Four new earthworm species of the genus Amynthas Kinberg (Oligochaeta: Megascolecidae) from the island of Hainan and Guangdong Province, China

This paper describes four new species of morrisi-group earthworms from Hainan and Guangdong Provinces, China: Amynthas instabilis sp. nov, Amynthas dilatatus sp. nov, Amynthas infuscuatus sp. nov. and Amynthas qiongzhongensis sp. nov. All four species have two pairs of spermathecal pores in 5/6–6/7; male pores in XVIII, 0.33 circumference ventrally apart, each on the top of a slightly raised porophore, surrounded by several tiny genital papillae, in a pulvinate pad with three to six circular folds. Characters of the spermathecae, prostate glands and other anatomical features easily distinguish the new species from earthworms previously reported from the morrisi-group. http://zoobank.org/urn:lsid:zoobank.org:pub:545E9152-C826-44A2-949E-402434D8493D.


Introduction
The morrisi-group is defined as Amynthas with intersegmental spermathecal pores, first spermathecal pores at 5/6, two thecal segments and holandry. Sims and Easton (1972) included 30 species in this group and, since 1972, nine more species from the morrisigroup have been reported from China: Amynthas nanulus (Chen et Yang 1975) (Chen 1938), Amynthas monoserialis (Chen 1938) and Amynthas sinuosus (Chen 1938). Since then, Sun et al. (2009) have reported four new species while Zhao et al. (2013) reported three new species. In total, 17 species of the morrisi-group have been recorded from Hainan. There are no earlier reports of morrisi-group earthworms from Guangdong Province, which is located just north of Hainan Province and separated from it by Qiongzhou Strait. During the years 2006, 2010 and 2011, we made a broad earthworm collection during field surveys in southern China, and discovered more new species of the morrisi-group.
In this paper, we describe four new species from Xiangtoushan National Nature Reserve of Guandong, Jianfengling National Nature Reserve and Diaoluoshan National Nature Reserve of Hainan, including Amynthas instabilis sp. nov., Amynthas dilatatus sp. nov., Amynthas infuscuatus sp. nov. and Amynthas qiongzhongensis sp. nov. Amynthas instabilis sp. nov. was the only species found in both Hainan and Guangdong Provinces.

Material and methods
The earthworms collected in 2006 were anaesthetized in 10% ethanol solution, and preserved in 10% formalin solution. The earthworms collected in 2010-11 were anaesthetized in 10% ethanol solution, and preserved in 95% ethanol solution. Holotypes and paratypes are deposited in the Shanghai Natural History Museum.
Sequences were aligned with Clustal X, and then pairwise distances between these species were calculated using the Kimura two-parameter model of DNA evolution with MEGA 5.

Results
The COI mitochrondrial DNA gene, considered as a barcode for earthworm identification (Huang et al. 2007;Novo et al. 2010), is an effective complement to morphological analyses.
Comparisons of COI gene sequences among A. instabilis sp. nov., A. qiongzhongensis sp. nov. and A. carnosus, A. robustus and A. triastriatus (Table 1), which belong to other species-groups and have similar male pores and genital markings and different numbers of spermathecal pores, indicated that the new species greatly differ from these last species. Pairwise distances of COI (Table 2) for A. instabilis, A. qiongzhongensis and other species show very low intraspecific values (0-0.16%), whereas distances between A. instabilis and A. qiongzhongensis were 13.59-13.79%. Among A. instabilis and the other three species they were 18.74-21.74%. Among A. qiongzhongensis and the other three species they were 17.05-19.72% . According to Chang and James (2011), values above 10-15% most probably indicate different species. Although the evaluation criterion is uncertain, it is clear that the new species and other species in Table 2 have large genetic divergences. Also, there is little difference between individuals of the same species.

Etymology
The species is named after its characteristic of variable numbers of genital papillae in the male pore region.
Male pores one pair in XVIII, 0.33 circumference apart ventrally, each on the top of a slightly raised, oval porophore in a pulvinate pad with six circular folds, some paratypes with three circular folds. Two tiny oval genital papillae, present at anterior and posterior of porophore, each male pore is medial to these two tiny papillae ( Figure 1A); some paratypes with three tiny oval genital papillae surrounding left male pore ( Figure 1D). Genital papillae numbers in male pores region are variable among specimens.
Female pore single in XIV, rounded, milky white.
Male sexual system holandric, testis sacs two pairs, in X-XI. Seminal vesicles two pairs, extending in XI-XIII, the posterior pair larger than the anterior one and extending to XIII. Prostate glands developed, inserting in XVIII and extending to XVII and XX, coarsely lobate, prostatic duct U-shaped, slightly thicker at the distal part ( Figure 1C). No accessory glands observed.

