Description of a new species of AplECTana (Nematoda: Ascaridomorpha: Cosmocercidae) using an integrative approach and preliminary phylogenetic study of Cosmocercidae and related taxa Author Chen, Hui-Xia Key Laboratory of Animal Physiology, Biochemistry and Molecular Biology of Hebei Province, College of Life Sciences, Hebei Normal University, 20 East Road of 2 nd South Ring, Yuhua District, 050024 Shijiazhuang, Hebei Province, People’s Republic of China liangliangex369@126.com Author Gu, Xiao-Hong Author Ni, Xue-Feng Author Li, Liang text Parasites & Vectors 2021 165 2021-03-18 14 1 1 10 http://dx.doi.org/10.1186/s13071-021-04667-9 journal article 10.1186/s13071-021-04667-9 1756-3305 11074724 Aplectana xishuangbannaensis n. sp. Type host: White-spotted thigh tree-frog Polypedates megacephalus (Hallowell) ( Anura : Rhacophoridae ). Type-locality: XiShuangBanNa Tropical Botanical Garden ( 21°41 ′ N , 101°25 ′ E ), Yunnan Province , China . Type specimens: Holotype : male ( HBNU–N-2020A009L ); allotype : female ( HBNU–N-2020A010L ); paratypes : 41 males , 122 females ( HBNU–N-2020A011L ). Site of infection : Intestine. Prevalence and intensity of infection: 12.1% ( 11 P. megacephalus infected out of 91 examined) were infected with intensity of 1–88 (mean 15.0) nematodes. 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 ) [ 19 ], details of the new species have been submitted to ZooBank. Te Life Science Identifier ( LSID ) of the article is urn:lsid:zoobank.org:pub:09F4B1EF-C3AF42E6-80E6-B734D6B084B8. Te LSID for the new name Aplectana xishuangbannaensis is urn:lsid:zoobank. org:act: 5E4C6C18-7B72-4C28-BD28-6964C6D8F0A3 . Etymology: Te specific epithet refers to the type location XiShuangBanNa Tropical Botanical Garden, Yunnan Province , China . Description General Small-sized, whitish nematodes. Body cylindrical, maximum width at about region of middle body. Cuticle with fine transverse striations and longitudinal stockade-like ornamentation ( Fig. 1a–c ). Somatic papillae small, distributed irregularly over body surface ( Figs. 1a–c, e, i , 2b ). Lateral alae extending from 60–70 posterior to base of lips as far as about middle of tail in both sexes ( Fig. 1b, f, i ). Oral aperture simple, triangular, surrounded by 3 small lips, each with inner flanges ( Figs. 1a, b, d , 2b ). Dorsal lip with pair of large double cephalic papillae; subventral lips with single large double cephalic papilla and amphid each ( Figs. 1a , 2b ). Oesophagus divided into anterior short pharynx, cylindrical corpus, slightly narrower isthmus and terminal posterior bulb with valves ( Fig. 2a ). Nerve ring located at about 1/2 of oesophageal length. Excretory pore slightly anterior to of oesophageal bulb ( Fig. 2a ). Tail of both sexes conical, with long filamentous tip ( Figs. 1e–g, i , 2c, f, h ). Male [Based on 10 mature specimens; Figs. 1b, d–h , 2a, f–h ]: Body 2.32–2.72 (2.49) mm long, maximum width 139–178 (158). Oesophagus 317–426 (374) long, representing 12.6–16.1 (15.0) % of body length; pharynx + corpus + isthmus 248–356 (307) long, bulb 59–69 (67) × 50–59 (54) ( Fig. 2a ). Nerve ring 158–198 (176) and excretory pore 257–376 (334) from anterior extremity, respectively ( Fig. 2a ). Posterior end of body distinctly curved ventrally ( Figs. 1e , 2f ). Spicules small, similar in shape and length, 139–178 (161) long, distal end pointed, representing 5.98–7.09 (6.47) % of body length ( Fig. 2g ). Gubernaculum absent. Caudal papillae: 6 pairs of precloacal, 3 pairs paracloacal (distinguishable from somatic papillae) and 4 pairs postcloacal papillae. Single median, ventral precloacal papilla present ( Figs. 1g , h , 2h ). Tail 198–248 (230) long, representing 8.26 ‒ 9.84 (9.26) % of body length ( Figs. 1e–g , 2f, h ). Female [Based on 10 mature specimens; Figs. 1a, c, i , 2b–e ]: Body 3.54–3.86 (3.65) mm long, maximum width 248–297 (272). Oesophagus 416–446 (431) long, representing 11.0–12.6 (11.8) % of body length; pharynx + corpus + isthmus 347–366 (356) long, bulb 69–79 (74) × 50–69 (62). Nerve ring 208–228 (215) and excretory pore 347–386 (366) from anterior extremity, respectively. Vulva transverse slit, 1.60–2.10 (1.89) mm from anterior extremity, at 44.8–54.5 (51.8) % of body length. Ovaries two, located anterior to vulva. Vagina muscular ( Fig. 2d ). Uteri amphidelphic, full of eggs in different stages of development; egg oval, large, with smooth surface, 149–297 (205) × 99–238 (146) ( n = 20) ( Fig. 2e ). Tail 347–406 (384) long, representing 9.78 ‒ 11.1 (10.5) % of body length ( Figs. 1i , 2c ). Table 1 Representatives of Cosmocercoidea used for phylogenetic analyses related to information on host, locality and GenBank ID

