Morphology, complete mitochondrial genome, and molecular phylogeny of Rhabdias macrocephalum n. sp. (Nematoda: Rhabdiasidae) from Diploderma splendidum (Reptilia: Agamidae)

Species of the genus Rhabdias Stiles & Hassall, 1905 are common parasitic nematodes occurring in the lungs of amphibians and reptiles worldwide. In the present study, Rhabdias macrocephalum n. sp. is described using integrated morphological methods (light and scanning electron microscopy) and molecular approaches (sequencing of the nuclear 28S and ITS regions, and mitochondrial cox1, cox2, and 12S genes) based on specimens collected from the green striped tree dragon Diploderma splendidum (Barbour & Dunn) (Reptilia: Agamidae) in China. The complete mitochondrial genome of R. macrocephalum n. sp. was sequenced and annotated: it is 14,819 bp in length, including 12 protein coding genes (missing atp8), 22 tRNA genes, 2 rRNA genes and three non-coding regions. The gene arrangement of R. macrocephalum n. sp. is different from all of the currently available mitogenomes of nematodes and represents a novel type of mitochondrial gene arrangement reported in Nematoda. Molecular phylogenetic results based on the ITS + 28S data support the monophyly of Entomelas, Pneumonema, Serpentirhabdias, and Rhabdias, and showed R. macrocephalum n. sp. forming a most basal lineage in Rhabdias.

It is not easy to precisely identify specimens of Rhabdias to species level based only on morphological characters, due usually to a lack of males and the extraordinary morphological similarity in females.Recently, some genetic data [i.e., large nuclear ribosomal DNA (28S), internal transcribed spacer (ITS), mitochondrial cytochrome c oxidase subunit 1 (cox1), and 12S small subunit ribosomal RNA gene] and mitochondrial genomes have been successfully used to identify species, discover sibling or cryptic species, and evaluate evolutionary relationships of Rhabdiasidae [1,15,16,28,31,36,46,47,52].However, the current genetic database, especially the mitogenomes for the rhabdiasid nematodes, remains very insufficient.To date, only R. bufonis and R. kafunata have been reported for the complete mitochondrial genomes in the Rhabdiasidae [28,52].
In the present study, a new species of Rhabdias collected from the green striped tree dragon Diploderma splendidum (Barbour & Dunn) (Reptilia: Agamidae) in China was precisely identified using integrated morphological methods (light and scanning electron microscopy) and molecular approaches (sequencing of the nuclear 28S and ITS regions and mitochondrial cox1, cox2, and 12S genes).Additionally, in order to enrich the mitogenomic data and reveal the patterns of mitogenomic evolution of the Rhabdiasidae, the complete mitochondrial genome of this new species was sequenced and annotated.Moreover, in order to determine the phylogenetic position of this new species within Rhabdias, phylogenetic analyses were performed based on the 28S + ITS sequences, using maximum likelihood (ML) and Bayesian inference (BI), respectively.

Morphological observation
In 2021, a total of 26 nematode specimens of Rhabdias were sent to the author's (Li L.) laboratory for species identification, which were recovered from the lung of a dead green striped tree dragon D. splendidum by a local veterinarian in Qinzhou, Guangxi Zhuang Autonomous Region, China.Specimens were fixed and stored in 80% ethanol until the morphological study.For light microscopy, nematode specimens were cleared in 50% glycerin, then examined and photographed using a Nikon Ò optical microscope (Nikon ECLIPSE Ni-U, Nikon Corporation, Tokyo, Japan).For scanning electron microscopy (SEM), the anterior and posterior ends of specimens were transferred to 4% formaldehyde solution, then post-fixed in 1% OsO 4 , dehydrated via an ethanol series and acetone and critical point dried.The specimens were coated with gold and examined using a Hitachi S-4800 scanning electron microscope (Hitachi Ltd., Tokyo, Japan) at an accelerating voltage of 20 kV.All measurements in the text are in micrometers unless otherwise stated.Type specimens were deposited in the College of Life Sciences, Hebei Normal University, Hebei Province, and the National Zoological Museum, Beijing, China.

