The megascolecid earthworms (Annelida, Oligochaeta, Megascolecidae) in the Phu Quoc island, Vietnam, with descriptions of three new species

Abstract The megascolecid earthworms of the Phu Quoc island are intensively investigated. Twelve species in three genera (Lampito Kinberg, 1867, Amynthas Kinberg, 1867, and Metaphire Sims & Easton, 1972) are recorded. Of these, Metaphire doiphamon Bantaowong & Panha, 2016 is recorded for the first time in Vietnam, and three species are newly described, namely Amynthas catenatussp. nov., A. phuquocensissp. nov., and A. poropapillatussp. nov. An identification key to 12 megascolecid species is provided as well.

To improve the knowledge of the earthworms of Vietnam, this study documents the diversity of the family Megascolecidae from Phu Quoc island; three new species are described.

Material and methods
Earthworms were collected manually in 34 sites in three habitats: natural forests, industrial plantations of Piper, Acacia, and other trees, and residential gardens (Fig. 1). After their collection, specimens were killed using 2% formalin and fixed in 4% formalin for 24 hours and then transferred to new 4% formalin for morphological examination and long-term preservation. Transverse body sections were processed using the classical method of haematoxylin and eosin. Selected segments were cleaned and dehydrated using graded ethanol concentrations. Segments were treated with paraffin, then cut using a Sakura Accu SRM 200CW microstome. The cut sections were stained using haematoxylin and eosin Y (Feldman and Wolfe 2014) and then transferred onto glass slides.
Material was examined under a Motic Digital microscope (model DM143-FBGG-C), and dissected from the dorsal side for internal observation. Colour images were taken using a camera attached directly to the microscope. Line drawings and colour images were improved and grouped into finished figures using Photoshop CS6.
All specimens including holotypes and paratypes are housed in the Laboratory of Zoology, Can Tho University. Some are shared with the laboratory of Department of Applied Biology, Kien Giang University.
Remarks. The species is widely distributed in sandy soils in coastal areas of Vietnam (Nguyen 2014;Nguyen et al. 2016).
Habitats. The species was found in the residential gardens and natural forests. They live in leaf litter and humid rocks.
Remarks. Thai et al. (2004) reported this species from the Phu Quoc island, but with question mark "Pheretima californica?". Comparing to M. californica previously recorded in the mainland of Vietnam, the population of the Phu Quoc island has several differences: septum 10/11 absent, intestinal caeca simple, and testis sacs connected. However, simple intestinal caeca were also reported widely in Myanmar and Taiwan (Gates 1972;Chang et al. 2009  Diagnosis. Medium-sized. Prostomium epilobous. First dorsal pore in 12/13. Three pairs of spermathecal pores in lateroventral intersegments 6/7/8/9. Male pores located inside copulatory pouches in xviii. Two pairs of genital markings in xvii and xix, in line with male pores. Intestinal caeca lobuled. Holandric. Testis sacs in xi, separated. Septa 8/9/10 absent.
Male pores located inside copulatory pouches in xviii. Ventral distance between two openings of copulatory pouches about 0.3× body circumference. Two pairs of dish-shaped genital markings present in 17/18 and 18/19, in line with the openings of copulatory pouches.
Habitats. The species was found in all habitats, but more in residential gardens. They live in clay soils in the depth of 15-30 cm.
Remarks. Nguyen (2014) also recorded this species in the mountainous region of An Giang province under the name "Metaphire sp. 4". Compared to the description of M. doiphanon by Bantaowong et al. (2016), our specimens differ slightly in the absence of septum 10/11, diverticula attached to the middle of ampulla duct, separated testis sacs, and intestinal caeca slightly lobuled ventrally. On the contrary, M. doiphanon has thick septum 10/11, diverticula attached to the base of ampulla duct, connected testis sacs, and simple intestinal caeca.

Metaphire houlleti (Perrier, 1872)
Habitats. The species was found mainly in industrial tree plantations and residential gardens. It was collected in leaf litter and sometimes in upper soil surface (0-10 cm).
Remarks. The specimens collected in Phu Quoc island have the seta a on segments iii-v being bigger than that on other segments.

