Morphological and molecular characteristics of Parasitodiplogaster religiosae n. sp. (Nematoda: Diplogastrina) associated with Ficus religiosa in China

A new nematode species of the genus Parasitodiplogaster was recovered from syconia of Ficus religiosa at the Guangxiao Temple, Guangzhou, China. It is described herein as P. religiosae n. sp. and is characterised by possessing the longest and thinnest spicule of all currently described males in the genus, an elongated laterally “ε-shaped” and ventrally rhomboid-like gubernaculum, a stoma without teeth, consisting of a ring-like cheilostom with indistinct anteriolateral projections, a tube-like gymnostom and a funnel-like stegostom, monodelphic with a mean vulval position of 66%. There are three pre-cloacal and six post-cloacal male genital papillae with the arrangement P1, P2, P3, (C, P4), P5, P6d, P7, P8, P9d, Ph. This new species was easily differentiated from other members of the genus by DNA sequences of partial small subunit rRNA gene (SSU) and the D2-D3 expansion segments of the large subunit rRNA gene (LSU). Phylogenetic analysis also corroborated its reasonable placement within a well-supported monophyletic clade with other Parasitodiplogaster species and within the australis-group that includes P. australis and P. salicifoliae that are all associates of fig wasp pollinators (Platyscapa sp.) of figs of the subsection Urostigma.


Introduction
Ficus religiosa L., a monoecious fig, popularly known as the ashwattha tree, bo tree, bodhi tree, peepal tree, peepul tree, pippala tree or sacred fig, belongs to the genus Ficus, subgenus Urostigma, section Urostigma, and subsection Urostigma. It is native to India, southwest China and Mainland Southeast Asia, and has been introduced and cultivated worldwide as an ornamental tree in parks and gardens in subtropical and tropical areas with the potential for becoming an PLOS  invasive weed, if its pollinator is co-introduced and becomes established (https://www.cabi. org/isc/datasheet/241680). It is also grown for traditional medicine for various types of human disorders, such as asthma, diabetes, diarrhea, epilepsy, gastric problems, inflammation, infections and sexual disorders [1]. It is pollinated by the fig wasp, Platyscapa quadraticeps (Mayr) (Agaonidae)(http://www.figweb.org/Ficus/Subgenus_Urostigma/Section_Urostigma/ Subsection_Urostigma/Ficus_religiosa.htm). Parasitodiplogaster Poinar, 1979 was described as the first parasitic nematode genus associated with fig-pollinating wasps by Poinar [2]. There is an interesting fact that the nematodes Parasitodiplogaster (Diplogasteridae) and Schistonchus (Aphelenchoididae) which are vectored by pollinator wasps are in apparently species-specific associations in co-evolved complex of nematode parasites and fig wasp pollinators [3]. The genus Parasitodiplogaster has since been reported from fig wasps and the sycones of Ficus species in North and Central America, Africa and Australia with a current total of 16 described species including P. australis Bartholomaeus [2,[4][5][6][7][8][9]. Three morphospecies (Msp1-3) of Parasitodiplogaster associated with figs from the subgenus Pharmacosycea in Panama were discriminated by male tail characters and Pharmacosycea-associated Parasitodiplogaster species were referred to as the P. maxinema-group by genotype [10]. Kanzaki et al. [11] re-characterised three Parasitodiplogaster species (P. nymphanema, P. obtusinema and P. trigonema) from species of Ficus from the subgenus Urostigma, section Urostigma, and subsection Americana in Panama based on morphological and molecular profiles, and revealed that these three species belong to the P. laevigata-group because of molecular phylogenetic inferences and some typological characters, e.g., stomatal morphology. Until now, no Parasitodiplogaster species have been reported from figs from China. A recent survey on diversity of fig-associated nematodes in the Guangdong Province from 2016 to 2017 revealed an undescribed species of diplogastrid nematode from F. religiosa in Guangzhou, China. It is described here as Parasitodiplogaster religiosae n. sp. using morphological and molecular methods.

Ethics statement
Specific permissions were not required for the nematodes collected for the present study in Guangdong Province, China. The field used for nematode collection was not privately owned but open to the public and did not involve endangered or protected species.

