Integrative taxonomy reveals a new species of the genus Burmoniscus (Isopoda, Philosciidae) from the Xuefeng Mountains, China

Abstract Three species of the genus Burmoniscus are identified from the Xuefeng Mountains, central China, by integrating morphological and molecular approaches. Burmoniscuschuanyanensis Li, sp. nov. is described. Morphological photographs of the new species are provided.


Introduction
The genus Burmoniscus was erected by Collinge (1914) to allocate B. moulmeinus [a junior synonym of B. coecus (Budde-Lund, 1895)]. The genus is mainly defined by the pereonites with sulcus marginalis and gland pores, noduli laterales on pereonites 2 and 4 shifted from lateral margins, and epimera of pleon reduced and adpressed; the head with supra-antennal line and no frontal line, mandibles with molar penicil reduced to a single plumose seta, outer lobe of maxillule with 4+6 (5 apically cleft) teeth, in-ner lobe with two unequal penicils and a small posterior point; uropod protopodite grooved on outer margin; insertion of endo-and exopodite at the same level; pleopod exopodites without respiratory areas (Taiti and Ferrara 1986). To date, the genus comprises 73 species distributed worldwide, of which the Oriental, Afrotropical, and Australian regions have a greater diversity (Taiti and Ferrara 1986;Kim and Kwon 2002;Kwon and Kim 2002;Schmalfuss 2003;Taiti and Ferrara 2004;Jeon and Kwon 2009;Karasawa 2016).
Prior to this study, 21 Burmoniscus species have been recorded from China (Kwon and Jeon 1993;Kwon and Taiti 1993;Dai and Cai 1998;Nunomura and Xie 2000). Among them, most members were reported from southwestern and eastern China, but the species of central China are poorly known. Moreover, no record of the genus is known from the Xuefeng Mountains, the largest mountain range of Hunan Province, central China.
In general, the taxonomy of terrestrial isopods using multiple methods has proved to be an efficient strategy in delimiting species (Karasawa 2016;Zimmermann et al. 2018a, b;Bedek et al. 2019). In this context, we studied the specimens collected from the region of the Xuefeng Mountains by integrating morphological and molecular characters. Three Burmoniscus species are identified, including one new species.

Morphological study
Specimens were collected by hand with tweezers, fixed in absolute ethanol, and stored at −20 °C in a freezer. The appendages were dissected and mounted on micro-preparations in neutral balsam mounting medium. Photographs were taken with a digital camera Zeiss AxioCam Icc 5 attached to a digital microscope Zeiss Stereo Discovery V12. The line drawings were drawn by the GNU Image Manipulation Program (Montesanto 2015). All the specimens are deposited in the Insect Museum, Jiangxi Agricultural University, Nanchang, China (JXAUM).

DNA extraction, amplification, and sequencing
Genomic DNA was extracted from pereonites of the samples (Table 1) using the Ta-KaRa MiniBEST Universal Genomic DNA Extraction Kit. Parts of the mitochondrial cytochrome c oxidase subunit I (COI) were amplified using the primers LCO1490 and HCO2198 (Folmer et al. 1994). PCR amplifications were performed with an initial denaturation at 95 °C for 3 min, followed by 35 cycles of 30 sec at 94 °C, 30 sec at 50 °C, and 1 min 72 °C, with a final extension at 72 °C for 10 min. The PCR products were sequenced by using an ABI3730XL DNA Analyzer (Applied Biosystems). All sequences were deposited in DDBJ (DNA Data Bank of Japan), with accession numbers listed in Table 1.

Sequence processing and phylogenetic analysis
A total of 11 mitochondrial COI sequences were obtained. In addition, two Burmoniscus sequences and another two sequences representing the outgroups were downloaded from NCBI for phylogenetic analysis ( Table 1). The estimation of sequence divergences was conducted using the Kimura two-parameter model (K2P) method in MEGA X including all sites, with the pairwise deletion option (Kumar et al. 2018). Sequences were aligned in MAFFT 7 (Katoh and Standley 2013). The best-fit partition model was select using the BIC criterion with ModelFinder (Kalyaanamoorthy et al. 2017) on the platform of PhyloSuite (Zhang et al. 2020). ModelFinder identified the HKY+G+F/TPM2+R4+F model as the best fit for Bayesian (BI) analysis and maximum likelihood (ML) analyses. The BI analysis was conducted in MrBayes v. 3.2.6 (Ronquist et al. 2012). Four chains of Markov chain Monte Carlo (MCMC) were run simultaneously for a total of 10,000 generations; the sampling frequency was set to 100, and the number of runs and burnin fraction with 2 and 0.25, respectively. The ML trees were constructed based on the above best models with 1000 bootstrap replicates using IQ-TREE 2 (Minh et al. 2020).

