﻿Four new earthworm species of the genera Amynthas and Metaphire (Oligochaeta, Megascolecidae) from Hunan and Anhui provinces, China

﻿Abstract This paper describes four new species earthworms from Hunan and Anhui provinces, China, Amynthasxiangtanensis Qiu & Jin, sp. nov., Amynthastaoyuanensis Qiu & Jin, sp. nov., Amynthasxuanchengensis Jin & Li, sp. nov. and Metaphiredonganensis Jin & Jiang, sp. nov.Amynthasxiangtanensissp. nov., and A.taoyuanensissp. nov. belong to the Amynthascorticis group. Both have four pairs of intersegmental spermathecal pores in 5/6–8/9; male pores in segment XVIII, separated by 1/3 of body circumference, each on top of a slightly raised porophore, surrounded by several tiny genital papillae. Amynthastaoyuanensissp. nov. prostate glands are degenerated. Amynthasxuanchengensissp. nov. belongs to the Amynthasmorrisi group, it has two pairs of spermathecal pores in 5/6 and 6/7; male pores in XVIII, separated by 1/3 of body circumference, each on top of a slightly raised, circular porophore. Metaphiredonganensissp. nov. belongs to the Metaphirehoulleti group. It has three pairs of spermathecal pores in 6/7–8/9; male pores in XVIII, separated by 1/3 of body circumference, each on the bottom center of the longitudinal copulatory chamber.

In 2015, 2016, and 2019, we investigated earthworm diversity in the two provinces and more than 31 and 24 Megascolecidae species were recorded from Hunan and Anhui, respectively.In this paper, we describe three new species of the genus Amynthas and one new species of the genus Metaphire found in those surveys (Fig. 1).Among them, A. xiangtanensis sp.nov.and A. taoyuanensis sp.nov.belong to the A. corticis group with intersegmental spermathecal pores in 5/6-8/9, which is widely distributed in China (such as Hainan, Yunnan, and Guangxi provinces) (Sun et al. 2012(Sun et al. , 2018(Sun et al. , 2021;;Dong et al. 2019;Yuan et al. 2019).Amynthas xuanchengensis sp.nov.belongs to the A. morrisi group with intersegmental spermathecal pores in 5/6 and 6/7, which is widely distributed in China including Hainan, Yunnan, and Guangdong provinces (Zhao et al. 2013;Jiang et al. 2015;Sun et al. 2015Sun et al. , 2021)).Metaphire donganensis sp.nov.belongs to the M. houlleti group, with intersegmental spermathecal pores in 6/7-8/9, which is also widely distributed in China including Jiangxi, Gansu, and Fujian provinces (Feng 1984;Sun et al. 2018).DNA barcodes of the four new species are provided (PP497092-PP497100) in this paper.

Materials and methods
The earthworms were collected in 2015, 2016, and 2019.Specimens were anaesthetized in a 10% ethanol solution and preserved in a 95% ethanol solution.Holotypes and paratypes are deposited in the Shanghai Natural History Museum.Table 1.Specimens with molecular data used in this study.Some species have no molecular data in GenBank.Abbreviations, HT holotype, PT paratype.
DNA was extracted from several specimens of A. xiangtanensis sp.nov., A. taoyuanensis sp.nov., A. xuanchengensis sp.nov., M. donganensis sp.nov.by using the E.Z.N.A. Mollusc DNA Kit (Omega Bio-tek, Norcross, GA, USA).The gene cytochrome c oxidase subunit I (COI) was amplified by polymerase chain reaction (PCR).The PCR amplification mixture (50 μL total) consisted of 1 μL of DNA template, 2 μL of each primer, 35.4 μL double-distilled H 2 O, and 9.6 μL Trans Taq TM Polymerase High Fidelity containing 0.6 μL TransTaqTM HiFi DNA polymerase, 4 μL 2.5 mM dNTPs and 5 μL 10 × TransTaq TM HiFi Buffer I. Primers used in the research were COI,5'-GGTCAACAAATCATAAAGATATTGG-3' and 5'-TAAACTTCAGGGTGACCAAAAAATCA-3' (Folmer et al. 1994).The PCR was carried out as follows: 5 min at 94 °C followed by 32 cycles 94 °C for 30 s, 50 °C for 30 s and 72 °C for 60 s, with an extension of 10 min at 72 °C.Sequencing was performed in the Beijing Genomics Institute (Shanghai, China).COI sequences of the new species were submitted to the NCBI GenBank databases under the accession numbers provided in Table 1, and sequences of other similar known species were retrieved from GenBank (Table 1).All the DNA sequences were aligned using ClustalX 2.0 (Thompson et al. 1997).The genetic pairwise distances between these species were calculated using the Kimura two-parameter model (Kimura 1980) in MEGAX with 1000 bootstrap replicates (Kumar et al. 2018) Diagnosis.Size medium to large.Spermathecal pores in 5/6-8/9, separated by 1/3 of body circumference.Male pores in XVIII, separated by 1/3 of body circumference, each on the top of a slightly raised, circular porophore.Spermathecae four pairs in VI-IX, ampulla heart-shaped, duct thick and ~ 1/4 of ampulla.Diverticulum is ~ 2/3 of main pouch (duct and ampulla together), terminal 1/2 dilated into rod-shaped seminal chamber.Intestinal caeca are simple.Prostate glands are well developed.
