A new species of the water mite genus Sperchon Kramer, 1877 from China, with identifying Sperchon rostratus Lundblad, 1969 through DNA barcoding (Acari, Hydrachnidia, Sperchontidae)

Abstract A new species of the water mite genus Sperchon Kramer, 1877 from China, Sperchon fuxiensis Zhang, sp. n., is described and illustrated in this article. DNA barcoding for the new species is documented for future use. Descriptions of both male and female of Sperchon rostratus Lundblad, 1969 are given in the present study, and DNA barcoding for identifying S. rostratus is also discussed.

During checking of a recent collection of water mites, three species (S. plumifer, S. rostratus, and Sperchonopsis echphyma Prasad & Cook, 1972) and a new species (S. fuxiensis sp. n.) were found. The descriptions and illustrations of S. fuxiensis sp. n. are given herein. DNA barcoding for these four species is also provided. DNA barcoding for indentifying S. rostratus is discussed in the present study.

Materials and methods
Water mites were collected by hand netting and preserved in absolute ethanol in 1.5ml centrifuge tubes. The centrifuge tubes were transported to the laboratory and stored at -20°C°C. The information of the samples used in this study is given in Table 1.

Molecular analysis
For molecular examination, each mite was transferred in individual 1.5ml tubes, and washed several times with sterile deionized water. Non-destructive DNA extraction was done on the whole mite. The genomic DNA was extracted by using the DNeasy Blood & Tissue kit (Qiagen, Hilden, Germany), following the manufacturer's instructions. Then, the mites were fixed in absolute ethanol and stored at -20°C for morphological analysis.
The standard COI barcoding fragments (658bp) were amplified with the universal primers LCO 1490 (5'-GGTCAACAAATCATAAAGATATTGG-3') and HCO 2198 (5'-TAAACTTCAGGGTGACCAAAAAATCA-3') (Folmer et al. 1994). Primers were synthesized by Shanghai Sangon Biotechnology (Shanghai, China). All amplification reactions were done in a total volume of 25μl, containing 1-5μl DNA; 12.5μl 2×Taq PCR MasterMix (Tiangen, Beijing, China) and deionized water. The PCR amplification was performed with the following profile: 5 min at 94°C;35 cycles of 30 sec at 94°C, 30 sec at 51°C, 45 sec at 72°C; final extension 10 min at 72°C. PCR products were purified by using QIAquick Gel Extraction kit (Qiagen, Hilden, Germany). The pure segments were ligated into the pGEM-Teasy vector (Promega, Madison, WI, USA) and introduced into Escherichia coli DH5a cells. Bacteria were cultured in LB medium after blue/white selection, and then inserts were sequenced with M13 primers. Each insert was sequenced twice with ABI 3730 automated DNA sequencer by Shanghai Sangon. All sequences were submitted to BOLD and GenBank. The BOLD process ID and the GenBank accession numbers are provided in Table 1. All the sequence data were analysed by using MEGA (ver. 6; Tamura et al. 2013), and were aligned by ClustalW. Genetic distances within and between species were calculated with a K2P model. Phylogenetic trees were constructed with neighbur-joining (NJ) and maximum-likelihood (ML) using K2P model. The sequence of Sperchonopsis echphyma was used as the outgroup. Bootstrap values were obtained from 1000 replicates.
The type specimens are deposited in School of Life Sciences, Huaibei Normal University, China. All measurements are given in μm.
Diagnosis. Integument fine spinules arranged in hexagonal pattern; A1 smooth; excretory pore surrounded by a sclerotized ring; P-II with a long ventro-distal projection and one thick seta; third to fifth segments of leg I-IV with short plumose setae.
Description. Female (n = 2): Body oval in shape, 948 (965) in length, 837 (842) in width. Integument yellow in colour, covered with very fine spinules arranged in hexagonal pattern (Fig. 3). A1 short, smooth and thick, other dorsal setae long and thin. Chitinous plates and glandular plates on both dorsum and venter well developed as illustrated in Fig. 1 and Fig. 2. The heart-shaped platelet between D2 somewhat bluish. Coxae in four groups, surface of coxae reticulated. ACG 92 (98) in length, apodeme well developed. E2 laterally between ACG and PCG. PCG 220 (231) in length. E4 absent from CxIII. Distance between anterior end of ACG and posterior end of PCG 373 (380). Genital field 205 (207) in length, 171 (175) in width. Preand postgenital sclerites developed. Three pairs of genital acetabula, the first pair of genital acetabula elliptical, the second pair somewhat triangular, and the third pair rounded and larger than the anterior two pairs. V1 on small sclerites and without accompanying glandularia. Excretory pore between V2, and with a sclerotized ring.