Metaphire peguana (Rosa, 1890)
Habitats. The species was commonly found in residential gardens and industrial tree plantations. They live in leaf litter and upper soil layer.
Remarks. The species has been commonly found in southern Vietnam. However, it was erroneously identified as Metaphire californica (Nguyen 2014, as Pheretima california: sic!; Nguyen et al. 2016Nguyen et al. , 2017, but recently corrected (Nguyen et al. 2019). In addition, it has not been recorded from the small islands of Lai Son, An Son, and Hon Tre, which are located between Phu Quoc island and southern Vietnam (Nguyen et al. 2017). Diagnosis. Medium-sized. Prostomium epilobous. First dorsal pore in 12/13. Four pairs of spermathecal pores in lateroventral intersegments 5/6/7/8/9. Male pores located inside shallow copulatory pouches in xviii. Two pairs of genital markings present in xvii and xix. Intestinal caeca simple. Holandric. Testis sacs connected. Septum 8/9 thick, 9/10 absent.
Habitats. The species was only found in the residential gardens. Vietnamese name. Giun quắn.
Three pairs of spermathecal pores in dorsal intersegments 6/7/8/9, located near dorsal line. Genital markings absent in the spermathecal region. Male pores located inside copulatory pouches in xviii; ventral distance between two openings of copulatory pouches about 0.3× body circumference. No genital markings in the male region.
Habitats. The species was collected in the depth of 0-10 cm of sandy soils in bushes and residential gardens.
Metaphire dorsobitheca was previously known only in its type locality (Dak No, Dak Lak Province). This is first time that this species has been found elsewhere.
Etymology. "catenatus", an adjective in apposition, to emphasize the chained seminal chamber of diverticula.
Four pairs of spermathecal pores in lateroventral intersegments 5/6/7/8/9. No genital markings in the spermathecal region. Male porophores small in xviii, without copulatory pouches; ventral distance between two male pores about 0.25-0.28× body circumference. About 8-12 small genital markings ventrally arranged in two transverse lines in front of and behind the setal ring xviii.
Spermathecae paired in vi-ix. Ampulla clavate; ducts not clearly distinct from ampulla. Diverticula slightly waved, attached to the base of ampulla; seminal chamber small, chained-shaped with 2 or 3 parts. No accessory glands.
Holandric. Testis sacs in x and xi, connected. Seminal vesicles well developed in xi and xii. Ovaries in 12/13. Ovisacs invisible. Prostate glands deeply lobuled within xvi-xx; ducts U-shaped. No accessory glands.
Habitats. The species was only collected under leaf litter in Piper plantations. Vietnamese name. Giun buồng tinh hình chuỗi. Remarks. The new species can be assigned to the A. corticis group, which is characterized by having four pairs of spermathecal pores in 5/6/7/8/9 and holandric (Sims and Easton 1972). This group has 108 described species worldwide, of which 17 have been recorded in Vietnam (Gates 1972;Sims and Easton 1972;Chang et al. 2009;Nguyen et al. 2016;Xiao 2019).
Compared to A. corticis (Kinberg, 1867), the new species differs in its smaller size (length 51-54 mm, diameter 2.2-2.5 mm vs length 45-170 mm, diameter 3.0-6.0 mm), first dorsal pore in 5/6 (vs in 11/12), and absence of pre-clitellar genital markings (vs genital markings paired near spermathecal pores). In addition, A. catenatus sp. nov. has 8-12 small genital markings ventrally arranged in two transverse lines in presetal and postsetal xviii, a clavate spermathecal ampulla, slightly wavy diverticula and attached to the base of ampulla, and a small seminal chamber, which is chained-shaped with 2 or 3 parts. On the contrary, A. corticis has two pairs of small circular genital markings located next to male pores in presetal and postsetal xviii, an ovoid ampulla, and a blunt, ovoid diverticula with straight stalk.
Two pairs of spermathecal pores in ventral intersegments 7/8/9. Genital markings absent in the spermathecal region. Male pores located in the setal ring xviii, without copulatory pouches; ventral distance between two male pores about 0.28× body circumference. Genital markings roundly pad-shaped and variable in the male region, normally with two pairs in 17/18 and 18/19, or a pair in xix (near 19/20).
Habitats. The species was occasionally found in natural forests along the main road to the northern part of Phu Quoc island. They were collected in the upper soil layer (0-10cm).

