Description and Distribution of Three Criconematid Nematodes from Hangzhou, Zhejiang Province, China

Populations of Criconemoides parvus, Discocriconemella hengsungica, and Discocriconemella limitanea, isolated in Hangzhou, China from the rhizosphere soil of woody perennials were characterized morphologically and molecularly. The morphometric data of the Chinese populations were compared with populations from other regions of the world. DNA barcoding with the mitochondrial COI gene confirmed conspecificity of Chinese and Costa Rican populations of D. limitanea. Phylogenetic assessment using a near full-length 18S ribosomal DNA sequence provided weak support for a grouping of Criconemoides parvus from China and C. annulatus from western North America. The phylogenetic position of D. hengsungica from China and an unknown species of Discocriconemella from Thailand relative to D. limitanea suggests that the genus Discocriconemella is not monophyletic. The study provides the first record of D. hengsungica in China and confirms the presence of C. parvus previously reported from China. Biogeographic implications of these nematode distributions are discussed.


Three criconematids from China
Discocriconemella hengsungica was originally described from Korea, and is the only record of its occurrence (Choi and Geraert, 1975), but there is no molecular information available for this species.
Thus, the objectives of the study were to: (1) establish the identity of these three species by morphological and molecular characterization, (2) integrate the morphometric characterization of Chinese populations of D. limitanea and C. parvus with measurements reported from different countries, (3) evaluate the phylogenetic and biogeographic relationships of these species within Criconematidae using 18S and COI DNA sequence.

Materials and methods
Nematode detection and morphological observations: Soil samples were collected from undisturbed natural locations in the Hangzhou Botanical Garden. Nematodes were extracted from soil using a modified Cobb sieving and flotation-centrifugation method (Jenkins, 1964). Nematodes were killed and fixed in hot 4% formaldehyde, infiltrated with glycerin following the method of Seinhorst (1959), and mounted on slides for observation and preservation. The measurements and light micrographs of nematodes were accomplished using an ocular micrometer and a Zeiss Stemi 2000-C compound microscope.
Nematodes were also examined using a Hitachi TM-1000 scanning electron microscope (SEM). For the SEM examination, the nematodes were fixed in a mixture of 2.5% paraformaldehyde and 2.5% glutaraldehyde, washed three times in 0.1 M cocodylate buffer, post-fixed in 1% osmium tetroxide, dehydrated in a series of ethanol solutions and critical point-dried with CO 2 . After mounting on stubs, the samples were coated with gold. Specimens from Costa Rica were processed for SEM using the methods described in Powers et al. (2010).

Molecular analyses
DNA samples from China were prepared according to Zheng et al. (2003). Individual nematodes were transferred into an Eppendorf tube containing 16 µL ddH2O. Two microliters PCR buffer solution was added to each tube. Nematodes were crushed using a sterilized pipette tip, briefly spun and immediately frozen at −68 o C for at least 30 min. The tubes were heated to 85 o C for 2 min, briefly spun, followed by the addition of 2 µL proteinase K. The tubes were incubated at 56 o C for 1 to 2 hrs, followed by 10 min at 95 o C. After incubation, these tubes were cooled at 4 o C and used for PCR (Zheng et al., 2003). Several sets of primers (synthesized by Invitrogen, Shanghai, China) were used in the PCR analyses to amplify the near full-length18S region of rDNA and COI region. Two sets of primers: the forward 18S39F (5′-AAA GAT TAA GCC ATG CAT G-3′) and the reverse 18S977R (5′-TTT ACG GTT AGA ACT AGG GCG G-3′), the forward 18S900F (5′-AAG ACG GAC TAC AGC GAA AG-3′) and the reverse 18S1713R (5′-TCA CCT ACA GCT ACC TTG TTA CG-3′) for amplification of the nearly full-length 18S rRNA . For the amplification of COI the primers used were COI-F5-(5′-AATWTWGGTGTTGGAACTTCTT -GAAC-3′) and COI-R9-(5′ CTTAAAACATAATGRAAAT-GWGCWACWACATAATAAGTATC-3) (Powers et al., 2014). The 25-µl PCR was performed using 2x-TsingKe Master Mix DNA polymerase (Beijing TsingKe Biotech Co., Ltd) according to the manufacturer's protocol in a BIOER-XP thermocycler. The thermal cycler program for 18S and COI was as follows: denaturation at 95°C for 5 min, followed by 40 cycles (18S) or 50 cycles (COI) of denaturation at 94°C for 30 s, annealing at 50°C (18S) or 48°C (COI) for 30 s, and extension at 72°C for 90 s. A final extension was performed at 72°C for 5 min as described by Powers et al. (2014) and Olson et al. (2017). PCR products were separated and visualized on 1% agarose gels and stained with ethidium bromide. PCR products of sufficiently high quality were sent for sequencing by Invitrogen (Shanghai, China).

