Morphology, genetic characterization and molecular phylogeny of pinworm Skrjabinema longicaudatum n. sp. (Oxyurida: Oxyuridae) from the endangered Tibetan antelope Pantholops hodgsonii (Abel) (Artiodactyla: Bovidae)

Background The Tibetan antelope Pantholops hodgsonii (Abel) (Artiodactyla: Bovidae) is an endangered species of mammal endemic to the Qinghai-Tibetan Plateau. Parasites and parasitic diseases are considered to be important threats in the conservation of the Tibetan antelope. However, our present knowledge of the composition of the parasites of the Tibetan antelope remains limited. Methods Large numbers of nematode parasites were collected from a dead Tibetan antelope. The morphology of these nematode specimens was observed using light and scanning electron microscopy. The nuclear and mitochondrial DNA sequences, i.e. small subunit ribosomal DNA (18S), large subunit ribosomal DNA (28S), internal transcribed spacer (ITS) and cytochrome c oxidase subunit 1 (cox1), were amplified and sequenced for molecular identification. Moreover, phylogenetic analyses were performed using maximum likelihood (ML) inference based on 28S and 18S + 28S + cox1 sequence data, respectively, in order to clarify the systematic status of these nematodes. Results Integrated morphological and genetic evidence reveals these nematode specimens to be a new species of pinworm Skrjabinema longicaudatum (Oxyurida: Oxyuridae). There was no intraspecific nucleotide variation between different individuals of S. longicaudatum n. sp. in the partial 18S, 28S, ITS and cox1 sequences. However, a high level of nucleotide divergence was revealed between the new species and its congeners in 28S (8.36%) and ITS (20.3–23.7%) regions, respectively. Molecular phylogenetic results suggest that the genus Skrjabinema should belong to the subfamily Oxyurinae (Oxyuroidea: Oxyuridae), instead of the subfamily Syphaciidae or Skrjabinemiinae in the traditional classification, as it formed a sister relationship to the genus Oxyuris. Conclusions A new species of pinworm Skrjabinema longicaudatum n. sp. (Oxyurida: Oxyuridae) is described. Skrjabinema longicaudatum n. sp. represents the first species of Oxyurida (pinworm) and the fourth nematode species reported from the Tibetan antelope. Our results contribute to the knowledge of the species diversity of parasites from the Tibetan antelope, and clarify the systematic position of the genus Skrjabinema.


Background
The Tibetan antelope Pantholops hodgsonii (Abel) (Artiodactyla: Bovidae) is an endangered species of mammal endemic to the Qinghai-Tibetan Plateau. The population of the Tibetan antelope has been declined severely, because of the loss and fragmentation of its habitat and commercial poaching [1,2]. The latest estimate of the global population of the Tibetan antelope is

Morphological observations
For light microscopical studies, nematodes were cleared in lactophenol. Drawings were made using a Nikon microscope drawing attachment. For scanning electron microscopy (SEM), the anterior and posterior parts of specimens were re-fixed in 4% formaldehyde solution, post-fixed in 1% OsO4, dehydrated via an ethanol series and acetone, and then critical-point dried. Samples were coated with gold and examined using a Hitachi S-4800 scanning electron microscope at an accelerating voltage of 20 kV. Measurements (the range, followed by the mean values in parentheses) are given in micrometers (μm) unless otherwise stated.
subfamily Oxyurinae (Oxyuroidea: Oxyuridae), instead of the subfamily Syphaciidae or Skrjabinemiinae in the traditional classification, as it formed a sister relationship to the genus Oxyuris.

Conclusions:
A new species of pinworm Skrjabinema longicaudatum n. sp. (Oxyurida: Oxyuridae) is described. Skrjabinema longicaudatum n. sp. represents the first species of Oxyurida (pinworm) and the fourth nematode species reported from the Tibetan antelope. Our results contribute to the knowledge of the species diversity of parasites from the Tibetan antelope, and clarify the systematic position of the genus Skrjabinema.
Keywords: Tibetan antelope, Parasite, Nematoda, Morphology, Genetic data, Phylogeny 100,000-150,000 mature individuals (https ://www.iucnr edlis t.org/speci es/15967 /50192 544). This species is listed as "Near Threatened" in the IUCN Red List of Threatened Species ™ and also listed as Class I (Endangered in China) National Protected Wild Animal Species in China.
In the present study, some nematode specimens were collected from the digestive tract of the Tibetan antelope, which were identified morphologically as a new species of the genus Skrjabinema (Oxyurida: Oxyuridae) using light and scanning electron microscopy. The nuclear and mitochondrial DNA sequences, i.e. small subunit ribosomal DNA (18S), large subunit ribosomal DNA (28S), internal transcribed spacer (ITS) and cytochrome c oxidase subunit 1 (cox1), were also amplified and sequenced for molecular identification of this species. Moreover, in order to clarify the systematic status of the genus Skrjabinema, phylogenetic analyses were performed using maximum likelihood (ML) inference based on 28S and 18S + 28S + cox1 sequence data, respectively.

