Description of two new Cryptaphelenchus species from China and Japan

Summary – The study aims to provide the morphological and molecular characterisation of two newly discovered Cryptaphelenchus species, namely: C. curvatum n. sp. and C. tumidus n. sp. Morphologically both species are minute nematodes, possess medium-sized stylets with basal knobs laterally directed, and short post-uterine sacs (PUS). Cryptaphelenchus curvatum n. sp. was detected from the local Pinus branch samples and is characterised by having an equal proportion of adult males and females, medium-sized body lengths of females 353 (320-388) μ m and males 277 (256-293) μ m, lateral ﬁelds with four lateral lines, 8-10 μ m long stylet, PUS about one vulva body width long and conical posterior body end with a blunt pointed tip. Cryptaphelenchus tumidus n. sp. was detected in a packaging wood sample from Japan and is characterised by having rare male, short body lengths of females 236 (217-270) μ m and males 180 μ m, obscure lateral ﬁelds, stylet 6.8 (6.0-7.5) μ m, PUS about half vulva body width long and a slim conical posterior body end with a ﬁnely rounded tip. The phylogenetic trees were also constructed based on rRNA gene sequences (D2-D3 segments of the 28S, ITS and 18S) and discussed in detail. Since Cryptaphelenchus species offer multitrophic relationships among their insect vectors and associated microbiota, species recognition and molecular studies of recovered populations will facilitate researchers to determine whether these species might play any ecological roles in insect bionomics or the residing host tree.

is a genus of entomophilic nematodes in the subfamily Ektaphelenchinae; member species are reported to inhabit the outer bark of coniferous and deciduous trees (Hunt, 1993;Pedram, 2017;Kanzaki et al., 2022).The majority of bark beetles are known to harbour Cryptaphelenchus species, and their relationship is revealed as endophoretic (Kanzaki et al., 2022).Bionomically, Cryptaphelenchus species do not cause any wood decay; rather, they feed on fungal propagules/hyphae that may develop on food sources, faeces, or on the dead larvae/eggs of the bark beetles in their galleries, indicating their ecological roles in the host's micro-environments (Poinar, 1972;Hunt, 1993).
In the present study, two new Cryptaphelenchus species were detected in the Ningbo plant quarantine lab during inspections of local samples and imported packaging wood.Morphological and molecular characteristics of both new species were studied in detail and they were named as C. curvatum n. sp. and C. tumidus n. sp.The phylogenetic trees were also constructed based on rRNA gene sequences (D2-D3 expansion segments of 28S rRNA, ITS and 18S) and discussed in detail.Since Cryptaphelenchus species offer multitrophic relationships among their insect vectors and associated microbiota, species recognition and molecular studies of recovered populations will facilitate researchers to determine whether these species might have some influential roles in the host's eco-space.

SAMPLING, NEMATODE EXTRACTION AND MORPHOLOGICAL STUDIES
The nematodes were extracted from branches of declining Larix gmelinii in China and imported Japanese packaging wood samples according to a modified Baermann funnel technique.The Cryptaphelenchus individuals were handpicked from nematode suspension and processed for preliminary microscopic examinations.For preservation and detailed morphological studies, nematodes were heatkilled by hot 4% formalin solution and transferred to anhydrous glycerin as described by De Grisse (1969) and mounted on permanent slides.Imaging and morphometrics were acquired using a Zeiss AxioCam MRm digital camera attached to a Zeiss Imager Z1 microscopy software.

