Three new widely distributed and polymorphic species of Amynthas earthworms (Oligochaeta, Clitellata, Megascolecidae) from South China Author Sun, Jing 0000-0003-1326-1886 School of Materials and Environmental Engineering, Chengdu Technological University, Chengdu, 610031, China. & sun _ jing. 2005 @ aliyun. com; https: // orcid. org / 0000 - 0003 - 1326 - 1886 sun_jing.2005@aliyun.com Author Jiang, Ji-Bao 0000-0002-5137-6097 School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai, 200240, China & jibao @ sjtu. edu. cn; https: // orcid. org / 0000 - 0002 - 5137 - 6097 jibao@sjtu.edu.cn Author Wu, Juzhen 0000-0001-7002-8023 School of Materials and Environmental Engineering, Chengdu Technological University, Chengdu, 610031, China. & 403141272 @ qq. com; https: // orcid. org / 0000 - 0001 - 7002 - 8023 403141272@qq.com Author Yuan, Zhu 0000-0003-2631-6495 School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai, 200240, China & zhuyuan 0452 @ jibao @ sjtu. edu. cn; https: // orcid. org / 0000 - 0003 - 2631 - 6495 jibao@sjtu.edu.cn Author Qiu, Jiang-Ping School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai, 200240, China text Zootaxa 2021 2021-03-02 4938 4 457 474 journal article 7747 10.11646/zootaxa.4938.4.5 c9ee5fe0-60c0-4b08-9dc2-3467b51a997a 1175-5326 4574948 F3741DEB-5690-4BD4-9B98-18A4EA2A57CD Amynthas rusticanus Sun & Qiu , sp. nov. ( Fig. 2 ) Type material . Holotype : One clitellate specimen (C-YN201108-02A), China , Yunnan Province , Xishuangbanna Dai Autonomous Prefecture , Mengla County ( 21°35ʹ03ʺN , 101°34ʹ58ʺE ), 722m above sea level (asl), under fern and shrub, laterite; Jul. 20, 2011 ; J. B. Jiang , J. Sun , X. D. Lei , and H. W. Feng coll. Paratypes : A total of 29 specimens , as follows: Two clitellates (C-YN201108-02B), same collection details as holotype . One clitellate (C-YN201101-18), China , Yunnan Province , Yuanjiang National Nature Reserve ( 23°40ʹ09ʺN , 101°51ʹ05ʺ E ), 854 m asl , under fruit trees (longan, mango, and plum), lateritic red soil; Jul. 16, 2011 ; J. B. Jiang , J. Sun , X. D. Lei , and H. W. Feng coll. One clitellate (C-YN201107-11), China , Yunnan Province , Xishuangbanna Dai Autonomous Prefecture , Mengla County ( 21°36ʹ21ʺN , 101°34ʹ54ʺE ), 694 m asl , daisy under trees, laterite; Jul. 20, 2011 ; J. B. Jiang , J. Sun , X. D. Lei , and H. W. Feng coll. One clitellate (C-YN201109-10), China , Yunnan Province , Xishuangbanna Dai Autonomous Prefecture , Mengla County ( 21°24ʹ14ʺN , 101°37ʹ27ʺE ), 722 m asl , in tropical primary forest, laterite; Jul. 20, 2011 ; J. B. Jiang , J. Sun , X. D. Lei , and H. W. Feng coll. Five clitellates (C-SC201009-08), China , Sichuan Province , Yibin City , Changning County , Mount Zhuji ( 28°22ʹ55.3ʺN , 104°52ʹ17.8ʺE ), 311 m asl , under Metasequoia , red-yellow soil; Aug. 15, 2010 ; J. Sun , Z. W. Yi , and J. H. Yi coll. Two clitellates and two aclitellates (C-SCCZ2015003-04), China , Sichuan Province , Chongzhou City , Jinjiang County ( 30°40ʹ36ʺN , 103°40ʹ59ʺ E ), 594 m asl , in secondary forest, red-yellow soil; Jun. 22, 2015 ; J. Sun , Z. W. Yi , and J. Yu coll. Two clitellates (C-YN201720-01), China , Yunnan Province , Dali City , Cangshan National Nature Reserve ( 25°46ʹ37.8ʺN , 100°05ʹ42.0ʺE ), 2194 m asl , in brown soil; Aug. 6, 2017 ; Z. Yuan , J. B. Jiang , Y. Dong , Q. Zhao coll. Two clitellates (C-YN201735-10), China , Yunnan Province , Dehong Dai and Jingpo Autonomous Prefecture , Tongbiguan Provincial Nature Reserve ( 24°08ʹ46.1ʺN , 98°01ʹ32.3ʺE ), 957 m asl , in yellow cinnamon soil, 11 Aug. 2017 ; Z. Yuan , J. B. Jiang , Y. Dong , Q. Zhao coll. Nine clitellates (C-YN201756-05), China , Yunnan Province , Lincang City , Nangunhe National Nature