New species of earthworms belonging to the Metaphire formosae species group (Clitellata: Megascolecidae) in Taiwan Author Chang, Chih-Han Author Chuang, Shu-Chun Author Wu, Jia Hsing Author Chen, Jiun-Hong text Zootaxa 2014 3774 4 324 332 journal article 46278 10.11646/zootaxa.3774.4.2 2383d3fd-3ec2-4af2-8d97-94d35629f479 1175-5326 225988 1E9E30D8-392F-454B-B6F6-68E2276B35F9 Metaphire taiwanensis tsaii ssp. nov. Chang and Chen ( Fig. 3 ) Metaphire taiwanensis (part): Chang et al. 2008: 958, 967, Figs. 1 , 4, Tables 1 , 3–4; Chang & Chen 2008: 46–48, Fig. 1 L, Table 1 ; Chang et al. 2009 : 6, Fig. 2 L. [Chang & Chen (2008) cited the taxon as ‘ Metaphire taiwanensis subsp.’]. Type specimens. Holotype : MZNTU 14-05614 (mature), collected 24 Nov 2002 from Fushan Botanical Research Station, Ilan County, Taiwan by C.-H. Chang. Paratypes : MZNTU 14-00033 (mature), collected 1 September 1994 from Fushan Botanical Research Station, Ilan County, Taiwan by S.-S. Lu; MZNTU 14-03883 (mature), collected 18 May 2002 from Jiaosi, Ilan County, Taiwan by I-H. Chen; MZNTU 14-00028 (mature), collected 15 November 1994 from Fushan Botanical Research Station, Ilan County, Taiwan by S.-S. Lu. Other material examined. Four mature specimens. MZNTU 14-00026, 8 December 1994 from Fushan Botanical Research Station, Ilan County, Taiwan by S.-H. Lu; MZNTU 14-00029, collected 16 November 1994 from Fushan Botanical Research Station, Ilan County, Taiwan by S.-H. Lu; MZNTU 14-05319, collected 24 January 2002 from Fushan Botanical Research Station, Ilan County, Taiwan by C.-H. Chang; MZNTU 14-05903, collected 14 April 2003 from Yuanshan, Ilan County, Taiwan by W.-M. Lee. Distribution. Northern Taiwan . Recorded in Fushan Botanical Research Station and the surrounding regions in the Ilan County. Etymology. Noun in the genitive case, after Taiwanese earthworm taxonomist Dr. Chu-Fa Tsai. DNA barcodes of type material. Available for MZNTU 14-05614 ( Table 1 ) Diagnosis. Pheretimoids with lengths 248–343 mm , clitellum width 10–12 mm . Copulatory pouches present with an anterior oval pad similar in size to the porophore. Spermathecae four pairs in 6–9. No genital papillae in the spermathecal pore area. Testes proandric. Prostate gland lobular. Caeca simple. FIGURE 3. Metaphire taiwanensis tsaii ssp. nov. A. Ventral view of anterior body (fp, female pore; mp, male pore; cl, clitellum). B. Spermatheca (amp, ampulla; dv, diverticulum). C. Male pore area (op, oval pad). D. prostate gland with prostatic duct. Drawing of B, C, and D based on photographs from Fig. 26 in Chang & Chen 2008. Morphology. External characters. Length (mature) 248–343 mm , clitellum width 10–12 mm , segment number 152–188. Number of annuli per segment three in 5–9, five in 10–13, three in body segments behind 17. Prostomium prolobous. Setae 121–164 in 7, 132–135 in 20, 22–28 between male pores. First dorsal pore in 12/13. Clitellum 14–16, annular, smooth, length 10–11 mm , dorsal pore absent, setae absent. Preserved specimens bluish brown on dorsum, light brown on ventrum. Clitellum dark bluish brown on dorsum, greyish brown on ventrum. Spermathecal pores four pairs in 5/6-8/9, minute, invisible externally, distance between the paired pores about 0.4– 0.5 body circumference apart ventrally. No genital papillae in the spermathecal pore region. Female pore single, mid-ventral in 14. Male pores paired in 18, small, latero-ventral, in a slightly C-shaped copulatory pouch, bordered laterally by a thick skin surrounded by circular folds. Porophore circular, tuberculated. An oval pad situated anteriorly to the porophore, with size similar to the porophore. Circular folds surrounding porophore and oval pad, extending anteriorly to 17/18 and posteriorly to 18/19. Genital papillae absent in the male pore area. Internal characters. Septa 5/6–7/8 and 10/11–13/14 thickened, 8/9 membranous, 9/10 absent. Gizzard in 8. Intestine enlarged from 15. Intestinal caeca paired in 27, simple, long, surface slightly wrinkled, extending anteriorly to 23. Oesophageal hearts enlarged in 10–13. Spermathecae four pairs in 6–9. Ampulla elliptical, about 3–5 mm in length, with a short stalk about 2 mm long. Diverticulum small, with an oval, smooth seminal chamber and a short coiled or twisted stalk, reaching the basal 1/3 of the ampulla. Meronephridia tufted, attached to the anterior face of septa 5/6 and 6/7. 