Nematode materials
Syconia in phase B-C were collected from a F. religiosa tree from the Guangxiao Temple in Guangzhou, China on May 26, 2016. They were dissected open with a scalpel and placed in distilled water for 20 min. Nematodes were then handpicked alive into water for DNA extraction, amplification, and sequencing attempts, or collected, heat-killed at 65˚C for 2-3 min and placed into 4% formalin for measurements by light microscopy and then processed into 100% glycerol for preparation of permanent mounts [12].

Morphological observations
Typological characters of nematodes were observed by light microscopy, including the stomatal, pharyngeal and male tail characters according to Sudhaus & Fürst von Lieven [13] for diplogastrids. Measurements and drawings of nematodes were conducted with the aid of a drawing tube attached to a microscope (Leica DM2500) and a stage micrometer. The morphometric data was processed using Excel software [14]. Photomicrographs were taken using a camera (Nikon DS-Fi1) attached to a microscope (Nikon ECLIPSE 80i) and edited using Adobe Photoshop CS6.

Molecular profiles
Ten nematode males were separately picked into distilled water and their identity was confirmed with light microscopy before being placed into 50 μL of worm lysis buffer (WLB) containing Proteinase K for DNA extraction [15]. DNA samples were stored at -20˚C until used as PCR templates.
The rDNA SSU and LSU sequences from the new species were deposited into the GenBank database under the accession numbers MG729403 and MG729404, and compared with other nematode species in GenBank using the BLAST homology search program. The most similar sequences were downloaded for phylogenetic analysis. DNA sequences were aligned by Mega5.05 [19]. The model of base substitution in the SSU and LSU sets were evaluated using MODELTEST version 3.06 [20]. The Akaike-supported model, the proportion of invariable sites, and the gamma distribution shape parameters and substitution rates were used in phylogenetic analyses. Bayesian analysis was performed to confirm the tree topology for each gene separately using MrBayes 3.1.0 [21] running the chain for 1,000,000 generations and setting the 'burn in' at 1,000. The Markov Chain Monte Carlo methods within a Bayesian framework was employed to estimate the posterior probabilities of the phylogenetic trees [22] using the 50% majority-rule.

Nomenclatural acts
The electronic vision of this paper meets the requirements of the amended international code of zoological nomenclature (ICZN), and therefore the new name contained herein is available under that code from the electronic vision of this paper. This published work and the contained nomenclatural acts have been registered in the online registration system for the ICZN in ZooBank. The ZooBank LSID (life science identifiers) for this publication is: urn:lsid: zoobank.org:pub:8402E565-4C3D-4676-87D1-C06EC261634E. The related LSID information can be viewed through any standard web browser by appending the LSID to the prefix "http:// www.zoobank.org/References/".

Type materials
Holotype male, one male paratype and one female paratype deposited in the Department of Nematology, University of California, Riverside, CA, USA. One male paratype and one female paratype deposited in the USDA Nematode Collection, Beltsville, MD, USA. The remaining

Diagnosis and relationships
Parasitodiplogaster religiosae n. sp. is characterised by possessing the longest and thinnest spicule of all currently described males in the genus, an elongated laterally "ε-shaped" and ventrally rhomboid-like gubernaculum, a stoma without teeth, consisting of a ring-like cheilostom with indistinct squared anteriolateral projections, a tube-like gymnostom and a funnel-like stegostom, monodelphic and a mean vulval position of 65.5%. There are three pre-cloacal and six post-cloacal male genital papillae with the arrangement P1, P2, P3, (C, P4), P5, P6d, P7, P8, P9d, Ph. Its status as a distinct species is corroborated by DNA sequences of SSU and D2-D3 LSU (Figs 5 and 6).
The genus Parasitodiplogaster is separated into several species groups that align with different Ficus subsections and their associated pollinators [9]. The new species belongs to the australis-group associated with the Ficus subsection Urostigma and the fig wasp genus Platyscapa. Members of this group typologically share a relatively simple stoma (with no metastegostomatal teeth), single female gonad and very short and conical tail of males [9]. Currently, the group contains two species, P. australis and P. salicifoliae. The molecular phylogenetic status of the species also supports its inclusion in the group (Figs 5 and 6).
Parasitodiplogaster australis sp. n. is differentiated from all other species of the genus by having females with only one gonad, males with C-shaped spicules with an arcuate, slender gubernaculum, characteristic arrangement of the male caudal papillae and DNA sequence data. The generic diagnosis of Parasitodiplogaster is emended to include loss of a female gonad.