Internal characters.Septa 5/6-7/8 thick and muscular, 10/11-12/13 slightly thickened, 8/9 and 9/10 absent.Gizzard long bucket-shaped, in IX-X.Intestine enlarged distinctly from XV. Intestinal caeca paired in XXVII, extending anteriorly to XXIII, simple, smooth on both sides or sometimes weakly constricted on ventral margin (Fig. 2C).Four esophageal hearts in X-XIII, the latter three are more developed than the first pair.Male sexual system holandric, testis sacs two pairs in X and XI, well developed, left and right lobes separated on the ventral side.Seminal vesicles two pairs in XI and XII, well developed, left and right lobes separated on the ventral side.Prostate glands well developed, inserting in XVIII and extending to XIV and XXI, coarsely lobate, prostatic duct U-shaped, slightly thicker at the distal part, a large lumpy accessory gland beside the prostatic duct (Fig. 2B).Spermathecae four pairs in VI-IX, ampulla heart-shaped, ~ 2.5-4.5 mm long in holotype; ampulla duct is thick and ~ 1/4 of ampulla.Diverticulum is ~ 2/3 of main pouch (duct and ampulla together), slender, terminal 1/2 dilated into rod-shaped seminal chamber.A pair of large lumpy accessory glands present on the ventrum of IX, corresponding to the position of papillae (Fig. 2D).Sometimes another pair of large lumpy accessory glands present on the ventrum of VIII, corresponding to the position of papillae.
Etymology.The species is named after its type locality.
Remarks.Amynthas xiangtanensis sp.nov., with four pairs of spermathecal pores in 5/6-8/9, keys to the Amynthas corticis group (Sims and Easton 1972) which consists of 111 species (Nguyen et al. 2020a(Nguyen et al. , 2020b;;Sun et al. 2021;Li et al. 2024); it is similar to A. taoyuanensis sp.nov. in setae number, spermathecal pores, male pores, and simple intestinal caeca (Table 2).However, A. xiangtanensis sp.nov.(110-184*5.0-7.0,yellowish brown dorsum and pale yellowish brown ventrum) is easily distinguished from A. taoyuanensis sp.nov.(length 41-120*3.5-4.5, colorless) by its larger body size and pigmentation.In addition, the first dorsal pore of The new species is also fairly close to Amynthas corticis (Kinberg, 1867) by having medium to large size, spermathecal pores location and simple intestinal caeca (Table 2).However, the new species differs from A. corticis in pigmentation (yellowish brown dorsum and pale yellowish brown ventrum), first dorsal pore in 9/10, paired papillae within spermathecal pore region before setae in VIII or IX, male pores middle and round by 1/3C, two or more papillae near male pore, prostate glands in XIV-XXI with a large lumpy accessory gland, ampulla heart-shaped, diverticulum terminal 1/2 dilated into rod-shaped seminal chamber, paired large lumpy accessory glands on VIII or IX.Whereas A. corticis has a greenish brown dorsum and an unpigmented ventrum, first dorsal pore in 11/12, paired papilla before or after setae in some or all near spermathecal pores, male pores small and circular to transverse elliptical disc by 1/4C-1/3C, one or more papillae near male pore, prostate glands in XVII-XX without accessory glands, ampulla ovoid, diverticulum straight stalked, terminally dilated into a blunt ovoid seminal chamber, accessory glands stalked, coelomic, bound down to the parietes or retained within body wall.Another similar species with four pairs of spermathecal pores in 5/6-8/9 is Amynthas maximus Qiu & Dong, 2019.The two species share some similarities, such as body size, pigmentation, spermathecal pores, simple intestinal caeca (Table 2).However, A. xiangtanensis sp.nov.has first dorsal pore in 