Molecular procedures
A total of three female specimens were randomly selected for the molecular analysis.Genomic DNA from each individual was extracted using a Column Genomic DNA Isolation Kit (Shanghai Sangon, Shanghai, China), according to the manufacturer's instructions.DNA was eluted in elution buffer and kept at À20 °C until use.The primers and cycling conditions for amplifying different target regions by polymerase chain reaction (PCR) are provided in Table 1.All PCR reactions were performed in 50 lL consisting of 10 mM Tris HCl at pH 8.4, 50 mM KCl, 3.0 mM MgCl 2 , 250 lM of each dNTP, 50 pmol of each primer, and 1.5 U of Taq polymerase (Takara Bio Inc., Kusatsu, Shiga, Japan) in a thermocycler (model 2720; Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA).
PCR products were checked on GoldView-stained 1.5% agarose gel and purified by the Column PCR Product Purification Kit (Shanghai Sangon).Sequencing for each sample was carried out for both strands using a DyeDeoxyTerminator Cycle Sequencing Kit v.2 (Applied Biosystems).The 28S, ITS, cox1, cox2, and 12S sequences obtained herein were deposited in the National Center for Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov).

Mitochondrial genome sequencing, assembly, and annotation
A total of 30 Gb clean genomic data were generated using the Pair-End 150 sequencing method on the Illumina NovaSeq 6000 platform by Novogene (Tianjin, China).The complete mitochondrial genomes were assembled using GetOrganelle v1.7.2a [12].Protein coding genes (PCGs), rRNAs, and tRNAs were annotated using MitoS web server (http://mitos2.bioinf.uni-leipzig.de/index.py)and MitoZ v2.4 [33].The open reading frame (ORF) of each PCG was confirmed manually by the web version of ORF finder (https://www.ncbi.nlm.nih.gov/orffinder/).The "lost" tRNA genes ignored by both MitoS and MitoZ, were identified using BLAST based on a database of the existing tRNA sequences of nematodes.The secondary structures of tRNAs were predicted by ViennaRNA module [9], building on MitoS2 [2] and RNAstructure v6.3 [40], followed by manual correction.MitoZ v2.4 was used to visualize and depict gene element features [33].The base composition, amino acid usage, and relative synonymous codon usage (RSCU) were calculated by Python script, which refers to Codon Adaptation Index (CAI) [23].The total length of the base composition included ambiguous bases.The base skew analysis was used to describe the base composition of nucleotide sequences.The complete mitochondrial genome of this new species obtained was deposited in the NCBI database (http://www.ncbi.nlm.nih.gov).

Phylogenetic analyses
Phylogenetic analyses of rhabdiasid nematodes were performed based on the ITS + 28S sequences using maximum likelihood (ML) with IQ-TREE [34] and Bayesian inference (BI) with MrBayes [41].Caenorhabditis elegans (Rhabditida: Rhabditidae) was chosen as the out-group.The in-group included 46 rhabdiasid species representing six genera.Detailed information on species included in the phylogenetic analyses is provided in Table 2. Genes were aligned separately using the MAFFT v7.313 multiple sequence alignment program under the iterative refinement method of E-INS-I [13].In addition, partially ambiguous bases were manually inspected and removed.The aligned and pruned sequences were concatenated into a matrix by PhyloSuite v1.2.2.The TVM + F+I + I+R2 model was selected for ML analyses.The GTR + F+G4 models were selected for BI analyses.Reliabilities for ML inference were tested using 1000 bootstrap replications, and BIC analysis was run for 5 Â 10 6 MCMC generations.

Etymology:
The specific name refers to the inflated cephalic end of the present specimens.

Genetic characterization
Three partial 28S sequences of R. macrocephalum n. sp.obtained here are all 551 bp, with no nucleotide divergence detected.Pairwise comparison of the partial 28S sequences of R. macrocephalum n. sp.obtained here with that of Rhabdias available in GenBank, displayed 1.45% (R. pseudosphaerocephala, MH516124; R. breviensis, MH516101) to 3.58% (R. tarichae, MH023521) nucleotide divergence.Two partial ITS sequences of R. macrocephalum n. sp.obtained here are both 700 bp, with no nucleotide divergence detected.Pairwise comparison of the partial ITS sequences of R. macrocephalum n. sp.obtained here with that of Rhabdias spp.available in GenBank, displayed 7.80% (R. breviensis, MH516064) to 16.8% (R. stomatica, MW522544) nucleotide divergence.Three partial cox1 sequences of R. macrocephalum n. sp.obtained here are all 655 bp, with no nucleotide divergence detected.Pairwise comparison of the partial cox1 sequences of R. macrocephalum n. sp.obtained here with that of Rhabdias spp.available in GenBank, displayed 8.70% (R. nipponica, LC671281) to 15.4% (R. lamothei, KC130747) nucleotide divergence.Two partial 12S sequences of R. macrocephalum n. sp.obtained here are both 474 bp, with no nucleotide divergence detected.Pairwise comparison of the partial 12S sequences of R. macrocephalum n. sp.obtained here with that of Rhabdias spp.available in GenBank, displayed 8.91% (R. engelbrechti, MG428408) to 11.5% (R. mariauxi, FN395318) nucleotide divergence.One partial cox2 sequence of R. macrocephalum n. sp.obtained here is 554 bp.Pairwise comparison of the partial cox2 sequence of R. macrocephalum n. sp.obtained here with that of Rhabdias spp.available in GenBank, displayed 11.2% (R. bufonis) to 13.2% (R. kafunata) nucleotide divergence.

