Morphological and Molecular Characterization of Phasmarhabditis huizhouensis sp. nov. (Nematoda: Rhabditidae), a New Rhabditid Nematode from South China

The genus Phasmarhabditis is an economically important group of rhabditid nematodes, to which the well-known slug-parasite P. hermaphrodita belongs. Despite the commercial use of Phasmarhabditis species as an attractive and promising approach for pest control, the taxonomy and systematics of this group of rhabditids are poorly understood, largely because of the lack of diagnostic morphological features and DNA sequences for distinguishing species or inferring phylogenetic relationship. During a nematode sampling effort for identifying free-living relatives of Caenorhabditis elegans in Huizhou City, Guangdong, China, a novel species belonging to the genus Phasmarhabditis was isolated from rotting leaves. Detailed morphology of the gonochoristic P. huizhouensis sp. nov. was described and illustrated. The adult female has a robust body, a relatively short and wide buccal capsule conjoined by a rhabditiform pharynx. Females are characterized by a short cupola-shaped tail end bearing a slender pointed tip, with the junction flanked by a pair of ‘rod-like’ phasmids. Males have an open peloderan bursa that is supported by 9 pairs of genital papillae and 1 terminal pair of phasmids. P. huizhouensis sp. nov. is morphologically very similar to the type species Phasmarhabditis papillosa but is distinguishable by its male caudal traits. The new species is readily differentiated from other taxa in the genus by its female tail shape. Molecular phylogenetic inferences based on small subunit (SSU) and the D2-D3 domain of large subunit (LSU) ribosomal DNA genes reveal that P. huizhouensis sp. nov. forms a unique branch in both phylogenies which is genetically related to P. hermaphrodita and other parasites such as Angiostoma spp. The host associations of P. huizhouensis sp. nov. and its ability to parasitize slugs are unknown.


Introduction
Guangdong Province, China (our sampling activity was conducted using rotting plant leaves, so no specific permissions were required, and the field studies did not involve endangered or protected species). Samples were placed onto a clean agar plate. Nematode individuals were picked out then cultured at room temperature using the C. elegans food (E. coli OP50). Measurements [20], drawings and descriptions were conducted from quiescent adult worms incubated at 4°C for 12h after recovery from 3-day culture plates. Differential interference contrast (DIC) images were taken with an Eclipse Ti Inverted Microscope (Nikon). Scanning electron microscopy (SEM) of P. huizhouensis sp. nov. was performed either using the Hitachi_S-450 scanning electron microscope at Hong Kong Baptist University or the Hitachi_S-3400N at China Agricultural University by the methods described previously [21]. Permanent slides were made using the specimens fixed in 3% formaldehyde, which were subsequently mounted in anhydrous glycerin under a paraffin seal after gradual dehydration of nematodes on a hot plate at 70°C [22].
The model of base substitution was evaluated using MODELTEST [31][32]. The estimates of base frequencies, the proportion of invariable sites, the gamma distribution shape parameters and substitution rates suggested by the Akaike Information Criterion were used in phylogenetic analyses. Bayesian analysis was performed to confirm the tree topology for each gene separately using MrBayes 3.1.0 [32] running the chain for 1 × 10 7 generations and setting the "burnin" at 2,5000 which is 25% of the run. The sampling and printing frequencies are 100 and 1000 respectively. We used the Markov Chain Monte Carlo (MCMC) method within a Bayesian framework to estimate the posterior probabilities of the phylogenetic trees using 50% majority rule [33]. Tracer was used for assessing and summarizing the posterior estimates of the various parameters sampled by the Markov Chain and confirming that the burnin was approaching stationarity (http://tree.bio.ed.ac.uk/software/tracer).

Nomenclatural acts
The electronic edition of this article conforms to the requirements of the amended International Code of Zoological Nomenclature, and hence the new names contained herein are available under that Code from the electronic edition of this article. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information can be viewed through any standard web browser by appending the LSID to the prefix "http://zoobank.org/". The LSID for this publication is: urn:lsid:zoobank.org: pub:47BE7062-4ECC-41B3-85CE-C3A53BE6C399. The electronic edition of this work was published in a journal with an ISSN, and has been archived and is available from the following digital repositories: PubMed Central, LOCKSS.