Parasite collection
A Tibetan antelope died naturally in the Hohxil National Nature Reserve, Qinghai Province, China. The digestive tract of this Tibetan antelope was sent to the Key Laboratory of Adaptation and Evolution of Plateau Biota (AEPB), Northwest Institute of Plateau Biology, Chinese Academy of Sciences for examination of parasites. Large numbers of nematode parasites were isolated from the caecum and colon. Specimens were fixed and stored in in 5% glycerine plus 70% ethanol until study.
Sequencing was carried out using a Dye Deoxy Terminator Cycle Sequencing Kit (v.2, Applied Biosystems, California, USA) and an automated sequencer (ABI-PRISM 377). Sequences were aligned using ClustalW2. The DNA sequences obtained herein were compared (using the algorithm BLASTn) with those available in the National Center for Biotechnology Information (NCBI) database (https ://www.ncbi.nlm.nih.gov).

Phylogenetic analyses
Phylogenetic trees were constructed using maximum likelihood (ML) inference with MEGA X software based on the partial 28S and 18S + 28S + cox1 sequence data, respectively. Pseudonymus spirotheca (Oxyurida: Thelastomatoidea: Pseudonymidae) was treated as the outgroup. The ingroup includes the representatives of the Oxyuridae with the 28S and 18S + 28S + cox1 sequence data available in the GenBank database. We used a builtin function in the software MEGA X to select a bestfitting substitution model for the present sequences according to the Bayesian information criterion. The K2 (Kimura 2-parameter) + G model for the 28S sequence data, and the HKY (Hasegawa-Kishino-Yano) + G + I model for the 18S+28S+cox1 sequence data were identified as the optimal nucleotide substitution model, respectively. Nodal support for ML trees were tested using 1000 bootstrap replications, and bootstrap values exceeding 80% were showed in the phylogenetic trees.

Site in host
Caecum and colon.

Prevalence and intensity
A single Tibetan antelope examined with 124 worms.

ZooBank registration
To comply with the regulations set out in Article 8.5 of the amended 2012 version of the International Code of Zoological Nomenclature (ICZN) [17], details of the new species have been submitted to ZooBank. The Life Science Identifier (LSID) of the article is urn:lsid:zoobank.org:pub:9194626F-7C3B-445C-BD36-0AF06E39C46F. The LSID for the new name Skrjabinema longicaudatum is urn:lsid:zoobank. org:act:3A5AB2D4-5B82-4CBE-8CF8-76B97783694E.

Etymology
The specific epithet is derived from a combination of the Latin words longus-(long) and caudatum-(cauda), and refers to the unusually long tail in the female of the new species.

Partial 18S region
Three 18S sequences of S. longicaudatum n. sp. obtained herein were all 678 bp in length and represent one genotype. There are two species of Skrjabinema with 18S sequence registered in GenBank, namely S. kamosika (AB699690) and Skrjabinema sp. (EF180060). Pairwise comparison of 18S sequences between S. longicaudatum n. sp. and the two species of Skrjabinema displayed 0.29-1.18% nucleotide divergence. The 18S sequences of S. longicaudatum n. sp. are deposited in the GenBank database under the accession numbers MW020179-MW020181.

Partial ITS region
Three ITS sequences of S. longicaudatum n. sp. obtained herein were all 1079 bp in length and represent one genotype. There are two species of Skrjabinema with ITS sequence registered in Gen-Bank, namely S. kamosika (AB699691) and Skrjabinema sp. (AB367796). Pairwise comparison of ITS sequences between S. longicaudatum n. sp. and the other two species of Skrjabinema displayed 20.3-23.7% nucleotide divergence. The ITS sequences of S. longicaudatum n. sp. are deposited in the GenBank database under the accession numbers MW020057-MW020059.

Partial 28S region
Three 28S sequences of S. longicaudatum n. sp. obtained herein were all 819 bp in length and represent one genotype. There is only one species of Skrjabinema, namely S. ovis (KY990019) with 28S sequence registered in Gen-Bank. Pairwise comparison of ITS sequences between S. longicaudatum n. sp. and S. ovis displayed 8.36% nucleotide divergence. The 28S sequences of S. longicaudatum n. sp. are deposited in the GenBank database under the accession numbers MW020098-MW020100.

Partial cox1 region
Three cox1 sequences of S. longicaudatum n. sp. obtained herein was 360 bp in length. There is no species of Skrjabinema with cox1 sequence registered on GenBank. The cox1 sequences of S. longicaudatum n. sp. are deposited in the GenBank database under the accession numbers MW021552-MW021554.