Molecular phylogeny of Rhabdiasidae
Phylogenetic results based on the ITS + 28S sequence data using ML and BI methods are almost identical (Fig. 7).The representatives of Rhabdiasidae were divided into four large monophyletic clades (Clade I, II, III, and IV).Clade I comprises species of Neoentomelas, Kurilonema, and Serpentirhabdias.Among them, Neoentomelas and Kurilonema have a closer relationship than Serpentirhabdias.Clade II includes representatives of Entomelas.Clade III contains species of Pneumonema, which showed a sister relationship with Clade IV, representing Rhabdias.In the genus Rhabdias, R. macrocephalum n. sp.formed a most basal lineage (Fig. 7).

Discussion
In the genus Rhabdias, a total of 21 species have been reported from lizards worldwide [5,19,37,48].Among them, only four species of Rhabdias were recorded from the lizards of Table 3. Annotations and gene organization of Rhabdias macrocephalum n. sp.Positive number in the "Gap or overlap" column indicates the length of intergenic sequence, and the negative number indicates the length (absolute number) that adjacent genes overlap (negative sign).The forward strand is marked as "+" and the reverse strand as "À".With the particular pattern of cuticular inflation, R. macrocephalum n. sp. is very similar to R. japalurae reported from Diploderma polygonatum Hallowell and D. swinhonis (Gunther) in Japan (Okinawa Island) and China (Taiwan Island), and R. mcguirei reported from Draco spilopterus (Wiegmann) in the Philippines [21,48].However, R. macrocephalum n. sp.can be differentiated from R. japalurae by having a distinctly shorter esophagus (0.87-0.98 mm long, representing 5.35-6.00% of body length in the new species vs 0.92-1.04mm long, representing approximately 8.90-9.40% of body length in R. japalurae) [21].The new species also differs from R. mcguirei by having a relatively shorter esophagus (esophageal length representing 5.35-6.00% of body length in the new species vs esophageal length representing 7.40-14.1% of body length in R. mcguirei) and different morphology of the tail (tail with distinct cuticular inflation and abruptly tapering from approximately 1/2 of region vs a tail with very narrow or inconspicuous cuticular inflation and abruptly tapering from anterior 1/3 of the region) [48].Moravec [37] described R. lacerate Moravec, 2010 from the common lizard Lacerta vivipara Jacquin (Squamata: Lacertidae) in north-western Slovakia.This species with a very small body length (only 1.22-1.34mm) and unique morphology of the tail tip (possessing 3 small cuticular spikes), is different from R. macrocephalum n. sp.Moreover, the other Rhabdias spp. reported from lizards are all collected from chameleonid and polychrotid hosts and distributed in tropical Africa, Madagascar, and Central America [3,4,19,[24][25][26][27]32].Additionally, R. macrocephalum n. sp.differs from all of these 21 Rhabdias spp. reported from lizards, including the four species parasitic in agamids, by having a conspicuously inflated cephalic extremity.

