﻿Microdousamblyrhynchos sp. nov., a new member of the small-toothed sleepers (Teleostei, Gobiiformes, Odontobutidae) from Guangxi, southern China

﻿Abstract Microdousamblyrhynchos, a new species, the second one in the genus, from the family Odontobutidae, is described from the Hongshui River, in the upper reaches of the Xijiang River of the Pearl River drainage, Baise City, Guangxi Zhuang Autonomous Region, southern China. This species is distinguished from its only congener, M.chalmersi, by the blunt snout (vs. pointed); mean snout length/head length ratio 0.27 (vs. 0.3); eye not extending outward (vs. protruding); mean interorbital width/head length ratio 0.25 (vs. 0.11). Additionally, the results of molecular phylogenetic analysis confirmed that M.amblyrhynchossp. nov. is distinct from its sister species, M.chalmersi.


Introduction
The family Odontobutidae comprises about 15-22 species in seven genera (Iwata 2011;Li et al. 2018). They are typically small, benthic, ambush predators that are endemic to freshwater ponds and creeks of eastern and southern Asia. This family can be distinguished from other gobiiforms by the following combination of characters: (1) six branchiostegal rays; (2) separated pelvic fins; (3) well-developed scapula excluding the cleithrum and proximal radials; and (4) longitudinally distributed lines of cephalic sensory papillae (Hoese and Gill 1993;Iwata 2011;Li et al. 2018). Li et al. (2018) revealed two clades within Odontobutidae using nuclear gene capture data, one of which includes three monophyletic genera: Microdous Li, 2018 (southern China and northern Vietnam) as sister to a clade consisting of Micropercops Fowler & Bean, 1920 (China, Japan and the Korean Peninsula) and Sineleotris Herre, 1940 (southern China and the Indo-China Peninsula).
Microdous Li, He, Jang, Liu & Li, 2018 was established for Phylipnus chalmersi Nichols & Pope, 1927 and distinguished from other genera of Odontobutidae by a unique combination of the following character states: (1) presence of suborbital bone; (2) presence of complete cephalic sensory canals; (3) small and cuspidal gill rakers; (4) gill openings extending to under the front part of the eyes; (5) absence of vertical bands on the sides; (6) absence of a dark band under the eye; and (7) presence of an irregular black fleck on the upper part of the base of the pectoral fin in preserved specimens (Li et al. 2018). In the present study, a new species in the previously monotypic Microdous is described from Guangxi, southern China. The new species is also supported by a molecular phylogenetic analysis of mitochondrial cytochrome c oxidase subunit I (COI) implemented to evaluate the position of the new species among closely related taxa.

Materials and methods
In total, eight specimens were collected from Lihong Village (24°26.21'N, 106°26.72'E; c. 870 m a.s.l.), Yuhong Town, Lingyun County, Baise City, Guangxi Zhuang Autonomous Region using fish traps (Fig. 1). Specimens were preserved in 95% ethanol in the field then transferred to 75% ethanol. The right pectoral fins of six specimens were