Phylogenetic analysis
Phylogenetic trees were constructed by maximum likelihood (ML) in MEGA version 6. Sequences were edited using CodonCode Aligner version 4.2 (http://www.codoncode.com/) and aligned using Muscle within MEGA version 6 (Tamura et al., 2013). Gap opening penalty was set at −400 with a gap extension penalty of 0. The general time reversible model with Gamma-distributed rates plus invariant sites (GTR+G+I) was determined to be the best substitution model by Bayesian Information Criterion using the Best Fit Substitution Model tool in MEGA 6.0. The ML trees used the all sites option for gaps and 200 bootstrap replications to assess clade support. The 18S tree used all the taxa previously presented in Powers et al. (2017)

Description
Female: Body cylindrical, ventrally arcuate after heat relaxation. The cephalic region is flat, continuous with the body contour. En face view, an oral disc with slightly elevated lateral pseudolips, oral aperture slitlike, with submedian lobes absent. Surrounding and apparently fused with the oral disc is a single labial annulus with dorsal and ventral indentations. Body annuli retrorse with posterior margins finely crenate, more prominent on the posterior body, anastomoses common in the middle of the body. Stylet is short with rounded basal knobs, DGO indistinct, and oesophagus criconematoid. Excretory pore at the base of the oesophageal bulb. Gonad monodelphic, outstretched, spermatheca oblong, filled with rodshaped sperm, vagina straight, vulva closed, anterior and posterior annuli around the vulva larger than the preceding body annuli; discontinuous annuli are more common near the region of the vulva. Tail conoid ending in a rounded terminus and the anus is indistinct.

Description
Female: Body cylindrical, ventrally curved after heat relaxation. Labial region a disc-like appearance in profile. En face view, does not show a discrete oral disc, instead the stylet appears to be located centrally in an inner rectangular area surrounded by a continuous, broad labial annulus with deep ventral and dorsal indentations forming two pairs of dorsal and ventral lobes combined with distinct lateral bulges. The oral disc and amphid apertures are indistinct due to amphidal excretions in SEM images. The labial annulus is separated from the body annulus by a high neck or collar. Body annuli retrorse to angular, without anastomosis or interruptions. Stylet long and flexible with anchor-shaped knobs, DGO indistinct; oesophagus criconematoid. Excretory pore located near the middle of the oesophageal bulb. Gonad monodelphic, outstretched, some individuals with reflexed ovary, spermatheca rounded filled with spherical sperm, vagina straight, and vulva closed. Tail conoid broadly rounded, and terminal annuli displaced dorsally and the anus is indistinct.
Male: Not found. Locality and habitat: The population was found in the rhizosphere of Castanopsis sclerophylla (Lindl.) Schott from a Botanical garden in Hangzhou, Zhejiang Province, China on March 28, 2017. The geographical location of the sampling site is ″30°15′17″N; 120°07′01″E.
Differential diagnosis: In the original description of D. hengsungica six specimens were studied. Only one female was observed with a few anastomoses. No anastomoses were observed on the Chinese specimens. The spermatheca was described as filled with sperm but no males were found. Similarly, the Chinese population had specimens with sperm-filled spermatheca, but no males were found. The original description lacks information on the morphology of the labial disc, position of excretory pore and anus, shape of vagina and vulva. Morphology of the Chinese population fits well with the characters included in the original description except for the complete absence of anastomoses. Morphometrically, the Chinese population is slightly longer (307-382 µm vs. 285-315 µm) with relatively longer stylets (100.3-113.5 µm vs. 104-108 µm) and less annuli from vulva to tail terminus (RV = 9-10 vs. 13-14). The slight morphometric differences could be attributed to fewer specimens studied in the original description and geographical variability.

