Research Article |
Corresponding author: JiangPing Qiu ( jpq@sjtu.edu.cn ) Academic editor: Fredric Govedich
© 2019 Yan Dong, Michelle Man Suet Law, JiBao Jiang, JiangPing Qiu.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Dong Y, Law MMS, Jiang J, Qiu J (2019) Three new species and one subspecies of the Amynthas corticis-group from Guangxi Zhuang Autonomous Region, China (Oligochaeta, Megascolecidae). ZooKeys 884: 23-42. https://doi.org/10.3897/zookeys.884.30988
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Three new species and one subspecies of the genus Amynthas are described from Guangxi Zhuang Autonomous Region, China: The new species are: Amynthas maximus Qiu & Dong, sp. nov. and Amynthas tortuosus Qiu & Dong, sp. nov., and Amynthas shengtangmontis Dong & Jiang, sp. nov., the subspecies is Amynthas shengtangmontis minusculus subsp. nov. All have four pairs of spermathecal pores in 5/6–8/9, which indicates that they should belong to the corticis-group. Their morphological characteristics are compared to other similar species in the corticis-group from China and other Asian countries, such as Amynthas pulvinus Sun & Jiang, 2013, Amynthas homosetus (Chen, 1938), Amynthas corticis (Kinberg, 1867), Amynthas dorsualis Sun & Qiu, 2013, and Amynthas carnosus (Goto & Hatai, 1899). In addition, the results presented are confirmed by the pairwise comparison of COI barcode sequences. The pairwise distances between each new species and the other eighteen corticis-group species are greater than 14.7% on average. Furthermore, the pairwise distance between A. shengtangmontis shengtangmontis and A. shengtangmontis minusculus is 10.7–11.4%.
Earthworm, cytochrome c oxidase subunit I gene, new species
The genus Amynthas Sims & Easton, 1972 is the dominant genus of Megascolecidae in China (
Guangxi Zhuang Autonomous Region is located in the southeast edge of Yunnan-Guizhou plateau, and west of Guangzhou-Guangxi hilly land. The landforms in Guangxi include mountains, hills and plains. Guangxi has a subtropical monsoon climate and the Pearl River, the Yangtze River, the Red River, and the coastal water systems flow through it. Guangxi has an average annual temperature between 16.5–23.1 °C, which is suited to the survival and dispersal of earthworms. In order to investigate the diversity of earthworms in China, we conducted a field survey in Guangxi Zhuang Autonomous Region in 2013 and have found both a number of described species and also species that are new to science. The previously described species are Amynthas dissimilis Qiu & Jiang, 2018 (
All of the newly described species and subspecies have four pairs of spermathecal pores in 5/6-8/9; hence, they belong to the Amynthas corticis-group.
The earthworms were collected in 2013, anaesthetized in 10% ethanol solution, and preserved in 99% ethanol solution. DNA was extracted from several specimens of A. maximus, A. tortuosus, A. shengtangmontis shengtangmontis, and A. shengtangmontis minusculus using the EZNA Mollusk DNA Kit (Omega Bio-tek, Norcross, GA, USA). The gene cytochrome oxidase subunit I (COI) was amplified. 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 1 min, with an extension of 10 min at 72 °C. Primers used in the research were: 5'-GGTCAACAAATCATAAAGATATTGG-3'and 5'-TAAACTTCAGGGTGACCAAAAAATCA-3' (
Sequences were aligned with ClustalX (Thompson 1997), and then pairwise distances between these species were calculated using Kimura two-parameter model of DNA evolution with MEGA 5 (
Species with molecular data used in this study. Abbreviations: HT holotype, PT paratype.
Species | Species No. | Locality | Reference | GenBank Acc. No |
---|---|---|---|---|
Amynthas maximus sp. n. (HT) | C-GX201304-01A | China: Guangxi | This paper | MG450707 |
Amynthas tortuosus sp. n. (HT) | C-GX201306-06A | China: Guangxi | This paper | MG450708 |
Amynthas tortuosus sp. n. (PT) | C-GX201301-09 | China: Guangxi | This paper | MK606425 |
Amynthas tortuosus sp. n. (PT) | C-GX201305-07 | China: Guangxi | This paper | MK606426 |
Amynthas shengtangmontis shengtangmontis sp. n. (HT) | C-GX201312-03A | China: Guangxi | This paper | MG450709 |
Amynthas shengtangmontis minusculus subsp. n. (HT) | C-GX201316-02A | China: Guangxi | This paper | MG450710 |
Amynthas shengtangmontis minusculus subsp. n. (PT) | C-GX201316-02B | China: Guangxi | This paper | MK606427 |
Amynthas fuscatus (Goto & Hatai, 1898) | Japan: Tokyo | Minamiya, submitted to GenBank in 2010 | AB542475 | |
Amynthas pulvinus Sun & Jiang, 2013 | C-HN201115-08 | China: Hainan | Sun et al. 2014 | JQ905266 |
Amynthas robustus (Chen, 1936) | C-SC201009-01 | China: Sichuan |
|
KF179573 |
Amynthas corticis (Kingberg, 1867) | C-HN201035-02 | China: Hainan |
|
KF205966 |
Amynthas carnosus (Goto & Hatai, 1899) | C-HN201002-01 | China: Hainan |
|
KF205962 |
Amynthas mirifius Sun & Zhao, 2013 | C-HN201103-02 | China: Hainan |
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JQ905265 |
Amynthas micronarius (Goto & Hatai, 1898) | Japan: Tokyo | Minamiya, submitted to GenBank in 2010 | AB542498 | |
Amynthas alexandri (Beddard, 1900) | Thailand |
|
KU565178 | |
