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Description of a new species of Aplectana (Nematoda: Ascaridomorpha: Cosmocercidae) using an integrative approach and preliminary phylogenetic study of Cosmocercidae and related taxa
Parasites & Vectors volume 14, Article number: 165 (2021)
Nematodes of the family Cosmocercidae (Ascaridomorpha: Cosmocercoidea) are mainly parasitic in the digestive tract of various amphibians and reptiles worldwide. However, our knowledge of the molecular phylogeny of the Cosmocercidae is still far from comprehensive. The phylogenetic relationships between Cosmocercidae and the other two families, Atractidae and Kathlaniidae, in the superfamily Cosmocercoidea are still under debate. Moreover, the systematic position of some genera within Cosmocercidae remains unclear.
Nematodes collected from Polypedates megacephalus (Hallowell) (Anura: Rhacophoridae) were identified using morphological (light and scanning electron microscopy) and molecular methods [sequencing the small ribosomal DNA (18S), internal transcribed spacer 1 (ITS-1), large ribosomal DNA (28S) and mitochondrial cytochrome c oxidase subunit 1 (cox1) target regions]. Phylogenetic analyses of cosmocercoid nematodes using 18S + 28S sequence data were performed to clarify the phylogenetic relationships of the Cosmocercidae, Atractidae and Kathlaniidae in the Cosmocercoidea and the systematic position of the genus Aplectana in Cosmocercidae.
Morphological and genetic evidence supported the hypothesis that the nematode specimens collected from P. megacephalus represent a new species of Aplectana (Cosmocercoidea: Cosmocercidae). Our phylogenetic results revealed that the Cosmocercidae is a monophyletic group, but not the basal group in Cosmocercoidea as in the traditional classification. The Kathlaniidae is a paraphyletic group because the subfamily Cruziinae within Kathlaniidae (including only the genus Cruzia) formed a seperate lineage. Phylogenetic analyses also showed that the genus Aplectana has a closer relationship to the genus Cosmocerca in Cosmocercidae.
Our phylogenetic results suggested that the subfamily Cruziinae should be moved from the hitherto-defined family Kathlaniidae and elevated as a separate family, and the genus Cosmocerca is closely related to the genus Aplectana in the family Cosmocercidae. The present study provided a basic molecular phylogenetic framework for the superfamily Cosmocercoidea based on 18S + 28S sequence data for the first time to our knowledge. Moreover, a new species, A. xishuangbannaensis n. sp., was described using integrative approach.
The superfamily Cosmocercoidea is a group of zooparasitic nematodes and currently comprises three families, namely, Atractidae Railliet, 1917, Cosmocercidae Railliet, 1916, and Kathlaniidae Lane, 1914 [1,2,3]. Among them, Cosmocercidae is the largest family, including approximately 200 nominal species, which are mainly parasitic in the digestive tract of various amphibians and reptiles worldwide [4,5,6]. The evolutionary relationships of the Cosmocercidae and the other two families are not yet resolved. Based on morphological and ecological traits, some previous studies [1, 6, 7] considered that the Cosmocercidae represents the ancestral group in Cosmocercoidea.
The present knowledge of the molecular phylogeny of Cosmocercoidea/Cosmocercidae is still very limited. To date, several studies [8,9,10,11] have provided molecular phylogenetic analyses to solve the systematic status of some genera in Cosmocercoidea using different genetic data. However, due to the paucity and inaccessibility of suitable material of Cosmocercoidea/Cosmocercidae for genetic analysis, all of these molecular phylogenetic studies have included only small numbers of representatives of these taxa.
To clarify the phylogenetic relationships of the Cosmocercidae and the other families Atractidae and Kathlaniidae in Cosmocercoidea, and the systematic position of the genus Aplectana in Cosmocercidae, phylogenetic analyses including the most comprehensive taxon sampling of Cosmocercoidea to date were performed using maximum likelihood (ML) inference and Bayesian inference (BI) based on 18S + 28S sequence data. Moreover, a new species of Aplectana was described using an integrative approach.
A total of 91 Polypedates megacephalus (Hallowell) (Anura: Rhacophoridae) collected in the XiShuangBanNa Tropical Botanical Garden, Yunnan Province, China, were investigated for nematode parasites. Nematode specimens were isolated from the intestine of this host and then fixed and stored in 80% ethanol until study.