Description
Adults. Cuticle surface smooth or finely annulated. Lateral field longitudinally striated, often bearing 7-11 lines (ridges or incisures) with 3 prominent ridges or bands in the middle (Fig 3F-3H). Lip region flattened anteriorly, slightly higher in females than males. Six lips bearing two circles of sensilla arranged in three pairs: one dorsal and the others sub-ventral, encircling a triangular mouth opening (Fig 3A-3C). Each lip with one apically-inserted, papilliform sensillum. Lateral lips present a pair of small, pore-like amphidial openings, located near the labial sensilla. The other four sub-lateral lips each with 1 papilliform cephalic sensillum posterior to the labial one (Fig 3A-3C). Stoma capsule relatively wide and short (15-24 μm × 5-8 μm), isomorphic/isotropic. Anterior part of the stoma (Cheilostom) cuticularized; gymnostom together with prostegostom and mesostegostom forming main part of the stoma, cylinder-like and slightly funnel-shaped; metastegostom ridges present, but lacking teeth or denticles; telostegostom short (Fig 2D and 2E). Anterior part of pharynx (= pro-+ meta-corpus) slightly longer than posterior (= isthmus + basal bulb). Metacorpus muscular, forming swollen median bulb (25-52 μm in diam.). Isthmus distinct. Nerve-ring encircling posterior region of isthmus. Basal bulb pyriform (39-71 μm in diam.) with valvular apparatus, but posterior structure of haustrulum obscure in many specimens. Pharyngo-intestinal valves (cardia) present. Excretory pore observed behind the base of basal bulb by light microscopy in a few females, but only ca 106 μm from anterior end revealed by SEM examination for one male (Fig 3D). Measurements are listed in Table 1.
Female. Body robust, tapering gradually to a blunt anterior end. Gonad didelphic, amphidelphic; anterior branch on the right of intestine, but posterior arm to the left. Vulva located near the middle of entire body (V = 51-54) with lips protruding (Fig 2F). Paired ovaries reflexed opposite to the vulva. Germ cells in both distal parts of ovaries arranged in a central rachis with proximal cells in elongation. Maturing oocytes proximally located in a single row at reflexed regions of both ovaries. Oviduct relatively long (127-221 μm). One or more aggregations of sperm cells visible occasionally but small (24-53 μm × 20-43 μm), each containing two or more sperm cells. Uteri often with numerous embryos irregularly arranged. Many embryos usually carrying unhatched larvae. Both distal parts of uteri (20-69 μm × 36-73 μm) filled with sperm cells, sometimes visible. Tail short and cupola-shaped with a slender pointed tip in the middle ( Fig 2G). The pointed tip varying in length from 48 to 64 μm. Phasmids 'rod-like' papilliform in SEM (1.6-2.2 μm in diam.), flanking the junction between the cupola-shaped tail end and its slender tip (Fig 3I-3K).

Type locality
The type specimens of Phasmarhabditis huizhouensis sp. nov. were derived from Strain ZZY0412, which was established from individuals isolated from rotting leaves in Kowloon Peak of Huidong County, Huizhou City, Guangdong Province, P.R. China (22°59 0 N, 114°43 0 E) by rearing on NGM agar plate seeded with colibacillus Escherichia coli strain OP50.

Type materials
Fifteen permanent slides containing paratype males and females of Phasmarhabditis huizhouensis sp. nov. strain ZZY0412 were deposited in the United States Department of Agriculture Nematode Collection (USDANC), Beltsville, MD, USA (T-6557p to T-6571p). Additional specimens fixed in 3% formalin were deposited in the laboratory of the Department of Biology, Hong Kong Baptist University, Hong Kong, China.
Living worms of the type isolate ZZY0412 were cryogenically preserved and deposited in the Department of Biology, Hong Kong Baptist University, Hong Kong, China.

Diagnosis and relationships
Phasmarhabditis huizhouensis sp. nov. is characterized by the following features, including gonochoristic reproductive mode, large female body size (L = 1.3-2.3 mm; 'a' value = 12.6-16), longitudinally striated lateral field with 3 distinct ridges/bands, relatively short and wide buccal capsule with simple metastegostom ridges, and a rhabditiform pharynx. Female didelphicamphidelphic reproductive system carrying numerous embryos, vulva mid-body located (V = 51-54); a short cupola-shaped tail end conjoining a slender pointed tip which is flanked by a pair of 'rod-like' phasmids. Male open peloderan bursa medium-developed that is supported by 9 pairs of genital papillae and 1 terminal pair of phasmids; spicules paired, with small forklike distal termini (obtuse tips divided by shallow indentation) and a short gubernaculum.
Phasmarhabditis huizhouensis sp. nov. morphologically belongs to the genus because of its large female body size, relatively short and wide buccal capsule, longitudinally striated lateral field, female 'rod-like' phasmids, and male open peloderan bursa as well as its 10 pairs of bursal papillae. Within the genus Phasmarhabditis, P. huizhouensis sp. nov. is clearly distinguished from P. hermaphrodita, P. neopapillosa and P. tawfiki by the female/hermaphroditic tail shape (a short cupola joining a slender pointed tip vs. a relatively short conoid gradually tapered to a  pointed terminus) [14,17]. P. huizhouensis sp. nov. shares natural habitats (e.g. isolated from decaying organic substances) and the female tail shape (cupola-shaped with a slender tip) with P. papillosa, but the new species can be differentiated from P. papillosa by a significantly longer spicule length (61-82 μm vs. ca 30 μm for P. papillosa, as measured from Fig 1B [15]) and a less-developed peloderan bursa (88-95 μm × 69-78 μm vs. 141 μm × 107 μm-the size for P. papillosa in Fig 1B [15]). In addition to these Phasmarhabditis species, there are remaining taxa of P. incilaria, P. mairei and P. pellio in the genus Pellioditis sensu Sudhaus [3]. P. huizhouensis sp. nov. is different from P. pellio in female body size: L = 1.3-2.3 mm vs. 0.79-0.90 mm, and male spicule length: 61-82 μm vs. only 21-26 μm for P. pellio [34]. The new species differs from P. incilaria in the stoma morphology (without ornamentation on metastegostom ridges vs. bearing bristle-like denticles) [5]. The description of P. mairei is unavailable and this species was considered to be 'species inquirenda' by Andrássy [5]. Within the genus Pellioditis sensu Andrássy [5], P. huizhouensis sp. nov. is distinguished from all species in the genus by the typology of stoma (relatively short and wide without ornamentation on glottoid apparatus vs. at least four times as long as its width with warts on metastegostom ridges).