Amynthas andersoni (Michaelsen, 1907) | Thailand |
|
KU565179 | |
Amynthas comptus (Gates, 1932) | Thailand |
|
KU565187 | |
Amynthas exiguus (Gates, 1930) | Thailand |
|
KU565189 | |
Amynthas formosae (Michaelsen, 1922) | India | Farooqui, submitted to GenBank in 2019 | LC458750 | |
Amynthas longicauliculatus (Gates, 1931) | Thailand |
|
KU565195 | |
Amynthas szechuanensis vallatus (Chen, 1946) | C-SC201102-05 | China: Sichuan |
|
KF205477 |
Amynthas mediocus ( |
C-GD201108-02 | China: Guangdong |
|
KF205405 |
Amynthas wulinensis Tsai et al., 2001 | Taiwan island |
|
DQ224182 | |
Amynthas yunlongensis (Chen, 1977) | C-GZ201101-06 | China: Guizhou |
|
KF179581 |
Amynthas stricosus Qiu & Sun, 2012 | C-HN201104-04 | China: Sichuan |
|
JX315345 |
Holotype :1 clitellate (C-GX201304-01A): China, Guangxi Zhuang Autonomous Region, Shiwan Mountain Nature Reserve (21.50299°N, 107.3035°E), 449 m asl, black sandy soil under bryophytes in a subtropical evergreen forest, 12 May 2013, JP Qiu, Y Hong, JB Jiang, LL Zhang, Y Dong legit. Paratypes: 8 clitellates (C-GX201304-01B): same date as for holotype.
Dimensions 145–170 mm by 5.8–6.2 mm at clitellum, clitellum taupe in 2/5 XIV–XVI, 78–101 segments. First dorsal pore in 13/14. Setae numbering 33–38 at III, 32–36 at V, 29–33 at VIII, 18–22 at XX, 50–65 at XXV; 9–13 between male pores; setae between spermathecal pores numbering 14–16 at VI, 10–14 at VII, and 18–22 at VIII. Four pairs of spermathecal pores ventrally in 5/6–8/9. Seven or eight (three specimens have seven papillae, and four specimens have eight papillae) postsetal genital papillae arranged in two rows in VI–IX, 0.33 circumference ventrally apart from each other. One pair of male pores in XVIII, each on the top of a central, round porophore surrounded by three or four circular ridges, with one presetal indented-topped genital papilla medial of each porophore. Ampulla elongate oval, stout duct as long as 3/5 ampulla. Diverticulum slightly shorter than main pouch, a little twist in the middle, terminal 2/5 dilated into a swollen, club-shaped seminal chamber. Prostate glands well developed.
A comparison of characters of A. maximus sp. nov., A. dorsualis, 2013, A. carnosus, A. corticis and A. wulinensis.
Characteristics | A. maximus sp. nov. | A. dorsualis | A. carnosus | A. corticis | A. wulinensis |
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Body length (mm) | 145–170 | 121–? | 110–340 | 45–170 | 128–174 |
Body width (mm) | 5.8–6.2 | 2.7–? | 4.0–9.0 | 3.0–6.0 | 5.6–6.1 |
Pigment | |||||
dorsum | Light purple brown before clitellum, from light purple brown to brown after | Dark grey before clitellum, dark brown after | Dark brown or purple | Greenish brown | Whitish purple |
ventrum | No pigment before clitellum, yellowish after | Light grey before VII, no pigment after | Dark brown or purple | No | Whitish gray |
First dorsal pore | 13/14 | 13/14 | 12/13 | 10/11 or 12/13, usually at 11/12 | 11/12 |
Clitellum locality | 2/5XIV–XVI | 1/10XIV-7/10XVI | XIV–XVI | XIV–XVI, occasionally shorter | XIV–XVI |
Spermathecal pores | 4 pairs, in 5/6–8/9, 0.33C | 4 pairs, in 5/6–8/9, dorsally, 0.6C | 4 pairs in 5/6–8/9 or 3 pairs in 6/7–8/9, 0.33C | 4 pairs, 5/6–8/9, 0.33C | 4 pairs, 5/6–8/9, ventral, 0.29C |
Male pores | Middle, round, surrounded by 3–4 circular ridges, 0.4C | Slightly raised, glandular, surrounded by 5–6 elliptic circular folds, 0.33C | Round or elliptic | Small, circular to transverse elliptical disc, 0.24–0.30C | Round or oval-shaped on setal line with depressed center, 2–3 circular folds, 0.24–0.28C |
Papillae preclitellum | 7–8 post-setal indented-topped genital papillae arranged in two rows in VI–IX | Invisible | 8 papillae just overhead 8 spermathecal pores, 2 pairs preclitellar arranged on VIII and IX | Paired presetal and/or postsetal in some or all, near spermathecal pores | Absent |
Papillae postclitellum | 2 presetal indented-topped genital papillae medial of porophore | Invisible | 2 paired presetal genital on XVIII and IX, 1 pair postsetal genital on XVIII | Present or absent, occasionally one or more near male pore | Oval-shaped, medial to male pore in each of XVII and XIX, occasionally XX |
Prostate glands | XVII–XIX | XVI–XX | Well developed | XVII–XX, rudimentary or absent | XV–XX, racemose, follicular |
Spermathecae | About 1.6 mm long, ampulla long-oval; duct as long as 3/5 ampulla | About 2.2 mm long, ampulla heart-shaped; duct as long as 2/5 ampulla | Ampulla oval or pear-shaped, duct equal to or slightly shorter than ampulla | Ampulla ovoid | Very short and stout stalk |
Diverticulum | Shorter, lightly twist in middle, terminal 2/5, swollen, club-shaped seminal chamber | Shorter, terminal 1/5, ovoid plump seminal chamber | One-third to half of ampulla, slender stalk and a wider seminal chamber | Blunt ovioid, straight stalk | Oval, shining white seminal chamber, a slender and straight stalk |
Accessory glands | 1 or 2 stalked accessory glands observed near ventral median line in VI, VII, VIII, IX | Invisible | – | Stalked, coelomic, bound down to parietes or retained within body wall | Paired in XVII and XIX, sessile, flowery |
External characters
