Description of Longidorus cheni sp. n. (Nematoda, Longidoridae) from China

Abstract Longidorus cheni sp. n., an amphimictic species recovered from the rhizosphere of Larix principis-rupprechtii and Pyracantha fortuneana in Shanxi and Beijing, China, is described and illustrated. The taxonomic position of L. cheni sp. n. among other species within the genus was elucidated using morphometric and molecular data, and phylogenetic relationships were inferred using D2–D3 expansion domains of 28S and 18S rRNA genes by Bayesian Inference (BI) method. The new species is characterised by females with a medium body size (L = 4.9–6.6 mm), a lip region slightly expanded, broadly rounded frontally and laterally, the amphidial fovea broad and symmetrically bilobed at base, odontostyle long and slender (143–168 μm), odonthophore slightly swollen at the base, tail short bluntly conoid to rounded. Guide ring located far posterior from the oral aperture (70–93 μm). Males with two ad-cloacal pairs of supplements preceded by a row of 10–14 ventromedian supplements, with robust spicules measuring 111–126 μm along the median line. Three juvenile stages were present, tail shape of J1 elongate conoid while in J2 and J3 the tail gradually becomes bluntly rounded. Codes for identifying the new species are: A6-B3-C5-D2-E2-F3-G1-H1-I2-J2-K2. Longidorus cheni sp. n. belongs to a group of species with a guide ring at the mid-odontostyle position that have a predominantly Asiatic origin. It differs from all of them by a combination of morphological characters and unique sequences of partial 18S and D2–D3 region of 28S rRNA genes. The percentage dissimilarities in partial 18S and D2–D3 28S rRNA genes of L. cheni to the closest species (L. litchii, L. fangi, L. jonesi and L. juglans) were 1.5 %–1.8 % and 16.8–18.3 %, respectively.


Introduction
Longidorids, despite their long history of research (the first species of the family Longidorus elongatus (de Man, 1876) was described almost one hundred and fifty years ago) continue to attract the attention of scientists due to their high species diversity, wide distribution, and economic importance. The valid Longidorus Micoletzky, 1922 species described to date reached 167 (Peneva et al. 2013, Xu et al. 2017 with the proposed synonymy of two species (Sturhan 2014, Tanha Maafi et al. 2015. Present records of Longidorus distribution in mainland China reported by Guo et al. (2011) and Xu et al. (2017) include 16 species, half of which were originally described from the country: L. jiangsuensis Xu & Hooper, 1990, L. fangi Xu & Cheng 1991, L. henanus Xu & Cheng 1992, L. litchii Xu & Cheng 1992, L. hangzhouensis Zheng, Peng, Robbins & Brown, 2001, L. camelliae Zheng, Peneva & Brown, 2000, L. asiaticus Triscuizzi, Archidona-Yuste, Troccoli, Fanelli, Luca, Vovlas & Castillo, 2015and L. juglans Xu, Ye, Wang, Zeng & Zhao, 2017 In a survey during August 2014 and May 2015, a new species of Longidorus was recovered from native conifers growing in a mountainous region of Shanxi and evergreen shrubs growing in a botanic garden in Beijing, the localities situated in northern and northeastern China, respectively. Molecular approaches and phylogenetic studies in combination with morphometric characters are used as a taxonomic standard for species identification and delimitation (Gutiérrez-Gutiérrez et al. 2013, Peneva et al. 2013, Archidona-Yuste et al. 2016. The study aims to characterise this undescribed nematode species based on morphological characters coupled with molecular data and infer the phylogenetic relationships with the other species of genus Longidorus.

Nematode sampling
Specimens examined in this study were extracted from soil samples collected from the rhizosphere of Larix principis-rupprechtii Mayr. from Shanxi and Pyracantha fortuneana (Maxim.) from Beijing, China. Five hundred grams (500 g) of soil were mixed and washed using a decanting and sieving technique (Brown and Boag 1988). The extract was left for two days on a Baermann funnel and the suspension was collected afterwards. Collected nematodes were examined under a stereomicroscope and Longidorus specimens were picked out and transferred to Syracuse dishes for storage. For morphometric studies the nematodes were killed, fixed with hot formalin, and processed to glycerine (Seinhorst 1959) as modified by De Grisse (1969). The micrographs, measurements, and drawings of nematodes were completed with the help of Nikon eclipse Ni-U 931845 compound microscope. All measurements were presented in micrometres (μm) and expressed as a mean ± standard deviation.

