﻿Description of the first species of Scutigerella (Symphyla, Scutigerellidae) from China, with mitogenomic and genetic divergence analysis

﻿Abstract Scutigerellasinensis Jin & Bu, sp. nov. from China is described and illustrated. It is characterized by a deeply emarginated posterior margin of tergite 2, less differentiated marginal setae on all tergites, absence of seta a3 around the antennal base, and 6–8 setae on the first tergite. The complete mitochondrial genome of the new species is also analyzed and compared with the mitogenome of Scutigerellacauseyae. In the reconstructed Neighbor-Joining tree based on COI gene sequences, S.sinensissp. nov. clusters with S.causeyae, however, with big distances. The genetic divergence among S.sinensissp. nov. and congeners, species of Hanseniella and Scutigerella, and both families of Symphyla was analyzed using COI gene sequences.


Introduction
The class Symphyla Ryder, 1880 is a monophyletic group of myriapods with worldwide distribution (Szucsich and Scheller 2011). However, both taxonomic and molecular studies are widely missing. There are only about 250 species reported worldwide (https://www.itis.gov/; Bu 2020, 2023) and ten species recorded in China until now Jin and Bu 2018, 2020, 2023. Only 65 mitochondrial gene sequences and two complete mitogenomes are available in Gen-Bank (accessed in February 2023). The sub-cosmopolitan genus Scutigerella Ryder, 1882 is the second largest group of the family Scutigerellidae. It includes 36 valid species registered in the Integrated Taxonomic Information System (ITIS; https://www. itis.gov/) (accessed in February 2023). However, Scutigerella gratiae (Ryder, 1881) is missing in the database of ITIS, but it is recorded in the virtual research environment Myriatrix, The Fellegship of the Rings (2020 onwards) (http://myriatrix.myspecies. info), thus 37 valid Scutigerella species in total. The occurrence of Scutigerella in China (Hunan Province) was only once mentioned in a monograph (Zhang and Wang 1992), but the species remained undetermined.
In recent years, many specimens of Scutigerellidae Bagnall, 1913 were obtained from Shanghai and Beijing and were carefully studied, and most of them belong to the genus Hanseniella Bagnall, 1913. Among those specimens, one species of Scutigerella was identified as new to science and is described in the present paper. It is also the first species of the genus from China. In order to provide further evidence for the new species and clarify its taxonomic position, its complete mitogenome was sequenced and analyzed. In addition, the phylogenetic relationship and genetic divergence of symphylans were analyzed based on DNA barcode sequences.

Sample collection and taxonomy
Soil and litter samples from broad-leaf and bamboo forests from Dajinshan Island, Shanghai were collected during several ecological surveys of soil fauna between 2015-2018, and specimens were extracted using Berlese-Tullgren funnels and preserved in 80% ethanol. Materials from Beijing were collected in Yuan-Ming Yuan Imperial Garden by Mr Rui-Qing Wang in 2021. They were mounted on slides using Hoyer's solution and dried in an oven at 50 °C. Morphological observations were performed under a phase contrast microscope (Leica DM 2500). Photographs were taken with a digital camera installed on the microscope (Leica DMC 4500). Line drawings were done using a drawing tube. All specimens are deposited in the collections of the Shanghai Natural History Museum (SNHM), Shanghai, China.

Molecular analyses
The specimens used for the experiment were collected by Ya-Li Jin and Yun Bu from Dajinshan Island on 11 November, 2017. Samples were preserved in absolute ethanol at -20 °C for DNA extraction. Prior to DNA extraction, a single individual was mounted on a temporary slide using absolute ethanol to confirm the species identification. One specimen, preserved in alcohol, was sent to Shanghai Yaoen Biotechnology Co., Ltd, China, where all laboratory procedures, including DNA extraction and library construction were made following custom procedures. DNA was extracted from a single individual of the species using the TIANamp MicroDNA extraction kit (Tiangen Co., Ltd, China). Libraries were constructed using KAPA Hyper Prep Kit (Roche). An Illumina NovaSeq platform was used to produce paired-end reads with 150 bp length. Approximately 10 Gb of data from the species was generated and used to assemble the mitogenomes.

Sequence analysis
NovoPlasty v.3.8.3 (Dierckxsens et al. 2016) was used to assemble the mitogenome using the COI sequence from Scutigerella causeyae Michelbacher, 1942 retrieved from GenBank as a seed (accession number NC008453). Genes annotation was performed using MitoZ v.2.4-alpha (Meng et al. 2019). The final mitogenome sequence with annotations and the raw sequencing data were submitted to the National Center for Biotechnology Information database (NCBI), accession numbers are listed in Table 1. The mitogenomic data of Scutigerella causeyae were downloaded from GenBank (https:// www.ncbi.nlm.nih.gov/), and the length, genes arrangement, nucleotides content, and other genomic features were compared with Scutigerella sinensis sp. nov. In order to make a comprehensive analysis of genetic divergences among symphylans, DNA barcode sequences (COI gene, 658 base pairs) of 26 sequences of the family Scutigerellidae and one sequence of the family Scolopendrellidae Newport, 1844 (outgroup) were downloaded from GenBank and analyzed. The detailed information and accession numbers of the 28 sequences analyzed in this study are listed in Table 1. To infer the position of the new species described, the Neighbor-Joining tree was constructed based on COI gene sequences by MEGA X (Kumar et al. 2018) with the Jukes-Cantor model (Jukes and Cantor 1969) and 1000 bootstrap replicates. The genetic distance (K2P-distance) was calculated using MEGA X (Kimura 1980;Kumar et al. 2018) and the genetic divergence was analyzed for different taxonomic levels of Symphyla.