Gene
Molecular analyses of the partial 28S, ITS, cox1, and 12S sequences of R. macrocephalum n. sp.displayed no nucleotide divergence among different individuals, but showed a high level of genetic divergence between this new species and other Rhabdias spp. in these genetic makers, which also supports the hypothesis that the present material represents a new species of Rhabdias.Rhabdias macrocephalum n. sp.represents the ninth species of Rhabdias reported in China.The current mitogenomic database for rhabdiasid nematodes remains very limited.Recently, the complete mitogenomes of R. kafunata and R. bufonis have been sequenced [52], which represented the only two rhabdiasid species with the mitogenomic data reported.The composition of the mitogenome of R. macrocephalum n. sp.[including 12 PCGs (missing atp8), 22 tRNA genes, and 2 rRNA genes] is identical to that of R. kafunata and R. bufonis, but the size of the complete mitogenome of R. macrocephalum n. sp.(14,819 bp) is slightly smaller than that of R. kafunata (15,437 bp) and R. bufonis (15,128 bp).Moreover, there are only three non-coding regions in the mitogenome of R. macrocephalum n. sp., but R. kafunata and R. bufonis have six and four non-coding regions in their mitogenomes, respectively.The mitogenomes of R. macrocephalum n. sp., R. kafunata, and R. bufonis all displayed a strong nucleotide compositional bias toward A + T (75.8-77.5%).To date, there have been 62 types of gene arrangements reported for the mitogenomes of nematodes [52].The mitogenome of R. macrocephalum n. sp.showed a high level of gene rearrangement, which is different from that of R. kafunata, R. bufonis, and all of other mitogenomes of nematodes available so far, and represented a novel type of gene arrangement reported in Nematoda.
Recently, Zeng et al. [52] provided a basic molecular phylogenetic framework for the Rhabdiasidae based on ITS + 28S sequence data, and determined the systematic position of the Rhabdiasidae in the order Rhabditida using mitogenomic phylogeny.The present phylogenetic results agreed well with this study [52] and also supported the monophyly of Entomelas, Pneumonema, Serpentirhabdias, and Rhabdias.It is interesting that the present phylogenetic results displayed R. macrocephalum n. sp.forming a most basal lineage in the genus Rhabdias, being a sister to all other Rhabdias species.In the present phylogeny, only R. nicaraguensis Bursey, Goldberg & Vitt, 2007 was collected from a lizard host [4]; however, this species nested in these Rhabdias species collected from amphibians in South and North America, and did not display a close affinity with the new species.Additionally, R. macrocephalum n. sp.showed a distant relationship to the Eurasian Rhabdias species (i.e., R. bufonis, R. kafunata, R. nipponica, R. kongmonthaensis, R. bulbicauda, and R. bermani).The patterns of parasite-host switching and geographical distributions during the evolutionary history of Rhabdias ancestors is still an unsolved mystery.A more rigorous molecular phylogenetic study that includes broader representatives of Rhabdias species, especially these species collected from lizard hosts, is need to solve the above-mentioned issue.

Figure 2 .
Figure 2. Line drawings of Rhabdias macrocephalum n. sp.from Diploderma splendidum in China.A: anterior part of body, lateral view; B: posterior part of body, lateral view; C: cephalic extremity, lateral view; D: cephalic extremity, apical view; E: region of vulva, lateral view; F: eggs.

Figure 3 .
Figure 3. Scanning electron micrographs of Rhabdias macrocephalum n. sp.from Diploderma splendidum in China.A: anterior part of body, lateral view; B: tail, ventral view; C: magnified image of lateral cuticular pore; D: cephalic extremity (single papilla on each lip arrowed), apical view; E: magnified image of amphid; F: mid-body at level of vulva, sublateral view; G: egg with developed larva; H: magnified image of lateral cuticular pores on the tail; I: magnified image of lateral cuticular pores on the middle of body.Abbreviations: sl, submedian lip; ll, lateral lip.

J
.-L.Zeng et al.: Parasite 2024, 31, 48 the family Agamidae, including R. japalurae Kuzmin, 2003, R. singaporensis Bursey, Hoong & Goldberg, 2012, R. mcguirei Tkach, Kuzmin & Brown, 2011, and R. odilebaini Kuzmin, Tkach & Bush, 2012[5,19,21,48].Rhabdias macrocephalum n. sp.can be easily distinguished from R. singaporensis by having a much longer esophagus (0.87-0.98 mm long, representing 5.35-6.00% of body length in R. macrocephalum vs 0.497-0.689mm long, representing approximately 4.00% of body length in the latter) and different location of the excretory pore (just posterior to the nerve ring in the new species vs at the level of esophageal-intestinal junction in R. singaporensis)[5].The new species is also different from R. odilebaini by having a particular pattern of cuticular inflation (cuticular inflation very narrow or inconspicuous in the anteriormost part and distinctly widening posteriorly from the level of nerve ring or mid-length of esophagus in the new species vs cuticle distinctly inflated to form a vesicle swollen in the anteriormost part of the body) and distinctly shorter tail (0.26-0.33 mm long, representing 1.52-2.02% of body length in the new species vs 0.36-0.50mm long, representing 3.10-3.50% of body length in R. odilebaini)[19].

Figure 5 .
Figure 5. Relative synonymous codon usage (RSCU) of Rhabdias macrocephalum n. sp.Codon families (in alphabetical order, from left to right) are provided below the horizontal axis.Values at the top of each bar represent amino acid usage in percentage.

Figure 6 .
Figure 6.Linearized representation of the nematode mitochondrial gene arrangement of nematodes.The non-coding regions are not indicated.

Table 1 .
The primers and cycling conditions for amplifying different target regions by polymerase chain reaction (PCR) in the present study.

Table 2 .
Detailed information on the representatives of Rhabdiasidae with their genetic data included in the phylogenetic analyses.

Table 4 .
Base composition and skewness of Rhabdias macrocephalum n. sp.