Distribution of trichodorid species in mainland China with description of Trichodorus hangzhouensis sp. nov. (Nematoda, Triplonchida)

Abstract Seven trichodorid species including a new one (Trichodorus hangzhouensissp. nov., T. nanjingensis, T. pakistanensis, T. cedarus, Paratrichodorus porosus, Nanidorus renifer and N. minor) were recovered from the rhizosphere of different hosts in 13 provinces of China. Each of the recovered species was characterized based on morphology and molecular data using rRNA gene sequences. Trichodorus hangzhouensissp. nov. is characterized by its males having medium-sized onchiostyle (46–49 µm) and three ventromedian cervical papillae (CP) anterior to the secretory-excretory (S-E) pore, CP1 located opposite the anterior part of isthmus, S-E pore opposite the isthmus or anterior end of pharyngeal bulb, spicules slightly ventrally curved, relatively small, 33.2 (32.0–34.5) µm long, wider slightly marked capitulum, lamina partially striated without bristles at striation; and females having rounded triangular sclerotized vaginal pieces with tips directed towards vulva, 1.5–2.0 µm sized, at about 1 µm apart, vulva pore-like in ventral view. Phylogenetic analysis based on D2-D3 28S rRNA gene sequences differentiated the new species among Trichodorus species from Europe, Asia and USA which formed a large clade. A review of the distribution of Trichodorus, Nanidorus and Paratrichodorus species revealed that T. cedarus, T. nanjingensis, T. pakistanensis and P. porosus are the most widespread species recorded from different provinces of China. This is the first extensive study of trichodorid species occurring in China.

Previously, morphological identification alone rendered difficulties due to the mixed species complexes, phenotypic variation (such as shape of some sclerotized structures, e.g., stylet, male spicules, vagina with its sclerotized pieces), overlapping diagnostic characters and uniformity in general appearance; however, DNA-based strategies have made it possible to overcome the limitations of the morphological approach only and provided useful insights into trichodorid taxonomy (Subbotin et al. 2019). In recent years several nematologists have successfully applied rRNA genes sequence (18S, 28S, and ITS) analyses for studying the phylogenetic relationships of trichodorid species (Duarte et al. 2010, Zhao et al. 2013, Pedram et al. 2015, 2017. The importance of sequence-based studies for species identification and the lack of molecular data of known species from China has led us to compile a detailed report of trichodorids occurring in the country.
The agricultural land of China represents 10% of the total arable land in the world. About 75% of the lands are cultivated areas used for food production such as rice, wheat, potatoes, tea, soybean, various fruits, tea and sugarcane (Guo 2008). Over the past years, there have been preliminary surveys of trichodorid nematodes in China associated with some of the above-mentioned crops, however many of the occurrence records of these nematodes were incomplete or overlooked. Yin and Feng (1981) reported Trichodorus species from the southern provinces of Guangdong and Hunan, however, the first official trichodorid survey started with Liu and Cheng (1990). Gradually, several studies on the distribution of trichodorids in China have been published with records from Fujian, Yunnan, Zhejiang and Guangdong provinces reporting the occurrence of six Trichodorus, two Nanidorus and two Paratrichodorus species (Xu and Decraemer 1995, Zheng et al. 2004, Zhao et al. 2005, Chi et al. 2011. Considering the potential importance of trichodorids in China, an extensive survey of various biotopes was carried out during the recent years. The objectives of the present study are to: i) characterize morphologically and molecularly recovered trichodorid species including one new Trichodorus species; ii) evaluate the phylogenetic relationships of the new species with other members of the genera based on their 18S rRNA, D2-D3 expansion domain of 28S rRNA and ITS2 of rRNA gene sequences and iii) summarize the geographic distribution of Trichodorus, Nanidorus and Paratrichodorus species in China, in addition, providing a comprehensive list of the past records and present findings of trichodorid nematodes.

