Description of Hemienchytraeus wuhanensis sp. nov. (Annelida, Clitellata, Enchytraeidae) from central China, with comments on species records of Hemienchytraeus from China

Abstract Hemienchytraeus wuhanensissp. nov. is described from hardwood forest soil in Wuhan, China. This moderately sized enchytraeid species of 6–9 mm body length is characterized by: (1) an oesophageal appendage with tertiary branches, (2) three pairs of secondary pharyngeal gland lobes in V, VI, VII, (3) five pairs preclitellar nephridia, from 5/6 to 9/10, (4) dorsal vessel originating in clitellar segments, (5) a girdle-shaped clitellum, (6) a relatively small male reproductive apparatus without seminal vesicle, and (7) spermathecae that extend to VI–VII. DNA barcodes of paratype specimens of the new species are provided. Previous species records of Hemienchytraeus from China are critically discussed.

To date, 24 species have been reported worldwide (Schmelz and Römbke 2005;Dózsa-Farkas and Hong 2010;Schmelz et al. 2015). These species are mainly distributed in America (12 species), Asia (11 species), Africa (3 species), and Europe (2 species). Six species have been reported from China so far: H. stephensoni Cognetti, 1927, H. bifurcatus Nielsen & Christensen, 1959, H. loksai Dózsa-Farkas, 1989, H. theae Prabhoo, 1960, H. planisetosus Xie et al., 1999, and H. brachythecus Xie et al., 1999(Wang and Cui 2007. Of these, the latter two are only known from China. In this paper, we add a new member to this list, which was collected from Wuhan, China. We describe the morphology of the species and compare it with congeners. We also provide COI sequences of Hemienchytraeus wuhanensis sp. nov. and calculate genetic distances using the sequences of Hemienchytraeus spp. available in GenBank. Finally, we comment on species finds of Hemienchytraeus spp. in China.

Materials and methods
Soil samples were collected at forest sites at the Huazhong Agricultural University and Wuhan University, Wuhan, in April 2019. The samples were directly scooped using a steel shovel to a depth of ca 15 cm, placed in a breathable cloth bag and taken to the laboratory and stored at 4 °C. Worms were extracted from soil using a standard hot wet funnel extracting device (O'Connor 1962;Healy and Rota 1992). All worms were examined and identified alive. Body size, colour, movement, and maturity were observed with a Zeiss Stemi 508 stereomicroscope. Other characters were examined, measured, and photographed with a Zeiss Axio Imager A2 microscope using differential interference contrast optics and a Zeiss Axiocam 305 color digital camera with ZEN 2011 Blue Version software. The specimens were then anaesthetized in 30% ethanol and preserved in 75% ethanol (Dózsa-Farkas and Hong 2010). For taxonomic observation, some mature specimens were stained with borax-carmine, dehydrated in an ethanol series from 70% to absolute, mounted temporarily in clove oil and permanently mounted in neutral balsam (Dózsa-Farkas et al. 2015;Zhang et al. 2018).
Drawings from whole mounts were made with the help of an Olympus drawing tube. Type material is deposited in the Museum of Aquatic Organisms (MAO), Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China.
Total genomic DNA was extracted from five entire individuals respectively, using TIANamp Micro DNA Kit (Tiangen Biotech, Beijing, China). The COI gene was amplified from each DNA extract with primers LCO1490 (5'-GGTCAACAAATCAT-AAAGATATTGG-3') and HCO2198 (5'-TAAACTTCAGGGTGACCAAAAAAT-CA-3') (Folmer et al. 1994). These five specimens, of which no morphological parts are left, are part of the type series, as paratypes. Eight COI gene sequences of four different species in genus Hemienchytraeus were downloaded from GenBank, alignments were trimmed (resulting alignments were 591bp), aligned and K2P genetic distances were calculated using MEGA-X (Kumar et al. 2018).
Etymology. Named after the city where the species was found. Distribution and habitat. Mineral soil and organic layers under camphor trees near a narrow, tarred road at Mount Shizi, Huazhong Agricultural University; mineral soil and organic layers under pine trees at Mount Luojia, Wuhan University. The two hills are about 10 km apart, with little human disturbance.
Brain about as long as wide (117 mm long, 93 mm wide, in vivo), slightly indented anteriorly, deeply incised posteriorly (Figs 2B, 3A). Oesophageal appendage arising from mid-dorsal region of pharynx in III as an unpaired root with large proximal chamber; following section longer than proximal chamber, with thick, meandering canal; two primary branches, longer than root, with smaller canal; each primary branch bifurcating into two short, secondary branches; each secondary branch bifurcating into four or more tertiary branches, the latter difficult to distinguish. Secondary and tertiary branches of same diameter, thinner than primary branches (Figs 2E, 3B, C). All three pairs of pharyngeal glands united dorsally, primary ventral lobes in V and VI. Three pairs of secondary pharyngeal gland lobes in V, VI and VII, small in VII (Figs 2D, 3D). Dorsal vessel from XII-XIII, blood colorless.