9/10, clitellum XIV-XVI, spermathecal pores, paired papillae before setae in VIII or IX, male pores without ridges, separated by 1/3 of body circumference, two or more papillae near male pore, prostate glands in XIV-XXI with a large lumpy accessory gland, spermathecae larger, ampulla heart-shaped, duct ~ 1/4 of ampulla, diverticulum terminal 1/2 dilated into rod-shaped seminal chamber, paired large lumpy accessory glands on VIII or IX.While A. maximus has first dorsal pore in 13/14, clitellum in 2/5XIV-XVI, fewer setae at VIII and XX, two pairs papillae after setae in VII and VIII, male pores surrounded by 3-4 circular ridges, separated by 2/5 of body circumference, paired papillae before setae medial of male pore, prostate glands in XVII-XIX with accessory glands invisible, spermathecae smaller, ampulla elongate-oval, duct 3/5 of ampulla, diverticulum terminal 2/5 dilated into a swollen, club-shaped seminal chamber, one or two stalked accessory glands on VI, VII, VIII and IX.
Regarding the pigmentation, clitellum, the position of spermathecal and male pores, simple intestinal caeca, and characteristics of the spermathecae, the new species is similar to Amynthas tortuosus Qiu & Dong, 2019.However, the two species are distinguished by body size, the position of the first dorsal pore, setae number, the number and position of papillae within the spermathecal pore and male pore region, the characteristics and ventral distance of male pores, the position of prostate glands and the existence or nonexistence of accessory glands, and the characteristics of the diverticulum and accessory glands (Table 2).

Amynthas taoyuanensis
Internal characters.Septa 5/6-7/8 thick and muscular, 10/11-12/13 slightly thickened, 8/9 and 9/10 absent.Gizzard spherical, in IX-X.Intestine enlarged distinctly from XV. Intestinal caeca paired in XXVII, extending anteriorly to XXIV, simple, smooth on both sides (Fig. 3B).Four esophageal hearts in XI-XIII, well developed.Male sexual system holandric, testis sacs two pairs, in X and XI, well developed, left and right lobes separated on the ventral side.Seminal vesicles two pairs, extending in XI and XII, well developed, left and right lobes separated on the ventral side.Prostate glands degenerated, prostatic duct U-shaped inserting in XVIII, several small lumpy accessory glands in center of ventral XVI, XVII, and XVIII; the position is consistent with the position of the ventral mastoid on the body surface.Spermathecae four pairs in VI-IX, ampulla oval-shaped, ~ 0.6-1.8mm long in holotype; ampulla duct is thick and ~ 1/2 of ampulla.Diverticulum is ~ 2/3 of main pouch (duct and ampulla together), terminal 3/4 dilated into bag-shaped chamber.Two pairs of large lumpy accessory glands present on the ventrum of VII and VIII (Fig. 3C).Sometimes pair of large lumpy accessory glands present on the ventrum of VII (specimen HU201601-03 and HU201602-04).
Etymology.The species is named after its type locality.
Remarks.Amynthas taoyuanensis sp.nov., with four pairs of spermathecal pores in 5/6-8/9, also belongs to the Amynthas corticis group.Amynthas taoyuanensis sp.nov. is close to Amynthas xiangtanensis sp.nov. in setae number, the position and characteristics of spermathecal pores and male pores, and the simple intestinal caeca.However, the differences between the two new species are body size, pigmentation, the position of the first dorsal pore, the position of clitellum, the position of papillae within spermathecal pores and male pore region, the existence or nonexistence of prostate glands, and the characteristics of the spermathecae and diverticulum (Table 2).
