Two new species of the genus Hessebius Verhoeff, 1941 from China (Lithobiomorpha, Lithobiidae)

Abstract Two new species, Hessebius luquensis sp. n. and Hessebius ruoergaiensis sp. n., are described based on material from Qinghai-Tibet Plateau. A key to the Chinese species of Hessebius is presented. The partial mitochondrial cytochrome c oxidase subunit I (COI) barcoding gene was amplified and sequenced for nine individuals of both species and the dataset was used for molecular phylogenetic analysis and genetic distance determination.


Two new species of the genus Hessebius
from China (Lithobiomorpha, Lithobiidae)

Introduction
Hessebius was created by Verhoeff (1941) to receive two Turkish species and was substantiated by Eason (1981). Its main character is the massive expansion and projection of the dorsolateral ridge of the female gonopod, according to Zalesskaja (1978) and Eason (1981).
The centipedes of China have been poorly studied. Up to now, three species of Hessebius have been recorded (Ma et al. 2014): H. jangtseanus (Verhoeff, 1942) distributed in Sanshenggou, Wolong Town, Wenchuan Country, Aba City, Sichuan Province; H. longispinipes Ma, Pei & Zhu, 2009 recorded in Xinjiang Uyghur Autonomous Region (Barkor country, Hami City) and H. multiforaminis Pei, Ma, Zapparoli & Zhu, 2010 recorded in Tibet Autonomous Region (Pulan country, Pulan Town, Ali City). Considering the geographic distribution of the species of Hessebius in China, their main habitat preference seems to be steppes, deserts or sub-deserts, and they are all seem to be native species. The known localities of Hessebius in China are shown in Figure 1.

Materials and methods
Specimen collection and preparation: The specimens were all collected by hand, preserved in 95 % alcohol, and deposited in the collections of Northwest Institute of Plateau Biology, Chinese Academy of Sciences. Characters were examined using SZ61 Olympus stereoscope and took pictures with a SX-3 (Shanghai optical instrument factory) camera. Terminology for external anatomy follows Bonato et al. (2010). Each specimens are numbered from 1 to 12 according to collection quantity and prefix with the abbreviation of the locality. The abbreviations used are: T, TT  DNA extraction and sequencing protocols: Standard DNA extraction and amplification methods were performed. Total DNA was extracted from a single leg removed from each specimen samples using MicroElute Genomic DNA kit (OMEGA), after overnight incubation at 65 °C. Polymerase chain reactions (PCRs) were conducted using Mastercycler pros PCR (Eppendorff) in total reaction volumes of 39-μL volumes containing 5-60 ng template DNA, 1μL; ddH2O 28μL; 10×Buffer 5μL (Takara, Dalian,  Ma, Pei and Zhu, 2009;circle = Tibet H multiforaminis Pei, Ma, Zapparoli and Zhu, 2010;triangle = Sichuan H jangtseanus (Verhoeff, 1942); pentagon = Luqu H luquensis sp. n.; square = Sichuan H ruoergaiensis sp. n. China); 0.5mm/L dNTPs 2.5μL (Takara, Dalian, China); 5U/μL Taq polymerase 0.5μL (Takara, Dalian, China); Forward Primer 1μL; Reverse Primer 1μL (synthesized by Sangon Biotech from Shanghai). An 686 bp fragment of COI was amplified using the primers LCO 1490/LCO 2198 (Edgecombe et al., 2002). PCR was performed as follows: initial denaturing at 95 °C for 10 min; followed by 35 cycles of 95 °C for 30 s, 44 °C for 30 s, and 72 °C for 90 s and a final extension at 72 °C for 10 min. The PCR products were purified using a purification kit (DC28106 250 Preps, QIAGEN, GERMAN). Sequencing reactions were implemented using ABI Prism BigdyeTM Terminator Cycle Sequencing Ready Reaction Kit on ABI 3730XL sequencer, with the PCR primers.
The GenBank accession numbers of all nine new sequences were MG515155-MG515163 (Hessebius COI). Sequence identities were confirmed with BLAST searches (Altschul et al. 1997). In order to eliminate indicators of nuclear mitochondrial pseudogenes (numts), such as indels, stop codons, and double peaks in sequence chromatograms, the whole dataset was translated into amino acids using the 'invertebrate' code in MEGA6 (Tamura et al. 2013) ; internal stop codons were absent in our dataset; gaps were absent.