Materials and methods
Soil sampling, nematode extraction, and morphological identification Two-thousand and fifty-two soil samples have been collected from 13 provinces of China. Nematodes were extracted from soil samples using a modified Baermann funnel method and modified Cobb' sieving and flotation-centrifugation method (Jenkins 1964). For morphological studies, nematodes were killed by hot formalin solution and processed to glycerine according to Seinhorst (1959) as modified by De Grisse (1969). Morphological observation, measurements, and photomicrographs were made using a Leica CTR 5000 compound microscope with differential interference contrast (DIC). Measurements were expressed as mean ±standard deviation (range). Species diagnoses were made following the polytomous key of Decraemer and Baujard (1998). Original descriptions were used for species added to trichodorid genera.

Phylogenetic analyses
The partial sequences of 18S, D2-D3 fragment of 28S and ITS2 of rRNA gene of Trichodorus hangzhouensis sp. nov. were compared with those of other species of fam. Trichodoridae available in GenBank using the BLAST homology search program. The sequence data sets used in this study were selected based on previously published studies (Zeng et al. 2014, Subbotin et al. 2019) and were used in phylogenetic analyses. Three separate 18S, 28S and ITS2 datasets were prepared. Multiple sequence alignment of each dataset was made using the Q-INS-i algorithm of MAFFT V.7.205 (Katoh and Standley 2013). The sequence alignments were edited by BioEdit (Hall 1999). The best fitted model of DNA evolution was obtained using jModelTest V.2.1.7 (Darriba et al. 2012) with the Akaike information criterion (AIC). General time-reversible model with invariable sites and a gamma-shaped distribution (GTR + I + G) was used for the 28S, 18S and ITS2 rRNA genes to reconstruct the phylogenies. Bayesian analysis was used to infer a phylogenetic tree by MrBayes 3.1.2 (Ronquist and Huelsenbeck 2003).
Model parameters were unlinked and the overall rate was allowed to vary across partitions. The number of generations for the total analysis was set to 10 million, with the chain sampled every 1000 generations and the burn-in value was 25%. The Markov chain Monte Carlo method within a Bayesian framework was used to estimate the posterior probabilities of the phylogenetic trees using the 50% majority rule (Larget and Simon 1999). Posterior probabilities (PP) were given on appropriate clades. The consensus trees were visualized using FigTree V1.4.3 (Stöver and Müller 2010).

Taxonomy
Trichodoridae Thorne, 1935 Trichodorus hangzhouensis sp. nov. http://zoobank.org/F0E3BA04-6CB9-4333-9477-1E2DCB844DB7 Description (Figs 1-3, For measurements see Table 1). Male. Body cylindrical with posterior end slightly curved ventrally. Cuticle slightly swollen upon fixation, 2.0-2.5 µm thick at mid-body. Lip region dome-shaped with double papillae (composed of outer labial and cephalic papillae). Amphidial aperture post-labial, slit-like, amphidial fovea cup-shaped. Stoma narrow, refractive strengthening rods 4-5 µm long. Nerve ring surrounding the anterior part of isthmus. Slender mid part of pharynx gradually widening to form a pharyngeal bulb. Five pharyngeal gland nuclei visible, the first ventrosublateral pair obscure. Pharyngeal bulb offset from intestine. Cardia conoid, difficult to observe. Three ventromedian CP present anterior to the secretory-excretory pore (S-E), the latter opposite isthmus or anterior end of pharyngeal bulb. CPl situated opposite the end of pharyngostom to mid-isthmus, distance of CPl-CP2, CP2-CP3 and CP3-SE becomes gradually shorter. Lateral cervical pores not clearly seen. Reproductive system typical of the genus, i.e., with a single anterior outstretched testis, short germinal zone, seminal vesicle packed with large round sperm cells with fibrillar structure and a sausage-shaped central nucleus. Spicules paired, relatively short 33.2 (32.0-34.5) µm, in holotype 34.5 µm, slightly ventrally curved. Capitulum widened, slightly marked, lamina partially striated, tapers gradually to the distal end, no bristles at striation. Gubernaculum having a keel-like thickening and proximal end visible between spicules (Fig. 1E). Three ventromedian precloacal supplements (SP) present. The posterior-most one (SP1) at the level of spicule capitulum, the SP2 slightly less than, or equal to one spicule length anterior to the SP1. The anterior most (SP3), 1.0-1.5 times spicule length apart from SP2. Cloacal lip rounded; slightly protruded, post-cloacal papillae not prominent. Tail short, conoid, less than one cloacal diameter long with one pair of subterminal subventral pores.
Female. Body straight or slightly curved upon heat relaxation. Anterior region similar to that of male except for secondary male characteristics. S-E pore located opposite isthmus or anterior part of pharyngeal bulb. Reproductive system didelphic amphidelphic with reflexed ovaries. Two finely granular oviduct cells at the tip of reflexed ovary, sperm round in shape distributed in the distal part of the uteri. Vagina well developed, pars proximalis vaginae barrel shaped in lateral optical view extending less than half corresponding body diameter. Sclerotized vaginal pieces (= pars refringes vaginae) rounded triangular with tips directed towards vulva, pieces 1.5-2.0 µm sized, at about 1.0 µm distance from each other, vulva pore-like in ventral view. Copulatory plug observed in uterus of two specimens. One pair of sublateral body pores almost opposite the vulva. Tail terminus conoid to rounded, anus subterminal, caudal pores subventral, immediately posterior to anus.
The other known Trichodorus, Nanidorus and Paratrichodorus species sequenced during this study clustered with their respective species available through GenBank database, thus supporting their identity.