Vietnamese name. Giun phú quốc.
Variations. The body size and number of genital markings in the male region are variable among collected specimens. Specimens can be divided into two groups based on body size. The group of smaller specimens (n = 15) present the following ranges: length = 71-85 mm, diameter = 2.5-2.9 mm, segments = 116-145. The larger specimens (n = 8) present: length = 96-145 mm, diameter = 3.1-4.7 mm, segments = 124-145. Both two groups were together at the same site.
Genital markings are also variable in the male region. Genital markings have two pairs located in 17/18 and 18/19 (Type 1), but reduced to three genital markings (Type 2), one pair either in xvii or in xiv (Type 3 or Type 4), or a single one in xix (Type 5) (Fig. 12).
Remarks. The new species can be assigned into the Amynthas aeruginosus group, which is characterized by having two pairs of spermathecal pores in 7/8/9 and holandric (Sims and Easton 1972). This group is known to have about 70 species distributed mainly in Southeast and East Asia. Of these, about 11 species have been recorded in Vietnam (Nguyen et al. 2016).
Within the A. aeruginosus group, the new species is fairly similar to A. phimpheti Hong, Inkavilay &James, 2018 andA. antethecus Hong, Inkavilay &James, 2018 in having two pairs of genital markings in the male region, but absent in the spermathecal region, and a simple intestinal caeca. However, these species can be distinguished by the positions of the first dorsal pore and the spermathecal pores and genital markings in the male region, the presence of septum 10/11, presence of accessory glands, and position of testis sacs (summarized in Table 2).
Regarding the arrangement of genital markings in the male region, the new species is slightly similar to A. platycorpus (Thai, 1982) andA. binhgiaensis (Le, 1994) by having two pairs of genital markings in 17/18 and 18/19. However, these species can be distinguished by number and position of spermathecal pores, first dorsal pore, genital markings in the spermathecal region, presence of septum 10/11, type of intestinal caeca and testis sacs (summarized in Table 3).  Etymology. "poropapillatus", an adjective in apposition, to emphasize the position of male pores on the genital markings.
Two pairs of spermathecal pores in intersegments 7/8/9. Male pores behind the setal ring xviii, on the posterior edge of genital markings, without copulatory pouches; ventral distance between two male pores about 0.33× circumference. Two pairs of genital markings present in xvii and xviii, slightly concave inside body wall.
Holandric. Testis sacs in xi, separated. Seminal vesicles well developed in xi and xii. Ovaries in 12/13. Ovisacs invisible. Prostatic glands deeply lobuled in xvi-xxi; ducts long. Two pairs of accessory glands present.
Habitats. The species was found only in natural forests in northern part of Phu Quoc island. It was collected in the upper soil layer (0-10 cm) of clay soils.
Vietnamese name. Giun nhú phụ chứa lỗ đực.  Hong, Inkhavilay & James, 2018 in having the spermathecal pores located ventrally, the genital markings paired in xviii, the genital markings in spermathecal region absent, and a simple intestinal caeca. However, these three species can be distinguished by the first dorsal pore, number and position of genital markings in the male region, position of male pores, presence of septa 8/9 and 10/11, and intestinal origin (summarized in Table 4).
Key to megascolecid species recorded in Phu Quoc island, southern Vietnam

Conclusion
As Phu Quoc island has a large area, the 12 species recorded at present may not reflect the true biodiversity of earthworms in this island. More intensive surveys likely will reveal additional new species and result in better understanding of the earthworm biodiversity of Vietnam.