﻿Two new Drawida (Oligochaeta, Moniligastridae) earthworms from Vietnam

﻿Abstract Two new earthworm species are described, namely Drawidaangiangsp. nov. and Drawidacochinchinasp. nov. The former can be recognized by having male pores on spiniform penises in intersegment 10/11, an erect and sac-shaped spermathecal atrium, glandular prostate, the capsule coiled one round, the vas deferens strongly coiled but small, two large, round, genital markings on segments ix–x, and three gizzards in xiii–xv. The latter species is distinguished in having the male pores placed on highly elevated, backwardly directed, conical penises in 10/11, a slender spermathecal atrium, a glandular prostate, a somewhat folded capsule, the vas deferens strongly coiled as a bunch and equal size to the testis sacs, a pair of genital markings located closely anterior to the penises with 1–3 additional ones in xi–xii, and three or four gizzards in xiii–xvi. The DNA barcode fragment of the COI gene was extracted for each species, and the COI genetic distances and phylogenetic analysis also supported two new species..


Introduction
To date, the earthworms of Vietnam are well known with 245 species and subspecies described (Nguyen TT et al. 2016a, 2016bNguyen QN et al. 2020;Lam et al. 2021), of which 232 belong to the species-rich family Megascolecidae; the family Moniligastridae has been reported with only five species, although Vietnam is located in the region of origin of this family (Sims and Easton 1972). Five Drawida Michaelsen, 1900 species are Drawida annamensis Michaelsen, 1934, D. beddardi (Rosa, 1890, D. delicata Gates, 1962, D. chapaensis Do & Huynh, 1993, and D. langsonensis Do, 1993in Do & Huynh, 1993. Three species are only known from Vietnam: D. annamensis, D. chapaensis, and D. langsonensis (Nguyen TT et al. 2016b).
Most recent research on earthworms in Vietnam focuses mainly on the family Megascolecidae, especially the pheretimoid group. There are no works on the family Moniligastridae or the genus Drawida in Vietnam. This work, therefore, aims to contribute to a better knowledge of the genus Drawida through descriptions of two new species.

Specimen collecting and preservation
Earthworms were manually searched for and collected in Vietnam for a decade during the rainy season, September to November, in 2010-2020. After collection, specimens were cleaned with tap water, killed in 2% formalin, temporally fixed in 4% formalin for 12 hours, then transferred to fresh 4% formalin for long-term preservation. Specimens for molecular study were preserved in 95% ethanol. Specimens including holotypes and paratypes were deposited in Laboratory of Zoology, Department of Biology, Can Tho University (CTU). Some were shared with the Department of Soil Ecology, Institute of Ecology and Biological Resources (IEBR), Hanoi, Vietnam.

Morphological examination
Material was examined under a Motic Digital microscope (model: DM143-FBGG-C) and dissected from the dorsal side for internal observations. Transverse body sections were processed using the classical method of Hematoxylin & Eosin. Selected segments were cleaned and dehydrated using graded ethanol concentrations. Segments were imbedded with paraffin, then cut using a microtome Sakura Accu SRM 200CW. The cut sections were stained using Hematoxylin & Eosin Y (Feldman and Wolfe 2014) and transferred onto glass slides and mounted.
Color images were taken using a camera attached directly to the microscope. Line drawings and color images were improved and grouped into plates using Photoshop CS6.