110°X
ijiang River Hongshui River clipped for molecular analysis, and then the specimens were fixed in 10% formalin and transferred into 75% ethanol for morphological examination. All specimens were assigned with a collection number (CL) to facilitate sample tracking. Five specimens (holotype and four paratypes) were used to collect morphological measurements and most counts except for the number of gill rakers and vertebrae (Table 1). Three paratypes were used in the molecular analysis. Seven specimens were used to calculate snout length/ head length ratios in order to examine whether the snout was blunter in the new species than in that of M. chalmersi.
Morphological measurements, counts and observations followed Wu et al. (2008). Nomenclature of cephalic sensory pores followed Akihito (1986). Each specimen was sexed by morphology of the urogenital papilla (Miller 1992). Two non-type specimens were dissected for counts of the number of gill rakers and vertebrae, and for examination of the vomerine region.
Genomic DNA was extracted using an Ezup Column Animal Genomic DNA Purification Kit (Sangon, Shanghai, China). Partial sequences (~ 1500 bp) of cytochrome c oxidase subunit I (COI), covering whole barcoding region was amplified from three paratype specimens (SOU1801010-1, SOU1801010-2, SOU1801010-3) as well as from comparative specimens ( Table 2) using Vazyme 2× Taq Plus Master Mix II (Sangon, Shanghai) with a forward primer, CCATTTTACCTGTGRCAATCACACG, and a reverse primer CAGAGCGGTTATGTRTCTGGCTTGAA according to Zhou et al. (2022). The following thermal cycles was followed: an initial denaturation at 94 °C for 5 min, 30 cycles of denaturation at 94 °C for 30 sec, primer annealing at 55 °C for 30 sec, extension at 72 °C for 1 min, and the final extension was 7 min at 72 °C (Zhou et al. 2022). The amplified products were checked on 1% agarose gels before sending to Azenta (Suzhou, Jiangsu, China) for sequencing. The sequence of three paratype specimens were lodged in GenBank with accession numbers OP536373, OP536374 and OP536375.
Description. Morphometric and meristic data for the holotype and paratypes are presented in Table 1. The maximum standard length (SL) was 76.2 mm.
Body stout, slightly compressed posteriorly. Head large, slightly depressed. Eye large, located in anterior half of head, not protruding outward. Several rows of tiny conical teeth on both jaws. Tiny, slender, teeth-like dermal projections in vomerine region (Fig. 5A). Gill opening extending to under anterior part of eye. Cephalic sensory canals complete (Fig. 6). Urogenital papilla distinct, rest behind the anus opening (Fig. 7).
Posterior tip of dorsal fin reaching origin of second dorsal fin when depressed. Second dorsal fin ends distinctly anterior of origin of caudal fin. Caudal fin and pectoral fin large, elliptical. Pelvic fins well separated, rear tip not reaching anus. Interopercle and subopercle naked. Ctenoid scales on dorsal and lateral side of body, ventral side of body posterior to anus and opercle, transforming ctenii present. Small cycloid scales on predorsal area, cheek, nape, preopercle, base of the pectoral fin, breast and abdomen.
Coloration in life. Head black and dark brown with black dots on cheek. Body side yellowish, several irregular dark patches and orange dots on the side. Back dark brown. Unpaired fins possessing several inconspicuous stripes of dark spots and white edge. Pectoral fins and pelvic fins transparent and dusky. An irregular black fleck on upper part of base of pectoral fin. Ventral side of abdomen pale, with dull and inconspicuous dark patches. Urogenital papilla dark brown (Fig. 3A).
Coloration preserved. Head dark brown with black dots on the cheek. Body side brown, orange dots absent. Back brown. Irregular black fleck on upper part of base of pectoral fin dull and inconspicuous. Ventral side of abdomen light brown. Urogenital papilla whitish (Fig. 3B).
Sexual dimorphism. Urogenital papilla elongate with a wide base, tapering and with a narrow tip in male; truncated in female (Fig. 7). Cephalic sensory canals system. Anterior extension in front of interorbital with three pairs of pores A, B and C, and a single interorbital pore D. A pair of pores E lateral to pore D. Lateral section of oculoscapular canal with a series of seven pairs of pores: F to L (terminal). Preopercular canal extending to ventral side of preopercle, with five pairs of pores: M (dorsal) to Q (ventral) (Fig. 6). Cephalic sensory papillae. Neuromast numerous, small and densely set in mostly longitudinally arranged rows (Fig. 6).
Biology. Mircodous amblyrhychos can be found in small creeks or rivers with slow moving, clear water and rocky bottom. Some remains of chitin exoskeleton of crustaceans were found in the anus opening of the holotype, suggesting that M. amblyrhynchos is carnivorous. The male's head would turn black with several orange dots on the body sides during their spawning seasons.
Etymology. This species is named for its blunt snout distinguishing it from M. chalmersi. The species name derives from Greek ambly meaning dull or blunt and rhynchos meaning snout.
Phylogenetic analysis. The COI tree with three individuals of M. amblyrhynchos and representative species of all available Odontobutidae genera except for Terateleotris is shown in Fig. 8. Rhyacichthys aspro Valenciennes, 1837 was used as the outgroup following Li et al. (2018). All genera of Odontobutidae were monophyletic in the tree.     All M. amblyrhynchos specimens formed a clade distinct from M. chalmersi. The pdistance between M. amblyrhychos and M. chalmersi was 10.7% (Suppl. material 1), suggesting that M. amblyrhynchos is a distinct species of the genus Microdous.