Reserve ( 23°38ʹ49.0″N , 99°20ʹ36.6ʺE ), 1429 m asl , in brown soil; Aug. 16, 2017 ; Z. Yuan , J. B. Jiang , Y. Dong , Q. Zhao coll. Four clitellates (C-YN201730-03), China , Yunnan Province , Nujiang Lisu Autonomous Prefecture , Gaoligongshan National Nature Reserve ( 26°32ʹ31.5ʺN , 98°54ʹ50.4ʺE ), 1867 m asl , in brown soil; Aug. 9, 2017 ; Z. Yuan , J. B. Jiang , Y. Dong , Q. Zhao coll. One clitellate (C-YN201752-01), China , Yunnan Province , Lincang City , Yongde Daxueshan National Nature Reserve ( 23°58ʹ14.9ʺN , 99°23ʹ57.8ʺE ), 1572 m asl , in brown soil; Aug. 15, 2017 ; Z. Yuan , J. B. Jiang , Y. Dong , Q. Zhao coll. Two clitellates (C-YN201606- 03), China , Yunnan Province , Pu’er City , Jingdong Yi Autonomous County ( 24°25ʹ09.7ʺN , 100°52ʹ44.9ʺE ), 1180 m asl , in brown soil; Jun. 11, 2016 ; X. Gao , Y. F. Lu , J. Z. Jiang , J. Long coll. Etymology. The species name is derived from the Latin word “ rustica ”, meaning rural, because the holotype was collected in a rural area of Yunnan Province , China . In Chinese, Amynthas rusticanus is ØTũḂḋ. Diagnosis. Small-sized; three pairs of spermathecal pores in 5/6–7/8, or two pairs of spermathecal pores in 5/6–6/7; male porophore resembles an oval pad, surrounded by 2–3 shallow skin folds; no genital papillae in the spermathecal pore and male pore regions; prostate gland developed in XVI–XX; spermathecal diverticulum shorter than the main spermathecal axis by ¼, stalk straight or twisted into two zigzags, distal ¼ dilated into a heart- or oval-shaped seminal chamber. Description. External characters : Preserved specimens light fuchsia before clitellum on dorsum, lacking pigment on the rest of the body. Dimensions 40–59 mm by 1.9–2.0 mm at clitellum; segments 91–92. No secondary annulations in any segment. Prostomium ½ epilobous. First dorsal pore in 12/13. Clitellum annular in XIV–0.17XVII in the holotype , XIV–XVI in paratype C-SCCZ2015003-04; smooth, swollen, no setae on the ventral side; dorsal pores absent on clitellum. Setae uniformly distributed, 34–46 on III, 46–60 on V, 50–60 on VIII, 46–48 on XX, 44–50 on XXV; 8–10 between male pores; setal formula AA=1.2–2.0AB, ZZ=1.7–2.0ZY. Spermathecal pores tiny, hard to locate; three pairs in 5/6–7/ 8 in holotype , rarely two pairs in 5/6–6/7 as in paratype C-SCCZ2015003-04; intersegmental, separated ventrally by about 0.33 of circumference ( Fig. 2a , sp.p). In holotype , no genital papillae in spermathecal pore region; in paratype C-SCCZ2015003-04, one small genital papilla posterior and medial to each of the left spermathecal pores in VI and VII. Male pores in XVIII, separated ventrally by 0.33 of circumference, slightly elevated from the skin sheath; glandular raised porophore resembles an oval pad, surrounded by 2–3 shallow skin folds ( Fig. 2a , mp). No genital papillae in the male pore region. Female pore single, mid-ventral on XIV, milk-white, elliptical. Internal characters : Septa 8/9–9/10 absent, 5/6–7/8 thickened and muscular, 10/11–13/14 thicker than those following. Dorsal blood vessel single, continuous onto pharynx; esophageal hearts 4 pairs in X–XIII, the first pair slimmer than the others. Gizzard ball-shaped, in VIII–X; intestinal swelling in XVI; intestinal caeca simple, originating in XXVII and extending forward to XXII, finger-shaped sacs, smooth on dorsal and ventral margins. Male organs: testis sacs in X and XI, oval, two lobes separated ventromedially; seminal vesicles in XI, XII, developed, left and right lobes connected ventromedially with a slim tube; prostate gland developed, in XVI–XX, its duct in XVIII, horizontally situated, stout and uniform ( Fig. 2b ); no visible accessory glands on XVIII. Spermathecae paired in VI–VIII