Three new species of octothecate pheretimoid earthworms from Taiwan, with discussion on the biogeography of related species

Three new species of octothecate Metaphire pheretimoid earthworms are described from Taiwan. They have close phylogenetic relationships with five described species, Metaphire paiwanna, Metaphire bununa, Metaphire riukiuensis, Metaphire taiwanensis, and Amynthas formosae. These eight species are all octothecate. They have similar body size, colour, prostate glands and caeca, and the seven species belonging to the genus Metaphire have C‐shaped male pores. Excluding M. riukiuensis, which has been found only in the Ryukyu Islands, the other seven species are endemic species in Taiwan. Each of them has a distinct geographical distribution. Accordingly, we inferred that the ancestor of these eight species, including M. riukiuensis, came to Taiwan from the continent of East Asia during the Pleistocene. A few of their descendants went northward to the Ryukyu Islands and were isolated. The others remained in Taiwan, dispersed, and were isolated by geographical barriers such as mountains and rivers. Subsequent allopatric speciation events in Taiwan and in the Ryukyu Islands then generated these eight species.


Introduction
Taiwan, a continental island on the west edge of the Pacific Ocean, is separated by the 170-km wide Taiwan  During the period between 1964 and 1999, research on the earthworm fauna of Taiwan was suspended. In 1999, 26 earthworm species were reviewed (Shih et al. 1999). However, 29 more species were reported in the next 5 years (Tsai CF et al. 1999, 2000a, 2000b, 2000c, 2004Tsai SC et al. 2000;Chang et al. 2001; Chuang and Chen

External characters
Length (mature) 215-425 mm, clitellum width 13.9-15.6 mm, segment number 129-189. Number of annulets (secondary segmentation) per segment three in 5-9, five in 10-13, and three in body segments behind 17. Prostomium epilobous. [30][31][32][33][34][35] between male pores. First dorsal pore in 12/13. Clitellum 14-16, smooth, saddle-shaped, length 9.8-14.1 mm, dorsal pore absent, setae absent. Spermathecal pores four pairs in 5/6-8/9, lateral, distance between the paired pores about 0.5 body circumference ventrally apart. No genital papillae in the spermathecal region. Female pore single, situated on the medio-ventral in 14. Male pore paired, situated on setal line close to lateral border of 18. Each male pore area is C-shaped, with the opening of the C facing the ventral setal line, bordered by a thick skin wall, which has several folds on its lateral side. The male pore area is enlarged, with length about twice the length of 18, extending to the setal line of 17 and 19. The male aperture is situated on the end of the ventral setal line, with one oval pad on each side. The two oval pads are linked by a vertical bar-shaped structure extending from the male aperture. These structures are sometimes partially covered by the skin wall bordering the male pore area. Genital papillae absent in the male pore area.
Live specimens bluish brown or dark purplish grey with metallic lustre on dorsum, reddish brown on ventral. Preserved specimens purplish brown on dorsum, light greyish brown on ventral.
Spermathecae four pairs in 6-9. Ampulla large, about 3.5-6.2 mm in length, with a stalk about 0.5-1.3 mm in length. The spermathecal diverticulum is short, beyond the middle of spermathecae, with a small oval seminal chamber on the tip. Nephridia tufted, attached to the post-segmental septa, surrounding the segmental chambers anterior to the 6/7 septum. Ovaries paired in 13, medio-ventral, close to the 12/13 septum.
Testis sacs paired in 10 and 11, the anterior pair oval, smooth, medio-ventral in front of 10/11, the posterior pair is much larger than the anterior one, filling the space between septa. Sperm ducts meeting in 12. Seminal vesicles paired in 11 and 12, the anterior pair included in the posterior testis sac, both pairs moderate in size. Prostate glands paired in 18, large, lobular, extending to 17.

Habitat and behaviour
This species lives in the mountains below the altitude of 2000 m, where the vegetation is evergreen broadleaf forest, deciduous broadleaf forest or mixed coniferous-broadleaf forest, according to the altitude. Because it can be found both in virgin forest and secondary forest, it might tolerate low levels of human activity. This species is an anecic species, having permanent vertical burrows. It is active around the upper layer of soil or on the ground at night. Although it is occasionally active after rain, it usually stays in the soil at day. When resting, it is usually found more than 30 cm deep below the ground, and the deepest case we found this species is in 80 cm.