Capitulum with a long rostrum, length 213 (219). Chelicera total length 219 (226), basal segment length 158 (164), claw length 61 (62), ratio of basal segment / claw length 2.6. Dorsal lengths of the palpal segments: P-I, 22 (23); P-II, 123 (127); P-III, 172 (178); P-IV, 178 (183); P-V, 36 (37). P-I short and without seta. P-II with a long ventro-distal projection bearing one long setae. Approximately ten setae on the lateral and dorsal side of P-II and none of them plumose. The ventral side of P-III nearly straight and without seta, four short smooth setae on the lateral and dorsal side. P-IV with two small peg-like ventral setae, one larger almost in the middle, another one near the ventral distal end.
Legs  -6, 193 (198). Third to fifth segments of leg I-IV with rather short plumose setae in longitudinal rows (Fig. 9). Ambulacrum with two claws. Claws with well protruded claw-blade and two small claws, a long dorsal and a shorter ventral one (Fig. 8).
Etymology. The species is named after the village where it was collected.
Remarks. Due to the shape of the integument, P-II with a very long ventrodistal projection, excretory pore surrounded by sclerotized ring, and third to fifth segments of leg I-IV with plumose setae, the new species is similar to S. hispidus Koenike, 1895 and Sperchon indicus Kumar, Kumar & Pesic, 2007(Kumar et al. 2007, Tuzovskij 2010. However, the new species differs from the two species in the shape of the acetabula. In S. hispidus and S. indicus, three pairs of acetabula are large and arranged densely, whereas in the new species, the three pairs of acetabula are relatively small and arranged loosely with large gaps. The new species also differs from S. hispidus and S. indicus in the shape of apodemes of anterior coxae, which are indistinct in S. hispidus and S. indicus, but well developed in the new species. Besides, E4 is situated on CxIII in S. hispidus and S. indicus, but absent from CxIII in the new species. Distribution. China (Anhui Province).  432 (441-476) in width, color yellow-brown. Integument with very fine spinules arranged in hexagonal pattern (Fig. 12). Chitinous plates in dorsum and venter well developed as illustrated in Fig. 10 and Fig. 11. All glandularia and O2 surrounded by a platelet. A1 short and smooth, other dorsal setae thin and long. Coxae in four groups, surface of coxae reticulated. ACG 136 (138-152) in length, posterior apodeme indistinct. E2 laterally between  in length, widely separated. E4 absent from CxIII. Distance between anterior end of ACG and posterior end of PCG 329 (347-361). Genital field 135 (142-156) in length, 121 (128-137) in width, with a small and rounded platelet in front. Three pairs of genital acetabula, first and second pairs of acetabula elongate and oval, third pair more or less rounded. Pre-and postgenital sclerite not developed. V1 without accompanying glandularia but on sclerites of medium size. Excretory pore slightly anterior to V2, and surrounded by a welldeveloped sclerotized ring.
Legs Remarks. Sperchon rostratus was first described from Burma by Lundblad (1969). However, the description and illustration given in the literature are short and insufficient. The species was subsequently recorded from China (Guizhou Province, Taiwan), Iran, and Turkey (Smit 1995, Boyaci 2007, Pešić and Vafaei 2009. Although the species has been reported many times, an illustration of the idiosoma for the male was given only once (from Turkey) (Boyaci 2007).
Due to the shape of integument, E4 absent from CxIII, P-II with one thin seta, and P-IV with two small peg-like setae, the female from China shows a general conformity with S. rostratus, a species previously reported from China, however, the morphological characters of the male show obvious differences between the specimens in our study and the Turkish specimens. It is obvious that the platelets of the dorsum and venter of S. rostratus are large and close together (Fig 10-11), but small and arranged loosely in the Turkish specimens (see details of S. rostratus in Boyaci 2007). In addition, the pre-and postgenital sclerites are small in our specimens but relatively large in Turkish specimens, and the pregenital sclerite is somewhat crescent-shaped in Turkish specimens. Additionally, our specimens possess a rounded platelet in front of the genital field, which is absent in the Turkish specimens.
Although there are many differences between the male of S. rostratus in our study and the Turkish specimens, considering most characters of our specimens (eg., the shape of integument, E4 absent from CxIII, P-II with one thin seta, P-IV with two small peg-like setae and same habitat of the female), we attribute the male specimens to S. rostratus. In order to test whether the male and the female are conspecific, we used DNA barcoding technology for S. rostratus. The results are given below (see Results of molecular analysis).

