Description of a new species of Aplectana (Nematoda: Ascaridomorpha: Cosmocercidae) using an integrative approach and preliminary phylogenetic study of Cosmocercidae and related taxa

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.


Background
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.

Parasite collection
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.

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 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.

Molecular procedures
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′-CGC GAA TRG CTC ATT ACA ACAGC-3′) and the reverse primer 18S-R (5′-GGG CGG TAT CTG ATC GCC -3′) [12]. The partial 28S region of nuclear rDNA was amplified by PCR using the forward primer 28S-F (5′-AGC GGA GGA AAA GAA ACT AA-3′) and the reverse primer 28S-R (5′-ATC CGT GTT TCA AGA CGG G-3′) [13]. The ITS-1 region of nuclear rDNA was amplified by PCR using the forward primer SS1 (5′-GTT TCC GTA GGT GAA CCT GCG-3′) and the reverse primer SS2R (5′-AGT GCT CAA TGT GTC TGC AA-3′) [14]. The partial cox1 region was amplified by PCR using the forward primer COIF (5′-TTT TTT GGT CAT CCT GAG GTT TAT -3′) and the reverse primer COIR (5′-ACA TAA TGA AAA TGA CTA ACAAC-3′) [15]. The cycling conditions were described by the previous study [9]. 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.
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.

Phylogenetic analyses
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 [18]. 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. Etymology: The specific epithet refers to the type location XiShuangBanNa Tropical Botanical Garden, Yunnan Province, China.

Partial cox1 region
Three cox1 sequences of A. xishuangbannaensis n. sp.

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. The representatives of Orientatractis and Rondonia formed Clade IV, representing the family Atractidae.

Discussion
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 [22-24]. 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].
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. 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 [43]. 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) [1] and Gibbons (2010) [44], 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) [45], Skrjabin et al. (1961) [5] and Chabaud (1978) [1] 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.