Remarks
Metaphire yuanpowa is a common species in the mountain area of northern Taiwan. It is usually found moving across the road at night, or at early morning after rain. This species resembles the other two sympatric species, Amynthas aspergillum and A. formosae, in body size, shape and colour, but it is easily distinguished by external characters in the field. The male pore area of M. yuanpowa is enlarged, with an obvious oval pad on each side of the male pore, while the other two species do not have these characters. The spermathecal pores of A. formosae are medio-dorsal in 5/6-8/9, and the pores are evident with the naked eye through the lighter colour of the pore borders (Michaelsen 1922). Amynthas aspergillum has many papillae on the male pore area. Furthermore, A. aspergillum was recognized as a peregrine species in Taiwan (Tsai CF et al. 2000a) and is usually found in cultivated soil, especially in the grassland of public parks (personal observation), while M. yuanpowa and A. formosae are often found on the mountain slope, where the soil is less disturbed.
Metaphire yuanpowa shares most of its characters with M. paiwanna and M. bununa, including body size, colour, number of spermathecae, and the position and morphology of prostate gland and intestinal caeca (Tsai CF et al. 2000c). However, the male pore area of M. paiwanna and M. bununa has only one oval pad between the male aperture and the anterior end of the male pore area, and the male pore area of M. paiwanna also has a horizontal ridge (Tsai CF et al. 2000c). Moreover, M. yuanpowa has testis sacs paired in 10 and 11, while both M. paiwanna and M. bununa have testis sacs paired only in 10.
Except for number of spermathecae, M. yuanpowa is similar to M. trutina, which has only three pairs of spermathecae ) and belongs to the houlleti-group in the genus Metaphire (Sims and Easton 1972). It is not yet known if the two species are distantly related due to the difference in the number of spermathecae or closely related due to the similarity of other characters. According to our field survey, M. yuanpowa is restricted to the west edge of the Shei-Shan Mountain Range, but M. trutina is found only in the east edge of the Shei-Shan Mountain Range. Although they seem to be allopatric, the detailed distribution pattern of the two species is still unknown. A comparative study based on cytochrome c oxidase subunit I gene sequence is now underway to unravel the phylogenetic relationship of these two species.
The species name ''yuanpowa'' is given to describe the male pore area of this species. The male pore area of this species is like the Chinese ''yuanpau'', which is a kind of currency in the past in China.

External characters
Length (mature) 335-429 mm, clitellum width 10.1-14.9 mm, segment number 132-177. Number of annulets (secondary segmentation) per segment three in 5-9, five in 10-13, and three in body segments behind 17. Prostomium prolobous. Setae 103-114 in 7, 120-131 in 20, 19-23 between male pores. First dorsal pore in 12/13. Clitellum 14-16, smooth, saddleshaped, length 13.8-14.4 mm, dorsal pore absent, setae absent. Spermathecal pores four pairs in 5/6-8/9, ventral, distance between the paired pores about 0.4 body circumference ventrally apart. No genital papillae in the spermathecal region. Female pore single, situated on the medio-ventral in 14. Male pore paired, situated on setal line close to lateral border of 18. Each male pore area is C-shaped, with the opening of the C facing the ventral setal line, bordered by a thick skin wall, which has several folds on its lateral side. The male pore area is enlarged, with length about twice the length of 18, extending to the setal line of 17 and 19. The male aperture is situated on the end of the ventral setal line, partially covered by the skin wall bordering the male pore area. The region covered by the skin wall is swollen, forming a smooth appearance. A very small pad presents on the posterior end of the male pore area. Genital papillae absent in the male pore area. Live specimens bluish brown or dark purplish grey with metallic lustre on dorsum, reddish brown on ventral. Preserved specimens purplish brown on dorsum, light greyish brown on ventral.
Spermathecae four pairs in 6-9. Ampulla large, about 3.5-5.5 mm in length, with a stalk about 0.5-1.4 mm in length. The spermathecal diverticulum is short, beyond the middle of spermathecae, with a small oval seminal chamber on the tip. Nephridia tufted, attached to the post-segmental septa, surrounding the segmental chambers anterior to the 6/7 septum. Ovaries paired in 13, medio-ventral, close to the 12/13 septum.
Testis sacs paired in 10, oval, smooth, medio-ventral in front of 10/11. Seminal vesicles paired in 11, filling the space between septa. Some specimens have a pair of very small vestiges of seminal vesicles in 12. Prostate glands paired in 18, large, lobular, extending to 17.