in holotype , about 1.7 mm long; spermathecal ampulla peach-shaped, 0.5 mm long, duct long and straight, 1.2 mm long; diverticulum shorter than the main spermathecal axis by ¼, its stalk straight or twisted into two zigzags, distal ¼ dilated into a heart- or oval-shaped seminal chamber; no nephridia on spermathecal ducts. Variation. The configuration of spermathecae varies between two and three pairs, of which three pairs are the dominant group. DNA barcodes. GenBank accession numbers KF205471 (C-YN201108-02A, holotype ) , MF541643 (CYN201101-18, paratype ) , MF541642 (C-YN201107-11, paratype ) , MF541641 (C-YN201109-10, paratype ) , MF541644 (C-SCCZ2015003-04, paratype ), MH 845520 (C-YN201720-01, paratype ), MH 845485 (C-YN201735- 10, paratype ), MH 845459 (C-YN201756-05, paratype ), MH 845505 (C-YN201730-03, paratype ), MH 845441 (C- YN201752-01, paratype ), MH 837684 (C- YN201606-03, paratype ) . FIGURE 2. Amynthas rusticanus Sun & Qiu sp. nov. , holotype. (a) Ventral view showing spermathecal pores (sp) and male pores (mp); (b) do., prostate gland; (c) do., spermathecae. Scale bar = 1 mm. Remarks. A. rusticanus sp. nov. was collected from 10 sites in two provinces in southeast China : Yunnan Province (Xishuangbanna, Yuanjiang National Nature Reserve, Cangshan National Nature Reserve, Tongbiguan Provincial Nature Reserve, Nangunhe National Nature Reserve, Gaoligongshan National Nature Reserve, Yongde Daxueshan National Nature Reserve, and Jingdong Yi Autonomous County) and Sichuan Province (Mount Zhuji and Jinjiang County). The new species keys to the Amynthas hawayanus -group ( Sims & Easton 1972 ). The hawayanus group is characterized by six thecal spermathecae in 5/6–7/8 and by holandry. To date, the group comprises 64 species ( Nguyen et al. 2020b ). Within the A. hawayanus -group, the morphological character of A. rusticanus sp. nov. can be described as “mediocre” because of its small body size, no representative and stable genital papillae outside, with simple style intestinal caeca and developed prostate glands. However, A. rusticanus sp. nov. has a relatively breadth geographical distribution in Southwest China , which recommend us to look for species with a wide geographical range as well as relatively similar morphology characters for comparison. Amynthas gracilis (Kinberg, 1867) is meeting our requirement. A. gracilis is a cosmopolitan species, and had been reported in 9 provinces in China , including eight spots in Southwest China ( Xu & Xiao 2011 ). A. rusticanus sp. nov. has some similarities to the cosmopolitan species A. gracilis . Considering the polymorphic nature of the new species, several previously described morphs of A. gracilis were selected for comparison: the Sichuan morph described by Chen (1931), the Taiwan morph described by Chang et al. (2009 a), the Burma morph described by Gates (1972) , and the US morph described by Blakemore (2009) and Chang et al. (2016) . The present new species is similar to A. gracilis in the following characters: 1) pigmentation is present dorsally and absent ventrally, regardless of the preserving fluid used; 2) the clitellum may not always occupy the exact boundaries of XIV–XVI, instead failing to reach the boundaries or extending beyond; 3) there are three pairs of spermathecal pores in 5/6–7/8; 4) the intestinal caeca are simple; 5) the spermathecal diverticulum has a small, ovate seminal chamber and slender stalk, except in the case of the US morph specimens from the National Museum of Natural History, Smithsonian Institution, Washington , DC ( Chang et al. 2016 ); 6) the prostate glands are developed. However, there are several differences: 1) the new species is smaller than all morphs