Molecular analysis
The best scores from the query of 1633-bp SSU gene fragment of P. huizhouensis sp. nov. against the GenBank database are the sequences from 3 isolates of unidentified Phasmarhabditis species, 6 isolates of P. hermaphrodita and 1 isolate of P. neopapillosa. SSU sequence alignments between P. huizhouensis sp. nov. and the most similar isolates revealed that the minimal sequence divergence is from an unidentified species ITD046 from North America (KM510210) [8], with 2.88% divergence over complete coverage. The four US P. hermaphrodita isolates of ITD290 (KM510209), ITD272 (KM510208), ITD207 (KM510207), and ITD056 (KM510206) [8] have a uniform divergence of 3.55% over complete coverage. The sequence of P. neopapillosa (FJ516754) gives 95% coverage yielding 2.94% divergence. The best match to the 902-bp D2-D3 sequence of P. huizhouensis sp. nov. from GenBank is another unidentified Phasmarhabditis species EM434 (EU195967), with 5.78% divergence over 95% coverage. D2-D3 sequence alignments between P. huizhouensis sp. nov. and the four US P. hermaphrodita isolates of ITD290 (KM510296), ITD272 (KM510195), ITD207 (KM510194), and ITD056 (KM510193) [8], yielded only 59% coverage with a uniform divergence of 7.84%. Comparisons of the sequences of the SSU and D2-D3 domain of the LSU rDNA genes between P. huizhouensis sp. nov. and its relatives further support that our rhabditid nematode is a novel species. ]. Using C. sinica as an outgroup taxon, P. huizhouensis sp. nov. is in a highly supported monophyletic clade with many isolates of Phasmarhabditis, 2 isolates of Agfa and 4 isolates of Angiostoma. This clade is sister to Pellioditis. The phylogenetic inferences based on two rDNA genes are largely consistent with each other. Both phylogenies reveal that P. huizhouensis sp. nov. is grouped with other nominal Phasmarhabditis species but in a unique branch.