Habitat and behaviour
This species lives in the mountains below the altitude of 1500 m, where the vegetation is evergreen broadleaf forest or deciduous broadleaf forest. Because it can be found both in virgin forest and secondary forest, it might tolerate low levels of human activity. This species is an anecic species, having permanent vertical burrows. It is active around the upper layer of soil or on the ground at night. Although it is occasionally active after rain, it usually stays in the soil during the day. When resting, it is usually found more than 30 cm deep below the ground.

Remarks
The male pore area of M. nanaoensis is C-shaped, and with respect to body size, colour, number of spermathecae, and position and shape of prostate gland and intestinal ceaca, M. nanaoensis resembles M. yuanpowa, M. paiwanna and M. bununa. However, the male pore area of M. yuanpowa has an oval pad on each side of the male aperture, and the width between the paired spermathecal pores of M. yuanpowa, which is about 0.5 body circumference apart, is wider than that of M. nanaoensis. The major difference between the two species is that M. yuanpowa is holandric, while M. nanaoensis is protandric. Metaphire paiwanna and M. bununa are also protandric species, but the male pore areas of these two species are different from that of M. nanaoensis. The male pore area of M. paiwanna and M. bununa has an oval pad between the male aperture and the anterior end of the male pore area, and the male pore area of M. paiwanna also has a horizontal ridge (Tsai CF et al. 2000c). These differences are easily seen under a dissection microscope, or even with the naked eye.
The species name ''nanaoensis'' is given referring to Nanao in Ilan County in Taiwan, where the species was first collected.

External characters
Length (mature) 291-408 mm, clitellum width 12.9-14.7 mm, segment number 122-191. Number of annulets (secondary segmentation) per segment three in 5-9, five in 10-13, and three in body segments behind 17. Prostomium prolobous. Setae 122-144 in 7, 134-156 in 20, 24-30 between male pores. First dorsal pore in 12/13. Clitellum 14-16, smooth, saddle-shaped, length 12.8-15.5 mm, dorsal pore absent, setae absent. Spermathecal pores four pairs in 5/6-8/9, lateral, situated above the lateral-midline, distance between the paired pores about 0.55 body circumference ventrally apart. No genital papillae in the spermathecal region. Female pore single, situated on the medio-ventral in 14. Male pore paired, situated on setal line close to lateral border of 18. Each male pore area is slightly Cshaped, with the opening of the C facing the ventral setal line, bordered by a thick skin wall. The appearance of the anterior part of the skin wall is tubercular. The male pore area is enlarged, with length about twice the length of 18, extending to the setal line of 17 and 19, surrounded by circular folds. The male aperture is situated on the extended line of the ventral setal line, slightly posterior to the middle of the male pore area. A horizontal ridge extends from the setal line, backward to the male aperture. An oval pad is situated behind the setal line of 17, close to the anterior end of the male pore area. The oval pad is linked to the male aperture through a seminal groove. Genital papillae absent in the male pore area. Live specimens dark purplish grey with metallic lustre on dorsum, reddish brown on ventral. Preserved specimens purplish brown on dorsum, light greyish brown on ventral.
Spermathecae four pairs in 6-9. Ampulla large, about 3.6-5.6 mm in length, with a stalk about 1.2-1.9 mm in length. The spermathecal diverticulum is short, usually shorter than one-third of spermathecae length, with a small oval seminal chamber on the tip. Nephridia tufted, attached to the post-segmental septa, surrounding the segmental chambers anterior to the 6/7 septum. Ovaries paired in 13, medio-ventral, close to the 12/13 septum.
Testis sacs paired in 10 and 11, the anterior pair oval, smooth, medio-ventral in front of 10/11, the posterior pair is much larger than the anterior one, filling the space between septa. Sperm ducts meeting in 12. Seminal vesicles paired in 11 and 12, the anterior pair included in the posterior testis sac, both pairs moderate in size. Prostate glands paired in 18, large, lobular, extending to 17.