: Light purple-brown pigment on pre-clitellum dorsum, no pigment on ventrum. Pigment from light purple-brown to brown on post-clitellum dorsum, light yellowish on ventrum. Clitellum taupe in 2/5 XIV–XVI. Dimensions 160 mm by 6.0 mm at clitellum, 92 segments. Prostomium ½ epilobous. First dorsal pore in 13/14. Setae numbering 36 at III, 34 at V, 31 at VIII, 20 at XX, 60 at XXV; 12 between male pores; Setae numbering 15 at VI, 12 at VII, 20 at VIII between spermathecal pores. Setae formula: AA = 1.1-1.4AB, ZZ = 1.2-2.0ZY. Clitellum annular, yellowish, in 2/5 XIV–XVI, setae not visible externally. Four pairs of spermathecal pores in 5/6–8/9, ventral, eye-like, 0.4 circumference ventrally apart from each other. Seven or eight (three specimens have seven papillae, and four specimens have eight papillae) postsetal genital papillae arranged in two rows in VI–X, 0.33 circumference ventrally apart from each other. One pair of male pores in XVIII, 0.4 circumference apart ventrally, each on the top of a central, round porophore surrounded by three or four circular ridges, with one presetal indented-topped genital papilla medial of each porophore (Figure
Internal characters. Septa 5/6–7/8, 10/11–13/14 thick and muscular, 8/9–9/10 absent. Gizzard bucket-shaped, in VIII–X. Intestine enlarged distinctly from XV onwards. Intestinal caeca paired in XXVII, extending anteriorly to XXII, transition state, ventral margin smooth, four pointed saccules in dorsal margin. Four pairs of esophageal hearts in X–XIII, developed. Ovaries in XIII. Four pairs of spermathecae in VI–IX, short, approx. 1.6 mm long, ampulla elongate-oval; duct as long as 3/5 ampulla. Diverticulum slightly shorter than main pouch (ampulla and duct), a little twist in middle, terminal 2/5 dilated into a swollen, club-shaped seminal chamber (Figure
The species is named after its large accessory glands observed in the spermathecal area.
Amynthas maximus sp. nov. keys to the corticis-group in
We also compare the new species with Amynthas corticis (Kinberg, 1867) which has been recognized as the typical species in the corticis-group. They share several common characters such as body size, pigment, clitellum extent, setal number, and both have stalked accessory glands. Other than that, the first dorsal pore in the new species is in 13/14, but in 10/11 or 12/13, usually in 11/12 in A. corticis. The diverticulum of A. maximus sp. nov. has a small twist in the middle compared with A. corticis, which has a long stalk. Moreover, A. maximus sp. nov. always exhibits genital markings in the male pore region, whereas in A. corticis, these markings are occasionally absent.
We further compare the new species with another species Amynthas dorsualis Sun & Qiu, 2013 described from Hainan, China, and its clitellum also occupies fewer than three segments. In our results, we find the two species share several common characters, including the first dorsal pore location, setal formula, male pore characters, and in the diverticulum being shorter than the main pouch. However, the morphological dissimilarity of the two species is substantial. For instance, the locations of spermathecal pores are different between A. dorsualis and A. maximus sp. nov. In A. dorsualis, the spermathecal pores are located on the dorsum, while the pores are located on the ventrum in A. maximus sp. nov. The distance between male pore is shorter on the ventral side in A. dorsualis than Amynthas maximus sp. nov. In addition, A. dorsualis has no genital markings and no accessory glands, whereas A. maximus sp. nov. exhibits genital markings near the spermathecal pores and the male pores region, and stalked accessory glands are present in spermathecal pores region.
The body size of A. maximus sp. nov. is similar to A. wulinensis described from Taiwan Island. But the other characters of A. maximus sp. nov. differ from A. wulinensis. Amynthas maximus sp. nov. has no pigment before clitellum, yellowish after clitellum on dorsum, first dorsal pore in 13/14, 0.33C between spermathecal pores, seven or eight indented-topped genital papillae in VI–IX and accessory glands in spermathecal pores region. In contrast, A. wulinensis has whitish gray on dorsum, first dorsal pore in 11/12, 0.29C between spermathecal pores, no genital papillae observed in spermathecal pores region, and accessory glands observed in male pores region. Table
Holotype: 1 clitellate (C-GX201306-06A): China, Guangxi Zhuang Autonomous Region, Shiwan Mountain Nature Reserve (21.84739°N, 107.88989°E), 553 m asl, black soil besides road, 13 May 2013, JP Qiu, Y Hong, JB Jiang, LL Zhang, Y Dong legit. Paratypes: 7 clitellates: 2 clitellates (C-GX201306-06B): same data as for holotype. 1 clitellate (C-GX201301-09): China, Guangxi Zhuang Autonomous Region, Shiwan Mountain Nature Reserve (21.48588°N, 107.57018°E), 130 m asl, black sandy soil at riverside, 11 May 2013, JP Qiu, Y Hong, JB Jiang, LL Zhang, Y Dong legit. 4 clitellates (C-GX201305-07): China, Guangxi Zhuang Autonomous Region, Shiwan Mountain Nature Reserve (21.50396°N, 107.53350°E), 494 m asl, black sandy soil besides road, 13 May 2013, JP Qiu, Y Hong, JB Jiang, LL Zhang, Y Dong legit.