DNA EXTRACTION, PCR AND SEQUENCING
DNA samples were extracted from freshly isolated nematodes recovered from branch samples and packaging wood.Briefly, single individuals were transferred separately to PCR tubes containing 8 μl ddH 2 O and 1 μl 10× PCR buffer.The tubes were stored at −70°C for at least 2 h, thawed at room temperature and 1 μl proteinase K (100 μg ml −1 ) added.Subsequently, the tubes were incubated at 65°C for 1-2 h and at 95°C for 10 min.Three sets of primers (synthesised by Invitrogen) were used in the PCR analyses to amplify the partial 18S, ITS, and D2-D3 expansion segments of 28S of rRNA.
PHYLOGENETIC ANALYSES D2-D3 expansion segments of 28S rRNA, ITS rRNA and 18S rRNA sequences of the newly detected Cryptaphelenchus species were obtained in this study.These sequences and the available Cryptaphelenchus spp.sequences were used for phylogenetic analyses.Outgroup taxa for each dataset were chosen following previously published studies of Xue et al. (2019), Pedram et al. (2020) and Fanelli et al. (2022).Multiple sequence alignments of the different genes were made using the FFT-NS-2 algorithm of MAFFT V.7.450 (Katoh et al., 2013).Sequence alignments were visualised using BioEdit (Hall, 1999).Sequences were edited manually and poorly aligned positions were trimmed using a light filtering strategy (up to 20% of alignment positions).Phylogenetic analyses of the sequence datasets were based on Bayesian inference (BI) using MrBayes 3.1.2(Ronquist & Huelsenbeck, 2003).The best-fit model of DNA evolution was obtained using JModelTest V.2.1.7 (Darriba et al., 2012) with the Akaike Information Criterion (AIC).The bestfit model, the base frequency, the proportion of invariable sites, and the gamma distribution shape parameters and substitution rates in the AIC were then used in MrBayes for the phylogenetic analyses.The general time-reversible model with a gamma-shaped distribution (GTR + G) for the D2-D3 segments of 28S rRNA, the transversion model with invariable sites and a gamma-shaped distribution (TrN + I + G) model for ITS rRNA, and TrN + G model for 18S gene, were run with four chains for 4 × 10 6 generations, respectively.A combined analysis of the two ribosomal genes was not undertaken due to some sequences not being available for all species.The Markov chains were sampled at intervals of 100 generations.Two runs were conducted for each analysis.After discarding burn-in samples of 30% and evaluating convergence, the remaining samples were retained for in-depth analyses.The topologies were used to generate a 50%  (Rambaut, 2014).

Female
Body slender, slightly tapering towards both ends, ventrally curved forming 'C' shape.Cuticle with distinct annuli (annuli 1.0-1.5 μm wide), having four lateral lines.Lip region semicircle, separated from rest of body by a shallow depression.Stylet delicate with narrow lumen, conus forming ca 40% of total length, basal knobs small and laterally directed.Procorpus slender, metacorpus (median bulb) oval, valve plates, central sclerotised, pharyngeal glands 20-30 μm long, single nucleus was observed in the dorsal gland, overlapping intestine dorsally.Junction of pharynx and intestine immediately at base of metacorpus.Nerve ring at ca 4-7 μm posterior to median bulb.Excretory pore slightly posterior to the nerve ring, located 60-75 μm from anterior end, hemizonid posterior to pore.Intestine simple, posteriorly ending in a blind sac, rectum and anus absent.Gonad monodelphic, occupying 30-40% of body length, composed of an outstretched ovary with two rows of oocytes, indistinct oviduct, spermatheca distinct, oblong, with sperm cells, crustaformeria and uterus indiscernible from each other, uterus thick-walled, vagina slightly directed anteriorly, sclerotisation around vagina not observed, postvulval uterine sac (PUS) ca one vulval body diam.(VBD) long, vulva a transverse slit, vulval lips not protruding.No specific structure seen at junction of uterus and PUS.Vulva to body end 67-87 μm or 6-7 VBD long.Body posterior to the end of blind sac conical, terminus bluntly pointed.

Male
Common.Body slender.General morphology of the anterior body parts similar to that of female.Gonad monorchic, occupying 35% of body length, testis outstretched with developing spermatocytes in a single row, sperm in vas deferens fine.Posterior body end strongly ventrally bent.Spicules arcuate, separate, capitulum with a slight depression in the middle, condylus, high, broadly rounded, well developed; rostrum triangular, 3-4 μm long with sharply pointed tip; spicule blade (calomus-lamina complex) arcuate with pointed terminus.Ventral apophysis present, seemingly separated from spicule, short crescent in shape with a short and blunt dorsal projection in lateral view, somewhat wing-like in ventral view where it is slightly compressed and flattened.Seven papilliform caudal papillae (one single and three pairs) are present, comprising a single mid-ventral precloacal papilla (P1), located 5-7μm anterior to cloacal aperture, first paired papillae (P2) subventral, located at the middle of P1 and cloacal aperture, second (P3) and third (P4) subventral pair located near tail terminus, P3 is larger, while P4 is very small.Tail conoid ending to a bluntly rounded terminus.Bursa and gubernaculum absent.