The new species is similar to Amynthas homosetus (Chen, 1938) in terms of body size, position and characteristics of spermathecal pores and male pores, and simple intestinal caeca.Whereas, the new species differs from A. homosetus in being unpigmented, the first dorsal pore in 10/11, clitellum in XIV-2/3XVI, 13 or 14 setae between male pores, the ventral distance of spermathecal pores is 1/3C, papillae paired after setae in VI, VII, or VIII, two paired or more papillae in XVI, XVII, XVIII, or XIX, prostate glands degenerated, spermathecae ~ 0.6-1.8mm long, ampulla oval-shaped, duct is thick and ~ 1/2 of ampulla, diverticulum terminal 3/4 dilated into bag-shaped seminal chamber, two paired large lumpy accessory glands on VII or VIII.Amynthas homosetus is dark chocolate-colored anteriorly and grey on other parts of dorsum, grey on the ventrum, the first dorsal pore in 12/13, clitellum without setae in XIV-XVI, nine setae between male pores, the ventral distance of spermathecal pores is 1/4C, papillae invisible within spermathecal pores and male pore regions, prostate glands developed in XVI-XXI, spermathecae heart-shaped, diverticulum seminal chamber ovoid (Table 2).
Amynthas taoyuanensis sp.nov. is also close to Amynthas stricosus Qiu & Sun, 2012 by body size, pigmentation, setae number, the characteristics of spermathecal pores and male pores, the ventral distance of male pores, simple intestinal caeca.However, the new species is distinguished from A. stricosus by the first dorsal pore, the position and existence or nonexistence setae of clitellum, and the ventral distance of spermathecal pores; A. taoyuanensis sp.nov.has paired papillae after setae in VI, VII, or VIII, but A. stricosus has no papillae; in addition, A. taoyuanensis sp.nov.has two paired papillae or more in XVI, XVII, XVIII, or XIX, but A. stricosus has a single or paired after setae in XVII, XIX, and XX; prostate glands of A. taoyuanensis sp.nov.are degenerated with several lumpy accessory glands in XVI, XVII, and XVIII, while developed in XVI-XX with accessory glands invisible in A. stricosus; furthermore, spermathecae of A. taoyuanensis sp.nov.are ~ 0.6-1.8mm long, ampulla oval-shaped, duct is thick and ~ 1/2 of ampulla, whereas ~ 1.6 mm long, ampulla heart-shaped, gradually slender duct as long as ampulla in A. stricosus; diverticulum of A. taoyuanensis sp.nov. is ~ 3/4 of main pouch, terminal 3/4 dilated into bag-shaped seminal chamber, but as long as main pouch, slender, terminal 2/5 dilated into band-shaped seminal chamber in A. stricosus; two paired large lumpy accessory glands on VII or VIII in A. taoyuanensis sp.nov., but no accessory glands in A. stricosus (Table 2).
According to body size, pigmentation, setae ventrally in clitellum, the position and characteristics of spermathecal pores and male pores, simple intestinal caeca, and the characteristics of spermathecae, A. taoyuanensis sp.nov. is somewhat similar to Amynthas genitalis Qiu & Sun, 2012.However, the new species is characterized by the first dorsal in 10/11, clitellum in XIV-2/3 XVI, 19-22 setae between spermathecal pores (VIII), papillae paired within spermathecal pore region, two paired or more papillae in XVI, XVII, XVIII, or XIX, prostate glands degenerated with several lumpy accessory glands in XVI, XVII, and XVIII, diverticulum ~ 3/4 of main pouch, terminal 3/4 dilated into bag-shaped seminal chamber, two paired large lumpy accessory glands on VII or VIII; whereas A. genitalis has 8-11 setae between spermathecal pores (VIII), no papillae within spermathecal pore region, paired papillae before setae annulet in XVIII and XIX, after setae annulet in XVII and XVII, prostate glands developed with accessory glands invisible in XVII-XX, diverticulum longer than main pouch, slender, terminal 0.29 dilated into rod-shaped seminal chamber, no accessory glands (Table 2).
