﻿A new species of Otostigmus (Chilopoda, Scolopendromorpha, Scolopendridae) from China, with remarks on the phylogenetic relationships of Otostigmuspolitus Karsch, 1881

﻿Abstract Through a combination of morphological and DNA data, a new scolopendrid centipede from southern and southwestern China was revealed: O.tricarinatussp. nov. The species belong to the politus group but has three sharp tergal keels. Validation of phylogenetic status was performed through molecular analysis of the cytochrome c oxidase subunit I (COI), 16S rRNA, and 28S rRNA sequences from 16 Otostigmus species. Otostigmustricarinatussp. nov. was found to be two populations and varied in the number of spines on the ultimate prefemur, the sutures on a sternite, and a pore-free median longitudinal strip in the pore field. The Yunnan-Guizhou plateau population of O.tricarinatussp. nov. was sister to the clade O.polituspolitus + O.politusyunnanensis + Guangxi population of O.tricarinatussp. nov. with strong support from both BI (bayesian inference) and ML (maximum likelihood) analyses (PP = 1, BS = 97%).


Introduction
The largest genus in the subfamily Otostigminae (Pocock, 1891), Otostigmus Porat, 1876, has approximately 110 recognised species.Otostigmus is classified into three subgenera: O. (Otostigmus), O. (Parotostigmus) Pocock, 1896, and O. (Dactylotergitius) Verhoeff, 1937(Schileyko et al. 2020).Lewis (2010) arranged the subgenus O. (Otostigmus) into nine species groups that were based on the following characteristics: the presence or absence of keels on tergites, the number of antennal articles, the number of antennal articles which lacked numerous setae, sternite tuberculation, the number of tarsal spurs on legs, the ultimate leg prefemur characteristics and the coxopleural process of leg-bearing segment 21 with or without a dorsal spine.
In this study we describe a new species, Otostigmus tricarinatus sp.nov., which belongs to the politus group.The phylogenetic status of the new species was validated through the molecular analysis of cytochrome c oxidase subunit I (COI), 16S rRNA, and 28S rRNA sequences which were derived from 18 Otostigmus species.

Species sampling and morphological examination
Material was collected from different provinces in China (Table 1, Fig. 2).These specimens were preserved in 75% ethanol, and genomic DNA was extracted from the leg tissue.An Olympus E-M10 II camera was used to capture live colour patterns.Holotypes and paratypes of the new species were maintained deposited at the Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, China (CMMI).
The morphological terminology follows Bonato et al. ( 2010).The taxonomic characteristics were observed using an Olympus SZ16 stereomicroscope.Helicon Focus 6.7.1 was used to create a multi-focused montage of pictures.The ArcMap 10.7.1 software tool was used to produce the maps.

DNA extraction and fragment amplification
Legs from each specimen were used to extract genomic DNA by using a Promega Wizard SV Genomic DNA Purification Kit (Promega, USA).Polymerase chain reaction (PCR) was used to amplify the cytochrome c oxidase subunit I (COI), mitochondrial ribosomal gene 16S, and nuclear ribosomal DNA 28S fragments.