A comparison of characters of A. tortuosus sp. nov., A. carnosus, A. corticis, A. homosetus, A. exiguus aquilonius, and A. stricosus.
Characteristics | A. tortuosus sp. nov. | A. carnosus | A. corticis | A. homosetus | A. exiguus aquilonius | A. stricosus |
---|---|---|---|---|---|---|
Body length (mm) | 55–86 | 110–340 | 45–170 | 116 | 39–63 | 72–97 |
Body width (mm) | 2.5–2.8 | 4.0–9.0 | 3.0–6.0 | 5.2 | 1.9–2.6 | 2–2.8 |
Pigment | ||||||
dorsum | Purple brown before clitellum, light purple brown after | Dark brown or purple | Greenish brown | Dark chocolate on anterior, grey on other parts | Dark reddish brown | No pigment |
ventrum | Light purple brown before clitellum, no after | Dark brown or purple | No | Grey | Light gray on ventrum | No pogment |
First dorsal pore | 13/14 | 12/13 | 10/11 or 12/13, usually at 11/12 | 12/13 | 6/7 | 11/12 or 12/13 |
Clitellum locality | XIV–XVI | XIV–XVI | XIV–XVI, occasionally shorter | XIV–XVI | XIV–XVI | XIV–XVI |
Setal formula | AA=1.2-2.0AB, ZZ=1.4-2.0ZY | – | ||||
Spermathecal pores | 4 pairs, in 5/6–8/9, 0.25C | 4 pairs in 5/6–8/9 or 3 pairs in 6/7–8/9, 0.33C | 4 pairs, 5/6–8/9, 0.33C | 4 pairs, in 5/6–8/9, 0.25C | 4 pairs, 5/6–8/9, ventral, 0.45C | 4 pairs, 5/6–8/9, 0.40C |
Male pores | Middle, round, surrounded by 3–4 rhombic ridges, 0.25C | Round or elliptic | Small, circular to transverse elliptical di sc,0.24–0.30C | Roundish glandular area, about 1.5mm in diameter, 0.25C | Round, smooth, slightly elevated with a male aperture inconspicuous on lateral concave area, 0.23–0.30C | on a coniform glandular disc surrounded by a round pad, 0.33C |
Papillae preclitellum | Four pairs of postsetal genital papillae in VI–IX | 8 papillae just overhead 8 spermathecal pores, 2 pairs preclitellar arranged on VIII and IX | Paired presetal and/or postsetal in some or all, near spermathecal pores | Invisible | presetal and postsetal, widely paired in 7, 8 and 9, number highly variable | Invisible |
Papillae postclitellum | 2 presetal crescent indented-topped genital papilla medial of male pores | 2 paired presetal genital on XVIII and IX, 1 pair postsetal genital on XVIII | Present or absent, occasionally one or more near male pore | Invisible | presetal and postsetal, widely paired in XVII, XVIII and XIX, number highly variable | postsetal, single or paired in XVII, XIX and XX |
Prostate glands | XVII–XXII | Well developed | XVII–XX, rudimentary or absent | XVI–XXI | XVI–XX, wrinkled | XVI–XX, coarsely lobate |
Spermathecae | About 2.4mm long, ampulla slender heart-shaped; duct short | Ampulla oval or pear-shaped, duct equal to or slightly shorter than ampulla | Ampulla ovoid | – | Ampulla peach-shaped, stalk straight, much shorter than ampulla | About 1.6mm long; ampulla heart-shaped, gradually slender duct as long as ampulla |
Diverticulum | About 2.0mm long, terminal 4/5, swollen, S-shaped twisted seminal chamber | One-third to half of ampulla, slender stalk and a wider seminal chamber | Blunt ovioid, straight stalk | Shorter, seminal chamber ovoid and whitish | Shorter, seminal chamber rudimentary or absent, straight or slightly bent | As long as main spermathecal axis, slender, terminal 0.4 dilated into a band shaped chamber |
Accessory glands | 1 stalked accessory gland observed near the ental part of each spermatheca | – | Stalked, coelomic, bound down to parietes or retained within body wall | Invisible | round, stalked observed in spermathecal pores and male pores region | Invisible |
Dimensions 55–86 mm by 2.5–2.8 mm at clitellum, 55–83 segments. First dorsal pore in 13/14. Setae numbering 24–26 at III, 34–36 at V, 34–36 at VIII, 32–36 at XX, 36–40 at XXV; 8–9 between male pores; setae between spermathecal pores numbering 9–12 at VI, 10–12 at VII, 12–13 at VIII. Four pairs of spermathecal pores in 5/6–8/9, eye-like. Four pairs of postsetal genital papillae in VI–IX, 0.20 circumference ventrally apart from each other. One pair of male pores in XVIII, each on the top of a central, round porophore surrounded by three or four rhombic ridges, with one presetal crescent indented-topped genital papilla medial of each male pore. Ampulla slender, heart-shaped; duct short. Diverticulum shorter than main pouch, terminal 4/5 slightly dilated into a swollen, S-shaped twisted seminal chamber. Prostate glands well developed.
