid uplift between 5.0 and 2.5 Ma, different populations of the ancestral species were
rapidly isolated by the mountains and the rivers that formed due to orogenesis. This isolation resulted in genetic differentiation and ultimately caused speciation of the M. formosae species group.
The endemic land fauna diversity in Taiwan was generally attributed to be consequences of multiple dispersal-isolation events between Taiwan and the surrounding regions (Lin et al., 2002; Ota, 1997; Ota et al., 2002; Tu et al., 2000; Yeh et al., 2004), while the contribution of the CMR and vicariance events during Penglai Orogeny to speciation of animals on this island has never been well investigated before. Our present study is the first case that demonstrates within-island speciation of animals through vicariance events caused by orogenesis in Taiwan. Consid-ering the diverse endemic invertebrate fauna awaiting being dis-covered and investigated, the present case may be one of the tremendous amounts of similar stories among the endemic fauna on this island.
Acknowledgments
We are grateful to the persons who kindly assisted us in the col-lection of earthworm samples. We are also grateful to Drs. S. James, C.-F. Tsai and H.-P. Shen and Mr. C.-C. Huang for their helpful com-ments on the manuscript. This study was supported by the Na-tional Science Council of Taiwan (NSC92-2621-B-002-019) to J.-H. Chen.
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