Species	Host	Locality	GenBank ID	References
18S	28S
Aplectana xishuangbannaensis n. sp.	Polypedates megacephalus (Hallowell)	China	MW329041	MW329038	Present study
Aplectana sp.	Hylarana spinulosa (Smith)	China	MW329991	MW364062	Present study
Cosmocerca ornata (Dujardin, 1845)	Hylarana spinulosa (Smith)	China	MW326676	MW326675	Present study
Cosmocerca simile Chen, Zhang, Feng & Li, 2020	Bufo gargarizans Cantor	China	MN839758	MN833301	Chen et al. [ 10 ]
Cosmocerca sp.1	Hoplobatrachus chinensis (Osbeck)	China	MW329987	MW329989	Present study
Cosmocerca sp.2	Bufo melanostictus Schneider	China	MW329990	MW329988	Present study
Cosmocercoides pulcher Wilkie, 1930	Bufo japonicus formosus	Japan	LC018444	LC018444	Tran et al. [ 46 ]
Cosmocercoides qingtianensis Chen, Zhang, Nakao & Li, 2018	Bufo gargarizans Cantor	China	MH178321	MW325956	Chen et al. [ 47 ]; Present study
Cosmocercoides tonkinensis Tran, Sato & Luc, 2015	Acanthosaura lepidogaster (Cuvier)	Vietnam	AB908160	AB908160	Tran et al. [ 46 ]
Cruzia americana Maplestone, 1930	Didelphis virginiana Kerr	USA	U94371	U94757	Nadler and Hudspeth [ 13 ]
Falcaustra sp. _T	Lithobates catesbeianus (Shaw) ; Indotestudo elongate (Blyth)	Japan;China	AB818380	MF094270	Hasegawa et al. [ 48 ]; Li et al. [ 49 ]
Megalobatrachonema hainanensis Chen, Zhang & Li, 2019	Amolops hainanensis (Boulenger)	China	–	MH545569	Chen et al. [ 9 ]
Megalobatrachonema terdentatum (Linstow, 1898)	Lissotriton vulgaris (Linnaeus)	Germany	–	MN444705	Sinsch et al. [ 50 ]
Megalobatrachonema wangi Chen, Zhang, Sinsch, Scheid, Balczun & Li, 2020	Quasipaa exilispinosa (Liu & Hu)	China	MW325957	MN245660	Present study; Chen et al. [ 11 ]
Orientatractis moraveci Cavalcante, Silva, Santos, Chagas-Moutinho & Santos, 2016	Pimelodus blochii Valenciennes	Brazil	KX524513	KX524514	Cavalcante et al. [ 51 ]
Rondonia rondoni (Travassos,1920)	Pterodoras granulosus ( Doradidae ); Pimelodus blochii Valenciennes	Peru; Brazil	DQ442679	KX524512	Wijova et al. [ 52 ]; Cavalcante et al. [ 51 ]
Ascaris lumbricoides Linnaeus,1758	Homo sapiens Linnaeus	USA	M74585	U94751	MÜller et al. [ 53 ]; Nadler and Hudspeth [ 13 ]

Genetic characterization Partial 18S region Tree 18S sequences of Aplectana xishuangbannaensis n. sp. (accession numbers MW329041–MW329043) obtained were all 1539 bp long, representing only one genotype. Tere is no species of Aplectana with 18S sequenced registered in GenBank. Pairwise comparison between A. xishuangbannaensis n. sp. and the other species of Cosmocercidae regarding the 18S sequences available in GenBank, including Cosmocerca simile (MN839758–MN839760), Cosmocercoides dukae (FJ516753), C. pulcher (LC018444, MH178322– MH178326), C. qingtianensis (MH032769–MH032771, MH178319–MH178321), C. tonkinensis (AB908160), C. wuyiensis (MK110872), Nemhelix bakeri (DQ118537) and Raillietnema sp. (DQ503461), displayed 1.88– 3.77% nucleotide divergence. Partial ITS-1 region Tree ITS-1 sequences of A. xishuangbannaensis n. sp. (accession numbers MW329035–MW329037) obtained were all 554 bp long, representing only one genotype. Tere are two species of Aplectana with ITS sequences available in GenBank, including A. chamaeleonis (MN907375 ‒ MN907378) and Aplectana sp. ’ Neyraplectana ’ PNLS-530 (MH836325). Pairwise comparison between A. xishuangbannaensis n. sp. and the previously mentioned taxa showed 46.67 and 45.47% nucleotide divergence, respectively. Pairwise comparison between A. xishuangbannaensis n. sp. and the other species