Another similar species with four pairs of spermathecal pores in 5/6-8/9 is Amynthas recavus Yuan & Jiang, 2019.The two species share some similarities, such as the position of male pores and simple intestinal caeca.However, the two species are distinguished by body size, pigmentation, the first dorsal pore position, setae number, the ventral distance of spermathecal pores, existence or nonexistence of papillae within spermathecal pore region, the characteristics of male pores and papillae within male pore region, prostate glands, the characteristics of spermathecae and diverticulum, and the existence or nonexistence of accessory glands (Table 2).Diagnosis.Size small.Spermathecal pores in 5/6 and 6/7, separated by 1/3 of body circumference.Male pores in XVIII, separated by 1/4 of body circumference, each on the top of a slightly raised, circular porophore.Spermathecae two pairs in VI and VII, ampulla heart-shaped, duct is thick and ~ 1/2 of ampulla.Diverticulum as long as main pouch (duct and ampulla together), terminal 1/2 dilated into ovoid-shaped seminal chamber.Intestinal caeca are simple.Prostate glands are developed.
Internal characters.Septa 5/6-7/8 thick and muscular, 10/11-12/13 slightly thickened, 8/9 and 9/10 absent.Gizzard spherical in IX-X.Intestine enlarged distinctly from XV. Intestinal caeca paired in XXVII, extending anteriorly to XXVI, simple, smooth on both sides (Fig. 4C).Four esophageal hearts in X-XIII, not well developed.Male sexual system holandric, testis sacs two pairs, in X and XI, well developed, left and right lobes separated on the ventral side.Seminal vesicles two pairs, extending in XI and XII, well developed, left and right lobes separated on the ventral side.Prostate glands well developed, inserting in XVIII and extending to XVI and XIX, coarsely lobate, prostatic duct U-shaped, slightly thicker at the distal part (Fig. 4B).No accessory glands observed.Spermathecae two pairs in VI-VII, ampulla heart-shaped, ~ 0.7 mm long in holotype; ampulla duct is thick and ~ 1/2 of ampulla.Diverticulum as long as main pouch (duct and ampulla together), terminal 1/2 dilated into ovoid-shaped seminal chamber.No accessory glands observed (Fig. 4D).
Etymology.The species is named after its type locality.
Another similar species with two pairs of spermathecal pores in 5/6 and 6/7 is Amynthas fucatus Zhao & Qiu, 2013.The two species share some similarities, such as clitellum in XIV-XVI, the position of spermathecal pores and male pores, invisible papillae within spermathecal pore region, and simple intestinal caeca.By contrast, the new species and A. fucatus can be separated on the basis of smaller body size and fewer setae at VIII.In addition, A. xuanchengensis sp.nov. is unpigmented, but dark red brown before clitellum and pale after clitellum in dorsum, pale red brown before clitellum and pale after clitellum in ventrum in A. fucatus; the first dorsal pore of A. xuanchengensis sp.nov. in 12/13, but 11/12 in A. fucatus; the ventral distance of spermathecal pores and male pores are 1/3C and 1/4C in A. xuanchengensis sp.nov., but 2/5C and 2/5C in A. fucatus; spermathecal pores of A. xuanchengensis sp.nov.are inconspicuous, but obvious in A. fucatus; male pores of A. xuanchengensis sp.nov.not surrounded by folds, but A. fucatus surrounded by three to five folds; invisible papillae within male pore region of A. xuanchengensis sp.nov., whereas two papillae on the inner side of male pore, another paired in XVIII in A. fucatus; prostate glands in XVI-XIX of A. xuanchengensis sp.nov., while XV-XXI with accessory glands invisible in A. fucatus; spermathecae ~ 0.7 mm long, ampulla heart-shaped in A. xuanchengensis sp.nov., nevertheless spermathecae longer, ampulla elongated ovoid in A. fucatus; diverticulum is as long as main pouch, terminal 1/2 dilated into ovoid-shaped seminal chamber in A. xuanchengensis sp.nov., but diverticulum ~ 1/3 of main pouch, straight, terminal 3/4 dilated into virgulate seminal chamber in A. fucatus (Table 3).