DNA Extraction, amplifications, and sequencing
DNA was extracted from a single adult nematode, carefully handpicked from nematode suspensions, transferred onto a glass slide containing a 13 μl H 2 O, and cut into two pieces using a sterilised scalpel. The nematode fragments were pipetted up to 10 μl and transferred to Eppendorf tubes with 8 μl Mg+ free buffer and 2 μl proteinase K (Ye et al. 2004). PCR tubes were centrifuged at 12000 rpm for 2 minutes and immediately frozen at -70 °C for at least 30 minutes. Subsequently, each tube was incubated for 65 °C for 3 hours and nematode was digested at 75 °C for 60 minutes and 95 °C for 10 minutes. Finally, the DNA suspensions were cooled down at 8 °C and stored at -20 °C until use. A total of 25 μl PCR mixture was prepared containing 2.5 μl LA buffer, 2 μl dNTP, 1.5 μl each primer (synthesized by Takara Company, Shanghai, China) and 3 μl DNA template, 0.3 μl LATaq and 14.2 μl distilled water. All PCR reactions were conducted in the S1000 thermal cycler (BIO-RAD). Fragments of 18S and 28S region were amplified using two sets of primers: forward primer SSU_F_07 (5' AAA GAT TAA GCC ATG CAT G 3') and reverse primer SSU_R_81 (5' TGA TCC ACC TGC AGG TTC AC 3') (Gutiérrez-Gutiérrez et al. 2011) and forward primer D2A (5' ACA AGT ACC GTG AGG GAA AGT TG 3') and reverse primer D3B (5'-TCG GAA GGA ACC AGC TAC TA-3') (De Ley et al. 1999), respectively. The thermal cycling protocol consisted of denaturation at 95 °C for 5 minutes, followed by 35 cycles of denaturation at 94 °C for 30 seconds, annealing at 55 °C for 45 seconds, extension at 72 °C for 2 minutes and a final extension at 72 °C for 10 minutes. After DNA amplification, 2.5 μl aliquots of PCR products were analysed by gel electrophoresis in 1 % agarose gel (100V, 400 mA, 30 minutes) stained in ethidium bromide for 10 minutes and DNA were visualized under UV illumination. Amplified PCR products were purified following the instructions as described in the nucleic acid purification kit of AXYGEN (catalogue No. AP-GX-250) of the AXYGEN Biotechnology Co., Ltd. Hangzhou, China. Purified DNA were ligated to pUCM-T vector and transformed in to DH 5alpha competent cells. The transformants were screened on an ampicillin agar LB plates containing 400 ml IPTG, X-Gal and left at 37 °C overnight. White colonies were selected, transferred to 5 ml LB containing 100 mg/ml ampicillin, and incubated at 37 °C for 16-24 hours. PCR amplification was further confirmed with the primer insertion and expected band; four clones were sequenced per population. Sequencing was done at the SANGON Biotechnology Co., Ltd. Since the clones were identical, only one sequence for each gene has been deposited in GenBank sequence database with the following accession numbers: KY284157 and KF270638 for D2-D3 expansion domains of 28S rDNA, KF261570 and MG656980 for the 18S rDNA region.