Habitat and behaviour
This species lives in the mountains between the altitudes of 500 and 2000 m, where the vegetation is evergreen broadleaf forest or deciduous broadleaf forest, according to the altitude and latitude. Because it can be found both in virgin forest and secondary forest, it might tolerate low levels of human activity. This species is an anecic species, having permanent vertical burrows. It is active around the upper layer of soil or on the ground at night. Although it is occasionally active after rain, it usually stays in the soil during the day. When resting, it is usually found more than 30 cm deep below the ground.

Remarks
Metaphire tahanmonta has a length greater than 300 mm, width over 10 mm, four pairs of spermathecae, and C-shaped male pores with an oval pad and a horizontal ridge. These characters are shared with M. paiwanna. However, M. paiwanna is protandric (Tsai CF et al. 2000c), while M. tahanmonta is holandric. In pheretimoid earthworms, most species have a holandric condition, while rare species have a protandric condition (Sims and Easton 1972;Easton 1979;Tsai CF et al. 2000c). Accordingly, it is reasonable to regard the holandric specimens as a different species.
Regarding body size, position of seminal vesicles, number of spermathecae, and holandry, M. tahanmonta shares the same characters with M. yuanpowa. However, the male pores of the two species are very different. The male pore of M. tahanmonta has a horizontal ridge and only one oval pad, while that of M. yuanpowa has no horizontal ridge and two oval pads on each side of the male aperture. Therefore, they are easily distinguished by their external characters.
The species name ''tahanmonta'' is given referring to Mt Tahan in Pingtung County, Taiwan, where this species was collected.