Results of molecular analysis
The ten nucleotide sequences of 658 bp obtained belong to four species (S. fuxiensis, S. plumifer, S. rostratus, and Sperchonopsis echphyma) and two genera (Sperchon and Sperchonopsis). Sequence of S. fuxiensis is documented as DNA barcoding for future use, the others were constructed for a phylogenetic tree and analysed for genetic distances. Phylogenetic tree based on neighbour-joining (NJ) and maximum-likelihood (ML) gave the same result, with minor difference in bootstrap support values only ( Figure 20). The male and female of S. rostratus were clustered in a clade together with S. plumifer. Genetic distances (K2P) for barcode region of CO I between the species analysed in this study were shown in Table 2. The sequence divergence between the both sexes was 0.6%-1.1% in S. rostratus, and 0.9-1.2% in S. plumifer, respectively. The intraspecific sequence divergence was 0-1.2% (average is 0.62%) in S. plumifer, and 0.2-1.1% (average is 0.73%) in S. rostratus, respectively (Table 2). Interspecific divergence between S. plumifer and S. rostratus was 15.7%-17.1% (average is 16.53%). Intergeneric divergence between Sperchon and Sperchonopsis was 32.0%-36.6%.

Discussion
In this study, the female of S. rostratus coincided with the species as previously reported, but the male specimens showed differences in the size of chitinous plates and genital field. In order to verify whether the male specimens belong to S. rostratus, we attempted to use the molecular identification known as "DNA barcoding" to construct a polygenetic tree and analyse genetic distance. Our attribution of the male specimens to S. rostratus was supported by molecular data. Phylogenetic tree showed that the male and the female of S. rostratus in our study could cluster in the same clade. In addition, the divergence between the male and the female was 0.6%-1.1%, which approximately agrees with the divergence of both sexes of S. plumifer (0.9-1.2%). The morphological characters of the male S. rostratus showed obviously differences between China and Turkey. Many characters of the Chinese male specimens in our study, such as with extended and fused chitinous plates in dorsum and venter, preand postgenital sclerite weakly developed, and with a rounded platelet in front of the genital field (Figures 10-11), are typical characters of the male sex. On the contrary, most characters of the Turkish male specimens, e.g., with smaller and unfused chitinous plates, the pregenital sclerite is crescent-shaped and without a rounded platelet in front of the genital field, are the typical characters of the female sex (Di Sabatino et al. 2010). In our study, the Chinese male specimens also match the female of S. rostratus with the support of molecular data. Therefore, the male specimens of S. rostratus re- ported from Turkey are female and not male. Although S. rostratus has been reported many times, the description and illustration are still incomplete. Our study represents the first description and illustration of both sexes.
In recent years, the research of molecular identification and phylogeny have been reported in many genera of water mites, such as Brachypodopsis, Hygrobates, Monatractides, Neumania, Torrenticola, and Unionicola (Ernsting et al. 2010, 2016). However, previous research rarely involved molecular analysis of both sexes.  reported that divergence value between the female and male of Torrenticola lundbladi is 0.15% (different in one nucleotide), which is much lower than the divergences in our study (0.6%-1.1% in S. rostratus and 0.9-1.2% in S. plumifer). However, only one female and one male of T. lundbladi were compared in the their study, so that the molecular data is hardly representative.  reported that an appropriate threshold for DNA barcoding sequence should certainly be high enough to separate only specimens that very likely belong to different species. Their research also suggested the 10× rule threshold value method, in which interspecific divergences should be nearly ten times higher than intraspecific divergences ). In our study, the ratio of interspecific divergences to intraspecific divergences in the genus Sperchon is 22.64-26.66, which indicates that the DNA barcoding of COI gene may be a useful tool for identifying the Sperchon species.
The instable divergences among the different species of water mites may be ascribed to limited data and researches on DNA barcoding for water mites. More molecular researches on water mites are required to solve this problem.