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 ends of nematodes 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 in parentheses) are given in micrometers (μm) unless otherwise stated. Type specimens were deposited in the College of Life Sciences, Hebei Normal University, Hebei Province, P.R. China.
Genomic DNA from each sample was extracted using a Column Genomic DNA Isolation Kit (Shanghai Sangon, China) according to the manufacturer’s instructions. The partial 18S region was amplified by polymerase chain reaction (PCR) using the forward primer 18S-F (5′-CGCGAATRGCTCATTACAACAGC-3′) and the reverse primer 18S-R (5′-GGGCGGTATCTGATCGCC-3′) . The partial 28S region of nuclear rDNA was amplified by PCR using the forward primer 28S-F (5′-AGCGGAGGAAAAGAAACTAA-3′) and the reverse primer 28S-R (5′-ATCCGTGTTTCAAGACGGG-3′) . The ITS-1 region of nuclear rDNA was amplified by PCR using the forward primer SS1 (5′-GTTTCCGTAGGTGAACCTGCG-3′) and the reverse primer SS2R (5′-AGTGCTCAATGTGTCTGCAA-3′) . The partial cox1 region was amplified by PCR using the forward primer COIF (5′-TTTTTTGGTCATCCTGAGGTTTAT-3′) and the reverse primer COIR (5′-ACATAATGAAAATGACTAACAAC-3′) . The cycling conditions were described by the previous study . PCR products were checked on GoldView-stained 1.5% agarose gels and purified with the Column PCR Product Purification Kit (Shanghai Sangon, China). Sequencing was carried out using a DyeDeoxy Terminator Cycle Sequencing Kit (v.2, Applied Biosystems, Foster City, CA, USA) and an automated sequencer (ABI-PRISM 377). Sequencing of each sample was carried out on both strands. Sequences were aligned using ClustalW2. The DNA sequences obtained herein were deposited in the National Center for Biotechnology Information (NCBI) database (http://www.ncbi.nlm. nih.gov) and compared (using the BLASTn algorithm) with those available in the GenBank database.
Phylogenetic trees were constructed based on the 18S + 28S sequence data using maximum likelihood (ML) in IQ-TREE and Bayesian inference (BI) in MrBayes 3.2 [16, 17]. Ascaris lumbricoides Linnaeus, 1758 (Ascaridida: Ascaridoidea) was used as the outgroup. The ingroup included 16 cosmocercoid species belonging to 8 genera in 3 different families: Cosmocercidae, Atractidae and Kathlaniidae. The detailed information of nematode species included in the phylogenetic analyses, is provided in Table 1. We used a built-in function in IQ-TREE to select a best-fitting substitution model for the sequences according to the Bayesian information criterion . The TIM3e + G4 model for 18S + 28S sequence data were identified as the optimal nucleotide substitution model. Reliabilities for the ML tree were tested using 1000 bootstrap replications, and the BI tree was tested using 50 million generations, and bootstrap values exceeding 70% were shown in the phylogenetic tree.
Family Cosmocercidae (Railliet, 1916)
Genus Aplectana Railliet & Henry, 1916
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) , details of the new species have been submitted to ZooBank. The Life Science Identifier (LSID) of the article is urn:lsid:zoobank.org:pub:09F4B1EF-C3AF42E6-80E6-B734D6B084B8. The LSID for the new name Aplectana xishuangbannaensis is urn:lsid:zoobank.org:act:5E4C6C18-7B72-4C28-BD28-6964C6D8F0A3.
Etymology: The specific epithet refers to the type location XiShuangBanNa Tropical Botanical Garden, Yunnan Province, China.
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).
[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).
[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).
Partial 18S region
Three 18S sequences of Aplectana xishuangbannaensis n. sp. (accession numbers MW329041–MW329043) obtained were all 1539 bp long, representing only one genotype. There 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
Three ITS-1 sequences of A. xishuangbannaensis n. sp. (accession numbers MW329035–MW329037) obtained were all 554 bp long, representing only one genotype. There 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.