Phylogenetic analyses: The sequences were aligned with Clustal X2.0 (Chenna et al. 2003). The aligned sequences were edited using the program BioEdit 7.0.9.0 (Hall 1999) by hand. The substitution model selection was implemented in jModelTest 2.1.4 (Darriba et al. 2012), the TIM2+G model was selected for all datasets by likelihood ratio tests either under the Akaike Information Criterion (AIC 14337.6710) or under the Bayesian Information Criterion (BIC 14617.1521). Topology was reconstructed under the TIM2+G model of nucleotide evolution in MrBayes. Bayesian inference (BI) was used to generate a phylogenetic hypothesis of the DNA haplotypes. BI was performed in MrBayes 3.2 (Ronquist and Huelsenbeck 2003) with 3,000,000 generations, sampling trees every 300 generations. Two independent runs each with four simultaneous Monte Carlo Markov chains (MCMC) were carried out. The first 25 % of generations were discarded as 'burn-in'. The convergence of chains was confirmed until average standard deviation of split frequency is below 0.01 (0.008300) and the potential scale reduction factor (PSRF) is close to 1.0 for all parameters. In phylogenetic analysis Anopsobius neozelanicus Silvestri, 1909 was used as outgroup.
Distance analysis: The analysis involved 27 nucleotide sequences (Appendix 1). Codon positions included were 1st+2nd+3rd. All ambiguous positions were removed for each sequence pair. There were a total of 632 positions in the final dataset. Evolutionary analyses were conducted in MEGA6 (Tamura et al. 2013). All pair-wise intra-and inter-specific distances were produced to evaluate species divergence in Hessebius. Habitat. Speciemens were collected under stones along roadside on steppes from Luqu. Etymology. The name is derived from the locality Luqu where the species is discovered. Luqu country is situated in the eastern edge of the Tibetan Plateau standing on the junction of Gansu, Qinghai and Sichuan Provinces. Diagnosis. Body length 8.5-12.3 mm; head slightly widened; antennae of 20 antennomeres; 7-10 ocelli arranged in three rows; Tömösváry's organ oval, almost equal in size to neighboring ocelli (Fig. 2B); lateral margins of forcipular coxosternite slanting; anterior margin with 2 + 2 sharp teeth and with setiform porodonts; tergites without triangular posterolateral process, a line of setae along posterior border of TT 8 and 10; legs 14 and 15 thicker than anterior ones in both sexes; a dorsal furrow on the tibia of legs 14-15 on male; coxal pores 3-6, round, arranged in one row; female gonopods with two moderately long, bullet-shaped spurs, the second article of the female gonopods having a massive process; terminal claw of the third article simple, with a small triangular protuberance on basal ventral side; male gonopods short and small.
Colour (based on specimens in 95 % ethanol): antennomeres yellow; tergites pale yellow, with brown margin; cephalic plate and T 1 brown; pleural region and sternites pale yellow; distal part of forcipules dark brown, maxillipede coxosternum and S 15 yellow; legs pale yellow with gray hue, pretarsal claw dark brown.
Cephalic plate smooth, convex, slightly longer than wide; short to long setae scattered along the marginal ridge of the cephalic plate; setae on head shield symmetrically arranged, three pairs between antennocellar and transverse suture, two pairs behind these sutures; frontal marginal ridge with clear transverse suture; projection of lateral marginal conspicuously discontinuous; posterior marginal ridge slightly concave without median thickening.
Antennae length 3.46 mm, extending back to anterior margin of T 5, consisting of 20 elongate antennomeres covered with dense pale setae. The basal two articles enlarged, then elongate and tapering. The first article wider than long, the second article has the equal width to length, terminal article approximately 2.5 times length to width. Setation: the first two articles has fewer setae than succeeding articles especially on anterior side, then increasing, till 6 or 7, the density become constant.
Forcipular coxosternites subtrapezoidal, coxosternite with narrow, straight or slightly recurved dental margin; 2+2 teeth on dental margin, small, blunt knobs with independent sclerotization from coxosternite; porodont setiform towards its apex, much stouter than a seta at its base; no shoulders lateral to the porodont; 3 to 4 long setae along the slope, some setae scattered on ventral side of coxosternite.
Sternites trapeziform, setae scattered very sparsely on the surface; four pairs of long bristles approximately symmetrical on the anterior corner and margin, one pair on the posterior corner; among long bristles there's small pairs of short bristles; short to long setae along posterior border, in some individuals SS 13, 14, 15 more dense.