Molecular phylogenetic relationships
Partial SSU and D2-D3 LSU expansion domains were sequenced for molecular characterization. Bayesian analysis of the relative placement of Parasitodiplogaster religiosae n. sp. among other Parasitodiplogaster species was performed. The tree inferred from SSU (Fig 5) using  [25]) are grouped in a monophyletic clade with 100% pp; iii) Parasitodiplogaster religiosae n. sp. is clustered in a highly supported (99% pp) monophyletic clade with the above-mentioned nine Parasitodiplogaster populations and three Teratodiplogaster populations. Unfortunately, the other members of the australis-group (P. australis and P. salicifoliae) have not been sequenced for SSU and were not available for analysis.  The tree inferred from D2-D3 of LSU (Fig 6) using Koerneria cf. luziae and Mononchoides sp. WY2009 (= Pristionchus sp.; see Susoy et al. [26]) as outgroups suggested that: i) Parasitodiplogaster religiosae n. sp., all 20 of the selected species (taxa) of Parasitodiplogaster and two species of Teratodiplogaster are in a monophyletic clade with 100% pp; ii) all the selected populations of Parasitodiplogaster are separated into four groups including the laevigata-group associated with figs and fig wasps associated with the subsection Americana, the maxinemagroup (subsection Pharmacosycea), the australis-group (subsection Urostigma) and the sycophilon-group (subsection Galoglychia), and P. religiosae n. sp. is clustered in the australisgroup together with P. australis and P. salicifoliae with 100% pp.

Discussion
Morphologically P. religiosae n. sp. belongs to the Parasitodiplogaster australis-group (P. australis + P. salicifoliae) which have only one ovary in the females (vs two ovaries in all of the other described species in the genus). However, it possesses the longest spicule relative to all other described species in the genus (by more than 2X). Phylogenetically, P. religiosae n. sp., P. australis and P. salicifoliae are putative sisters in a single clade and clustered into the "australisgroup" (Fig 6), but its sequences are unique. Even though the "australis-group" comes out of figs from the same Section and Subsection (Urostigma; Urostigma) their collection sites are geographically widely disparate (from China, Australia, and South Africa, respectively). The 10.4% of molecular sequence differences in D2-D3 LSU between P. religiosae n. sp. and P. australis, and 8.7% between and P. salicifoliae helped to confirm that P. religiosae n. sp. is a distinct species from P. australis and P. salicifoliae. The genus Parasitodiplogaster was phylogenetically separated into four groups (Fig 6) as previously reported [9][10][11]. Parasitodiplogaster religiosae n. sp. is clearly clustered into the "australis-group" with P. australis and P. salicifoliae (Fig 6). The species within this group share the same Ficus subsection Urostigma and fig wasp genus Platyscapa and the characters of possessing stomas without teeth and females being monodelphic when compared with Parasitodiplogaster species in the other three groups. The possession or lack of metastegostomatal of teeth is confusing because Poinar [2] and Poinar & Herre [4] reported no teeth in the other three groupings, but re-descriptions have confirmed this to be incorrect [8,10,11,27]. In addition, Bartholomaeus et al. [6] reported two large metastegostomatal teeth in P. australis which upon subsequent re-examination of fresher specimens showed the putative teeth to actually be cheilostomal anteriolateral projections, not metastegostomatal teeth (Nutsumi Kanzaki and Robin M. Giblin-Davis Unpubl. Obs.). Among species of the australis-group there are distinct differences in the spicule, gubernaculum and stomatal morphology and genital papillae number, e.g., the longest and thinnest spicule in P. religiosae n. sp., and 9 pairs of the papillae in P. religiosae n. sp. vs 8 in P. salicifoliae and 7 in P. australis. Based on the morphological, molecular and host association data, species in the australis-group continue to appear to comprise a naturally derived lineage within Parasitodiplogaster with generic attributes. Wöhr et al. [9] suggested that P. salicifoliae and P. australis might comprise a new genus. The inferred placement of P. religiosae n. sp. further supports this hypothesis, but more extensive biogeographical study, fig (subsection Urostigma) and fig wasp (Platyscapa) host sampling, and SSU sequencing of P. salicifoliae and P. australis are required for this action in the future.