Three new species and one subspecies of the Amynthas corticis-group from Guangxi Zhuang Autonomous Region, China (Oligochaeta, Megascolecidae)

Abstract 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 minusculussubsp. 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%.

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 , Amynthas anteporus , Amynthas marsupiformis , Amynthas crassitubus Qiu & , and Amynthas stabilis . In this paper, we describe three new species and a subspecies of Amynthas which were collected from the Shiwan Mountain National Nature Reserve (22.6750°~22.07167°N, 107.49972°~108.21972°E) and the Dayao Mountain National Nature Reserve (25.11667°~25. 23334°N, 113.18333°~113.26667°E) in Guangxi Zhuang Autonomous Region, China. Distributions of known species in Guangxi Zhuang Autonomous Region and sampling points for this paper are shown in Figure 1.
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.

Materials and methods
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'-GGT-CAACAAATCATAAAGATATTGG-3'and 5'-TAAACTTCAGGGTGACCAAAAAAT-CA-3' (Folmer et al. 1994), or 5'-GGTCAACAAATCATAAAGATATTGG-3' and 5'-TATACTTCTGGGTGTCCGAAGAATCA-3' (Bely and Wray 2004). Sequencing was performed in the Beijing Genomics Institute (Shanghai, China). Sequencing was submitted to NCBI GenBank and accession numbers were shown in Table 1. All holotypes and paratypes are deposited in the Shanghai Natural History Museum.
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 (Tamura et al. 2011). Images were produced using the Affinity Photo and SketchBook software.  (Chen, 1946) C-SC201102-05 China: Sichuan Sun 2013, in Chinese KF205477
Description. External characters: Light purple-brown pigment on pre-clitellum dorsum, no pigment on ventrum. Pigment from light purple-brown to brown on postclitellum 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 1A). Single female pore in XIV, ovoid. 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 1B). One or two stalked accessory glands observed near ventral median line in VI-IX. Holandric: two pairs of testis sacs in X and XI, separated from each other, well developed. Two pairs of seminal vesicles in XI and XII, developed. Prostate glands undeveloped, inserting in XVIII and extending from XVII-XIX, coarsely lobate, prostatic duct Ishaped, of uniform thickness. No accessory glands observed in male pore region.
Etymology. The species is named after its large accessory glands observed in the spermathecal area.
Remarks. Amynthas maximus sp. nov. keys to the corticis-group in Sims and Easton (1972) with four pairs of spermathecal pores intersegmentally in 5/6-8/9. Amynthas maximus sp. nov. is similar to Amynthas carnosus (Goto & Hatai, 1899) as re-described by Chang et al. (2016) with respect to body size, the distance between spermathecal pores and male pores, shorter diverticulum than main spermathecal axis, and no accessary glands near prostates. In contrast, the pigment on its ventrum is lighter than A. carnosus and other differences include the first dorsal pore, clitellum location, spermathecal pores, and male pores characters. The first dorsal pore in A. maximus sp. nov. is located in 13/14, versus 12/13 in A. carnosus; the clitellum occupies less than three segments; four pairs of spermathecal pores while sometimes three pairs in A. carnosus; the porophore is surrounded by three or four circular ridges, but no ridges are present in A. carnosus; several accessory glands observed in the spermathecal region in the new species but none in A. carnosus.
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 ven- 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 2 Table 3 Material Diagnosis. 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.
Description. 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, 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 2B). One stalked accessory gland observed medial to each spermathecal duct. Holandric: two pairs of testis sacs in X-XI, separated from each other, developed. Two pairs of seminal vesicles in XI-XII, well developed. Prostate Etymology. The species is named after the crooked shape of its diverticulum. Remarks. 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 (Chen 1938) described from Hainan Island, which has a very similar distance between the male pores and spermathecal pores, and similar setal numbers. The differences between the two species are as follows: the body size of A. tortuosus is much smaller than A. homosetus; the size of prostate glands is larger in A. tortuosus; the shape of the seminal chamber is S-shaped twisted in A. tortuosus, while it is ovoid in A. homosetus; and several genital markings are present in the spermathecal pore region and male pore region in A. tortuosus sp. nov., while these markings are absent in A. homosetus.
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 3C), and diverticulum as long as main chain, U-shaped twisted in the middle, terminal 1/3 dilated into a chili-shaped seminal chamber ( Figure 3B). One round semitransparent accessory gland presents near the medial area of each spermatheca. Holandric: two pairs of testis sacs in X-XI, separated from each other, well developed. Two pairs of seminal vesicles, in XI-XII, well developed. Prostate glands developed, thick, inserting in XVIII and extending from XV to XXII, coarsely lobate, prostatic duct U-curved, slightly thicker at the ental part. Two stalked accessory glands near the medial area of the distal part of the prostatic duct.
Etymology. The species is named after the name of the collection site Shengtang Mountain, a famous peak of the Dayao Mountain National Nature Reserve.
Remarks. Amynthas shengtangmontis sp. nov. keys to the corticis-group in Sims and Easton (1972). In terms of morphology, it is closely related to A. carnosus, A. corticis, and Amynthas pulvinus Sun & Jiang, 2013 (described from Hainan Island). Body size, body pigment, setal numbers, the first dorsal pore location, and simple intestinal caeca are similar among the four species.
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 4).  Diagnosis. 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 indentedtopped 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.
Remarks. 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.
Etymology. The subspecies is named after its small body size, compared to the nominate species.

Molecular results
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 5. Studies show that pairwise distances of COI of interspecies in the same genus are 17-23% (Sun 2013), 16-23% (Huang et al. 2007), 15-16% (Admassu et al. 2006, 16-22% (Novo et al. 2009), and 15-28% (Chang et al. 2008). In general, pairwise distances between three new species and the other eighteen corticis-group species are greater than 14.7%. Together with the different morpho-   logical characters of each, we could conclude that A. maximus, A. tortuosus, A. shengtangmontis shengtangmontis, and A. shengtangmontis minusculus are different from the previously described species and each other. Since the pairwise distance between A. shengtangmontis shengtangmontis and A. shengtangmontis minusculus is 10.7%-11.4%, which is more than 1% and less than 15%, by definition we conclude that both subspecies are valid.