Partial 28S region
Three 28S sequences of A. xishuangbannaensis n. sp. (accession numbers MW329038–MW329040) obtained were all 740 bp long, representing only one genotype. There 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
Three cox1 sequences of A. xishuangbannaensis n. sp. (accession numbers MW327586–MW327588) obtained were all 384 bp long, representing only one genotype. There is no species of Aplectana with cox1 sequence registered in GenBank. Pairwise comparison between A. xishuangbannaensis n. sp. and the other species of Cosmocercidae regarding the cox1 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 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 . Clade III included species of Falcaustra and Megalobatrachonema, which represent the family Kathlaniidae. The representatives of Orientatractis and Rondonia formed Clade IV, representing the family Atractidae.
The genus Aplectana (Cosmocercoidea: Cosmocercidae) is a group of zooparasitic nematodes, with approximately 50 nominal species mainly parasitic in various amphibians, and rarely occurring in reptiles worldwide [4, 5, 20,21,22]. The absence of rosette papillae or plectanes in males and presence of somatic papillae, lateral alae and two prodelphic ovaries, uteri containing numerous eggs of normal size in females, allocate the present specimens to the genus Aplectana. To date, only four species of Aplectana have been reported in China, namely A. hainanensis Bursey, Goldberg & Grismer, 2018, A. hylae Wang, 1980, A. macintoshii (Stewart, 1914) and A. paucipapillosa Wang, 1980 [22,23,24]. Lacking a gubernaculum, the new species can be easily distinguished from the four above-mentioned species (the four species all possessing a gubernaculum) [20, 22, 23].
In the genus Aplectana, A. akhrami (Islam, Farooq & Khanum, 1979), A. artigasi Puga & Torres, 1997, A. chilensis Lent & Freitas, 1948, A. crossodactyli Baker, 1980, A. crucifer Travassos, 1925, A. delirae (Fabio, 1971), A. dubrajpuri Sou & Nandi, 2015, A. hoplobatrachusia Sou, Sow & Nandi, 2018, A. meridionalis Lent & Freitas, 1948, A. papillifera (Araujo, 1977), A. praeputialis (Skrjabin, 1916), A. tarija Ramallo, Bursey & Goldberg, 2007, and A. vercammeni Le Van Hoa, 1962, have no gubernaculum [20, 22, 25,26,27,28,29,30,31,32,33,34,35], similar to the new species.
Aplectana xishuangbannaensis n. sp. differs from A. dubrajpuri and A. meridionalis in the different position of the excretory pore (situated at anterior end of oesophageal bulb vs at 1/2 between nerve ring and oesophageal bulb in the latter two species). With only one pair of precloacal papillae, A. tarija, which has six pairs of precloacal papillae, can be easily differentiated from the new species. Aplectana artigasi, A. chilensis, A. crucifer, A. praeputialis, A. vercammeni and A. hoplobatrachusia differ from A. xishuangbannaensis n. sp. by having relatively longer spicules (spicules representing 9.10–15.2% of body length in the former species vs spicules representing 5.98–7.09% of body length in A. xishuangbannaensis n. sp.). Aplectana papillifera can be easily distinguished from the new species by having a larger female body (5.90–8.50 vs 3.54–3.86 mm in A. xishuangbannaensis n. sp.), relatively shorter female tail (representing 4.47‒5.59% of body length in A. papillifera vs representing 9.78‒11.1% of body length in the new species) and a different arrangement and number of caudal papillae (precloacal: 10 pairs; paracloacal: 1‒2 pairs; postcloacal: 8 pairs in the former vs precloacal: 6 pairs; paracloacal: 3 pairs; postcloacal: 4 pairs in A. xishuangbannaensis n. sp.).
The new species differs from A. crossodactyli by having relatively longer spicules (vs representing 3.78–4.64% of body length in A. crossodactyli) and fewer precloacal papillae (6 pairs in the new species vs 20 pairs in the latter). Aplectana xishuangbannaensis n. sp. can be easily distinguished from A. akhrami by having a different position of the vulva (vulva from anterior extremity at 44.8–54.5% of body length in the new species vs vulva from anterior extremity at 29.0–30.6% of body length in A. akhrami) and a much longer female tail (vs 0.16 mm, representing 4.44–5.33% of body length in A. akhrami).
Currently, the specific diagnosis of Aplectana spp. remains based on morphology, and the genetic data of these parasites are severely limited. Based on the genetic analysis of A. xishuangbannaensis n. sp., no intraspecific nucleotide differences in 18S, ITS-1, 28S and cox1 regions among different individuals were noted, but a high level of interspecific genetic variation in these regions among species of the other genera in the Cosmocercidae was clear.