Legs: tarsal articulations only visible with shallow ventral suture on 1 st to 11 th , distinct on 12 th and 13 th , well-defined on legs 14 and 15; leg pairs 14 and 15 thicker and longer with sparse setae in contrast to legs 1-13; pretarsus of legs 1-13 with a slightly curved, long, principal claw and smaller and thinner anterior and posterior accessory spines, anterior accessory spines long and slender, 0.33-0.5 the length of principal claw, posterior one stouter, 0.25 the length of principal claw, forming slightly larger angles with tarsal claws; accessory apical spines on the 14th vestigial, absent on the 15th; abundant glandular pores on surfaces of femur, tibia and tarsus of legs 14 and 15; short to moderately long setae scattered over the surface of legs 1-13, latter half of tarsi generally more setose with two rows of setae along ventral side, fewer setae on legs 14-15.
14 th and 15 th legs: swollen, 15th leg 30% of body-length, tarsus 1 4.3 times longer than wide, tarsus 2 48% length of tarsus on leg 15. Data on the leg plectrotaxy are compiled in Table 1. In the male the 14th and 15th tibia has a dorsal sulcus extending along its whole length.
Coxal pores on legs 12-15, circular; inner pores smaller. Distance between pores 2-3 times bigger than diameter of pore; formula 4, 4, 4, 4. Coxal pores set in a shallow groove arranged in a row with short to long setae scattered over the surface of apophysis.
Female S15 generally trapeziform, straight posteromedially; sternite of genital segment well sclerotised, wider than long; sternite of genital segment with posterior margin moderately concave between condyles of gonopods, except for a small, median approximately circular bulge, distal lightly sclerotised; short to long setae scattered over the surface of genital segment and lateral margins.
Female gonopods divided into three articles, the first article moderately broad, bearing 11-17 short to moderately long setae, arranged in three rows; the first article also bearing 2+2 moderately long, bullet-shaped spurs, inner spur slightly smaller and more anterior than the outer (Fig. 2D); the second article with 6 setae arranged in one rows (Fig. 2F); dorsolateral ridge of second article with a massive expansion projecting distally over the base of the third article (Fig. 2F), six short blunt spines along the dorsolateral ridge, one on the ventral side of dorsodistal projection (Fig. 2F); three moderately long setae on third article; dorsolateral setae one on the first article, eight on the second article four of which short and blunt and four on the third (Fig. 2E); one long dorsomedial setae on each article (Fig. 2E); terminal claw simple, slender and sharp, having small triangular protuberance on ventral side (Fig. 2D). Male S15: subsemicircular, well chitinized, long setae scattered sparsely over its surface and posterior margins. Male genitalia: first genital sternite wider than long, well chitinized; posterior margin quite deeply concave between the gonopods, no bulge medially; 24 short to medium setae scattered sparsely over its surface and at lateral margins, second genital sternite with abundance seta; gonopod of a single small article with 2 seta on its surface, apically slightly chitinized, flat (Fig. 2H).
Remarks. The female of H. luquensis sp. n. is mostly similar to Hessebius longispinipes Ma, Pei and Zhu, 2009, but can be readily distinguished by the following characters: more antennomeres (20 + 20, vs. 18 + 18 in Hessebius longispinipes), more ocelli in three rows, a bulge exists near the base of the porodont; 14 th accessory spines present, apical claw of female gonopods with triangular protuberance only on the ventral side and the apex of the male gonopod flat versus hemisphere in H. longispinipes.
Diagnosis. Body length 9.2-10.0 mm; antennae composed of 19-20 antennomeres; 7-10 dark ocelli on each side; Tömösváry's organ ovate to round, larger to the adjoining ocelli; 2+2 triangular sharp prosternal teeth; porodonts long and strong, lateral to lateral tooth; posterior angles of all tergites round; legs 14 and 15 thicker than anterior ones; coxal pores 3-5, ovate to round, arranged in one row; female gonopods with 2 bullet-shaped spurs, the second article of the female gonopods extend-ing backwards bearing 5 lateral spines; terminal claw of the third article simple, with inconspicuous triangular ventral accessory denticles; male gonopods short and small with 2 long setae.
Colour: body pale yellow; antennae and distal part of forcipules brown; cephalic plate, TT 1, 2 dark and median and posterior parts of TT 3-14 dark forming a line; pleural region and SS pale yellow with dark hue; legs pale yellow with dark hue excluding tarsus yellow.
Antennae: 41.6% of body-length with 20 moderately elongate articles; the basal one wider than long; the 8, 9, 10, 11 elongate; the ultimate one is three times longer than wide. Abundance setae scattered on the surfaces of from the first to the last.