Molecular results
COI sequences of five paratype specimens of H. wuhanensis sp. nov. were successfully acquired and submitted to GenBank with accession numbers. This is the fourth species of Hemienchytraeus of which DNA sequences are available (Table 1), the other three being H. quadratus, H. koreanus, and H. jeojunensis Dózsa-Farkas & Hong, 2010, all from South Korea. Clear genetic gaps were observed among the four species with high interspecific distances (7.0-21.9%) and low intraspecific distances (0%) among H. wuhanensis sp. nov. specimens based on the K2P distances of COI sequences (Table 2). Interestingly, among the three species from South Korea, the one with lowest genetical distance to H. wuhanensis sp. nov., H. koreanus, is also the one which is most similar morphologically to the new species (see below).  remarks Three non-sexual characters have been shown to be very useful for the distinction of Hemienchytraeus species: oesophageal appendage (branching pattern, relative branch length), secondary pharyngeal gland lobes (number, position, size), and preclitellar nephridia (number, position) (Schmelz et al. 2009). Indeed, these three characters in H. wuhanensis suffice to distinguish it from all other species, even from those with an incomplete description, because details of the oesophageal appendage are known in all species, the only exception being H. brasiliensis (Cognetti, 1900), a species of uncertain identity (incertae sedis) according to Schmelz and Römbke (2005). Further useful characters include the origin of the dorsal blood vessel, presence/absence of a seminal vesicle, shape and size of spermathecae, sperm funnels and male glandular bulbs, and distribution pattern of clitellar gland cells; the latter is fully known only in recently described species. Considering the three above-mentioned non-sexual diagnostic characters, the new species is most similar to H. loksai Dózsa-Farkas, 1989, which also has an oesophageal appendage with tertiary branches, three pairs of secondary pharyngeal gland lobes in V, VI, VII, and five pairs of preclitellar nephridia, from 5/6 to 9/10. However, in H. loksai the secondary pharyngeal glands increase in size from IV to VII. The species was described from Ecuador and has been recorded from China (Xie et al. 1999). Further conspicuous differences of H. loksai from the new species include larger body size (length >12 mm, 49-55 segments), a postclitellar origin of the dorsal blood vessel, larger spermathecae (extending to IX-X), very large sperm funnels (up to 800-900 μm long), and a huge seminal vesicle (extending into XIV-XVII).
One more species of Hemienchytraeus has oesophageal appendages with tertiary branches, i.e., H. brachythecus Xie et al., 1999. This species is also similar to the new species in the absence of a seminal vesicle. Conspicuous differences of H. brachythecus include a very short spermatheca, confined to V, two pairs of secondary pharyngeal gland lobes in V and VI, and first pair of preclitellar nephridia in 6/7. Three pairs of secondary pharyngeal gland lobes are also known in H. koreanus Dózsa-Farkas &Hong, 2010, andin H. siljae Schmelz &Römbke, 2005. H. koreanus resembles the new species also in the position of the preclitellar nephridia (5/6-9/10) and in a girdleshaped clitellum. Conspicuous differences of H. koreanus include a postclitellar origin of the dorsal blood vessel, large spermathecae, and the presence of a seminal vesicle.
H. siljae resembles the new species in several characters, for example the girdleshaped clitellum, the absence of a seminal vesicle, and the approximate shape and size of spermathecae and sperm funnels. Conspicuous differences include a more posterior origin of the dorsal blood vessel (XIV), four pairs of preclitellar nephridia, from 6/7 to 9/10, and an oesophageal appendage with three elongate secondary branches on each side, without tertiary branches.
Hemienchytraeus bifurcatus Nielsen & Christensen, 1959 originally described from Denmark, has been considered a "species inquirenda" (Schmelz and Römbke 2005), because the original description is incomplete with respect to secondary pharyngeal gland lobes, preclitellar nephridia, and details of the clitellum. A validation of H. bifurcatus is difficult because type material is lost, and efforts to obtain fresh material at the type locality have so far been unsuccessful (Schmelz and Römbke 2005). Hence, the records of this species from China (Liang and Xie 1992;Wang and Liang 2002) cannot be confirmed; those specimens may just as well belong to a new species.
The redescription of H. loksai by Xie et al. (1999) based on material from Hunan Province, China, agrees with the original description in conspicuous details (e.g., size of seminal vesicle and sperm funnels) but lacks information on the secondary pharyngeal gland lobes; furthermore, the first preclitellar nephridia are in 6/7, not in 5/6 as originally described. Material of H. loksai sensu Xie et al. (1999) should be reinvestigated to confirm the species identity of the specimens.
Finally, H. theae Prabhoo, 1961 described from India, and recorded from China by Liang and Xie (1992), was originally insufficiently described: secondary pharyngeal gland lobes, number and position of nephridia, details of the clitellum, and origin of the dorsal blood vessel are unknown. Reinvestigation of the type material present at the Zoological Survey of India (Prabhoo 1961) and comparison with the material un-derlying the record of Liang and Xie (1992) would be necessary to confirm the species identity of the Chinese specimens.
Despite these taxonomic uncertainties, the presence of at least seven species of Hemienchytraeus in China is beyond doubt. Actually, many more species of Hemienchytraeus are to be expected in this country, in view of the preference for tropical or subtropical soils of this globally distributed genus.