Notes on the earthworm species, Metaphire anomala (Michaelsen, 1907) (Clitellata, Megascolecidae) in Southern Vietnam, with descriptions of two new species Author Nguyen, Tung T. 55B94873-F7A0-461A-9C33-600FE5929A28 Department of Biology, School of Education, Can Tho University, Can Tho City, Vietnam. Department of Soil Ecology, Institute of Ecology and Biological Resources, Vietnam. Academy of Science and Technology, 18, Hoangquocviet, Caugiay, Hanoi, Vietnam. Graduate University of Science and Technology, Vietnam Academy of Science and Technology, 18, Hoangquocviet, Caugiay, Hanoi, Vietnam. thanhtung@ctu.edu.vn Author Lam, Dang H. F41F98AF-5FE5-4063-86E4-7D7AC3F50931 Department of Biology, School of Education, Can Tho University, Can Tho City, Vietnam. Department of Soil Ecology, Institute of Ecology and Biological Resources, Vietnam. Academy of Science and Technology, 18, Hoangquocviet, Caugiay, Hanoi, Vietnam. Graduate University of Science and Technology, Vietnam Academy of Science and Technology, 18, Hoangquocviet, Caugiay, Hanoi, Vietnam. lamhaidangct@gmail.com Author Nguyen, Anh D. FCE53D0A-593C-4ACC-800B-E0237DC185F4 Department of Soil Ecology, Institute of Ecology and Biological Resources, Vietnam. Academy of Science and Technology, 18, Hoangquocviet, Caugiay, Hanoi, Vietnam & Graduate University of Science and Technology, Vietnam Academy of Science and Technology, 18, Hoangquocviet, Caugiay, Hanoi, Vietnam. ducanh410@yahoo.com text European Journal of Taxonomy 2021 2021-04-21 746 94 111 http://dx.doi.org/10.5852/ejt.2021.746.1321 journal article 10.5852/ejt.2021.746.1321 b2f74aea-802a-48ea-95c5-36fb3f34bc82 2118-9773 4709138 90ACC242-A5C2-4644-B09F-E57F63D3FF88 Metaphire iranomala sp. nov. urn:lsid:zoobank.org:act: 2ECB8639-C770-4828-92D9-055E0778E422 Figs 1–3 , Tables 2–3 Pheretima anomala (non Pheretima anomala Michaelsen, 1907 ) – Thai et al. 2004: 758 . – Nguyen 2014: 108 . – Nguyen et al. 2017a: 893 , fig. 7; 2017b: 98, fig. 3 ; Nguyen et al. 2019: 120 , fig. 3. Metaphire anomala – Nguyen et al. 2016: 50 . Diagnosis Medium-sized worm, length 157–228 mm , diameter 6.5–8.0 mm, segments 85–145. Prostomium epilobous. Clitellum annular, within xiv–xvi. First dorsal pore in 12/13. Four pairs of spermathecal pores on ventrolateral intersegments 5/6/7/8/9. Male pores on the setal ring of segment xix; copulatory pouches present. Genital markings absent. Holandric. Testis sacs connected. Intestinal caeca simple. Septa 8/9/10 absent. Etymology The epithet ‘ iranomala ’ is formed by the prefix ‘ ir ’ and ‘ anomala ’ to emphasise the wrong name ‘ anomala ’ recorded in Vietnam . Material examined Holotype VIETNAM • mature spec .; Ba Ria-Vung Tau Province , Con Dao National Park ; 8°42′12″N ; 106°35′41″ E ; 120 m a.s.l. ; 19 Oct. 2019 ; Nguyen Thanh Tung and Nguyen Thi Bao Ngoc leg.; natural forest ; CTU-EW.020.h01 . Paratypes VIETNAM • 9 matures ; same collection data as for holotype; CTU-EW.020.p02 . Other material VIETNAM – Ba Ria-Vung Tau Province • 16 matures ; same collection data as for holotype; CTU-EW.020.03 • 10 matures ; Con Son Island ; 8°41′59″ N , 106°36′54″ E ; 10 m a.s.l. ; 18 Oct. 2019 ; Nguyen Thanh Tung and Nguyen Thi Bao Ngoc leg.; residential gardens ; CTU-EW.020.04 • 33 matures ; Con Son Island ; 8°39′53″ N , 106°34′00″ E ; 20 m a.s.l. ; 19 Oct. 2019 ; Nguyen Thanh Tung and Nguyen Thi Bao Ngoc leg.; natural forest ; CTU-EW.020.05 . – Kien Giang Province • 1 mature ; Da Dung mountain ; 10°25′07″ N , 104°28′46″ E ; Nov. 2010 ; Nguyen Thanh Tung leg.; natural forest ; CTU- EW.DNA.020.23 • 49 matures ; Lai Son Island ; 09°48′01″ N , 104°39′18″ E ; Nov. 2013 ; Trinh Thi Kim Binh leg.; natural forest ; CTU-EW.020.11 . – An Giang Province • 57 matures ; Cam mountain ; 10°30′36″ N , 105°00′09″ E ; Nov. 2010 ; Nguyen Thanh Tung leg.; natural forest ; CTU-EW.020.12 • 2 matures ; Cam mountain ; 10°30′36″ N , 105°00′09″ E ; Nov. 2010 ; Nguyen Thanh Tung leg.; natural forest ; IEBR-EW.020.22 . – Dong Nai Province • 2 matures ; Vinh Cuu Nature Reserve ; 11°06′46″ N , 107°03′12″ E ; Oct. 2019 ; Nguyen Quoc Nam leg.; natural forest ; IEBR-EW.020.18a • 3 matures ; Vinh Cuu Nature Reserve ; 11°06′46″ N , 107°03′12″ E ; Oct. 2019 ; Nguyen Quoc Nam leg.; natural forest ; CTU-EW.DNA.020.18b • 7 matures ; Cat Tien National Park ; 11°26′05″ N , 107°25′45″ E ; Oct. 2013 ; Le Van Nhan leg.; natural forest ; CTU-EW.020.07 . – Tay Ninh Province • 1 mature ; Duong minh Chau District ; 11°22′59″ N , 106°12′00″ E ; Sep. 2019 ; Nguyen Quoc Nam leg.; home garden ; IEBR-EW.020.16 • 6 matures ; Ba Den mountain ; 11°23′26″ N , 106°09′19″ E ; Sep. 2019 ; Nguyen Quoc Nam leg.; mango gardens ; CTU-EW.020.17 . Description Body cylindrical, large-medium size, length 157–228 mm , diameter 6.5–8.0 mm, segments 85–145, weight 3.0–8.4 gr. Dorsum slightly dark grey, ventrum paler. Prostomium epilobous. First dorsal pore in 12/13. Perichaetine, setae at pre-clitellar segments stouter and sparser than that at post-clitellar segments; setal numbers: 55–62 in viii, 65–71 in xxx, 10–16 between male pores on xix. Setal distance: aa> ab, zz> zy. Clitellum annular, within xiv–xvi, darkish brown, without dorsal pores and setae. Female pore single, mid-ventral in xiv. Four pairs of spermathecal pores on ventrolateral intersegments 5/6/7/8/9. Ventral distance between spermathecal pores ca 0.27–0.3 body circumference. Male pores on copulatory pouches in xix; ventral distance between male pores ca 0.2–0.3 body circumference. Genital markings absent in both spermathecal and male pores region. Septa 5/6/7/8 thickened, 8/9/10 absent, 10/11/12 thin. Oesophageal gizzard between 7/8 and 10/11. Intestine origin at xv; caeca simple, short within xxvii-xxiv. Last hearts in xiii. Pharyngeal micronephridia developed in 5/6/7. Lymph glands absent. Typhlosole simple, lamelliform. 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. Holandric. Testis sacs in x and xi, connected ventrally. Seminal vesicles well developed in xi and xii. Ovaries on septum 12/13 posteriorly. Prostate glands, deeply lobuled, paired in xvii–xxi; prostatic ducts C-shaped. Accessory glands massed, covered the copulatory pouches. DNA barcode COI barcode data (partial) is for the paratypes uploaded to GenBank under the accession numbers MW076191 , MW076192 , MW076193 , MW076194 , MW076195 , and MW076196 . The new species shares the identity of 88.13% and 88.33% with Metaphire anomala (KU262251, KU565252 , KU565253 , KU565254 ). 