DNA extractions, PCR, and sequencing
Total genomic DNA was extracted from several body segments using a DNeasy Blood & Tissue Kit (Qiagen TM). A fragment of the mitochondrial gene, cytochrome c oxidase subunit I (COI), was amplified using polymerase chain reaction (PCR). Universal primers LCO-1490and HCO-2198(Folmer et al. 1994 were used to amplify a 680 bp fragment of the COI region. PCR conditions for amplification of the COI gene were as follows: an initial denaturation at 95 °C for 2 minutes followed by 36 cycles of 95 °C for 20 seconds, 42 °C for 45 seconds, and 72 °C for 1 minute, and a final extension at 72° for 5 minutes. Successfully amplified samples were sent for purifying and sequencing at the FirstBase Company (Malaysia). The same primers for the initial PCR were also used as sequencing primers.
Each sequence chromatogram was manually checked using BioEdit v.7.1 (Hall 1999), and the identity confirmed by a BLAST search (Zhang et al. 2000). All confirmed sequences were aligned using multiple sequence alignment with the program ClustalX v. 2.0 (Larkin et al. 2007).
The K2P (Kimura 2 parameters) genetic distance was calculated in MEGA 7.0 (Kumar et al. 2016. The phylogenetic tree was reconstructed using a maximumlikelihood analysis with the best model chosen using ModelFinder (Kalyaanamoorthy et al. 2017) performed in IQTREE v.1.6.2 for Windows (Nguyen LT et al. 2015). The best model was GTR + F + I + G4 with BIC score = 12628.239 and -lnL = 6005.512.

