Notes on the earthworm species, Metaphire anomala (Michaelsen, 1907) (Clitellata, Megascolecidae) in Southern Vietnam, with descriptions of two new species

Integrative taxonomy was employed to exploit the differences between the known Metaphire anomala (Michaelsen, 1907) and other specimens collected in Vietnam. The results brought to light two new species, namely Metaphire iranomala sp. nov. and Metaphire decemtheca sp. nov. The former is easily recognised by having male pores on xix and four pairs of spermathecal pores on 5/6/7/8/9 while the latter is distinguished by having fi ve pairs of spermathecal pores on 4/5/6/7/8/9. The K2P distances of the fragment of the cytochrome c oxidase subunit I (COI) gene are 13.1% between M. iranomala sp. nov. and M. anomala (Michaelsen, 1907) and 18% between M. decemtheca sp. nov. and Metaphire grandiverticulata Nguyen & Lam, 2017. The intraspecifi c divergences are 1.5%–10.6% for M. iranomala sp. nov. and 2.1%–11.4% for M. decemtheca sp. nov.


Introduction
described a new species, namely Pheretima anomala, from only eight specimens collected from the Botanical Gardens of Sibpur, Calcutta (India). The species is very specifi c without spermathecae and male pores on segment xx. Later, Gates (1925) commented on the species based on specimens collected from Rangoon (Myanmar). His specimens were slightly different from the original description (length 116-162 mm vs 80-90 mm, papillae on xviii-xxii vs on xvii-xxiii). Gates (1925) also described a new species, P. insolita. The species contained six types (A, B, C, D, E, F) distinguishable in length, diameter, number of segments, location and numbers of papillae in the male region. Pheretima insolita was very similar to P. anomala in terms of traces of male pores on xx, but differs in having spermathecal pores on 5/6/7/8 and variations of papillae in the male region. Stephenson (1929) discussed all types of P. insolita and P. anomala. He commented that the original specimens (P. anomala) possibly acted as functional males while the functional females would be P. insolita. Stephenson (1929), therefore, suggested to unite all the forms of P. anomala and P. insolita as one species, P. anomala. Michaelsen (1907)'s specimens would constitute the forma typical.
The species Metaphire anomala has previously been reported in Southern Vietnam as 'so-called' Pheretima anomala Michaelsen, 1907. It is widely distributed in Southern Vietnam: Ba Ria-Vung Tau (Con Lon Isl.), Dong Nai (Cat Tien), Kien Giang (Lai Son Isl., Phu Quoc Isl.), An Giang (Nhon Mts, Phu Cuong Mts, Ba Doi Mts, Cam Mts, To Mts), Dong Nai (Vinh Cuu, Xuan Loc, Dinh Quan, Trang Bom, Cam My, Long Thanh, Cat Tien NP), Tay Ninh (Ba Den Mts) (Thai et al. 2004;Nguyen 2014;Nguyen et al. 2016Nguyen et al. , 2017aNguyen et al. , 2017bNguyen et al. , 2019Nguyen et al. , 2020. During our re-examination of previously and newly collected specimens housed at Can Tho University, we found that the specimens collected from Southern Vietnam were very different from their original description (Michaelsen 1907), Gates (1925Gates ( , 1972 and recent re-description (Bantaogwon et al. 2011). An integrative approach combining morphological and molecular data was used to confi rm the taxonomic status of the Vietnamese specimens. Two new species were discovered and named in this study.
Colour images were taken using a camera attached directly to the microscope. Line drawings and colour images were improved and grouped into plates using Photoshop CS6.

DNA extraction, PCR reaction and phylogenetic analysis
Total genomic DNA was extracted from the body walls of segments 30-35 using the DNAeasy Blood & Tissue Kit (QiagenTM). A 680bp fragment of the cytochrome c oxidase subunit I (COI) mitochondrial gene was amplifi ed using the polymerase chain reaction (PCR) method with the universal primer set (HCO-2190/COI-E and LCO-1498/LCO-1498m) (Folmer et al. 1994). The PCR conditions for amplifi cation were as follows: an initial denaturation at 95°C for 2 min followed by 36 cycles of 95°C for 20 s, 42°C for 45 s, and 72°C for 1 min, and a fi nal extension at 72°C for 5 min. PCR products were checked for potentially successful amplifi cation using electrophoresis in 1% Agarose-TBE 1X. Successfully amplifi ed PCR products were purifi ed and sequenced at FishBase, Inc. (Malaysia) on an Applied Biosystems automatic sequencer (ABI3130 XL). Each successful sequence was manually checked using BioEdit ver. 7.1 (Hall 1999) and confi rmed using BLASTN 2.6.0+ searches (Zhang et al. 2000). All confi rmed sequences were aligned with MUSCLE (Edgar 2004). All nucleotide sequences have been deposited in GenBank (NCBI).