External characters: Pre-clitellum, purple-brown pigment on dorsum, light purple-brown on ventrum. Post-clitellum, light purple-brown on dorsum, no pigment on ventrum. Clitellum taupe. Dimensions 76 mm by 27 mm at clitellum. 75 segments. Prostomium ½ epilobous. First dorsal pore in 13/14. Setae numbering 24 at III, 34 at V, 36 at VIII, 36 at XX, 40 at XXV; 8 between male pores; Setae between spermathecal pores numbering 11 at VI, 10 at VII, 13 at VIII. Setae formula AA = 1.2-2.0AB, ZZ = 1.4-2.0ZY. Clitellum annular, pale taupe, in XIV–XVI, setae not visible externally. Four pairs of spermathecal pores in 5/6–8/9, eye-like, 0.25 circumference ventrally apart from each other. Four pairs of postsetal genital papillae in VI–IX, 0.20 circumference ventrally apart from each other. One pair of male pores in XVIII, 0.25 circumference apart ventrally, each on the top of a central, round porophore surrounded by three or four rhombic ridges, with one presetal crescent indented-topped genital papilla in the center of each male pore region (Figure
Internal characters. Septa 5/6–7/8 thick and muscular, 10/11–12/13 slightly thickened, 8/9–9/10 absent. Gizzard ball-shaped, in VIII–X. Intestine enlarged distinctly from XVI onwards. Intestinal caeca paired in XXVII, simple, smooth, extending anteriorly to XXIV. Four pairs of esophageal hearts in X–XIII, the first pair very thin, the last three pairs developed. Ovaries in XIII. Four pairs of spermathecae in VI–IX, small, 2.4 mm long. Ampulla slender, heart-shaped; duct short. Diverticulum 2.0 mm long, slightly shorter than main pouch, terminal 4/5 slightly dilated into a swollen, S-shaped twisted seminal chamber (Figure
The species is named after the crooked shape of its diverticulum.
Amynthas tortuosus sp. nov. is a comparatively small earthworm and shares some similarities to A. carnosus and A. corticis. All of them have genital markings both on the spermathecal and the male pore regions and share similar setal numbers. However, the body size of A. tortuosus sp. nov. is very distinct from others. The new species has roughly half the body size of A. carnosus and A. corticis, both in length and width. The first dorsal pore is in 13/14 and the distance between the spermathecal pores and male pores is less than those of A. carnosus and A. corticis. Moreover, A. tortuosus sp. nov. has long diverticulum with an S-shaped twisted seminal chamber and eight stalked accessory glands observed near the spermathecal duct, rather than a straight or slender stalk and an absence of accessory glands in A. carnosus.
We also compare the new species with Amynthas homosetus (
The body size of A. tortuosus sp. nov. is similar to Amynthas exiguus aquilonius Tsai et al., 2001 described from Taiwan Island and Amynthas stricosus Qiu & Sun, 2012 described from Hainan Island. The first dorsal pore of the new species is in 13/14, but in 6/7 in A. exiguus aquilonius, and in 11/12 or 12/13 in A. stricosus. The new species has more closely spaced spermathecal pores than A. exiguus aquilonius and A. stricosus. Additionally, A. exiguus aquilonius has more genital papillae observed in spermathecal pores and male pores region than the new species. Accessory glands are observed in spermathecal pores and male pores region in A. exiguus aquilonius, but in the new species, accessory glands are only observed in spermathecal pores region. Furthermore, A. stricosus has a band shaped chamber, no genital papillae near spermathecal pores region, no accessory glands, and papillae observed in XVII, XIX and XX, but the new species has a S-shaped twisted seminal chamber, four pairs of genital papillae in spermathecal pores region, accessory glands near spermathecal duct, and no genital papillae observed in XVII, XIX and XX. Details of the comparison are showed in table 3.
Holotype: 1 clitellate (C-GX201312-03A): China, Guangxi Zhuang Autonomous Region, Dayao Mountain National Nature Reserve (23.97299°N, 110.11106°E), 1210 m asl, black sandy soil in bamboo forest, 15 May 2013, JP Qiu, Y Hong, JB Jiang, LL Zhang, Y Dong legit. Paratypes: 4 clitellates (C-GX201312-03A): same data as for holotype.
Dimension 100–134 mm by 4.2–5.1 mm at clitellum, 116–138 segments. First dorsal pore in 12/13. Setae numbering 26–28 at III, 24–32 at V, 26–32 at VIII, 33–40 at XX, 35–42 at XXV; 8–11 between male pores. Setae between spermathecal pores numbering 11–15 at VI, 10–13 at VII, 12–16 at VIII. Four pairs of spermathecal pores in 5/6–8/9. Four pairs of postsetal genital papillae arranged in VI–IX, 0.25 circumference ventrally apart from each other. One pair of male pores in XVIII, each on the top of a large raised, round porophore, surrounded by two circular ridges, with one presetal indented-topped genital papilla medial of each porophore. First ampulla of the three pairs is heart-shaped, duct stalked, diverticulum as long as main chain, U-shaped twist in the middle, terminal 4/5 dilated into a club-shaped seminal chamber. Ampulla of the fourth pair elongate-oval, duct as long as 1/6 ampulla, diverticulum as long as main chain, U-shaped twist in the middle, terminal 1/3 dilated into a chili-shaped seminal chamber. One round, semitransparent accessory gland presents near the medial area of each spermatheca. The prostate glands are developed.