of Cosmocercidae regarding the ITS sequences available in GenBank, including Cosmocerca japonica (LC052772 ‒ LC052782), C. longicauda (MG594349 ‒ MG594351), C. ornata (MT108302), Cosmocerca sp. LL-2020 (MT108303), C. simile (MN839761 ‒ MN839768), Cosmocercoides pulcher (MH178314–MH178318, LC018444), C. qingtianensis (MH178311–MH178313, MH032772–MH032774), C. tonkinensis (AB908160, AB908161) and C. wuyiensis (MK110871), displayed 28.53–47.52% of nucleotide divergence. Fig. 1 Scanning electron micrographs of Aplectana xishuangbannaensis n. sp. collected from Polypedates megacephalus (Hallowell) ( Anura : Rhacophoridae ) in Yunnan Province, China. a Cephalic end of female (somatic papillae (black arrows) and amphids (white arrows) arrowed), subapical view. b Anterior part of male (somatic papillae (black arrows) and lateral ala (white arrow) arrowed), lateral view. c Magnified image of somatic papilla and longitudinal stockade-like ornamentation of cuticle of female. d Cephalic end of male (inner flanges arrowed), subapical view. e Posterior end of male (precloacal papillae arrowed),lateral view. f Tail of male (lateral ala arrowed), lateral view. g Tail of male (four pairs of postcloacal papillae arrowed), ventro-lateral view. h Magnified image of single, median precloacal papilla. i Tail of female (somatic papillae arrowed), ventral view Fig. 2 Aplectana xishuangbannaensis n. sp. collected from Polypedates megacephalus (Hallowell) ( Anura : Rhacophoridae ) in Yunnan Province, China. a Anterior part of male, lateral view. b Cephalic end of female (somatic papillae arrowed), apical view. c Tail of female, lateral view. d Vulva,lateral view. e Egg. f Posterior end of male, lateral view. g Spicules. h Tail of male,ventral view. Scale bars : a , c‒h , 100 μm; b , 20 μm Partial 28S region Tree 28S sequences of A. xishuangbannaensis n. sp. (accession numbers MW329038–MW329040) obtained were all 740 bp long, representing only one genotype. Tere is only one species of Aplectana , Aplectana sp. ’ Neyraplectana ’ PNLS-530, with 28S sequence data (MH909070) available in GenBank. Pairwise comparison between A. xishuangbannaensis n. sp. and the previously mentioned taxon showed 20.67% of nucleotide divergence. Pairwise comparison between A. xishuangbannaensis n. sp. and the other species of Cosmocercidae with 28S sequences available in GenBank, including Cosmocerca simile (MN839755–MN839757), Cosmocercoides pulcher (LC018444) and C. tonkinensis (AB908160), displayed 16.78–17.94% of nucleotide divergence. Partial cox1 region Tree cox 1 sequences of A. xishuangbannaensis n. sp. (accession numbers MW327586–MW327588) obtained were all 384 bp long, representing only one genotype. Tere is no species of Aplectana with cox 1 sequence registered in GenBank. Pairwise comparison between A. xishuangbannaensis n. sp. and the other species of Cosmocercidae regarding the cox 1 sequences available in GenBank, including C. japonica (LC052756 ‒ LC052770), C. ornata (MT108304), Cosmocerca sp. LL-2020 (MT108305), C. simile (MN833301 ‒ MN833303), C. pulcher (MH178306–MH178310, LC052771) and C. qingtianensis (MH178303–MH178305, MH032775– MH032777), displayed 10.23–21.09% nucleotide divergence. Phylogenetic analyses Phylogenetic trees inferred from maximum likelihood (ML) and Bayesian inference (BI) showed that representatives of Cosmocercoidea were divided into four major clades ( Fig. 3 ). Clade I included the species of three genera Cosmocerca , Cosmocercoides and Aplectana , representing the family Cosmocercidae . Among the three genera, Cosmocerca displayed a closer relationship to Aplectana rather than Cosmocercoides . Clade II included only Cruzia americana (a common nematode parasite in the digestive tract of opossums), which belongs to the subfamily Cruzinae in the family Kathlaniidae according to the current classification [ 1 ]. Clade III included species of Falcaustra and Megalobatrachonema , which represent the family Kathlaniidae . Te representatives of Orientatractis and Rondonia formed Clade IV, representing the family Atractidae .