Molecular analysis
The genetic distance between new species and other Drawida ranges from 21.5% (D. cochinchina and D. japonica) to 29.3% (D. angiang and D. scandens).
The interspecific divergence among Drawida species ranges from 16.3% (D. impertusa and D. robusta) to 31.1% (D. nilamburensis and D. japonica). The average interspecific distance in the genus Drawida was previously reported as 22%, and the maximum one was 34.3% between D. impertusa and D. deshayesi (Thakur et al. 2021). The p-genetic distance was also known to range from 18% between D. koreana and D. japonica japonica to 24.82% between D. koreana and D. gracilis (Zhang et al. 2020).
Testis sacs, paired, large, located on posterior side of septum 9/10, much larger than coils of vas deferens; vas deferens twisted and strongly coiled, ending at ental ends of prostate capsule which basally connect to penial pouch. Ovaries on septum 10/11; ovisacs sac-shaped in xiii and xiv. Accessory glands in ix and x in correspondence with genital markings outside. Etymology. A noun in apposition, angiang, is used to emphasize the province where type specimens were collected.
Remarks. The species is very similar to D. angchiniana Chen, 1933 from northern China (Anhwei and Kiangsu) and South Korea (Jeju Island) (Michaelsen 1931;Chen 1933;Kobayashi 1937) by the presence of genital markings in the male region and Table 2. Character comparison between Drawida angiang sp. nov., D. cochinchina sp. nov., D. longatria Gates, 1925, D. ofunatoensis (Ohfuchi, 1938), and D. angchiniana Chen, 1933.  (1892), and Blakemore and Kupriyanova (2010) absence in the spermathecal region. However, the two species can be distinguished by the location of genital markings (ix and x vs x and xi), number of segments (170-221 vs 134-145), location of clitellum (x-xiv vs ix-xiv), and shape of the spermathecal atrium (long, enlarged distally vs short or cylindrical) ( Table 2). The new species, D. japonica (Michaelsen, 1892) and D. koreana Kobayashi, 1938 share several common characters, such as the presence of genital markings and accessory glands. However, the new species differs from those two species in having three gizzards from xiii, the spermathecal atrium erect, the vas deferens strongly coiled as a bunch, and the prostate cylindrical and somewhat folded, while D. japonica and D. koreana have two to three gizzards from xii, the spermathecal atrium short and small, the vas deferens not into a bunch (coiled or loosely twisted), and the club-shaped or thumb-shaped prostate ( Table 2).
Coloration: body light grey, uniformly color in both ventrum and dorsum. Prostomium undeveloped. No dorsal pores. Setae lumbricine, with eight setae distributed round body, setal formula aa: ab: bc: cd: dd = 6.2-7.0: 1: 7.0-8.5: 1: 33-35. Clitellum annular, within ix-xiv, reddish brown. Spermathecal pores paired, in ventro-lateral intersegmental furrow 7/8, close to seta c. Genital markings present, variable, one or two pairs in viii and ix, located between setae b and c, sometimes with an additional one or two in medio-ventral viii and ix, one pair anterior to penis in x, and additional 1-3 in xi-xii. Female pore hardly visible. Male pores located in intersegmental furrow 10/11, between setae b and c, closed to seta c, on the top of highly elevated, backwardly directed, conical penises in 10/11. Nephridiopores anterior margin of segments iv onwards, in d lines, especially clear from vii to xv.
Internal: Septa 5/6/7/8/9 thick, 9/10 and subsequence septa thin. Gizzards three or four in xiii-xvi. Last hearts in ix. Intestinal origin at xvi or xvii. Spermathecae paired, on viii, spermathecal ampulla oval; spermathecal ducts coiled and twisted, passing through septum 7/8, and ending at ectal end of atrium; Spermathecal atrium tubular, strongly coiled as a bunch in vii, mass larger than spermathecal ampulla. Prostate glandular, glandularity strongly reduced; prostatic capsule cylindrical, somewhat folded. Testis sacs paired, in x, large, sac-shaped; vas deferens strongly coiled as a bunch, equal in size to testis sacs, and entering testis sac at its ental end. Ovarian chamber complete, ovisacs well developed, in xii-xviii. Accessory glands present, but invisible. DNA character. The COI fragment was uploaded to GenBank with accession numbers ON303831, ON303832, ON303833. The new species has a close COI identity of 81% with D. ghilarovi (KY711506) Distribution. The species was also found in Kien Giang (Da Do, Da Dung, and Ta  Etymology. The noun cochinchina (= southern Vietnam) is used in apposition is to accentuate its wide distribution in southern Vietnam.
Remarks. The new species is very similar to D. longatria Gates, 1925 in having genital markings in 10/11, the presence of a spermathecal atrium, and the spermathecal ducts being twisted and strongly coiled. However, it differs from D. longatria in having prostate capsule cylindrical, somewhat folded, three or four esophageal gizzards in xiii-xvi, the spermathecal atrium in vii, testis sacs in 10/11, ovisacs well developed in xii-xvii, and having hidden accessory glands. On the contrary, D. longatria has the prostate capsule digitiform, four esophageal gizzards in xv-xviii, the spermathecal atrium in viii, testis sacs in 9/10, ovisacs in xi-xiv, and obvious accessory glands.
The new species is also similar to D. ofunatoensis (Ofuchi, 1938) in having paired genital markings and testis sacs in septum 10/11. However, it differs in having the clitellum within ix-xi, the spermathecal atrium and seminal ducts twisted and strongly coiled, and the prostate cylindrical and strongly folded. Drawida ofunatoensis has the clitellum located in x-xiii, the spermatheca lacking an atrium, and the male atrium globular.
The new species is somewhat similar to D. nepalensis in having the clitellum within ix-xiv, the presence of genital markings, spermathecal pores located median to c, and the vas deferens strongly coiled as a bunch. However, it differs from D. nepalensis in having the spermathecal atrium slender and strongly coiled as a bunch, one gizzard per segment (three to four within xiii-xvi), a folded cylindrical prostate, and hidden accessory glands. On the contrary, D. nepalensis has each gizzard pass through several segments (two to four within xii-xx), the spermathecal atrium stouter and sac-like, the prostate club-shaped, and obvious accessory glands.
Compared to the other five Drawida species recorded in Vietnam, D. annamensis, D. chapaensis, D. delicata, D. langsonensis, and D. beddardi, this new species is clearly distinguished by having paired genital markings and the spermathecal atrium and spermathecal ducts strongly twisted and coiled, while all other species have no genital markings and the spermathecal atrium and spermathecal ducts are simply undulated.

Conclusions
The discovery of two new species of Drawida brings the number of species in Vietnam to seven. However, due to the placement of Vietnam in region of origin of the genus Drawida, this number of species does not reflect the true biodiversity in this country. It is, therefore, suggested that additional intensive surveys are needed to reveal more new species awaiting discovery.