Diagnosis and relationships.
The new species is characterized by the male having a relatively short onchiostyle (46-49 µm ) and 3 ventromedian cervical papillae anterior to the S-E pore, CP1 located opposite isthmus, distance of CPl-CP2, CP2-CP3 and CP3-S-E becoming gradually shorter, S-E pore located opposite isthmus or anterior end of pharyngeal bulb, pharynx offset, spicules relatively short, slightly curved, 33.2 (32.0-34.5) µm long, with wider slightly marked capitulum, lamina partially striated and tapering gradually to the distal end, bristles at striation absent, three ventromedian precloacal supplements; female with barrel shaped vagina, vaginal scletorized pieces medium-sized (1.5-2.0 µm), rounded triangular with tips directed towards vulva, slightly separated from each other (c. 1.0 µm) , vulva pore-like in ventral view.
occurrence in China. In general, trichodorids have been recovered from many localities in China but some species are geographically concentrated in some areas, e.g., T. nanjingensis is recorded in high percentage in the northern area (Beijing), T. pakistanensis in the southeastern region (Fujian), T. cedarus in eastern regions (e.g., Zhejiang), and P. porosus is very common in Yunnan and Zhejiang provinces. Other species were found in a relatively few numbers. Two of the recorded species are known to transmit tobra viruses (N. minor and P. pachydermus) (MacFarlane et al. 2002).   12.3 ± 1.4(11-15) 11.8 ± 2.9 (8-17) 10.1 ± 1.7 (7-14) CP2-CP3 15.1 ± 0.7 (14-16) 8.9 ± 1.5 (5-10) CP3 to S-E pore (CP2 to S-E pore for T. nanjingensis) Figure 7. Phylogenetic relationships of Trichodorus hangzhouensis sp. nov. and other trichodorid species based on partial 18S rRNA gene sequences. The Bayesian tree was inferred under the general time-reversible model of sequence evolution with correction for invariable sites and a gamma-shaped distribution (GTR+I+G). Tripyla sp. served as an outgroup species. Posterior probability values exceeding 70% are given on appropriate clades.