TYPE HABITAT AND LOCALITY
The new species was recovered from branches of a declining Larix gmelinii, with blue staining from fungal colonisation.The tree was in Fushun city, Liaoning province, P.R. China.Insect galleries of presumed insect associates were not observed on the collected samples.The attempt to rear nematodes on Botrytis cinerea culture plates was unsuccessful.

TYPE MATERIAL
Holotype female, ten female and 11 male paratypes (slide nos C46-1 to C46-10) deposited in the nematode collection of Technical Centre of Ningbo Customs, Ningbo, P.R. China; and four female and four male paratypes (slide no.T560) deposited in the Canadian National Collection of Nematodes, Ottawa, ON, Canada.

ETYMOLOGY
The species epithet is derived from the Latin word curvatum representing ventrally curved characteristics of the female body.

Female
Cuticle swell irregular.Body slender, almost straight after fixation, slightly tapering towards both ends.Cuticle with distinct annuli (annuli 0.8-1.1 μm wide), lateral lines obscure.Lip region semi-circular, separated from rest of body by a shallow depression.Stylet delicate with narrow lumen, conus forming ca 40% of total length, basal knobs small and laterally directed.Procorpus slender, metacorpus (median bulb) oval, valve plates central, sclerotised, pharyngeal glands 20-30 μm long, overlapping intestine dorsally.Junction of pharynx and intestine immediately at base of metacorpus.Nerve ring at ca 3-6 μm posterior to median bulb.Excretory pore located 46-51 μm from anterior end, ca 4-8 μm posterior to nerve ring, hemizonid posterior to excretory pore.Intestine simple, posteriorly ending in a blind sac, rectum and anus absent.Gonad monodelphic, occupying 40-50% of body length, composed of an outstretched ovary with a single row of oocytes, indistinct oviduct and spermatheca, crustaformeria and uterus indiscernible from each other, uterus thick-walled, vagina slightly directed anteriorly, post-vulval uterine sac (PUS) ca 0.5 vulval body diam.(VBD) long, vulva a transverse slit, vulval lips not protruding.No specific structure seen at junction of uterus and PUS.Vulva to body end 59-74 μm or 7-8 VBD long.Body posterior to the end of blind sac conical, almost straight, terminus finely rounded.

Male
Rare; only one male was detected among this population.Body slender.General morphology of anterior part similar to that of female.Gonad monorchic, occupying 34% of body length, testis outstretched with developing spermatocytes in single row, sperm in vas deferens fine.Posterior body end strongly ventrally bent.Spicules arcuate, separate, capitulum with shallow depression condylus high and broad, well developed; rostrum triangular 2-3 μm long with pointed tip, spicule blade (calomuslamina complex) curved with pointed terminus.Since only one male was present, our observations suggest the presence of two pairs of subventral caudal papillae (P3 and P4) situated near the tail terminus.Apophysis not observed.Bursa and gubernaculum absent.Tail conical, dorsally convex, ventrally slightly concave with finely rounded tip.

TYPE HABITAT AND LOCALITY
The new species was recovered from Pinus sp.packaging wood from Japan with blue stain from fungal colonisation.Insect galleries of presumed insect associates were not observed on the collected samples.An attempt to rear nematodes on Botrytis cinerea plates was unsuccessful.

TYPE MATERIAL
Holotype female, 12 female and one male paratype (slide nos 152A-1 to 152A-6) deposited in the nematode collection of Technical Centre of Ningbo Customs, Ningbo, P.R. China.Three female paratypes (slide no.T708) were deposited in the Canadian National Collection of Nematodes, Ottawa, ON, Canada.

ETYMOLOGY
The species epithet is derived from the Latin word tumidus representing swollen cuticle of the female body.

DIAGNOSIS AND RELATIONSHIPS
Cryptaphelenchus tumidus n. sp. is characterised by the shorter body length of females 236 (217-270) μm and male 180 μm, lateral fields obscure, stylet delicate, medium-sized 6.8 (6.0-7.5)μm with small basal knobs, minute PUS half vulva body width long, slim conical posterior body end with a finely rounded tip, males rare.
In addition, the new species is also different from all the Cryptaphelenchus species by irregular body cuticle swelling.
Our phylogenetic analysis indicated that in all the trees C. tumidus n. sp.grouped with C. recticaudatus; however, both species are different in terms of morphology and place of origin.C. tumidus n. sp. was detected in Pinus wood material of Japan whereas C. recticaudatus was reported from Pinus elliottii located in the USA.The phylogenetic relationships of C. curvatum n. sp. is unresolved; we anticipate that a genome-wide sequence acquisition and detailed phylogeny of Cryptaphelenchus species will aid in finding the closely related species and their association with each other.