Amynthas xuanchengensis sp.nov.appears to be closely related to A. infuscuatus Jiang & Sun, 2015 in the combined characters of ventrum pigmentation, first dorsal pore in 12/13, clitellum in XIV-XVI, the position and characteristics of spermathecal pores, invisible papillae within spermathecal pore region, the position of male pores, simple intestinal caeca, and ampulla heart-shaped.Conversely, they still have some differences, such as body size, dorsum pigmentation, and setae number.Amynthas xuanchengensis sp.nov.smaller, without pigment on dorsum, but purple before VIII; pale brown after VIII in A. infuscuatus; four setae between male pores of A. xuanchengensis sp.nov., but 8-10 setae in A. infuscuatus; the ventral distance of male pores is 1/4C in A. xuanchengensis sp.nov., but 1/3C in A. infuscuatus; spermathecal pores of A. xuanchengensis sp.nov.are inconspicuous, but obvious in A. infuscuatus; male pores of A. xuanchengensis sp.nov.surrounded by no folds, but A. infuscuatus surrounded by three or four folds; invisible papillae within male pore region of A. xuanchengensis sp.nov., whereas two papillae on the inner side of male pore in A. infuscuatus; prostate glands in XVI-XIX of A. xuanchengensis sp.nov., while XVI-1/2XX with accessory glands invisible in A. infuscuatus; spermathecae ~ 0.7 mm long, duct is thick and ~ 1/2 of ampulla in A. xuanchengensis sp.nov., but spermathecae longer, duct slender, twice as long as ampulla in A. infuscuatus; diverticulum is as long as main pouch, terminal 1/2 dilated into ovoid-shaped seminal chamber in A. xuanchengensis sp.nov., but diverticulum longer than main pouch, slender, terminal 1/3 dilated into swollen seminal chamber in A. infuscuatus (Table 3).Amynthas xuanchengensis sp.nov.and Amynthas zonarius Sun & Qiu, 2015 share some common characters in pigmentation, first dorsal pore in 12/13, clitellum in XIV-XVI, the characteristics of spermathecal pores, invisible papillae within spermathecal pore region, the position of male pores, simple intestinal caeca.In contrast, A. xuanchengensis sp.nov.differs from A. zonarius in smaller body size and fewer setae.Additionally, the ventral distance of spermathecal pore and male pores are 1/3C and 1/4C in A. xuanchengensis sp.nov., but 2/5C and 1/3C in A. zonarius; male pores of A. xuanchengensis sp.nov.surrounded by no folds, but A. zonarius surrounded by five folds; invisible papillae within male pore region of A. xuanchengensis sp.nov.whereas one or two papillae on the inner side of male pore, sometimes another two on XVIII in A. zonarius; prostate glands in XVI-XIX in A. xuanchengensis sp.nov., while XVI-1/2XX with accessory glands invisible in A. zonarius; spermathecae ~ 0.7 mm long, ampulla heart-shaped, duct is thick and ~ 1/2 of ampulla in A. xuanchengensis sp.nov., whereas spermathecae longer, ampulla ovoid, duct as long as ampulla in A. zonarius; ovoid-shaped seminal chamber in A. xuanchengensis sp.nov., but band-shaped seminal chamber in A. zonarius (Table 3).

Genus
Internal characters.Septa 5/6-7/8 thick and muscular, 10/11-12/13 slightly thickened, 8/9 and 9/10 absent.Gizzard spherical, in IX-X.Intestine enlarged distinctly from XV. Intestinal caeca paired in XXVII, extending anteriorly to XXIII, simple, smooth dorsal margin, weakly constricted on ventral margin (Fig. 5C).Four esophageal hearts in X-XIII, the latter three are more developed than the first pair.Male sexual system holandric, testis sacs two pairs, in X and XI, well developed, left and right lobes connected on the ventral side.Seminal vesicles two pairs, extending in XI and XII, well developed, left and right lobes separated on the ventral side.Prostate glands well developed, inserting in XVIII and extending to XVI and XXII, strip lobate, prostatic duct U-shaped, slightly thicker at the distal part (Fig. 5B).No accessory glands observed.Spermathecae three pairs in VII-IX, ampulla heart-or rod-shaped, ~ 3.2-8.0mm long in holotype; ampulla duct is swollen and as long as ampulla.Diverticulum as long as main pouch (duct and ampulla together), slender and straight at proximal part, terminal 1/2 dilated into twisted in zigzag fashion.No accessory glands observed (Fig. 5E).
Etymology.The species is named after its type locality.
