Notes on Metaphire multitheca (Chen, 1938) (Oligochaeta, Megascolecidae) recorded from Vietnam, with descriptions of two new species

Abstract The paper deals with Pheretima multitheca multitheca Chen, 1938 recorded from Vietnam (non Pheretima multitheca Chen, 1938 now in Metaphire from Hainan Island). As a result, a new species, Amynthas erroneous sp. n., is revealed from materials which were previously misidentified as Pheretima multitheca multitheca. The new species is obviously distinguished from other Amynthas species by multiple spermathecal pores lateroventral in intersegments 5/6/7/8/9, and presence of two pairs of crescentic genital markings in xviii. In addition, another new species, Amynthas nhonmontis sp. n., is described and easily recognized by multiple spermathecal pores ventral in intersegments 5/6/7/8 and three pairs of genital markings in xvii, xix and xx.


Introduction
The species, Metaphire multitheca (Chen, 1938) was originally described from Hainan Island (China) in genus Pheretima by Chen (1938). The species is recognized by multiple spermathecal pores (more than two spermathecal pores per segment) in vi, vii and viii, two pairs of genital markings in 17/18 and 18/19, and presence of copulatory pouches. The species, was subsequently recorded from various parts of Vietnam by Nguyen (1994), Pham (1995Pham ( , 2010, Huynh (2005aHuynh ( , 2005b, and Nguyen and Tran (2008). However, all of these authors commented that the population from Vietnam has differences from the original description, such as absence of copulatory pouches, and multiple spermathecal pores in intersegments 5/6/7/8/9.
While examining new materials, we realized that all previous records have been misidentified as Pheretima multitheca Chen, 1938 requiring its naming as a new species. In addition, another new species having multiple spermathecae per segment is also described from Vietnam.

Material and methods
Examined specimens were previously collected from various parts of Vietnam and deposited in: SORC Soil Organism Research Center; HNUE Hanoi National University of Education; CTU Laboratory of Zoology, Cantho University, Cantho City, Vietnam.
Material for DNA barcoding was taken from holotype. The primer sets, LCO1490 and HCO2198, used in a wide range of invertebrate taxa were used to amplify a fragment of the cytochrome c oxydase subunit I gene (Folmer et al. 1994).
Holotype and paratypes are deposited in the Laboratory of Zoology, Cantho University (= CTU), Cantho City, Vietnam.

Taxonomic account
Remarks. Members of the genus can be easily recognized by the presence of intestinal caeca near xxvii, the absence of copulatory pouches, and often by absence of micronephridia on spermathecal ducts. It is noted that about 500+ nominal species have been recorded, but a considerable number are likely synonyms (Blakemore 2002(Blakemore , 2007.
Male pores located in porophores in xviii, without copulatory pouches; ventral distance between male porophores about 0.28× body circumference. Two pairs of large, crescentic genital markings in xviii, located in front of and behind male porophores.
Holandric. Testis sacs not separated, developed in x and xi. Seminal vesicles well developed within xi-xii, yellowish white. Oviduct poorly developed on septum 12/13 posteriorly; a pair of ovaries in xiii. Prostate glands racemose, paired in xvii-xix; prostatic ducts C-shaped. Two accessory glands present.
DNA. COI barcode data not yet available.
Habitat and ecology. The species was found in soils in which old growth trees had been grown. No other ecological data had been recorded.

No Characters
The new species is widely distributed in central parts and highlands of Vietnam. Surprisingly, it has never yet been found in northern and southern Vietnam.
Multiple spermathecal pores ventral in intersegments 5/6/7/8. No genital markings in spermathecal region. Male porophores flattened; male pores located in setal ring in xviii, without copulatory pouches. Ventral distance between male porophores about 0.3x body circumference. Genital markings usually three pairs in setal rings in xvii, xix and xx, rarely more or less than three pairs; genital markings arranged in longitudinal line with male pores.
DNA. COI barcode data (partial) is for holotype uploaded to GenBank with accession number KR676559.
Habitats and ecology. This species has been found only in Nhon mountain (Tinh Bien district) in An Giang province. Adult specimens were only collected during the rainy season (from October to March) in southern Vietnam and found in heavy clays at the foothill of Nhon mountain. Remarks. Amynthas nhonmontis sp. n. differs from other Amynthas species in having multiple spermathecal pores ventral in intersegments 5/6/7/8 and three pairs of genital markings in xvii, xix and xx. The new species is superficially similar to Polypheretima elongata (Perrier, 1872) due to external morphology: body cylindrical, multiple spermathecae per segment, presence and arrangement of genital markings near male pores. However, it is clearly distinguished from Po. elongata by presence of intestinal caeca, three spermathecal segments, absence of copulatory pouches.
Its DNA barcode is definitive for the new species (nearest BLAST result is Amynthas morrisi at 87%).

Conclusion
Two new species of the genus Amynthas Kinberg, 1867 are described. Both of them are characterised by multiple spermathecal pores per segment, lack of copulatory pouches, and presence of intestinal caeca from xxvii. To date, only eight species having multiple (more than two) spermathecae per segment have been found in Vietnam. They are arranged in three genera: Polypheretima Michaelsen, 1934, Amynthas Kinberg, 1867and Metaphire Sims & Easton, 1972 Polypheretima spiridonovi (Thai, 1996), from Khanh Hoa province, southern Vietnam (stat. n. from Blakemore 2007Blakemore , 2008 (Thai & Tran, 1986), stat. n. from Nghe An province, central Vietnam.