Discussion
Among trichodorids, Trichodorus, Nanidorus and Paratrichodorus are cosmopolitan genera, species of those genera have been reported from all the continents except Antarctica (Decraemer and Robbins 2007). Regional endemicity has been observed for Trichodorus and Paratrichodorus species. Subbotin et al. (2019) stated that Californian populations of Trichodorus may be endemic originating in the same region, and hypothesized that this is an apparent centre of speciation, in addition to the Iberian Peninsula (Decraemer et al. 2013) and Irano-Anatolian region (Pedram et al. 2015,  . The distribution of trichodorids throughout Asia (except for Iran) is not well documented but in the distribution data of EPPO 2014, trichodorids are either present or widespread in Afghanistan, Bahrain, China, India, Indonesia, Japan, Korea, Turkey, and Uzbekistan. Taxonomic and faunistic records presently list 10 trichodorids (six Trichodorus spp., two Paratrichodorus spp. and two Nanidorus spp.) from China, which represents a comparatively low diversity, and possibly reflects the relatively few studies conducted; so far about half of the territory of the country has been observed for this nematode group with different intensity of sampling. Trichodorus hangzhouensis sp. nov. seems to represent another endemic for China in addition to T. nanjingensis and T. guangzhouensis which are reported only for this country so far.
The D2-D3 region of the 28S rDNA gene has been shown to be of importance in trichodorid molecular taxonomy (Subbotin et al. 2019). The phylogenetic analysis inferred from this gene sequences revealed four highly supported clades. The first is the largest one and consists of Trichodorus species from Europe, Asia and the USA including T. hangzhouensis sp. nov., and corresponds to the Clade I according to Subbotin et al. (2019), the second clade includes Nanidorus species and Trichodorus species from Asia (corresponds to the Clade II (Subbotin et al. 2019)), the third clade consists of Paratrichodorus species distributed in USA, Europe and Asia (Clade III in Subbotin et al. 2019), the fourth clade includes Trichodorus and Monotrichodorus from the USA (Clade IV and V according to the same authors). These results are consistent also with other previously published studies (Asghari et al. 2018. It is interesting to mention that the three species occurring in southeastern Asia (T. cedarus, T. nanjingensis and T. japonicus) form a highly supported subclade within Clade II, while the new species is part of another phylogenetically more distant group (Clade I). However, in the ITS2 tree the position of T. nanjingensis differs substantially.
In addition, the trichodorid species molecularly characterized during this study (T. nanjingensis, T. cedarus, T. pakistanensis, P. porosus, N. minor, N. renifer) clustered with the known species from different countries; these results further validated their identity. It is also noted that the position of T. hangzhouensis sp. nov. differs more or less in the phylogenetic trees based on the different gene sequences, and this could be also caused by the incomplete sequence data for a given species.
All present, and most previous, phylogenetic reconstructions inferred from three different gene sequences (18S, D2D3 28S and ITS2) showed that Nanidorus and Paratrichodorus species each formed highly supported clades. Trichodorus species studied molecularly so far take three different positions based on D2D3 28S r RNA gene sequences: i) the large part containing only Trichodorus species and forming Clade I sensu Subbotin et al. (2019); ii) three species of southeastern Asian origin clustering together with Nanidorus species and iii) Californian species forming a highly supported clade.
All the three aforementioned genera occur in China. From our observations, trichodorids seem not host specific and can be found in various types of ecosystems. The occurrence of Trichodorus, Nanidorus and Paratrichodorus recovered from soils in China is quite low (4.1%) compared to trichodorid occurrence in other countries such as Great Britain (22%), Italy (9.6%), Iran (7%), Belgium (19.6), Portugal (32.6%) and Slovak Republic (33%) (Alphey and Boag 1976, Roca and Lamberti 1985, De Waele and Sturhan 1987, De Waele and Coomans 1991, Almeida 1993, Lišková and Sturhan 1999. In the past surveys concerning the stubby root nematodes, this group is reported being generally in somewhat low densities (Aballay and Eriksson 2006). The low density of trichodorids populations in the soil could be related to the sampling strategies (depth and intensity of sampling) or studied plants (crops or natural vegetation). Boag (1981) suggested that the distribution of trichodorids is correlated with soil moisture, particle size structure and seasonal fluctuation of temperature. De Waele and Coomans (1991) also recognized that geographic distribution of certain trichodorid species may be influenced by their habitat and found a relatively high presence of populations in soil with a pH <5.5.
In conclusion, this study provides a morphological and molecular characterization of T. hangzhouensis sp. nov. and three known trichodorid species together with updated records of this group in China. Among 164 populations recovered in China, the highest number of records is for P. porosus (42.6%) followed by T. nanjingensis (39.6%). The systematics and diagnostics of trichodorid nematodes are important because of regulatory and management issues attributed to this group of nematodes being vectors of tobra viruses. Thus, updated descriptions based on sufficient examination material and accurately identified specimens, coupled with molecular analysis are necessary for better understanding of the current distribution and host association of this complex group of nematodes.