Sequence and phylogenetic analyses
The D2-D3 28S and 18S rDNA sequences were compared with those of other nematode species deposited in GenBank database using BLASTn similarity search tool. The homologous sequences nearest to those of the new species were aligned using the GUIDANCE2 Server available at http://guidance.tau.ac.il/ with default parameters (Sela et al. 2015) and manually trimmed and edited in Mega 7 (Kumar et al. 2016). Bayesian Inference (BI) algorithm implemented in MrBayes 3.2.5 was used for phylogenetic relationships reconstructions Ronquist 2001, Ronquist et al. 2012). For further details, see Lazarova et al. (2016). The 50 % majority rule consensus BI trees of Longidorus and Paralongidorus spp. are based on a multiple sequence alignment data sets that included: a) 57 sequences and 700 total characters for D2-D3 28S rRNA gene and b) 48 sequences and 993 total characters for 18S rRNA gene.
Female. Body habitus G-shaped when relaxed by gentle heat (Fig. 1M) gradually tapering in both ends. Cuticle under light microscope with three distinct layers, the middle one consisting of several sub-layers, slightly refractive, most pronounced at labial and tail regions, the inner one thicker at labial and tail region with radial striations, cuticle 6 μm thick at post-labial area, 5 μm along the body and 11 μm in post-anal region. Nine lateral, three dorsal and five ventral body pores in the neck region. Lip region slightly expanded, broadly rounded frontally and laterally (Fig. 2D). Amphidial fovea broad and symmetrically bilobed at the base (Fig. 3B). Odontostyle long and slender with simple base, odontophore slightly swollen at the base (Fig. 3A), approx. 1/3 of the odontostyle length. Guide ring located far posterior from oral aperture (Fig. 4A-B). Pharynx dorylaimoid with anterior part more or less coiled, pharyngeal bulb comparatively short measuring 107-138 × 23-28 μm (Fig. 1O). Arrangement of pharyngeal glands normal, dorsal gland nucleus located at 23-34 μm and ventrosublateral gland nuclei at 48-54 μm from the beginning of pharyngeal bulb. Pharyngo-intestinal valve (cardia) hemispherical (Fig. 1P). Tail short dorsally convex and terminus bluntly conoid with two pairs of caudal pores (Fig. 2I). Vulva a transverse slit located slightly anterior from mid body (V = 40-48 %), vagina well developed extending nearly half of body diameter (Fig. 3D, E). Reproductive system amphidelphic with anterior and posterior branches almost equally developed (Fig. 3C). Sperms observed in the uteri of most females (Fig. 3F).   Male. Morphologically similar to female. Body G to spiral shape (Fig. 1M). Testes paired and fully developed. Sperms abundant and irregularly shaped (Fig. 3K). Spicules robust (Fig. 3J). Lateral guiding piece 32-35 μm. Two ad-cloacal pairs of supplements preceded by row of 10-14 ventromedian supplements (Fig. 3H). Tail ventrally curved bluntly conoid to hemispherical, 2-3 lateral pores on each side (Fig. 3H).
Juveniles. Three juvenile stages (J1-J3) distinctly separated by differences in the body length, odontostyle and replacement odontostyle length (Fig 1A-C). In the first stage juvenile, the anterior part of replacement odontostyle is inserted in the wall of odontophore (Fig 3A). Morphologically, juveniles resemble adults except for the smaller size and not developed reproductive system. Habitus assuming J shape does not change with the stage (Fig. 1I-K). Tail length does not change while anal width increases ( Fig. 1E-G) thus c' ratio decreases (Table 2), guide ring position becoming more posterior during successive stages ( Fig. 2A-C). First stage juvenile is characterized by a conoid tail becoming bluntly conoid in second to third stages (Fig. 2F-H).
Sequences and phylogenetic analyses. The length of PCR products based on gel images of the amplification of partial 18S and D2-D3 region of 28S RNA genes of L. cheni sp. n. (LDT235 and BJ07) was 844 bps and 856 bps, respectively. The sequences of both populations were identical. The phylogenetic relationships of L. cheni sp. n. with the closest species inferred from analyses of the partial 18S rDNA and D2-D3 expansion segments of 28S rDNA sequences using BI are presented in Figs 6 and 7, respectively. In general, the new species grouped with other Longidorus species of predominantly Asiatic origin in both phylogenetic reconstructions. In D2-D3 rDNA phylogenetic tree, L. cheni clustered in a Type habitat and locality. Specimens were recovered from soil around the roots of a conifer (L. principis-rupprechtii) and Chinese firethorn (P. fortuneana) in mountainous region of Shanxi and botanic garden in Beijing,China,GPS coordinates 37°50'815"N,111°27'253"E and 30°34'54.7"N,114°15'40.9"E, respectively. Type material. Holotype. Female slide no. LS5313 and paratypes (slides no. LS 5301-5312, LS 5314-5350) includes 12 females, 12 males and 52 juveniles deposited in the Nematode collection C602 Nematology laboratory of Zhejiang University, Hangzhou, China. One female, one male, three juveniles deposited at the nematode collection of the Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences, Sofia, Bulgaria.
Etymology. The species is named after Prof. Pinsan Chen, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, one of the pioneer plant nematologists in China.