Discussion
The five earthworms, Metaphire yuanpowa, M. nanaoensis, M. tahanmonta, M. paiwanna, and M. bununa are all octothecate. Among them, M. paiwanna and M. bununa have been inferred to have close phylogenetic relationships with M. riukiuensis from the Ryukyu Islands, Japan (Tsai CF et al. 2000c) and with M. taiwanensis, a gigantic earthworm from Taiwan (Tsai et al. 2004). The close resemblance in external characters between M. riukiuensis and A. formosae has also been mentioned (Ohfuchi 1957). These eight species present several common characters (Table I). They have similar body size and colour, their lateral hearts are enlarged in 10-13, and the position and morphology of prostate glands and caeca of these eight species are similar. Besides this, their habitats, burrows, casts, and behaviour are also similar. Excepting A. formosae, the other seven species have C-shaped male pores (copulatory pouches). Most of them have oval pads or vestiges of oval pads in their male pore area, and their male pores are covered with lateral skin folds. Due to the difference in male pore area and the absence of copulatory pouches, A. formosae belongs to the genus Amynthas, while the other seven species belong to the genus Metaphire. The close resemblance in morphological characters in the eight species implies their close phylogenetic relationship. This implication was preliminarily supported by our phylogenetic analysis based on mitochondrial 16S rRNA gene sequence data (Chang and Chen 2003). This result suggests that the presence or absence of copulatory pouches, which so far is the key character between Amynthas and Metaphire (Sims and Easton 1972), may be due to convergent evolution. If it is the case, the grouping of species into Amynthas and Metaphire does not relate to their phylogenetic relationships, and Amynthas and Metaphire may not be monophyletic groups.
According to Sims and Easton's (1972) grouping, M. yuanpowa and M. tahanmonta, like M. riukiuensis, belong to the octothecate holandric ignobilis-group within the genus Metaphire. On the other hand, M. nanaoensis, which should be grouped with M. paiwanna, M. bununa and M. taiwanensis, belongs to the octothecate protandric stephensoni-group (Tsai et al. 2004) within the genus Metaphire. However, comparing the morphology of the eight species, including M. riukiuensis, M. yuanpowa, M. tahanmonta, M. paiwanna, M. bununa, M. taiwanensis, M. nanaoensis, and A. formosae, we strongly suggest that they are more closely related to each other than to con-group earthworm species. For this reason, Sims and Easton's (1972) grouping is considered inapplicable in the discussion on the phylogeny and biogeography of these eight closely related species.
Excluding M. riukiuensis, the other seven species are all endemic to Taiwan. For this reason, the speciation events generating these species must have occurred in Taiwan. Because Taiwan emerged from the West Pacific Ocean near the continent of East Asia about 5 million years ago (Lin and Wu 1997), the ancestor of these species probably came to Taiwan during the Pleistocene (2,000,000,10,000 B.P.), when Taiwan was linked several times with the continent of East Asia through land bridges formed by sea-level alterations due to glaciations (Voris 2000;Lin et al. 2002). This is the only period in the past in which Taiwan was connected with the continent of East Asia by closure of the Taiwan Strait (Lin and Wu 1997). Such inference has been similarly drawn in mammals (Lai 1989;Hsu et al. 2000Hsu et al. , 2001Horng et al. 2003), birds (Yan 1989), reptiles (Lu and Chen 1989;Lin et al. 2002), amphibians (Lu andChen 1989), and insects (Lin and Wu 1997), and is also proposed by Tsai CF et al. (2000a) in earthworms. So far, it is reasonable to propose that the ancestor of these species might have come from south-east China during glaciations. According to this hypothesis, the sister group of these eight earthworm species should be found in south-east China if it is not extinct. However, the sister group of the eight species remains unrecognized in this study for two reasons. First, we examined all species reported from China in closely related groups proposed by Sims and Easton (1972). Some species of ignobilis-group and bucculenta-group (Sims and Easton 1972) within the genus Metaphire, such as M. bucculenta, M. bipapillata and M. ignobilis, were found in China (Gates 1935;Chen 1936). These species could be candidates as the sister group of the eight species. But none of them has large body size and has male pore areas similar to the latter. Second, till now, only a few samples have been recorded from the mountains in those south-eastern provinces of China such as Fu-Chan or Can-Ton. Such results give little information on the sister group. Therefore, no Chinese earthworms can be the reasonable sister group in this study. More phylogenetic studies with extensive sampling of the Chinese Metaphire earthworm species are necessary to find the sister group of these eight species. Accordingly, the allopatric speciation origins of the seven earthworm species in Taiwan result from dispersal of the ancestor species. Except for M. bununa, all the other six species in Taiwan are distributed below 3000 m in altitude. Because the high mountains run north to south in Taiwan, they provide several geographical barriers to animal dispersal from west to east and vice versa, and for gene flows between west and east populations. This pattern allows allopatric speciation to occur, resulting in closely related species occupying different areas in Taiwan (Lue and Lai 1997;Lin et al. 2002). The situation is much the same in the seven earthworm species. Amynthas formosae is restricted to the western hills of the Central Mountain Area, M. yuanpowa is restricted to the west edge of the Shei-Shan Mountain Range and M. nanaoensis is restricted to the north tip of the Central Mountain Range. Besides high mountains as geographical barriers, a large river is another barrier that earthworms will not be able to cross. This is also the pattern in the seven earthworm species in Taiwan. The southern boundary of M. nanaoensis is He-Ping River; and the northern boundary of M. tahanmonta is Lau-Nong River.
Except for the Ryukyu Islands, M. riukiuensis or related species have not been found in Japan. Metaphire riukiuensis has only been found in the southernmost island group of the Ryukyu Islands, Sakishima group (Ohfuchi 1957). These islands are separated from the northern parts of the Ryukyu Islands by Kerama Gap, and have never been linked with them by land bridges in the past (Lin et al. 2002). The geographical distribution of these species implies that the ancestor of M. riukiuensis came northward from Taiwan, rather than southward from Japan, though the land bridge between Taiwan and the Ryukyu Islands formed several times in the Pleistocene. This hypothesis is well supported by Ohfuchi (1957). He recognized that the earthworm species in the two southernmost groups, Sakishima group and Okinawa group, have intestinal caeca similar to those of the species in the Oriental region, in which South China and Taiwan are included (Hikida and Ota 1997;Lin and Wu 1997;Tzeng et al. 1997;Tsai CF et al. 2000a), rather than those of the species in the Palaearctic region, in which Japan is included (Cox and Moore 2000).
The eight earthworm species in Taiwan and Ryukyu Islands do not show any geographical trends in their character states. The character states of these species are mosaic, and concordance of state-shift does not occur among these species. Besides this, we are not able to recognize enough synapomorphic characters for cladistical analysis. All these phenomena impede the understanding of phylogenetic relationships among these eight earthworm species through morphological characters. For these reasons, further phylogenetic analyses based on molecular data are now being conducted.