Etymology
The epithet 'iranomala' is formed by the prefi x 'ir' and 'anomala' to emphasise the wrong name 'anomala' recorded in Vietnam.
Spermathecae paired in vi-ix. Spermathecal ampulla large, mango-shaped; duct about a quarter of ampulla length. Diverticula attached to the base of ampulla ducts; distal part strongly coiled, swollen into coiled sinusoidal seminal chambers. Spermathecal ducts without nephridia. Accessory glands absent in the spermathecal region.

Habitat
The species was found in leaf-litters or in the top-soil layer, especially in moist places (near streams) or in rocky holes with organic matter. Metaphire iranomala sp. nov. has a soft body, violet light skin when alive. Its moving behavior is similar to a caterpillar locomotion. The species was commonly located in hilly/mountainous areas, but occasionally found in deltas.

Variations
Metaphire iranomala sp. nov. has two slightly different morphological types. The fi rst type is more likely to be distributed in islands or in coastal provinces in Vietnam; the other type is found in mainland provinces.     Gates (1925Gates ( , 1972 and Stephenson (1929); 4 Michaelsen (1907).   (Cognetti, 1908) and M. isselii (Goto & Hatai, 1899) sharing location of male pores not in segment xix.

Characteristics
There are not many differences between the two types except the ventral distance between the male pores (0.2-0.22 vs 0.25-0.3). However, the COI genetic distance also distinguishes two types (see below).

Remarks
The new species has been previously identifi ed as Metaphire anomala. It is widely distributed in Southern Vietnam (Thai et al. 2004;Nguyen 2014;Nguyen et al. 2017aNguyen et al. , 2017bNguyen et al. , 2019Nguyen et al. , 2020. However, this species is very different from both the original description (Michaelsen 1907), and re-descri ption of M. anomala from Myanmar (Gates 1925(Gates , 1972, Thailand (Bantaowong et al. 2011) in the position of male pores, number and position of spermathecal pores, genital markings in male and spermathecal regions and body size. These differences are summarised in Table 2. A few Metaphire species have been known to exhibit male pores not in segment xviii. Only M. anomala has male pores on xx, whereas two other species, M. isselii Cognetti, 1908 andM. megascolidioides Goto &Hatai, 1899, have male pores on xix. The new species is similar to these two species by having male pores on xix, a fi rst dorsal pore in 12/13 and the absence of genital markings in the spermathecal region. However, the new earthworm species is clearly distinguished by body size, the number and position of spermathecal pores, the morphology of its male region, the status of septum 8/9, and they type of intestinal caeca. The differences are summarised in Table 3.

Etymology
The epithet 'decemtheca' emphasises the number of spermathecae of the new species.
Five pairs of spermathecal pores on ventrolateral intersegments 4/5/6/7/8/9. Ventral distance between spermathecal pores ca 0.38-0.45 body circumference. Genital markings absent in spermathecal region. Male pores on line with setal ring in xviii; copulatory pouches present; ventral distance between male pores is 0.38 body circumference. One pair of fl at ellipsoidal genital markings in xviii located next to male pores ventrally.
Spermathecal paired in v-ix. Spermathecal ampulla oval-shaped, ducts stout, short, about one-third of ampulla length. Diverticula shorter than ampulla, strongly and constrainedly waved, attached to the base of ampulla ducts; seminal chamber small oval-shaped. Spermathecal ducts without nephridia. Accessory glands absent in the spermathecal region.

DNA barcode
COI barcode data (partial) is for the paratypes uploaded to GenBank under the accession numbers MW076201, MW076202 and MW076203. The new species shares the identity of 85.8% and 86.1% with Amynthas sp. (KT252973, KT205464).

Habitat
The species was found in the leaf litter of soil layer at a depth of 0-10 cm, near streams, and scattered on Con Son Island.

Variations
There are two morphological types found on Con Son Island. The fi rst type was found in natural forests, with the presence of reddish-brown stripes, a bigger size (l = 133-170 mm, d = 5.0-6.5 mm), the fi rst dorsal pore in 13/14 and ventral distance between spermathecal pores ca 0.38-0.4 body circumference. The second type found in residential gardens and forest edges exhibited no stripes, a smaller size (l = 64-122 mm, d = 3.0-3.4 mm), the fi rst dorsal pore in 12/13 and ventral distance between spermathecal pores ca 0.43-0.45 body circumference. The COI genetic distance also showed the variations of 2.2-11.3%.