Remarks
In comparison to the other 30 species of the morrisi-group reported from China (19) and other Asian countries (11), Amynthas instabilis sp. nov. is closely related to Amynthas incongruus (Chen 1933) and Amynthas tripunctus (Chen 1946) from China, and to A. dilatatus sp. nov., A. infuscuatus sp. nov. and A. qiongzhongensis sp. nov. These six species share some similarities with similar male pores and genital markings, two pairs of intersegmental spermathecal pores in 5/6-6/7, and simple intestinal caeca. Table 3 illustrates in detail the differences among A. incongruus (Chen 1933), A. tripunctus (Chen 1946) and the four new species.
Amynthas instabilis sp. nov. differs from A. tripunctus (Chen 1946) by the presence of pigment on the dorsum, location of first dorsal pore, setae number in segment VIII, prostate glands and spermathecal characters. Amynthas instabilis sp. nov. has tan pigment on the dorsum, first dorsal pore in 11/12, setal number 52-62 at VIII, prostates glands less developed and diverticulum half as long as main pouch. In contrast, A. tripunctus has grey pigment on dorsum, first dorsal pore in 12/13, setae number fewer at VIII, more setae between male pores, well-developed prostate glands and a longer diverticulum.
Amynthas instabilis sp. nov. and A. incongruus (Chen 1933) can be separated on the basis of spermathecal characters. The ampulla of A. instabilis sp. nov. is heartshaped, a slender to stout duct is as long as ampulla. The ampulla of A. incongruus is ovoid or elongate spherical, duct either long and slender, or short stout, terminal three-quarters of diverticulum dilated into a twist chamber. In addition, A. incongruus has very small seminal vesicles and accessory glands, with prostate glands absent entirely and in a few cases well developed (Chen 1933).  A. tripunctus (Chen 1946) A. incongruus (Chen 1933) Body length (mm)*  Both clitellates were externally characterized and dissected.

Etymology
The species is named after the characteristics of the seminal chamber.

External characters
Male pores one pair in XVIII, 0.33 circumference ventrally apart from each other, each on the top of a slightly raised, rounded porophore in a pulvinate pad with three or four circular folds. each male pore is medial to these two tiny papillae (Figure 2A), some paratypes with four or five tiny oval genital papillae surrounding each male pore ( Figure 2D). In holotype, a pair of tiny conical genital papillae present on anterior border of segments XVIII, 0.2 mm apart from each other.
Female pore single in XIV, ovoid, milky white.
Male sexual system holandric, testis sacs two pairs, in X-XI. Seminal vesicles two pairs, extending in X-XII, the pair in XI larger than the other. Prostate glands developed, inserting in XVIII and extending to 1/2XVII and XX, coarsely lobate composed of three major lobes. Prostatic duct U-shaped, conspicuously curved at the distal part ( Figure 2C). No accessory glands observed.

Remarks
The present species is obviously different from the other species in Table 3 because of the long diverticulum with a swollen seminal chamber. In addition, A. dilatatus sp. nov. differs from A. tripunctus (Chen 1946) in the features of body length, segment number, setae number between male pores, location of first dorsal pore and prostate glands. Paratypes. 2 clitellates (C-HN020-02B) with the same data as for holotype. All three clitellates were externally characterized and dissected.

Etymology
The species is named after its dark pigment.

External characters
Male pores one pair in XVIII, 0.33 circumference ventrally apart from each other, each on the top of a slightly raised, oval porophore in a pulvinate pad with three or four circular folds. Two rounded genital papillae, flat-topped, present at inner ridge of porophore, anteromedial one larger than the other ( Figure 3A).
Female pore single in XIV, oval.
Intestinal caeca paired in XXVII, simple, smooth, brown, extending anteriorly about to XXIV. Oesophageal hearts in X-XIII. Ovaries in XIII. Spermathecae two pairs in VI-VII, ampulla ovoid, about 2.5 mm long in holotype; duct slender, twice as long as ampulla. Diverticulum slightly longer than main pouch, slender, terminal one-third dilated into a swollen chamber ( Figure  3B), milky white. No accessory glands observed.
Male sexual system holandric, testis sacs two pairs, in X-XI. Seminal vesicles two pairs, extending in X-XII, the anterior pair larger. Testis sacs and seminal vesicles separated from each other on ventrum. Prostate glands developed, inserting in XVIII and extending to XVI and half of XX, coarsely lobate composed of two major lobes. Prostatic duct U-shaped, slender ( Figure 3C). No accessory glands observed.

Remarks
Both A. infuscuatus sp. nov. and A. tripunctus have similar body length, setae, location of first dorsal pore, seminal vesicles and diverticulum length. However, A. infuscuatus sp. nov. is distinguished from A. tripunctus by its segment number, ampulla shape and seminal chamber length.
In comparison to A. incongruus, spermathecae of A. infuscuatus sp. nov. is quite different. Ampulla is heart-shaped, duct slightly shorter than ampulla, and terminal two-sevenths of diverticulum dilated into a swollen seminal chamber. Moreover, body length and first dorsal pore are also distinctive characters.
Although both have terminal third dilated into seminal chamber, A. infuscuatus sp. nov. and A. instabilis have no similar spermathecae. The ampulla of A. infuscuatus is ovoid, smaller than the heart-shaped ampulla of A. instabilis, but its ampulla duct and diverticulum are longer. Six individuals were dissected and 10 individuals were characterized externally only.

Etymology
The species is named after its type locality (Li-Miao Autonomous County of Qiongzhong, Hainan).