Molecular phylogeny of the family Cosmocercidae (Nematoda: Ascaridida), with description of a new species of Aplectana using an integrative approach

Nematodes of the family Cosmocercidae (Ascaridida: Cosmocercoidea) are mainly parasitic in the digest 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 of the Cosmocercidae and the other two families Atractidae and Kathlaniidae in the superfamily Cosmocercoidea, are still under debate. Moreover, the systematic position of some genera in Cosmocercidae remains unclear. has closer relationship to the genus Aplectana in the family Cosmocercidae, Our present study provided the basic molecular phylogenetic framework for the superfamily Cosmocercoidea based on 18S + 28S sequence data for the rst time. Moreover, a new species of Aplectana, A. xishuangbannaensis n. sp., was described using an integrative approach. 18S, ITS1, 28S and cox1 regions of n. sp. and in the intraspecic nucleotide differences in 18S, ITS-1, 28S and cox1 regions among different individuals, high level of interspecic genetic variation in these among of the other in Cosmocercidae. Our data of A.

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 the Cosmocercoidea.
Our present knowledge of the molecular phylogeny of the Cosmocercoidea/ Cosmocercidae is still very limited. To date, several studies [8][9][10][11] provided molecular phylogenetic analyses to solve the systematic status of some genera 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 the Cosmocercoidea / Cosmocercidae.
In order to clarify the phylogenetic relationships of the Cosmocercidae and the other families Atractidae and Kathlaniidae in the 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, respectively. 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. Specimens were isolated from the intestine of this host, then xed and stored in 80% ethanol until study.

Morphological observations
For light microscopical studies, nematodes were cleared in lactophenol. Drawings were made with the use of a Nikon microscope drawing attachment. For scanning electron microscopy (SEM), the anterior and posterior end of nematodes were re-xed in 4% formaldehyde solution, post-xed 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 College of Life Sciences, Hebei Normal University, Hebei Province, P. R. China.

Phylogenetic analyses
Phylogenetic trees were constructed based on the 18S + 28S sequence data using maximum likelihood (ML) inference with IQ-TREE and Bayesian inference (BI) with Mrbayes 3.2 [16,17]. Ascaris lumbricoides Linnaeus, 1758 (Ascaridida: Ascaridoidea) was treated as the outgroup. The ingroup included 16 cosmocercoid species belonging to 8 genera in three 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-tting substitution model for the sequences according to the Bayesian information criterion [18]. The TIM3e + G4 model for 18S + 28S sequence data were identi ed as optimal nucleotide substitution model. Reliabilities for ML tree was tested using 1000 bootstrap replications and BI tree was tested using 50 million generations, and bootstrap values exceeding 70% were showed in the phylogenetic tree.   (Figs. 1a, 2b). Oesophagus divided into anterior short pharynx, cylindrical corpus, slightly narrow isthmus and terminating posterior bulb with valves (Fig. 2a). Nerve ring located at about 1/2 of oesophageal length. Excretory pore suited at level of anterior of oesophageal bulb (Fig. 2a) (Fig. 3a). 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, more or less equal in length, 139-178 (161) long, distal end pointed, representing 5.98-7.09 (6.47) % of body length (Fig. 2g) Phylogenetic analyses (Fig. 3) Our phylogenetic trees using Maximum likelihood (ML) and Bayesian inference (BI) analyses both showed that the representative of Cosmocercoidea were divided into four large clades (Fig. 3)  Our phylogenetic results are largely congruent with the traditional classi cations of the Cosmocercoidea, which have been proposed based mainly on morphological characters and ecological traits, including the structure of the oesophagus, the presence or absence of precloacal sucker, the morphology of caudal papillae, the host-type, the form of female reproductive organs and the mode of reproduction [1,2,36].
The results supported the Cosmocercidae and Atractidae to be monophyletic groups with strong support from both Maximum likelihood (ML) and Bayesian inference (BI) analyses. However, the Kathlaniidae was not monophyly, and the representatives of the sampled Kathlaniidae were divided into two distinct clades (clade II including the genus Cruzia and clade III including the genera Falcaustra and Megalobatrachonema).
The systematic position of the subfamily Cruziinae has long been under debate. Travassos (1917) [40] erected the family Cruziidae for the reception of the genus Cruzia and considered this family has close relationship with the Kathlaniidae. Later, Ortlepp (1924) [41] treated it as a subfamily of Kathlaniidae. Subsequently, Khalil (1927) [42] rejected the validity of the family Cruziidae or subfamily Cruziinae, but he recognized the genus Cruzia to be a member of the Kathlaniidae. However, Skrjabin et al. (1961) [5] considered the Cruziidae/Cruziinae should be placed in the Atractoidea/Atractidae. Chabaud (1978)  Kathlaniidae. Our molecular phylogenetic results con icted with these traditional opinions, which supported that the subfamily Cruziinae should be moved out from the hitherto-de ned family Kathlaniidae and elevated to a separate family. The highly specialized structure of pharynx (the presence of unique pharyngeal lamellae) and the unique digestive system (the presence of intestinal caecum) of this group supported its full family-status [43]. However, only one species of the Cruziinae was included in our phylogenetic analyses, thus we do not make any immediate systematic changes in the Cosmocercoidea, because a more rigorous molecular phylogenetic study with broader representatives of the Cruziinae using different nuclear and/or mitochondrial genetic markers (including mitochondrial genomic data) is required to further ascertain its systematic position.
The Cosmocercidae currently includes approximately 200 nominal species placed in more than 20 genera, which is the largest family in the superfamily Cosmocercoidea [1,3,21,44]. However, the phylogenetic analyses at the generic level are generally lacking in the Cosmocercidae, due to the paucity and inaccessibility of suitable material. According to Chabaud (1978) [1] and Gibbons (2010) [44], the morphology of caudal papillae in male is one of the most important genetic diagnosis characters in the Cosmocercidae. Species of the genus Aplectana have no modi ed papillae (plectanes and/or rosette papillae), but species of the genera Cosmocerca and Cosmocercoides have modi ed papillae (plectanes and/or rosette papillae). Wilkie (1930) [45], Skrjabin et al. (1961) [5] and Chabaud (1978) [1] all considered these genera with modi ed papillae (plectanes and/or rosette papillae) seem to have close relationships in the Cosmocercidae. However, our molecular phylogenetic results supported the genus Cosmocerca has closer relationship to the genus Aplectana, rather than the genus Cosmocercoides in the Cosmocercidae, which are con icted with the traditional views on the systematics of these groups.

Conclusions
Our knowledge of the phylogeny of the Cosmocercoidea and its included families/genera is still far from comprehensive. The present molecular phylogenetic results supported that the subfamily Cruziinae should be moved out from the hitherto-de ned family Kathlaniidae and elevated to a separate family, and the genus Cosmocerca has closer relationship to the genus Aplectana in the family Cosmocercidae, Our present study provided the basic molecular phylogenetic framework for the superfamily Cosmocercoidea based on 18S + 28S sequence data for the rst time. Moreover, a new species of Aplectana, A. xishuangbannaensis n. sp., was described using an integrative approach.