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, large, surface tuberculated, with a large dorsal lobe. Prostate glands paired in 18, large, oval, extending to 17 and 19. Remarks. M. taiwanensis tsaii ssp. nov. corresponds to clade E2 of M. taiwanensis in Chang et al. (2008). The two subspecies, M. taiwanensis taiwanensis and M. taiwanensis tsaii , have an allopatric distribution. They diverged by 5.7% in the COI gene (Chang et al. 2008). Morphologically, M. taiwanensis taiwanensis is much larger than M. taiwanensis tsaii . The two subspecies also show different levels of allometric growth on their male pore areas compared to their body size: the male pore areas of the two subspecies are almost the same in size, but compared to their body size, the male pore area of M. taiwanensis taiwanensis is relatively smaller. It is also smoother and flatter with circular folds almost restricted to the ventral surface, whereas that of M. taiwanensis tsaii is more three-dimensional, with circular folds extending to the lateral side of the body. TABLE 1. GenBank accession numbers of DNA barcode sequences from type specimens of the Metaphire formosae species group. Species MZNTU cat. GenBank accession nos. Types References nos. M. feijani 14-07095 AY960809 holotype Chang et al. 2008 14-07099 AY962162 paratype Chang et al. 2008 M. tengjhihensis sp. nov. 14-05901 AY960801 holotype Chang et al. 2008 14-07175 AY962164 paratype Chang et al. 2008 M. nanaoensis truku ssp. nov. 14-07228 AY962150 paratype Chang et al. 2008 14-05342 AY960805 paratype Chang et al. 2008 M. tahanmonta 14-03993 AY962115 holotype Chang et al. 2008 14-05898 AY739335 paratype Chang & Chen 2005 The assignment of the formosae species group to either Metaphire or Amynthas has recently been highly debated. The two genera are distinguished by only one character, the copulatory pouch, a structure present in Metaphire and absent in Amynthas ( Sims & Easton 1972 ) . However, there are two distinguishable forms of copulatory pouches that seem to be non-homologous (James et al. 2005), and each of them has various degrees of reduction or “degradation”, which makes it difficult in some cases to decide between presence or absence of that character. James prefers to restrict Metaphire to species with well-developed copulatory pouches (James 2005; James et al. 2005) and assigns species with "degraded" pouches to Amynthas , while Blakemore (2010) prefers to restrict Amynthas to species with superficial male pores, arguing that all species with the derivative character state of nonsuperficial male pores should belong to Metaphire . As a result, species in the formosae species group were assigned to Amynthas and Metaphire by James and Blakemore, respectively. We follow the conventional practice of assigning species in the formosae species group to Metaphire ( Chang & Chen 2004 , 2005a , b ; Tsai et al. 2000 , 2003 , 2004 ), but recognize the different forms of copulatory pouches presented by Easton (1979) and James (James 2005; James et al. 2005), particularly the intramural and intracoelomic ones. Furthermore, we doubt that the assignment of species with the intramural form and the various “degradations” of both forms to either Amynthas or Metaphire is satisfying regarding preserving information of character states and evolution. Recent molecular phylogenetic studies strongly suggest that Metaphire is not monophyletic (Chang et al. 2008; James 2005); it is indeed polyphyletic (unpublished data) regardless of difference in opinions on the definition of Metaphire or Amynthas . Considering this nature and the viewpoints already discussed by the above authors, any further arguments on the Amynthas -or- Metaphire issue seem meaningless before someone takes on the task of revising the whole group.

14-05899 AY960800	paratype Chang et al. 2008
M. taiwanensis tsaii ssp. nov.	14-05614 AY962157	holotype Chang et al. 2008
Discussion

Hypotheses regarding synonyms of earthworms can usually be tested by comparing DNA barcodes. Blakemore et al. (2010) further recommended that any new earthworm taxa described should be accompanied by DNA barcodes from types to meet current standards. Although some of the DNA barcodes published by Chang and Chen and their coauthors ( Chang & Chen 2005b ; Chang et al. 2008) are from type specimens of the M. formosae species group, this connection has never been established either explicitly or implicitly until the present study ( Table 1 ). DNA barcodes from non-type specimens are also available for all species and subspecies in the M. formosae species group (see Chang et al. 2008). These genetic data, together with all the published morphological descriptions, enable unambiguous identification. Drawing a line between intraspecific and interspecific morphological variations is sometimes difficult in Amynthas and Metaphire . The studies regarding the M. formosae species group published after 2000 (as cited in this study) have collectively demonstrated an integrative taxonomic approach through which hypotheses regarding species are tested using morphological, molecular, biogeographical and, to some extent, ecological data. Considering the complexity of morphological variations and the huge numbers of described (and undescribed) species in Amynthas and Metaphire , and in order to prevent interminable arguments about synonyms, we strongly suggest preserving DNA-friendly samples on a regular basis and using an integrative taxonomic approach that combines morphological and molecular data when it comes to describing a new species of Amynthas or Metaphire that may raise debates about synonyms.