A comparison of characters of A. shengtangmontis shengtangmontis, A. shengtangmontis minusculus, A. carnosus, A. corticis. and A. pulvinus.
Characteristics | A. shengtangmontis shengtangmontis | A. shengtangmontis minusculus | A. carnosus (Goto & Hatai, 1899) | A. corticis (Kinberg, 1867) | A. pulvinus Sun & Qiu, 2013 |
Body length (mm) | 100–134 | 75–83 | 110–340 | 45–170 | 93.5 |
Body width (mm) | 4.2–5.1 | 3.0–3.2 | 4.0–9.0 | 3.0–6.0 | 3.4 |
Pigment | |||||
dorsum | From brown to no | Purple brown | Dark brown or purple | Greenish brown | Buff |
ventrum | From brown to no | No | Dark brown or purple | No | No |
First dorsal pore | 12/13 | 11/12 | 12/13 | 10/11 or 12/13, usually at 11/12 | 12/13 |
Clitellum locality | XIV–XVI | XIV–XVI | XIV–XVI | XIV–XVI, occasionally shorter | XIV–XVI |
Spermathecal pores | 4 pairs, in 5/6–8/9, 0.40C | 4 pairs, in 5/6–8/9, 0.40C | 4 pairs in 5/6–8/9 or 3 pairs in 6/7–8/9, 0.33C | 4 pairs, 5/6–8/9, 0.33C | 4 pairs, in 5/6–8/9, 0.33C |
Male pores | Large raised, round, surrounded by 2 circular ridges, 0.44C | Raised, elliptic, surrounded by 6 circular ridges, 0.40C | Round or elliptic | Small, circular to transverse elliptical disc, 0.24–0.30C | Slightly elevated round, 0.33C |
Papillae preclitellum | Four pairs of postsetal genital papillae arranged in VI–IX | Three pairs of postsetal genital papillae arranged in VI–VIII | 8 papillae just overhead 8 spermathecal pores, 2 pairs preclitellar arranged on VIII and IX | Paired presetal and/or postsetal in some or all, near spermathecal pores | Invisible |
Papillae postclitellum | 2 presetal indented-topped genital medial of porophore | 2 small indented-topped genital papillae medial of male pore | 2 paired presetal genital on XVIII and IX, 1 pair postsetal genital on XVIII | Present or absent, occasionally one or more near male pore | Rectangle-shaped, on 17/18–18/19 |
Prostate glands | XV–XXII | XVI–XXI | Well developed | XVII–XX, rudimentary or absent | XVII–XX |
Spermathecae | About 2.2–3.0mm long, ampulla of the first three pairs heart-shaped, duct stalk. Ampulla of the forth pair long-oval, duct as long as 1/6 ampulla | About 2.2–2.7mm long, duct as long as 1/2 ampulla | Ampulla oval or pear-shaped, duct equal to or slightly shorter than ampulla | Ampulla ovoid | About 2.4mm long, ampulla slender heart-shaped; duct short |
Diverticulum | As long as main chain, U-shaped twisted in middle, terminal 4/5, club-shaped seminal chamber of the first three pairs and terminal 1/3 dilated into a chilli-shaped seminal chamber of the forth pair | As long as main chain, terminal 1/2, long club-shaped seminal chamber | One-third to half of ampulla, slender stalk and a wider seminal chamber | Blunt ovioid, straight stalk | Shorter, terminal 1/5, small ovoid plump seminal chamber |
Accessory glands | 1 round semitransparent accessory gland present near the medial area of each spermatheca, 2 stalk accessory glands observed near the medial of the distal part of the prostatic duct | 6 semitransparent elliptic accessory glands observed near the distal part of the last three pairs spermathecae | – | Stalked, coelomic, bound down to parietes or retained within body wall | A pair, cling to body wall, irregular in shape, and extended from XVII–XIX |
External characters: Pigment from brown to no pigment on dorsum, from light brown to no pigment on ventrum. Dimensions 102 mm by 4.5 mm at clitellum, 117 segments. Prostomium ½ epilobous. First dorsal pore in segments 12/13. Setae numbering 26 at III, 26 at V, 29 at VIII, 36 at XX, 37 at XXV; 9 between male pores. Setae between spermathecal pores numbering 13 at VI, 12 at VII, 14 at VIII. Setal formula: AA = 1.0-1.4AB, ZZ = 2.0-2.2ZY. Clitellum annular, in XIV–XVI, setae not visible externally. Four pairs of spermathecal pores in 5/6–8/9, 0.40 circumference apart ventrally. Four pairs of genital papillae on VI–IX, 0.25 circumference ventrally apart from each other. One pair of male pores in XVIII, 0.40 circumference ventrally apart from each other, each on the top of a larger raised, round porophore, surrounded by two circular ridges, with one presetal indented-topped genital papilla medial of each porophore (Figure
Internal characters. Septa 5/6–7/8 thick and muscular, 10/11–11/12 slightly thickened, 8/9–9/10 absent. Gizzard bucket-shaped, wider below than above, in VIII–X. Intestine enlarged distinctly from XV. Intestinal caeca paired in XXVII, simple, smooth, extending anteriorly to XXI. Esophageal hearts in X–XIII. Ovaries in XIII, four pairs of spermathecae in VI–IX, 2.2–3.0 mm long. Spermathecae of two shapes: ampulla of the first three pairs heart-shaped, duct stalked, diverticulum as long as main chain, U-shaped twist in the middle, terminal 4/5 dilated into a club-shaped seminal chamber. The ampulla of the fourth pair elongate-oval, duct as long as 1/6 ampulla (Figure
The species is named after the name of the collection site Shengtang Mountain, a famous peak of the Dayao Mountain National Nature Reserve.