Discussion
Microdous differs from other genera of the Odontobutidae by the following characters: lateral line absent (present in Terateleotris); barbel-like projection on sensory papilla absent (present in Neodontobutis); prevomerine teeth absent (present in Perccottus); cephalic sensory canals complete (moderate in Micropercops, absent or reduced in Odontobutis); maximum head width/maximum head depth ratio slightly greater or equal to 1 (vs. maximum head depth/maximum head width far less than 1 in Sineleotris); and dark band under eye (vs. present in Sineleotris).
Our analyses using both morphological and molecular data clearly suggest that the specimens collected from Yuhong Town, Lingyun County, Baise City, Guangxi, southern China should be recognized as a new species belonging to the genus Microdous. This new species can be morphologically distinguished from its only congener by its wider interorbital width, blunt snout, and non-extending eyes. A single specimen (as Perccottus chalmersi) from Xiajia village, Lingyun town was described by Zheng (1981) that had a blunt snout and dark fins with several inconspicuous stripes formed of dark spots. This specimen might in fact be a record of M. amblyrhynchos; however, it was inaccessible for comparison due to its unknown whereabouts.
The genus Microdous was established by Li et al. (2018) for P. chalmersi and the etymology of the generic name is "small + teeth" in Greek, referring to "a curved band of fine teeth on the vomer" (Nichols and Pope 1927) or "slender and tiny teeth on the vomer region of the fish" (Li et al. 2018), which were also mentioned as "vomerine teeth" in other studies (Zheng 1981;Wu et al. 2008). This character state is present in both M. amblyrhynchos and M. chalmersi (Fig. 5). However, our stereomicroscopic observation of the vomerine region showed that these projections growing directly from skin are different from the prevomerine teeth in other species such as Perccottus glenii Dybowski, 1877. Similar "skin teeth" were also found in Neodontobutis hainanensis Chen, 2002and Sineleotris saccharae Herre, 1940(Chen and Zheng 1985Pan 1991). These vomerine teeth-like dermal projections are weak and tiny compared to the size of the fish, leaving their function as unclear.
Our phylogenetic analysis (Fig. 8) showed that Microdous is monophyletic, and the specimens of M. amblyrhynchos formed a clade distinct from M. chalmersi. Microdous was dated to the Miocene (17.9 Ma) and its origin was estimated as from southern China (Li et al. 2018). Biogeographical (Li 1981;Zhu 2016), palaeobotanical (Yao et al. 2009) and geological (Liang 2018) studies indicated that the Hainan Island was adjacent to the mainland several times due to plate movement, volcanic activity or sea level change since the Eocene epoch (30 Ma) (Liang 2018). Thus, the repeated connections and separations between Hainan Island and mainland could account for the divergence between M. chalmersi and M. amblyrhynchos.
The present-day distribution of genus Microdous includes southern China and northern Vietnam (Kottelat 2001 as Perccottus chalmersi). However, the Laotian species Sineleotris namxamensis (Chen & Kottelat, 2004) shares closest morphological similarities with the genus Microdous rather than the genus Sineleotris, such as the absence of a dark band under the eye and the arrangement of the cephalic sensory canal system. Thus, S. namxamensis would be better placed in the genus Microdous; the taxonomy of this species requires further research and molecular evidence.
Due to the rarity of the species, we know nothing about the reproduction, life cycle, and behavior of M. amblyrhynchos. Currently, the new species is only known from its type locality despite frequent surveys in Guangxi, suggesting that it is probably a species with a restricted distribution range. Habitat degradation at the type locality due to invasive species and illegal electric fishing may threaten the survival of this species. Therefore, we recommend that M. amblyrhynchos should be listed as a Vulnerable (VU) species [IUCN Red List criteria A1cde+B1b(iii)+D2].
According to Li et al. (2018), southern China maybe the region where the family Odontobutidae originated. This region has high Odontobutidae species diversity, including eight species of five genera. A new member of the genus Neodontobutis, N. lani (Zhou et al. 2022) was described recently from Longzhou Town, Guangxi, Zuojiang River basin, suggesting that more new species from these places may be uncovered.

Ethical approval
All animal procedures performed in this research were done so in accordance with the "Ethical Standards of the Shanghai Ocean University (2020)".