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 first type is more likely to be distributed in islands or in coastal provinces in Vietnam ; the other type is found in mainland provinces. Fig. 1. Metaphire iranomala sp. nov. , holotype (CTU-EW.020.h01). A1, A2 . Ventral view of male pores region. B . Lateral view of spermathecal pores region. C1, C2 . Right spermathecae. D1, D2 . Intestinal caecum. E . Testis sacs and seminal vesicles. F1, F2 . Right prostatic gland. G . Copulatory pouch transverse body section. Scale bars = 1 mm. Fig. 2. Metaphire decemtheca sp. nov. , holotype (CTU-EW.183.h01). A1, A2 . Ventral view of male pore region. B1, B2 . Right spermathecae. C1, C2 . Intestinal caecum. D . Ventral view of spermathecal pore region. E1, E2 . Lateral view of spermathecal pores region. F1, F2 . Left prostatic gland. G . Testis sacs and seminal vesicles. H . Male pore region transverse body section. Scale bars = 1 mm. Table 2. Comparison between M. iranomala sp. nov. and M. anomala ( Michaelsen, 1907 ) from Thailand, Myanmar and India (its original description).

Characteristics	Vietnam 1	Thailand 2	Myanmar 3	Original description (India) 4
Length	157–228	134–154	116–200	80–90
Diameter	6.5–8.0	4.6–6.7	3.0–7.0	5.0–5.5
Segment	85–145	116–126	119–130	130
Spermathecal pores	four pairs in 5/6/7/8/9	3 pairs in 5/6/7/8	absent or 5/6/7/8	absent
Pre-clitellar setae	46–53 (viii)	84–96	90–96 (viii)	70 (v)
Post-clitellar setae	62–71 (xxx)	58–67	61–70 (xx)	74 (xxv)
Male pores	xix	xx	xx	xx
Ventral distance between male pores	0.2–0.3	0.33	–	0.17
Genital markings	absent	3–6 pairs in xvii–xix, xxi–xxiii or absent	present in xvii–xix and xxi–xxiv	4 pairs in xviii– xix, xxi–xxii
Septum 8/9	absent	absent	present	present
Testis sacs	connected	separated	separated	separated

1 Freshly collected specimens; 2 Bantaowong et al. (2011) ; 3 Gates (1925 , 1972 ) and Stephenson (1929) ; 4 Michaelsen (1907) . Table 3. Comparison between M. iranomala sp. nov. , M. megascolidioides ( Cognetti, 1908 ) and M. isselii ( Goto & Hatai, 1899 ) sharing location of male pores not in segment xix.

Characteristics	M. iranomala	M. megascolidioides 1	M. isselii 2
Length	157–228	240	155–180
Diameter	6.5–8.0	15	10
Segment	85–145	118	94–115
Spermathecal pore	lateroventral, 5/6/7/8/9	lateroventral, 4/5/6/7/8/9	ventral, 6/7/8/9
Setal number between two male pores	10–16	9	0
Ventral distance between two male pores	0.2–0.3	?	<0.25
Genital markings in male region	absent	3 pairs in xvii, xviii and xx	2 pairs in 18/19 and 19/20
Septum 8/9	absent	absent	thin
Intestinal caeca	simple	manicate	simple
Intestinal origin	xv	xv	xvi

1 Goto & Hatai (1899) and Blakemore (2016b) ; 2 Cognetti (1908) . 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 identified as Metaphire anomala . It is widely distributed in Southern Vietnam ( Thai et al. 2004 ; Nguyen 2014 ; Nguyen et al. 2017a , 2017b , 2019 , 2020 ). However, this species is very different from both the original description ( Michaelsen 1907 ), and re-description of M. anomala from Myanmar ( Gates 1925 , 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 and M. 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 first 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 .