Our phylogenetic results are largely congruent with the traditional classifications of the Cosmocercoidea, which have been proposed based on morphological characters and ecological traits, including the structure of the oesophagus, the presence or absence of a precloacal sucker, the morphology of caudal papillae, the morphology of female reproductive organs and the reporductive strategies [1, 2, 36].
The systematic position of the subfamily Cruziinae has long been under debate. Our molecular phylogenetic results conflicted with the traditional classfication [1, 5, 40,41,42], which suggested that the subfamily Cruziinae should be moved out from the hitherto-defined family Kathlaniidae and elevated to a separate family. The highly specialized structure of the pharynx (the presence of unique pharyngeal lamellae) and the unique digestive system (the presence of an intestinal caecum) of this group support its full family status . However, a more rigorous molecular phylogenetic study with broader representatives of the Cruziinae using different nuclear and/or mitochondrial genetic markers is required to further ascertain its systematic position.
The Cosmocercidae currently includes about 200 nominal species allocated in more than 20 genera, representing the largest family within Cosmocercoidea [1, 3, 21, 44]. However, the phylogenetic relationships among genera within Cosmocercidae is poorly understood because of the lack of genetic data. According to Chabaud (1978)  and Gibbons (2010) , the morphology of caudal papillae in males is one of the most important characters for generic diagnosis in the Cosmocercidae. Species of the genus Aplectana have no modified papillae (plectanes and/or rosette papillae), but those of Cosmocerca and Cosmocercoides have this character. Wilkie (1930) , Skrjabin et al. (1961)  and Chabaud (1978)  considered these genera with modified papillae more closely related to each other than Aplectana. However, our results indicated that Cosmocerca is closer to Aplectana rather than Cosmocercoides, conflicting with the traditional systematics based on morphology.
The present study provided a preliminary molecular phylogenetic framework for the superfamily Cosmocercoidea based on 18S + 28S sequence data for the first time to our knowledge. The family Kathlaniidae is not a monophyletic group. Cruziidae is probably valid as a family for Cosmocercoidea. The genus Aplectana is closer to Cosmocerca than to Cosmocercoides, which is basal within Cosmocercidae. Moreover, morphological and genetic evidence both supported the hypothesis that our nematode specimens collected from P. megacephalus represent a new species of Aplectana, which is the fifth species in the genus reported from China. However, the phylogenetic aspects of Cosmocercoidea are far from being well understood.
Availability of data and materials
The nuclear and mitochondrial DNA sequences of Aplectana xishuangbannaensis n. sp. obtained in this study were deposited in GenBank database under the accession numbers MW329041–MW329043 (18S sequences), MW329035–MW329037 (ITS-1 sequences), MW329038–MW329040 (28S sequences) and MW327586–MW327588 (cox1 sequences). Type specimens of the new species were deposited in the College of Life Sciences, Hebei Normal University, Hebei Province, under the accession numbers HBNU–N-2020A009–11L, China.
Scanning electron microscopy
Polymerase chain reaction
Small subunit ribosomal DNA
Large subunit ribosomal DNA
Internal transcribed spacer
Cytochrome c oxidase subunit 1
Large double cephalic papillae
Single median precloacal papilla
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The authors are grateful to Dr. František Moravec (Institute of Parasitology, Biology Centre of the Czech Academy of Sciences, Czech Republic) and Professor Hideo Hasegawa (Faculty of Medicine, Oita University, Japan) for providing important literature. The authors also thank Dr. Gang Wang (XiShuangBanNa Tropical Botanical Garden, Yunnan Province, China) for helping to collect the frogs.
This study was supported by the Natural Science Foundation of Hebei Province (C2019205094), the Support Program for 100 Excellent Innovative Talents of Hebei Province (SLRC2019033), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB26000000) and the Youth Top Talent Support Program of Hebei Province for Dr. Liang Li and the Natural Science Foundation of Hebei Normal University for Dr. Hui-Xia Chen (L2020B15).
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Chen, HX., Gu, XH., Ni, XF. et al. Description of a new species of Aplectana (Nematoda: Ascaridomorpha: Cosmocercidae) using an integrative approach and preliminary phylogenetic study of Cosmocercidae and related taxa. Parasites Vectors 14, 165 (2021). https://doi.org/10.1186/s13071-021-04667-9
- Genetic data
- Molecular phylogeny
- New species