Phylogenetic analyses
Phylogenetic trees based on the partial 28S sequence data showed that representatives of the family Oxyuridae were divided into three monophyletic clades. Clade I included members of the genera Syphacia, Passalurus, Syphatineria, Syphabulea and Rauschtineria, representing the subfamily Syphaciinae. Clade II contained species of the genera Oxyuris and Skrjabinema, representing the subfamily Oxyurinae. Clade III included species of the genus Trypanoxyuris, representing the subfamily Enterobiinae (Fig. 4). Skrjabinema longicaudatum n. sp. displayed a sister relationship to S. ovis.
Phylogenetic tree constructed based on the 18S+28S+cox1 sequence data had similar topology to the phylogenetic results using the partial 28S sequence data, in which representatives of the Oxyuridae also divided into three monophyletic clades (Fig. 5). Species of Trypanoxyuris and Enterobius formed clade I,  -bars: a, f, 500 μm; b, e, g, h, i, 50 μm; c, 100
Skrjabinema ovis is the type-species of this genus, which has been widely reported from goats and sheep in Asia, Europe, America and Australia [21]. This It is difficult to identify and discriminate the pinworms using traditional methods due to their extraordinary morphological similarity and sometimes the male worms being unavailable [26]. Molecular approaches have been employed for identification and discrimination of pinworms in some previous studies [24,[26][27][28][29][30][31][32]. However, to date, genetic data of pinworms available in the GenBank database remain limited, which has hindered the further studies of DNA-based taxonomy, population genetics and phylogenetics of this group of nematode parasites.
In the present study, we amplified and sequenced the partial 18S, 28S, ITS and cox1 alignments of our specimens for future use in the molecular identification of this new species. There was no intraspecific nucleotide variation between different individuals of S. longicaudatum n. sp. in the partial 18S, 28S, ITS and cox1 sequences. However, a high level of nucleotide divergence was revealed between the new species and its congeners in 28S (8.36%) and ITS (20.3-23.7%) regions, respectively. The more slowly evolving 18S gene may be not suitable for species identification of Skrjabinema, because of very low level of interspecific nucleotide variation detected between different species of Skrjabinema (0.29-1.18%). However, the 18S gene could be chosen to provide resolution at higher taxonomic levels. It is the first time to report the cox1 sequence of Skrjabinema species.
The systematic position of Skrjabinema is still under debate. Skrjabin [22] placed this genus into the subfamily Syphaciinae Railliet, 1916 in Syphaciidae Skrjabin & Schikhobalova, 1951. Erkulov & Moldopiyazova [33] proposed a new subfamily Skrjabinemiinae for the genera Skrjabinema and Citellina. Hugot [34] reduced the family Syphaciidae to a subfamily in Oxyuridae and did not recognise the validity of Skrjabinemiinae. The present phylogenetic analyses based on the partial 28S and 18S+28S+cox1 sequence data supported the genus Skrjabinema to be a member of the subfamily Oxyurinae, with a sister relationship with the genus Oxyuris, which agrees well with recent molecular phylogenetic results [26]. Our present knowledge of the composition of the nematode parasites of the Tibetan antelope remains limited. In the light of available literature, only three species of nematodes have been recorded from the Tibetan antelope, including Nematodirus sp., Marshallagia mongolica Schumakoviech, 1938 and M. marshalli (Ransom 1907) (Rhabditida: Strongyloidea) [9][10][11]. Skrjabinema longicaudatum n. sp. represents the first species of Oxyurida (pinworm) and the fourth nematode species reported from the Tibetan antelope.

Conclusions
A new species of pinworm Skrjabinema longicaudatum n. sp. (Oxyurida: Oxyuridae) is described using light and scanning electron microscopy, based on specimens collected from the endangered Tibetan antelope. Skrjabinema longicaudatum n. sp. represents the first species of Oxyurida (pinworm) and the fourth nematode species reported from the Tibetan antelope. The nuclear and mitochondrial DNA sequences (i.e. 18S, 28S, ITS and cox1) were amplified and sequenced for molecular identification of this new species. Phylogenetic analyses using maximum likelihood (ML) inference based on 28S and 18S + 28S + cox1 sequence data suggested that the genus Skrjabinema should belong to the subfamily Oxyurinae (Oxyuroidea: Oxyuridae), instead of the subfamily Syphaciidae or Skrjabinemiinae in the traditional classification, as it formed a sister relationship to the genus Oxyuris. Our results contribute to the knowledge of the species diversity of parasites from the Tibetan antelope, provided useful genetic data for molecular identification and phylogeny of the Oxyuridae, and also clarified the systematic position of the genus Skrjabinema.