Discussion
The interrelationship between the genera Phasmarhabditis and Pellioditis remains unresolved, but in accord with both revisions by Andrássy (1983) and Sudhaus (2011) our study supported that the 'Papillosa' group of four known species comprising P. papillosa, P. hermaphrodita, P. neopapillosa and P. tawfiki, together with the new member of P. huizhouensis sp. nov. constitute a sound taxonomic group, based on an amalgamation of morphological and molecular data. The group is characterized by relatively short and wide buccal capsule, longitudinally striated lateral field with 3 or more ridges, relatively short female/hermaphroditic tail with prominent phasmids projecting from body contour, and male open peloderan bursa with 10 pairs of bursal papillae. However, the systematic relationships between these Phasmarhabditis taxa and their relatives in the subfamily Peloderinae are poorly understood, for example, two species of this subfamily Pellioditis marina and P. mediterranea sensu Andrássy [5] included by both phylogenetic trees (Figs 4 and 5) have been recently proposed fortransfer into another genus (Litoditis Sudhaus, 2011) [3]. This volatility of nematode systematics is a result of the scarcity of informative morphological/anatomical characters and a lack of DNA barcode sequences. The taxonomy of the whole Peloderinae group needs more work. Within the genus Phasmarhabditis, P. huizhouensis sp. nov. can be easily distinguished from other species except for P. papillosa by the female/hermaphroditic tail shape. P. huizhouensis sp. nov. is a morphologically cryptic species of P. papillosa, lacking typical autapomorphies except in males. The basis on which the former can be convincingly differentiated from P. papillosa are solely male caudal characters derived from available drawings for P. papillosa. Because of the previous inadequate morphological study and absence of DNA sequences for P. papillosa, further comparison at morphological as well as molecular levels between P. huizhouensis sp. nov. and P. papillosa is necessary when P. papillosa is recollected and sequenced. The excretory pore position of P. huizhouensis sp. nov. is significantly variable between females and a male observed in this study, which suggests the variability of excretory pore position for this species. However, a possibility of misidentification is unavoidable given the microscopic observation on the robust female adults in low magnifications. Thus, the excretory pore position of this species needs reexamination in the future.
Scanning electron micrographs of Phasmarhabditis hermaphrodita isolate ITD272 [8] and P. huizhouensis sp. nov. (Fig 3) reflect a number of apomorphic features shared by both species, e.g. the lip region characters (a triangular mouth opening formed by three pairs of lips with 6 labial and 4 cephalic sensilla, as well as small amphids) and lateral field morphology (longitudinally striated with 3 obvious middle-ridges). Female/hermaphroditic tail shapes are clearly distinct in the species but exhibit some degree of homology, i.e., a short conoid gradually tapered to a pointed terminus with prominent phasmids (see Fig 1F [8]) vs. a short cupola sharply to a pointed tip with 'rod-like' phasmids for P. huizhouensis sp. nov. (Fig 3I). Morphological features combined with molecular evidence are sufficiently for supposing a close systematic affinity in generic level between P. huizhouensis sp. nov. and P. hermaphrodita. However, other relatives of P. huizhouensis sp. nov. inferred from phylogenetic trees, such as Angiostoma and Agfa nematodes, are distantly related to the new species taxonomically. The genera belong to the distinct families Angiostomatidae and Agfidae respectively [35], and are clearly different from P. huizhouensis sp. nov. morphologically. P. huizhouensis sp. nov. differs from Angiostoma species in the type of stoma and esophagus (the former has a typically rhabditiform pharynx and a wide buccal capsule; the buccal capsule of Angiostoma spp. is wide or narrow which is conjoined by a pharynx with indistinct isthmus and a small-valvate or valveless basal bulb), cephalic sensory traits (6 labial as well as 4 cephalic sensory papillae vs. 6+6 papillae at the cephalic end), lateral field morphology (marked with longitudinal ridges vs. presence of lateral alae or lacking), and male caudal configuration (enveloped by an open peloderan bursa vs. an open leptoderan bursa or differing in the patterning of bursal papillae) [36][37][38]. P. huizhouensis sp. nov. is easily differentiated from Agfa flexilis by the morphology of the pharynx (a typically rhabditiform that is valvulated in the basal bulb for P. huizhouensis sp. nov. vs. a much longer one without valvular apparatus for A. flexilis) [39]. Nevertheless, molecular phylogenetic inference based on SSU rDNA sequences reveals that Phasmarhabditis isolates, Angiostoma species. and A. flexilis constitute a well-supported monophyletic clade (Fig 4), which conforms to a previous phylogenetic study among these genera [40]. Phylogenetic analysis based on the D2-D3 domain of LSU rDNA sequences in this study confirmed the close molecular relationship between these taxa (Fig 5). The reason is partly owing to the number of MOTUs (Molecular Operational Taxonomic Unit) which is insufficient to infer phylogenetic relationship between these genera, but the results reflect the inconsistencies between molecular phylogeny and traditional morphological classification. Revision of the systematics of the order Rhabditida based on previous morphological studies is needed.
Phasmarhabditis huizhouensis sp. nov. was isolated from rotting plant tissues, but is phylogenetically related to the slug-parasite P. hermaphrodita and other parasitic nematodes of Angiostoma spp. and Agfa species (Figs 4 and 5), suggesting that P. huizhouensis sp. nov. may be a facultative parasite. The ecology and host associations of P. huizhouensis sp. nov. remain unknown. It would be worthwhile to explore the potential of this Phasmarhabditis nematode to parasitize slugs as a local bio-control agent. Mass production of its dauer larvae would be essential if this species is proved to be capable of controlling slugs. In this study, the C. elegans bacterial food of E. coli OP50 was successfully used for culture of P. huizhouensis sp. nov., but no dauer stage was observed, suggesting that this bacterium may not be the best kind of food for this phasmarhabditid nematode, given that the growth rate of Phasmarhabditis species and its reproductive capacity for dauer larvae are strongly influenced by bacterial species [41]. The most suitable type of bacterial food for dauer larval formation and for mass production of P. huizhouensis sp. nov. require further study.