Cephalic plate wider than long, with clear transverse suture; median furrow on cephalic plate absent; lateral margin discontinuous, posterior margin slightly concave; moderately long setae scattered along marginal ridge and cephalic plate (Fig. 3A).
Two posterior large ocelli and eight smaller ocelli arranged in three rows (Fig. 3B). Tömösváry's organ ovate, larger than the adjoining ocelli, some distance from the adjoining ocelli, situated on ventral side of cephalic pleurite.
Prosternum: subtrapezoidal coxosternite with narrow, straight dental margin; 2+2 subtriangular teeth as extensions of the coxosternite teeth; median incision "U" shaped; long and strong setiform porodonts; lateral borders without shoulders; pretarsal section of forcipules slightly longer than tarsal section; 3 lines of short setae and 1 moderately long setae arrange on ventral side of coxosternite (Fig. 3D).
Sternites smooth, S1 subsquare, SS 2-14 trapeziform, posterolateral narrower than anterolateral. One to three pairs of setae symmetrical on anterior corners; one pair of setae on posterolateral margins; a few setae distributed along posterior margins; a band of setae on anterior margins of SS 2-7.
Legs: tarsal articulation on anterior pairs of legs fused on dorsal side of leg, distinct ventrally from 1 st to 13 th , on 14 th and 15 th leg divided into basitarsus and distitarsus; pretarsus claws moderately long, curved ventrally on all legs; anterior and posterior accessory spines present from the 1 st to the 14th leg, only posterior accessory spines on the 15th leg; anterior accessory spines long and straight, nearly half of length of the main claw, posterior accessory claws strong and curved, nearly a third length of the main claw; Legs 14-15 (Fig. 3A) thickened. Numerous short to long setae fairly evenly distributed on all sections along legs. Legs' plectrotaxy as in Table 2. In male, one comparatively obvious furrow on the dorsal side of the tibia of legs 14 and 15 (Fig. 3G). 15 th legs: approx. one third of body-length. Leg 15 basitarsus 129% length of distitarsus; basitarsus 84% length of tibia; tibia 2.6 times longer than maximal width, basitarsus 3.6 times, distitarsus 3.2 times. Basitarsus nearly the same length of distitarsus on leg 14.
Female: S15 subtrapeziform with short to long setae covered. The first genital sternite bears approx. 48 setae, posterior margin of which moderately embayed between gonopod articulations. Two long conical spurs on the female gonopod, the proximal ones smaller (Fig. 3E); Claw of female gonopod with small triangular ventral accessory denticles (Fig. 3F); five stronger and curved spines like thorn on distinct dorsodistal projection (Fig. 3F); 15 or 16 setae arranged in three rows on basal article of gonopod, six long setae on second article, 3 long setae on third (Fig. 3E).
Comparatively long setae distribute on male first genital sternite with fewer setae near S 15; posterior median margin of the first genital sternite deeply concave between gonopods; male gonopod short with two setae sometimes retracted from tergite of first genital sternite (Fig. 3H).
Remarks. Hessebius ruoergaiensis sp. n. is very similar to Hessebius jangseanus: the number of ocelli of both species are overlapped, but H. ruoergaiensis has fewer ocelli, no more than ten; fewer coxal pores in H. ruoergaiensis, no more than five; the distribution of accessory claw on the legs is the same in both species; however, the tibia of the 14 th and 15 th leg of H. ruoergaiensis have dorsal sulcus which is absent in H. jangseanus; the plectrotaxy of legs also similar but different.

Discussion
Both molecular analysis (Fig. 4) and morphology support that Hessebius luquensis sp. n. and H. ruoergaiensis sp. n. form new species and that the relationship is genetically close. The two species are morphologically similar, but can be readily distinguished using COI.
Generally speaking, in Lithobiomorpha, intraspecific distances are less than 10 %, while distances between species ranges often more than 10%. Sometimes the distance between the species from the same genus are larger than from different genus, such as 17.3 % (interspecific mean p-distance between Hessebius luquensis sp. n. and Hessebius ruoergaiensis sp. n.) vs 15.7 % (interspecific distances between Hessebius luquensis sp. n. and Lithobius (Ezembius) giganteus Sseliwanoff). This may indicate that each species have been evolved independently its habitat for a long time. Morphologically similar species, for instance species from the same genus, also have high similarity in gene that the branches which they represented joined together shown in phylogenetic tree (St. Clair and Visick 2010).