Amynthas shengtangmontis sp. nov. keys to the corticis-group in
In contrast, distance between the spermathecal pores and the male pores of the new species is 0.40C body circumference compared with 0.33C in A. carnosus, A. corticis, and A. pulvinus. In addition, there are eight postsetal genital markings on VI–IX in the new species, but the markings are present on V–VIII in A. carnosus, and there are more than two pairs of markings on VIII and IX in A. carnosus. The porophore of the new species is large, raised, round, and surrounded by two circular ridges, whereas the porophore is small in A. corticis and no genital markings apparent on the spermathecal pore region in A. pulvinus. Moreover, the new species has two different shapes of spermathecae, heart-shaped ampulla and diverticulum with club-shaped seminal chamber; and long-oval ampulla and diverticulum with chili-shaped seminal chamber, which are very different from those in the other species (Table
Holotype: 1 clitellate (C-GX201316-02A): China, Guangxi Zhuang Autonomous Region, Dayao Mountain Nature Reserve (24.16658°N, 110.24313°E), 1285 m asl, black sandy soil under bryophytes beside road, 16 May 2013, JP Qiu, Y Hong, JB Jiang, LL Zhang, and Y Dong legit. Paratypes: 6 clitellate (C-GX201316-02B): same date as for holotype.
Dimensions 75–83 mm by 3.0–3.2 mm at clitellum, 75–87 segments. First dorsal pore in 11/12. Setae numbering 21–26 at III, 18–21 at V, 27–32 at VIII, 29–34 at XX, 36–40 at XXV; 5–7 between male pores; Setae between spermathecal pores numbering 9–11 at VI, 10–12 at VII, 10–12 at VIII. Four pairs of spermathecal pores in 5/6–8/9. Three pairs of postsetal genital papillae arranged in VI–VIII, 0.13 circumference apart ventrally. One pair of male pores in XVIII, each on the top of a raised, elliptic porophore surrounded by six circular ridges, with one small indented-topped genital papilla medial of each male pore. Ampulla heart-shaped; stout duct as long as 1/2 ampulla. Diverticulum as long as main pouch, terminal 1/2 dilated into a long club-shaped seminal chamber. Prostate glands developed.
External characters: Purple brown pigment on dorsum, no pigment on ventrum. Dimensions 83 mm by 3.2 mm at clitellum, 87 segments. Prostomium ½ epilobous. First dorsal pore in 11/12. Setae numbering 26 at III, 21 at V, 32 at VIII, 34 at XX, 38 at XXV; 6 between male pores; setae between spermathecal pores numbering 10 at VI, 11 at VII, 12 at VIII. Setal formula AA = 1.2-1.4AB, ZZ = 1.4-1.8ZY. Clitellum annular in XIV–XVI, setae not visible externally. Four pairs of spermathecal pores in 5/6–8/9, 0.40 circumference ventrally apart from each other. Three pairs of postsetal genital papillae arranged in VI–VIII. One pair of male pores in XVIII, 0.40 circumference apart ventrally, each on the top of a raised, elliptic porophore surrounded by six circular ridges, with one small indented-topped genital papilla in the center of each male pore (Figure
Internal characters. Septa 5/6–7/8, thick and muscular, 10/11–11/12 slightly thickened, 8/9–9/10 absent. Gizzard bucket-shaped, wider below than above, in IX–X. Intestine enlarged distinctly from XV. Intestinal caeca paired in XXVII, simple, smooth, extending anteriorly to 1/2 XXIV. Esophageal hearts in X–XIII. Ovaries in XIII, four pairs of spermathecae in VI–IX, heart-shaped, 2.2–2.7mm long, duct as long as 1/2 ampulla. Diverticulum as long as main pouch, terminal 1/2 dilated into a long club-shaped seminal chamber. Six semitransparent elliptical accessory glands observed near the ental part of the last three pairs spermathecae (Figure
We compare A. shengtangmontis minusculus subsp. nov. with A. shengtangmontis shengtangmontis and find that they share similar characters of spermathecal pores, male pores, intestinal caeca, and prostate glands. However, there is a level of difference between them. For instance, A. shengtangmontis minusculus subsp. nov. has a smaller body size, fewer and more closely spaced genital papillae, longer spermathecal duct, accessory glands only observed in the spermathecal area. On the other hand, the first dorsal pore of A. shengtangmontis minusculus subsp. nov. is located in 11/12 compared to in 12/13 in A. shengtangmontis shengtangmontis. The pairwise distance of COI between A. shengtangmontis shengtangmontis and A. shengtangmontis minusculus subsp. nov. is 10.7%-11.4%, which is acceptable to differentiate subspecies.
The subspecies is named after its small body size, compared to the nominate species.
In addition to the morphological comparison between the species, we also compared the COI gene sequences of the three proposed new species and one subspecies and the results of the pairwise distances of COI, ranging from 10.7%-25.2%% are shown in Table
Percentage of pairwise distances obtained for the sequences of COI genes in Amynthas species.