Phylogenetic analysis and genetic distance
The genetic distance between Otostigmus species was calculated using the Kimura 2-parameter model in MEGA X (Kumar et al. 2018)  28S DNA sequences obtained from this study, as well as previous phylogenetic studies acquired from GenBank (Siriwut et al. 2018;Vu et al. 2020;Joshi and Karanth 2012;Vahtera et al. 2012Vahtera et al. , 2013)), were used as part of the phylogenetic analysis (Table 1).Three partitioned genes, COI, 16S, and 28S, were aligned in BIOEDIT 7. 1.3.0 (11/4/2011) (Hall 1999), using the CLUSTAL W tool (Thompson et al. 1994).Bayesian inference (BI) and maximum likelihood (ML) meth-ZooKeys 1168: 161-178 (2023), DOI: 10.3897/zookeys.1168.82750Tian-Yun Chen et al.: New species of Otostigmus and phylogenetic relationships of politus group ods were used in the construction of phylogenetic trees.Branch support was evaluated through standard statistical testing (bootstrap support and posterior probability).ML analysis was performed using the IQ-TREE 1.6.8software tool (Nguyen et al. 2015) in the PHYLOSUITE 1.2.2 platform (Zhang et al. 2020) with 500,000 ultrafast bootstraps (Hoang et al. 2018).MODELFINDER (Kalyaanamoorthy et al. 2017) selected GTR+F+I+G4 as the preferred substitution model.Furthermore, MODELFINDER was used to evaluate the best-fit substitution models of BI, with GTR+F+I+G4 being selected as the optimal substitution model.MRBAYES 3.2.6 (Ronquist et al. 2012) was used to run Bayesian analyses.10,000,000 generations were used, sampling every 1000 generations, and dividing 25% of the trees as burn-in.A split frequency of less than 0.01 was used to determine stationarity, and a consensus tree was constructed using the remaining trees.Etymology.The name refers to the characteristics of the tergites.The tri-compounded with the Latin carinatus refers to the three sharp keels on the tergites.

Order
Diagnosis.Antennae with 17 articles, basal three glabrous dorsally, the apical article with a well-developed lateral depression.TT 3-20 with three longitudinal keels.SS 2-19(20) with paramedian sutures occupying anterior 20-100% of sternites, a median depression, and two posterolateral depressions.Coxopleural process with 1-3 apical spines and none or one lateral spine, porefree median longitudinal strip in pore field from the posterior of sternite 21 to the end of coxopleural process.The ultimate leg prefemur typically with 0-7 spines, lacking corner spine.
Antennae with 17 articles, 3 glabrous dorsally, 2.5 glabrous ventrally, apical article double the length of the penultimate, with a well-developed lateral depression (Fig. 3A).Antennae reach the posterior margin of T2 when reflexed.Forcipular coxosternite slightly wider than long and lacking sutures/sulci.Coxosternal tooth-plates wider than long, with four teeth.Trochanteroprefemoral process bears one apical and one lateral tubercle.
The left coxopleural process has one apical and one lateral spine and the right one has two apical and one lateral spine (Fig. 3F, G).Pore-free median longitudinal strip in pore field from the posterior of sternite 21 to the end of coxopleural process.The ultimate legs are long and slender, with the left prefemur having one ventrolateral and one ventromedial spine.Right prefemur with one ventrolateral spine, one ventromedial spine, and one dorsomedial spine.Ultimate leg relatively long with dense setae (Fig. 3H).Dorsal surface of ultimate prefemur with convex granules, lacking corner spine.Legs 1-5 with two and legs 6-20 with one tarsal spur.Legs 1 and 2 with one tibial spur: leg 1 with one prefemoral and one femoral spur.
Remarks.Schileyko (1995) described "O.amballae Chamberlin, 1913" on specimens from Vietnam.The morphology of these specimens is different from that of the holotype of O. amballae (see Lewis 2002) and were found to be identical to O. tricarinatus sp.nov.that has three well-developed longitudinal keels in TT3-20, a very short coxopleural process with no dorsal spine, an ultimate prefemur with two ventral spines, and lacking a corner spine.The holotype of O. amballae possesses a low median keel at TT3-20, paramedian sutures each in a sulcus from approximately T13, with two lateral keels on each side of sutures; the coxopleural process is moderately long with a single dorsal spine; the ultimate prefemur with VL3, M3, VM2, DM2, and one corner spine.Lewis (2002) further noted that all specimens assigned to O. amballae by Schileyko (1995) should be reassessed.
The material here assigned to O. tricarinatus sp.nov.refers to two geographically separate groups: the Yunnan-Guizhou plateau population (Fig. 4) and the Guangxi population (Fig. 3).They are different in the following characteristics: 1) 80% paramedian sutures on sternite in the former population compared to 20% paramedian sutures on sternite in the latter; 2) the ultimate leg prefemur with 4-7 spines in the former population compared to 1-4 spines in the latter; 3) the pore-free median longitudinal strip in pore field absent in the former population while found from the posterior of sternite 21 to the end of coxopleural process in the latter population.Furthermore, phylogenetic analysis revealed that Guangxi population is a sister group to O. politus congeners + Yunnan-Guizhou plateau population and has strong node support from both ML and BI analyses (PP = 1, BS = 97%).They are considered to be the same species because no more reliable identification characteristics could find.