Species delimitation based on COI and morphological characters
All results are based on the pairwise analysis of the 15 sequences (Table 1). In this study, we acquired 11 COI sequences representing three Burmoniscus species. All the new sequences are 658 bp in length and were deposited in DDBJ (accession no. LC617864 to LC617874). The extremes of inter-and intraspecific distances are presented in Table 2. Both BI and ML analyses represent the same topologies and revealed three clades with high support values (Fig. 1). Three molecular operational taxonomic units (MOTUs)  (Taiti & Ferrara, 1983) LCS2028 China, Hunan, Lingcuishan LC617873 LCS2029 China, Hunan, Lingcuishan LC617874 B. dasystylus Nunomura, 2003 Japan, Wakayama, Shirahama AB626154 Ligidium ryukyuense Nunomura, 1983 Japan, Amami-Oshima AB626261 Ligia exotica Roux, 1828 China, Guangxi, Beihai KC762932 from the Xuefeng Mountains were detected in the molecular analyses, which are consistent with the results of morphological taxonomy (Figs 2-5) for B. chuanyanensis Li, sp. nov., B. mauritiensis (Taiti & Ferrara, 1983), and B. kathmandius . Diagnosis. Male pleopod 1 endopodite with distal portion bearing bilobated apical tip with one subapical spine-like projection between lobes.
Etymology. The new species is named after the national forest park where the type specimens were collected. Figure 4A-E Rennelloscia mauritiensis Taiti & Ferrara, 1983: 203, fig. 2. Burmoniscus mauritiensis (Taiti & Ferrara, 1983): Taiti and Ferrara 1986: 187;Kwon and Taiti 1993: 17. Remarks. In the examined specimens of this species, the body length of males were 4.0-7.0 mm, and of females 4.5-7.0 mm. Taiti and Ferrara (1983) have adequately described and illustrated this species. By morphology, it is easily distinguished by the unique characters of the male pleopod 1 endopodite, which is distinctively bent out-  (Taiti & Ferrara, 1983), male A habitus in dorsal view B telson in dorsal view C pleon in lateral view D pleopod 1 exopodite E pleopod 1 endopodite. wards near the apical area and densely covered with denticles (see Taiti and Ferrara 1983: fig. 2). Furthermore, the morphological characters of this species are identical to those of B. dasystylus Nunomura, 2003(see also Nunomura 2003a). Karasawa (2016) provided a COI sequence (accession no. AB626154) of B. dasystylus. In this context, we estimated the COI sequence divergences of B. mauritiensis and B. dasystylus based on our data (accession no. LC617873, LC617874) and the published sequence (accession no. AB626154). The result shows that their minimum interspecific distance is much smaller than the maximum intraspecific distance of B. mauritiensis (Table 2). In the phylogenetic analysis, they were also represented by a well-supported clade (Fig. 1). Thus, according to the original descriptions and molecular analyses, B. dasystylus is most probably a junior synonym of B. mauritiensis and that a re-examination of the type material is necessary to confirm the synonymy. Figure 5A-E Burmoniscus watanabei  Remarks. This species has the body length of males 3.5-5.0 mm, and of females 4.5-7.5 mm. Karasawa (2016) redescribed and illustrated the morphological characters in detail. It can be distinguished by the characters of the male pleopod 1 endopodite (Fig. 5E); however, morphological variations of the diagnostic characters have caused some taxonomic misinterpretation. Karasawa (2016) clarified the confusion through a combined morphological and molecular approach, and 11 species were considered to be junior synonyms of B. kathmandius. The present work identified the specimens from the Xuefeng Mountains by integrating morphological characters (Fig. 5A-E) and a phylogenetic analysis (Fig. 1). , male A habitus in dorsal view B telson in dorsal view C pleon in ventral view D pleopod 1 exopodite E pleopod 1 endopodite.

Discussion
In the original species descriptions, the male pleopod 1 generally provides the most significant diagnostic character, especially the apical portion of the endopodite, which shows a considerable difference in shape among the species. However, it is difficult to distinguish the interspecific morphological differences and intraspecific morphological variations in the closely related congeners. Thus, the assistance of the other methods is necessary to solve morphological problems (Karasawa 2016;Zimmermann et al. 2018a, b;Bedek et al. 2019). In this context, we examined the Burmoniscus species from the Xuefeng Mountains, central China, for the first time using an integrative taxonomical approach. Our results showed that the male apical structure of the pleopod 1 endopodite is a reliable diagnostic character. To delimit the species more precisely, we supplemented the molecular analyses and revealed three species, including a new species. Certainly, more species in this genus will be discovered in China with further sampling.