Description
Female: Body stout, ventrally arcuate after heat relaxation, lip region with disc-like appearance. En face view, a labial annulus with deep dorsal and ventral indentations, the oral opening appearing as a slit on a rounded oral disc flanked by two lateral amphidial apertures. The lateral edges of the labial annulus straight, lacking a central bulge. Body annuli retrorse, finely crenate edges, frequent anastomoses or discontinuous annuli that demarcate lateral lines. Stylet robust, anchor-shaped knobs, DGO indistinct. Oesophagus criconematoid. Excretory pore at the base of the oesophageal bulb. Gonad monodelphic, prodelphic, outstretched, spermatheca oblong  (Fig. 6C). The specimen had a body length of 258 µm, spicule of 19 µm, and gubernaculum of 5 µm. In the female, the relatively abrupt constriction of the post-vulval body was not described or illustrated in the original description, but the populations from Malaysia (Sauer and Winoto, 1975), Brazil (Rashid et al., 1986), India (Rahaman and Ahmed, 1994) and Ecuador (Talavera and Hunt, 1997) reported the narrowing of the post-vulval body profile. The South African population (Van den berg and Cadet, 1992) was reported to have distinct tooth-like projections on the margins of the ventral body annuli. The Brazilian population (Loof and Sharma, 1980) was reported to have a conspicuous break between the fourth and fifth annuli.
Morphometrically, the Congo population (Coomans, 1966) is slightly longer than the original description (260-280 µm vs. 207-228 µm). The Ivory Coast population (Luc, 1970)   52-53 µm, respectively) in relation to the original description. The two Brazilian populations reported by Loof and Sharma (1980) and Rashid et al. (1986) are a mixture of small and large specimens. These two populations also differ from each other morphometrically; the notable difference of these two populations from the original description is the variable body length (167- Ye et al.
Zhang et al.
Zhang et al.
Li et al.  vs. 207-228 µm) and longer stylet length (53-60 µm vs. 52-53 µm). Overall, the morphometrics are within the range of variation of the species according to the populations described by various authors. Five additional populations of D. limitanea from China have been reported from Guangzhou, Guangdong, Fujian and Yunan provinces. Nematodes from all of these populations have overlapping morphometric ranges, fit well within with the original description and confirm to the species as described by multiple authors (Luc, 1970;Rashid et al., 1986;Rahaman and Ahmed, 1994;Talavera and Hunt, 1997).

Molecular profiles and phylogenetic status
Several key systematic features of criconematid nematodes are revealed by the 18S and COI phylogenetic trees. First, in the 18S tree ( Fig. 9) which provides better resolution at the deeper nodes in the tree, there is strong bootstrap support (99%) for a clade that combines Discocriconemella limitanea from China with conspecific specimens from Costa Rica. This clade confirms the species identification and provides evidence of an amphi-Pacific disjunction, the first molecular data from a nematode to support this distribution pattern. Studies of many plant species suggest this is one of several intercontinental distribution patterns that link Asia and North America (Li and Wen, 2013;Fritsch et al., 2015). COI (Fig. 10) also supports this grouping at a lower support value (82%). Similarly, Criconemoides parvus groups with C. annulatus Cobb in Taylor, 1936 from western U.S. in the 18S tree, albeit at a relatively low support value (58%). There are no molecular data of C. parvus from North America, although the type locality is in the western state of Nevada. The placement of Discocriconemella hengsungica and an unknown Discocriconemella specimen from Thailand, in both 18S and COI trees, provides strong evidence that the genus Discocriconemella is not a monophyletic group. Discocriconemella hengsungica is a member of a larger criconematid clade that predominantly includes nematodes that possess scales or projections on the cuticle in at least one life stage. Xenocriconemella (De Grisse and Loof, 1965) is also a member of this group which adds evidence that cuticle projections are not reliable taxonomic characters in establishing the genera .
Overall, the addition of these species from China to a reference dataset of criconematid nematodes provides insight into the biogeography of nematodes in general. It is likely that additional collections of plant parasitic nematodes from Asia will also contribute to fundamental questions of angiosperm biogeography (Raven and Axelrod, 1974;Fritsch et al., 2015;Wen et al., 2016).