of A. gracilis ; 2) the first dorsal pore is found in 12/ 13 in the new species, whereas in A. gracilis it is found in 10/11 or 11/ 12 in the Taiwan morph and the US morph from the Smithsonian Institution, or 10/ 11 in the Sichuan morph, the Burma morph, and the US morph from Brookfield ( Blakemore 2009 ); 3) there are no setae on the clitellum of the new species, but a few setae are always present on the ventral side of segment XVI in the Sichuan and Burma morphs of A. gracilis ; 4) there are no genital markings in the spermathecal pore region of the new species, but they are present in A. gracilis in the Burma morph (segment VI-IX) and the US morph from Brookfield; 5) the male porophores are elevated and surrounded by 2-3 shallow skin folds in the new species, but the male pore is on top of a whorl-like elevation in the Sichuan morph of A. gracilis ; 6) there are no genital papillae in the male pore region of the new species, but one or more medial and postsetal genital papillae are present in the Sichuan, Taiwan , Burma , and US morphs of A. gracilis ; 7) the spermathecal duct is obviously longer than the ampulla in the new species, whereas it is shorter than the ampulla in the Taiwan , Burma , and US morphs of A. gracilis . On the other hand, Blakemore (2009) reported that the US morph of A. gracilis , in rare cases, has only two pairs of spermathecal pores; in this characteristic, it differs from the Sichuan morph, Taiwan morph, and Burma morph, but is similar to the new species. However, Chang et al. (2016) checked the specimens of A. gracilis preserved in the National Museum of Natural History, Smithsonian Institution, Washington , DC, and found that the US morph has three pairs of spermathecal pores. Considering the spermathecal pores (three pairs in 5/6–7/8), simple intestinal caeca and long spermathecal duct, A. rusticanus sp. nov. is also similar to the Chinese endemic species Amynthas limpidus (Chen, 1938) . However, they still have several differences: 1) A. rusticanus (length 40–59 mm by width 1.9–2.0 mm) is smaller than A. limpidus ( 150 mm by 6.0 mm) in body size; 2) the setae are moderate (34–46 on III, 46–60 on V, 50–60 on VIII, 46–48 on XX, 44–50 on XXV) in number in the new species, but extremely numerous (82 on III, 128 on VI, 142 on VII, 96 on XXV) in A. limpidus ; 3) there are no genital papillae in the spermathecal and male pore regions in the new species, but several genital markings are present in A. limpidus ; 4) the prostate gland is developed in the new species, but undeveloped (Chen used “very small” to describe it) on A. limpidus ; 5) the spermathecal diverticulum is shorter than the main spermathecal axis in the new species, but slightly longer than the main pouch in A. limpidus . Amynthas carnosus (Goto & Hatai, 1899) also keys to the A. hawayanus -group and is very common in Sichuan Province ( Xu & Xiao 2011 ), but it is not morphologically similar to the new species in having big body size (vs. small body size as length 40–59 mm by width 1.9–2.0 mm), two pairs of genital papillae on spermathecal pore region (vs. no genital papillae on spermathecal pore region predominantly), three pairs of genital papillae on male pore region (vs. no genital papillae on male pore region), and spermathecal diverticulum straight as well as half as long as the main spermathecal axis (vs. spermathecal diverticulum shorter than the main spermathecal axis by ¼, its stalk straight or twisted into two zigzags, and distal ¼ dilated into a heart- or oval-shaped seminal chamber). Besides, we have given a comparison of genetic similarity ( Table 2 ). The P-distance result (0.18) shows A. rusticanus sp. nov. and A. carnosus are different species.