Microdous amblyrhynchos, 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. amblyrhynchos sp. nov. is distinct from its sister species, M. chalmersi.

Freshwater sleepers, morphology, phylogeny, taxonomy, Xijiang River

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.

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 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.

Figure 1. 

The sampling location of Microdous amblyrhynchos sp. nov. in China (inset) indicated by the green triangle.

Type specimens were deposited at the Fish Collection of the Shanghai Ocean University (SOU), China. Collection numbers and voucher numbers (in parentheses) of specimens of the new species are as follows: CL3084-1 (= SOU1801010-1), CL3084-2 (= SOU1801010-2), CL3084-3 (= SOU1801010-3), CL3084-4 (= SOU1801010-4), CL3084-5 (= SOU1801010-5), CL3084-6 (= SOU1801010-6), CL3084-7 (= SOU1801010-7), CL3084-8 (= SOU1801010-8).

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.

Sequences of additional material were retrieved from the National Center for Biotechnology Information database (Table 2). All sequences were aligned and analyzed using MEGA11 (Tamura et al. 2021). A maximum-likelihood tree including all genera of Odontobutidae except for Terateleotris was reconstructed under the HKY+G model with 1000 bootstrap replications. The resulting tree was visualized in FigTree v. 1.4.4 (Rambaut 2018).

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, 2002 and Sineleotris saccharae Herre, 1940 (Chen and Zheng 1985; Pan 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.

All M. chalmersi specimens examined for morphological comparison were deposited in the Fish Collection of the Shanghai Ocean University, China with the registration tags 76V8791, 76V8792, 76V9228, from Qiongzhong, Hainan, May 1976; HN832384 from Changjiang, Hainan, May 1983, and 070504 from Wuzhishan, Hainan, May 1983; CL2076-1 from Wuzhishan, Hainan, August 2018.

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

We thank Mr Jiahu Lan for collecting the samples, Mr. Mingwei Zhou and Miss Fangxin Wang for assisting with molecular lab work. This work was supported by “Science and Technology Commission of Shanghai Municipality (19410740500)” to C. Li.