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | 22 | 23 | 24 | |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
S1 HT | ||||||||||||||||||||||||
S2 HT | 19.2% | |||||||||||||||||||||||
S2 PT | 19.2% | 0.0% | ||||||||||||||||||||||
S2 PT | 19.2% | 0.0% | 0.0% | |||||||||||||||||||||
S3 HT | 16.4% | 15.7% | 15.7% | 15.7% | ||||||||||||||||||||
S4 HT | 16.7% | 14.7% | 14.7% | 14.7% | 10.7% | |||||||||||||||||||
S4 PT | 16.9% | 15.4% | 15.4% | 15.4% | 11.4% | 0.2% | ||||||||||||||||||
A. fuscatus | 15.9% | 17.4% | 17.4% | 17.4% | 20.4% | 17.4% | 17.0% | |||||||||||||||||
A. pulvinus | 20.0% | 18.2% | 18.2% | 18.2% | 19.7% | 18.4% | 18.8% | 18.6% | ||||||||||||||||
A. robustus | 18.9% | 16.9% | 16.9% | 16.9% | 17.8% | 18.0% | 18.0% | 17.1% | 22.7% | |||||||||||||||
A. corticis | 15.6% | 18.0% | 18.0% | 18.0% | 18.2% | 18.7% | 18.8% | 18.0% | 16.3% | 18.0% | ||||||||||||||
A. carnosus | 19.8% | 17.4% | 17.4% | 17.4% | 20.2% | 20.0% | 19.9% | 18.3% | 18.7% | 16.7% | 16.7% | |||||||||||||
A. micronarius | 20.7% | 19.8% | 19.8% | 19.8% | 19.9% | 19.5% | 20.1% | 21.0% | 19.1% | 20.9% | 17.5% | 21.2% | ||||||||||||
A. alexandri | 24.3% | 21.3% | 21.3% | 21.3% | 23.7% | 22.4% | 22.0% | 21.3% | 21.5% | 20.7% | 21.5% | 20.8% | 22.6% | |||||||||||
A. andersoni | 23.2% | 20.1% | 20.1% | 20.1% | 23.0% | 21.0% | 20.9% | 18.9% | 21.2% | 19.6% | 17.3% | 18.4% | 20.9% | 23.6% | ||||||||||
A. comptus | 21.9% | 19.2% | 19.2% | 19.2% | 20.7% | 19.4% | 20.2% | 19.1% | 21.2% | 19.6% | 18.5% | 20.0% | 20.5% | 23.5% | 19.2% | |||||||||
A. exiguus | 18.2% | 18.0% | 18.0% | 18.0% | 18.1% | 18.5% | 18.7% | 18.2% | 18.6% | 18.7% | 18.5% | 19.1% | 18.3% | 24.2% | 21.3% | 22.1% | ||||||||
A. formosae | 21.4% | 21.0% | 21.0% | 21.0% | 19.3% | 21.9% | 22.0% | 23.2% | 19.8% | 22.7% | 20.3% | 22.7% | 20.8% | 20.5% | 24.4% | 24.1% | 23.5% | |||||||
A. longicauliculatus | 20.2% | 19.5% | 19.5% | 19.5% | 21.6% | 21.8% | 22.1% | 19.6% | 21.5% | 21.3% | 19.8% | 22.6% | 21.7% | 22.9% | 18.3% | 18.1% | 20.2% | 23.9% | ||||||
A. mirifius | 17.6% | 16.6% | 16.6% | 16.6% | 18.7% | 17.1% | 17.1% | 16.1% | 16.7% | 17.9% | 15.3% | 17.4% | 20.5% | 19.2% | 20.7% | 19.8% | 18.9% | 21.3% | 19.7% | |||||
A. szechuanensis | 22.5% | 20.4% | 20.4% | 20.4% | 22.6% | 20.4% | 20.5% | 19.3% | 22.4% | 21.8% | 18.2% | 19.8% | 21.5% | 24.7% | 19.3% | 18.9% | 20.0% | 24.0% | 18.3% | 21.4% | ||||
A. mediocus | 19.4% | 19.4% | 19.4% | 19.4% | 19.4% | 23.8% | 23.6% | 18.0% | 19.8% | 19.1% | 18.5% | 20.5% | 21.4% | 25.2% | 22.5% | 18.7% | 18.2% | 24.2% | 20.9% | 18.8% | 22.5% | |||
A. wulinensis | 20.2% | 18.3% | 18.3% | 18.3% | 21.8% | 18.4% | 18.4% | 19.4% | 20.9% | 20.2% | 17.8% | 19.7% | 18.2% | 25.1% | 20.7% | 21.5% | 20.4% | 23.0% | 23.2% | 19.9% | 19.2% | 19.7% | ||
A. yunlongensis | 19.7% | 18.6% | 18.6% | 18.6% | 18.4% | 16.8% | 17.3% | 19.2% | 18.0% | 17.4% | 17.8% | 17.9% | 20.3% | 21.3% | 22.2% | 19.8% | 17.1% | 21.0% | 20.0% | 17.0% | 16.2% | 20.3% | 20.5% | |
A. stricosus | 16.3% | 15.0% | 15.0% | 15.0% | 18.2% | 16.6% | 16.4% | 13.1% | 18.4% | 17.1% | 16.1% | 17.2% | 20.8% | 18.4% | 17.3% | 17.6% | 21.7% | 18.8% | 18.4% | 19.5% | 20.8% | 18.1% | 16.5% | 21.1% |
This study was supported by the National Natural Science Foundation of China grant No.41471204, No.41701272, and No.41771279. We are grateful to Professor Hong Yong from the College of Agriculture, Chonbuk National University, Associate Professor Qi Zhao and Dr. Zhang Liangliang from Shanghai Jiao Tong University for their assistance with the field work.