Figure 5 .
Figure 5. Maximum likelihood phylogenetic tree and Bayesian phylogenetic tree based on combined data for Otostigmus along with posterior probability (PP > 0.9 / BS > 70%) values for each node.
in North China and Korea, is geographically isolated from other politus-group members.The records of O. politus politus from Vietnam with a long ultimate prefemur and spine arrangement were 5-6VL, 5 VM, and 3DM(Schileyko 2007), which resembles O. politus yunnanensis rather than O. politus politus.
Table 2 lists the PCR primers and programs used.

Table 1 .
Otostigmus species vouchers and GenBank accession numbers.

Table 2 .
Tian-Yun Chen et al.: New species of Otostigmus and phylogenetic relationships of politus group Primers and programs of PCR.

Table 3 .
Mean K2P genetic distance between the politus group, aculeatus group and rugulosus group species based on COI sequences.

Table 4 .
Mean K2P genetic distance within the politus group, aculeatus group and rugulosus group species based on COI sequences.

. lewisi Discussion Vu
(Lewis 2002))employed a 638 bp COI loci for phylogenetic analysis of Vietnam Otostigmus centipedes and discovered that the "O.amballae" from Vietnam was not monophyletic.We made use of three loci, consisting of COI, 16S, and 28S rRNA, to construct a phylogenetic tree, and verified that the Vietnamese "O.amballae" specimens have sufficient support to form two clades: specimen IEBR-Chi 013 (Vietnam, Ta Xua) form a clade with Guangxi population of the newly discovered species O. tricarinatus sp.nov.andspecimenIEBR-Chi036(Vietnam,Me Linh) forms another clade with O. tricarinatus sp.nov.(Yunnan-Guizhouplateaupopulation).The morphological characteristics of O. tricarinatus sp.nov. is different from the holotype of O. amballae(Lewis 2002)in terms of keels on the tergite, a corner spine of the ultimate leg prefemur, and a dorsal spine in the coxopleural process.Combined with the phylogenetic results, we propose that all Vietnamese specimens previously assigned to O. amballae Chamberlin, 1913 must be referred to O. tricarinatus sp.nov.The presence of three well-developed longitudinal keels on TT3-20 is the main morphological character that distinguishes O. tricarinatus sp.nov.fromotherOtostigmus (Otostigmus) species.The keels on tergites are a widespread morphological characteristic in OtostigminaeKraepelin, 1903, andmay be found in the genera/subgenera Alipes, Edentistoma, Otostigmus (Parotostigmus) and O. (Otostigmus).Otostigmus scaber, O. rugulosus Porat, 1876, and O. amballae generally have tergal longitudinal keels Table 5 presents the morphological characters of O. tricarinatus sp.nov., O. scaber, O.amballae, and O. rugulosus.Phylogenetic analysis revealed that O. tricarinatus sp.nov., O. politus politus, O. politus yunnanensis formed a clade constituting the politus group.Otostigmus politus politus and O. politus yunnanensis are reciprocally monophyletic and form a clade which is sister to O. tricarinatus sp.nov.(Yunnan-Guizhou plateau population).Furthermore, O. tricarinatus sp.nov.(Guangxi population) is a sister clade of O. tricarinatus sp.nov.(Yunnan-Guizhou plateau population), O. politus politus, and O. politus yunnanensis.However, O. politus