Remarks
A few Metaphire species have been described with fi ve pairs of spermathecal pores, including M. megascolidioides and M. fordi Michaelsen, 1934. The new species is very similar to M. megascolidioides with its fi rst dorsal pore in 12/13 and an absence of genital markings in the spermathecal region. However, M. decemtheca sp. nov. is distinguished by having male pores on xviii, a pair of genital markings in xviii, separate intestinal caeca and a small size (l = 64-185 mm, d = 3.0-6.0 mm) whereas M. megascolidioides has male pores on xix, three pairs of genital markings in xvii, xviii and xx, manicate intestinal caeca and a larger size (l = 240 mm, d = 15 mm).
The new species is also distinguished from M. fordi by having a larger size (l = 64-185 mm, d = 3.0-6.0 mm vs l = 50-64 mm, d = 1-1.5 mm) and the fi rst dorsal pore in 12/13 (vs 11/12). Additionally, M. decemtheca sp. nov. has no genital markings in the spermathecal region, but does have one pair of large, round genital markings located next to the openings of copulatory pouches in xviii. By contrast, M. fordi has small circular papillae paired in both spermathecal and male regions.
In terms of the morphology of the male region, the new species is very similar to M. grandiverticulata Nguyen & Lam, 2017 by having a pair of large, round genital markings located next to the openings of its copulatory pouches and an absence of genital markings in the spermathecal region. However, M. decemtheca sp. nov. has fi ve pairs of spermathecal pores on 4/5/6/7/8/9, a larger size (l = 64-185 mm, d = 3.0-6.0 mm), connected testis sacs, oval-shaped spermathecal ampulla, stout ducts, strongly and constrainedly waved diverticula and a small oval-shaped seminal chamber. Meanwhile, M. grandiverticulata has four pairs of spermathecal pores on 5/6/7/8/9, a smaller size (l = 69-92 mm, d = 2.3-2.7 mm), separated testis sacs, a small heart-shaped spermathecal ampulla, extremely short muscular ducts, stout and long unwaved diverticula and a bullet-shaped seminal chamber.

Statement of DNA dataset
The COI dataset comprises a 609 bp fragment from 36 sequences of 8 earthworm species including an outgroup species, Polypheretima elongata (  (Table 4).
The species Metaphire iranomala sp. nov. has genetic variations of 0.2% to 9.4%. The maximum distance between two types is 9.4%, but it is considered to be less than the average distance of 16%. Therefore, it is suggested that two morphological types still refl ect one species. Similarly, the species M. decemtheca sp. nov. also has genetic divergences of 2.2% to 11.3% corresponding to morphological variations.
The K2P species divergences were reported, but were different for earthworm groups, for example, 13-15% for Allolobophora Eisen, 1874 (King et al. 2008

Phylogenetic relationship
A phylogenetic tree was reconstructed for a 609 bp dataset using the Likelihood ML and Inference BI analysis (Fig. 4) Nguyen et al. (2016) provided the comprehensive checklist of 212 earthworm species in Vietnam. Earthworms have been well surveyed in most areas in Vietnam, but not in highly mountainous or remote areas. For example, earthworms on islands still have been poorly recognised, except some data in Nam Du (Michaelsen 1934), Con Son (Thai et al. 2004), Lai Son, Hon Tre, Nam Du and Phu Quoc (Nguyen et al. 2017a(Nguyen et al. , 2020. Several species were also recently described from Lai Son Island and Phu Quoc Island (Nguyen et al. 2017a(Nguyen et al. , 2020. It is thus suggested that more intensive surveys in islands of Vietnam would bring more new taxa to discovery. Nguyen et al. (2016) also suggested some species should be revised or rechecked to confi rm their taxonomic status because those species, which were recorded in Vietnam, differentiated from original descriptions, e.g., Metaphire multitheca (Chen, 1938), M. anomala (Michaelsen, 1907). These records of M. multitheca and M. anomala in Vietnam have been corrected and found to be new species, M. erroneous Nguyen & Nguyen, 2015 and M. iranomala sp. nov.

Discussion
The high intraspecifi c divergences in Metaphire iranomala sp. nov. and M. decemtheca sp. nov. may suggest more Vietnamese species containing these cryptic forms, such as M. houlleti (Perrier, 1872) (as discussed in Nguyen et al. 2018). The cryptic speciation events have also been reported in many earthworm species using either mitochondrial data (King et al. 2008;James et al. 2010;Novo et al. 2010) or nuclear data (Rougerie et al. 2009). The COI barcode was also applied to discriminate earthworm species and to suggest cryptic forms (Jeratthitikul et al. 2017).