Remarks.Metaphire donganensis sp.nov.with three pairs spermathecal pores in 6/7-8/9, keys to the Metaphire houlleti group, which includes 44 species (Sims and Easton 1972;Feng 1984;Feng and Ma 1987;Qiu and Zhong 1993;Sun et al. 2018).Metaphire vulgaris agricola (Chen, 1930) is slightly akin to the new species in the respects of body size, first dorsal pore in 12/13, clitellum in XIV-XVI, setae number, the position and characteristics of spermathecal pores, simple intestinal caeca, and the characteristics of diverticulum.Instead, the difference between the two species is in the pigmentation, pale brown in M. donganensis sp.nov., earthy yellow in M. vulgaris agricola (Feng, 1981).Further, the ventral distance of spermathecal pore and male pores are 1/3C and 1/3 C in M. donganensis sp.nov., but 1/4C and 1/4C in M. vulgaris agricola; no papillae within spermathecal pore region of M. donganensis sp.nov., but paired papillae in VII of M. vulgaris agricola; no papillae within male pore region of M. donganensis sp.nov., while paired papillae on the inner side of male pore in M. vulgaris agricola; prostate glands well developed in XVI-XXII of M. donganensis sp.nov., nevertheless in XVII-XX with accessory glands invisible in M. vulgaris agricola; spermathecae ~ 3.2-8.0mm long, ampulla heart-or rod-shaped, duct swollen, as long as ampulla of M. donganensis sp.nov., while spermathecae ~ 4.5 mm long, ampulla pear-shaped, ampulla duct 1/2 of ampulla in M. vulgaris agricola; no accessory glands in M. donganensis sp.nov., while paired accessory glands in the VIII of M. vulgaris agricola (Table 4).
We compare the new species to Metaphire tschiliensis lanzhouensis (Feng, 1984), which has three pairs of spermathecal pores in 6/7-8/9.They share several common characters in the first dorsal pore position, clitellum in XIV-XVI, setae number, the position of spermathecal pores, the position and characteristics of male pores, no papillae within spermathecal pore region, simple intestinal caeca, and the characteristics of the diverticulum.Quite the contrary, the new species is smaller than M. tschiliensis lanzhouensis.Beyond that, coloration is pale brown in M. donganensis sp.nov., earthy yellow in M. tschiliensis lanzhouensis; spermathecal pores are obvious in M. donganensis sp.nov., but inconspicuous in M. tschiliensis lanzhouensis; no papillae within male pore region of M. donganensis sp.nov., but one in pouch of M. tschiliensis lanzhouensis; prostate glands well developed in XVI-XXII of M. donganensis sp.nov., while underdeveloped in XVII-XIX with a large lumpy accessory gland in M. tschiliensis lanzhouensis; spermathecae ~ 3.2-8.0mm long, ampulla heartor rod-shaped of M. donganensis sp.nov., but spermathecae ~ 4 mm long, ampulla spherical-shaped in M. tschiliensis lanzhouensis (Table 4).
Considering the three pairs of spermathecal pores in 6/7-8/9, we compared the new species with Metaphire viridis Feng & Ma, 1987.Both have first dorsal pores in 12/13, clitellum in XIV-XVI, the same position and characteristics of spermathecal pores, simple intestinal caeca, and the same characteristics of diverticulum.However, the new species differs from M. viridis in many respects.Metaphire donganensis sp.nov. is smaller than M. viridis with pale brown coloration; setae number of M. donganensis sp.nov. is less than M. viridis; the ventral distance of spermathecal

Figure 1 .
Figure 1.Distribution of four species in Hunan Province and Anhui provinces, China.

Diagnosis.
Size medium to large.Spermathecal pores in 6/7-8/9, separated by 1/3 of body circumference.Male pores in XVIII, separated by 1/3 of body circumference, each on the bottom center of the longitudinal copulatory chamber.Sometimes the copulatory chamber eversion is ridged.Spermathecae three pairs in VII-IX, ampulla heart-or rod-shaped, duct thick and as long as ampulla.Diverticulum as long as main pouch (duct and ampulla together), slender and straight at proximal part, terminal 1/2 dilated into twisted in zigzag fashion.Intestinal caeca are simple.Prostate glands are well developed.

Table 3 .
A comparison of characters of A. xuanchengensis sp.nov.and similar species of the Amynthas morrisi group.Abbreviations: sp, spermathecal pores, mp, male pores.

Table 4 .
A comparison of characters of M. donganensis sp.nov.and similar species of the Metaphire houlleti group.Abbreviations: sp, spermathecal pores, mp, male pores.

Table 5 .
Percentage of pairwise distances of COI genes between 13 species.