Metaphire decemtheca, Nguyen & Lam & Nguyen, 2021

Nguyen, Tung T., Lam, Dang H. & Nguyen, Anh D., 2021, Notes on the earthworm species, Metaphire anomala (Michaelsen, 1907) (Clitellata, Megascolecidae) in Southern Vietnam, with descriptions of two new species, European Journal of Taxonomy 746, pp. 94-111 : 103-107

Metaphire decemtheca sp. nov.

urn:lsid:zoobank.org:act:F717F325-F04E-409A-93B1-0A3997E11E9E

Figs 2–3

Diagnosis

Medium-sized worm. Prostomium epilobous. First dorsal pore in 12/13. Clitellum annular, within xiv– xvi. Five pairs of spermathecal pores on ventrolateral intersegments 4/5/6/7/8/9. Male pores on the setal ring of xviii; copulatory pouches present. A pair of genital markings in xviii. Holandric. Testis sacs connected. Intestinal caeca simple. Septum 8/9 absent or very thin, 9/10 absent.

Etymology

The epithet ‘ decemtheca ’ emphasises the number of spermathecae of the new species.

Material examined

Holotype VIETNAM • mature; Ba Ria-Vung Tau Province, Con Son Island ; 8°42′17″ N, 106°35′28″ E; 50 m a.s.l.; 19 Oct. 2019; Nguyen Thanh Tung and Nguyen Thi Bao Ngoc leg.; natural forest; CTU-EW.183.h01 .

Paratypes VIETNAM • 9 matures; same collection data as for holotype; CTU-EW.183.p02 .

Other material

VIETNAM – Ba Ria-Vung Tau Province • 38 matures; same collection data as for holotype; CTU-EW.183.03 • 23 matures; Con Son Island ; 8°43′50″ N, 106°37′31″ E; 10 m a.s.l.; 18 Oct. 2019; Nguyen Thanh Tung and Nguyen Thi Bao Ngoc leg.; natural forest; CTU-EW.183.04 • 55 matures; Con Son Island ; 8°43′59″ N, 106°37′21″ E; 10 m a.s.l.; 18 Oct. 2019; Nguyen Thanh Tung and Nguyen Thi Bao Ngoc leg.; residential gardens; CTU-EW.183.05 .

Description

Body cylindrical, medium-sized worms. Length 64–185 mm, diameter ca 3.0–6.0 mm, segments 79– 124, weight 1.1–3.9 gr. Dorsum slightly dark grey with reddish browns stripes or absent, ventrum paler. Prostomium epilobous. First dorsal pore in 12/13. Perichaetine; setae at pre-clitellar segments stouter and sparser than that at post-clitella segments; setal numbers: 38–51 in viii, 46–57 in xxx, 8–13 between two male pores on xviii. Setal distance: aa> ab, zz = zy. Clitellum annular (3/4xiv–3/4xvi), slightly brown, without dorsal pores and setae. Female pore single, mid-ventral in xiv.

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 flat ellipsoidal genital markings in xviii located next to male pores ventrally.

Septa 5/6/7/8 thick, 8/9 absent or membrane (if present), 9/10 absent, 10/11/12/13 thin. Oesophageal gizzard between 7/8 and 10/11. Intestine origin at xv; caeca simple, somewhat rudimentary lobulated dorsally, within xxvii–xxv. Last hearts in xiii. Pharyngeal micronephridia developed in 5/6/7. Lymph glands sac-like, present from 27/28. Typhlosole simple, lamelliform.

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.

Holandric. Testis sacs in x and xi, connected ventrally. Seminal vesicles developed in xi and xii. Ovaries on septum 12/13 posteriorly. Ovisacs developed on 12/13, poorly developed on 13/14. Prostate glands deeply lobuled within xvi-xx; duct S-shaped, enlarged basally. Accessory glands sessile massed corresponding to external genital markings.

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 first 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 first 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 first 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 five pairs of spermathecal pores, including M. megascolidioides and M. fordi Michaelsen, 1934 .The new species is very similar to M. megascolidioides with its first 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 first 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 five 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.

Molecular analysis

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 1 ). The nucleotide frequencies of A, T, G and C were 29.7%, 28.7%, 18.6% and 23.0%, respectively. The GC content was 41.6%. The combined dataset contained 210 (34.5%) parsimony informative and 216 (35.5%) variable sites.

Genetic distance

The average K2P distance is 16.0% between earthworm morphospecies. The K2P distance between species varies from 13.0% ( Metaphire iranomala sp. nov. and M. anomala ) to 23.5% ( M. peguana and M. grandiverticulata ). The intraspecific distance varies from 0.2% ( Polypheretima elongata , M. megascolidioides ) to 8.0% ( M. decemtheca sp. nov.). The new species, M. decemtheca sp. nov., has the K2P divergence of 17.7% with M. megascolidioides and of 18.3% with M. grandiverticulata ( 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 reflect 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), 13.2% for Eisenia fetida / andrei complex ( Römbke et al. 2016), more than 14% in Octolasion lacteum Örley, 1885 , Lumbricus rubellus Hoffmeister, 1843 ( Klarica et al. 2012), more than 18.7% in pheretimoid species ( Chang et al. 2008), 19.8% in the genus Lumbricus Linnaeus, 1758 ( James et al. 2010), 8.9 – 22.9% in the genus Pheretima Kinberg, 1867 , 14.2 – 21.9% in the genus Amynthas Kinberg, 1867 , 7.5 – 18.0% in the genus Pithemera Sim & Easton, 1972 and 11.7–21.0% in the genus Polypheretima Michaelsen, 1934 ( Aspe & James 2018). Recently, Jeratthitikul et al. (2017) also calculated the average species distance of 20% for Amynthas and Metaphire species in Thailand. Our calculation of 16% only applies to the Metaphire species.

Phylogenetic relationship

A phylogenetic tree was reconstructed for a 609 bp dataset using the Likelihood ML and Inference BI analysis( Fig. 4 ).Two new species are clearly separated from their congeners. Metaphire iranomala sp. nov. is a sister clade of M. anomala with strong supports of bootstrap and BI values (90% and 1.00 bpp, respectively). Two subclades of M. iranomala sp. nov. are also well supported with bootstrap and BI values (89% and 0.92 bpp, respectively).

Metaphire decemtheca sp. nov. is related to M. grandiverticulata and M. megascolidioides with moderate bootstrap support and strong BI values of 75% and 0.95 bpp. Within the clade of M. decemtheca sp. nov., two morphological variations also represent two linages: CTU-EW.183.06 and CTU-EW.183.p02 + CTU- EW.183.03. The relationship is well supported with bootstrap of 96% and BI value of 1.00 bpp.

Figures

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.

Fig. 3. The distribution of the two new species in Southern Vietnam.

Fig. 4. The phylogenetic diagram inferred from the 594 bp dataset using the Maximum Likelihood (ML) and Bayesian Inference (BI) analysis. The values at node show the bootstrap and BI. The information in parentheses are locations and number of spermathecal pores.