Data availability statement
After publication, mitogenome sequence and raw sequencing data will be available in GenBank (NCBI) at https://www.ncbi.nlm.nih.gov/ under the accession numbers OQ165321/PRJNA900014.
Diagnosis. Trunk with 15 tergites. Four macrosetae (a1-a4) around the antennal base, rarely seta a3 absent. Posterior margins of tergites emarginated. Last tergite with a deep cavity between cerci. First pair of legs with 4 segments, others with 5 segments. Styli present at the base of legs 3-12. Coxal sacs present at the base of legs 3-10.
Distribution. Sub-cosmopolitan (Szucsich and Scheller 2011 Table 2 Diagnosis. Scutigerella sinensis sp. nov. is characterized by absence of a3 seta around the antennal base, 6-8 setae on the first tergite, deeply emarginated posterior margin of tergite 2, 28-37 marginal and 41-57 inner setae on tergite 2, less differentiated marginal setae on all tergites, femur of first pair of legs without a conspicuous ventral process, posterior styli without a lateral seta, cavity of fifteenth tergite V-shaped, tarsus of last pair of legs moderately set with setae, cerci 2.7-3.4 times as long as width, cerci densely covered with subequal setae, cerci without expansion in terminal area. Material examined. Holotype: male (slide no. SH-DJS-SY2015009) (SNHM), China, Shanghai, Dajinshan Island, extracted from soil samples of bamboo forest, alt.   (Fig. 1A).
Sense calicles located on two ventral protuberances of last tergite, posterior to base of leg 12, with smooth margin around pit. Sensory seta inserted in cup center, extremely long (235-275 μm).
Etymology. This new species is named after the country of origin, from the Latin adjective sinensis, meaning Chinese.
Distribution. China (Shanghai, Beijing). Ecology. Our current investigation indicates that Scutigerella sinensis sp. nov. is a rare species in natural habitats with very low density. We found about ten individuals among several hundred symphylans from plots of different vegetation. This Scutigerella is often coexisting with other dominant species of Hanseniella and Symphylella in the upper soil layer (0-10 cm) or humus.
Remarks. The head chaetotaxy was briefly described in the previous studies of Scutigerella, usually with only shapes and numbers mentioned. The macrosetae around the base of the antenna of Scutigerella have been noticed and named by former researchers, and all species examined until now have a complete set of four macrosetae (a1-a4), which was deemed as a good diagnostic character in the taxonomy of Scutigerella (Hinschberger 1950;Juberthie-Jupeau and Tabacaru 1968;Scheller 1986). Scutigerella sinensis sp. nov. has three macrosetae (a1, a2, and a4) around the antennal base, with a3 seta absent, which can be easily distinguished from all other congeners. Our observation indicates this character is stable in both adults and juveniles and can be a unique feature of the new species. The present new species is most similar to the cosmopolitan species Scutigerella immaculata (Newport, 1845) in the shapes and chaetotaxy of tergites and legs, but differs in the absence of the a3 seta on the head (present in S. immaculata), number of marginal setae on tergite 2 and 3 (less than 50 in S. sinensis sp. nov. vs. more than 50 in S. immaculata), and the shape of the stylus (tapering in S. sinensis sp. nov. vs. cylindrical in S. immaculata).

Mitogenomic analysis
The mitochondrial genome of Symphyla until now was only known from two species: Scutigerella causeyae and one undetermined species of Symphylella (Podsiadlowski et al. 2007;Gai et al. 2008). In the present study, we sequenced the complete mitogenome of Scutigerella sinensis sp. nov. The mitogenome of S. sinensis sp. nov. is 14 512 bp long and contains the control region (CR) and all 37 genes typically found in Arthropoda (Fig. 4, Table 3). The nucleotide composition varies along its length, being AT-rich for the entire mitogenome with: A -36.41% (5284); T -34.46% (5001); C -19.41% (2817) and G -9.71% (1410). With an AT content of 81% the AT rich or control region is 297 bp long and is located between trnQ and trnM. Three different start codons were present in the protein coding genes: ATG (6×) and ATA (2×), canonical codons encoding Methionine, and ATT (5×) encoding Isoleucine. Two different stop codons were present: TAA (11×) and TAG (2×). No truncated stop codon was observed (Table 3).
Compared to the mitogenome of the congeneric species S. causeyae, the new sequence is 125 bp smaller and differs in the relative position of three tRNA genes (Q, M, I) located next to the control region. Additionally, the tRNA-Valine is located between the rRNAs, like in the inferred arthropod ground pattern (Staton et al. 1997) (Fig. 5). This is the first report of the occurrence of this ground plan in Scutigerella, which is an important similarity between the new species and the hypothetical ancestor. On other hand, the translocation between the tRNA genes P and T is shared between S. sinensis and S. causeyae, but not observed in the ground pattern; it can be a step toward for further studies on character evolution in Scutigerella.    and Scutigerellidae. Since most sequences downloaded from GenBank are from the individuals only primarily determined to family or genus levels and the validation of identification cannot be confirmed, we refrain from questioning the monophyletic status of Hanseniella and Scutigerella.

Genetic divergence
The pairwise genetic distance of 28 sequences of symphylan species based on the K2P model is given in the Suppl. material 1. The genetic distance between S. sinensis sp. nov. and other congeners is 0.2747 on average (0.2280-0.2946), which gives further support for our morphological identification, however the coverage of species does not allow us to draw too many conclusions. The genetic distances of the COI gene among different taxonomic levels of Symphyla are given in Table 4 (but determinations of species downloaded from GenBank are questionable).