Discussion
Predominantly, the aphelenchoidids gained attention due to the outbreaks of pine wilt disease caused by the interaction of long-horn beetles and Bursaphelenchus xylophilus (Steiner & Buhrer, 1934) Nickle, 1970(Futai, 2013;Togashi & Arakawa, 2003).Gradually, the research was expanded to other insect-related aphelenchs such as Cryptaphelenchus, and several species were included in the genus (Hunt, 1993).Studies have shown that the interaction between Cryptaphelenchus and beetles is endophoretic (Kanzaki et al., 2022).Presumably, nematodes curb the microbiota in the beetle's galleries and in return are transported to new favourable localities (Xue et al., 2019).Regarding food preference, two fungal species, Botrytis cinerea and Ophiostoma minus, were tested as pabulum for Cryptaphelenchus species (Kanzaki et al., 2022).Some Cryptaphelenchus species were reported to propagate successfully, indicating a mycophagous habit that enables Cryptaphelenchus to remain in the environment even in the absence of a suitable host (Kiontke et al., 2013;Pedram, 2017Pedram, , 2020;;Kanzaki et al., 2022).In the present study, the local and intercepted species were recovered from deciduous coniferous tree samples but remained unable to propagate on given fungal cultures.
Phylogenetic studies of Kanzaki et al. (2013) placed Cryptaphelenchus in clade 3; this clade comprises nematodes having versatile feeding behaviour, such as fungivory, predation and parasitism.Their studies also indicated that nematodes in this clade have particular importance as they may have originated from fungivorus nematodes of soil or dead wood and evolved to become endophores of insects.Recent studies showed the monophyly of Cryptaphelenchus species (Pedram et al., 2020).With that in mind, our phylogenetic analyses are only based on sequences of Cryptaphelenchus species.We did not observe any outliers; instead, the available species were appropriately arranged in distinct clades.Both new species are morphologically and molecularly different from each other and occupied different positions in the 28S tree, indicating their novel status in the genus Cryptaphelenchus.
Currently, the genus contains 24 nominal species and a recently transferred species, C. minutus.Initially, this species was described under Bursaphelenchus.However, Fanelli et al. (2022) performed a detailed morphological and molecular study and moved this species to Cryptaphelenchus.Unlike other Cryptaphelenchus species, C. minutus was found associated with a dying P. wallichiana Jackson tree in India (Walia et al., 2003) and subsequently reported from coniferous trees of Croatia, Italy, and Portugal (Carletti, 2008;Fonseca et al., 2012;Ðo d et al., 2013).However, none of the studies aimed to measure the potential damage of C. minutus.To this end, we advocate that Cryptaphelenchus species are not known to induce any bio-destruction of host trees and research is needed to unravel the bionomics of C. minutus.
In the Ningbo plant quarantine lab, the occurrence of each recovered nematode population is studied in detail to ascertain the species' status and to assess if the species is a regulated pest (Gu et al., 2008).Fortunately, Cryptaphelenchus species are not known to cause any plant diseases, but their correct identification will aid in future ecological studies.It is important to note that only C. paravaricaudatus was officially reported from China.The occurrence of C. curvatum n. sp. and C. tumidus n. sp.from woodlands of China and Japan indicates that more exploratory surveys are needed to assess the existing Cryptaphelenchus diversity in both countries and to determine if these species play any ecological role in insect bionomics or the residing host tree.

Fig. 5 .
Fig. 5. Phylogenetic relationships among Cryptaphelenchus spp.using the Bayesian 50% majority rule consensus tree as inferred from D2-D3 expansion domains of the 28S rRNA sequence alignment under the general time-reversible model with a gamma-shaped distribution (GTR + G).Posterior probabilities of more than 0.70 are given for appropriate clades.Newly obtained sequences in this study are shown in bold.The scale bar indicates expected changes per site.

Fig. 6 .
Fig. 6.Phylogenetic relationships among Cryptaphelenchus spp.using the Bayesian 50% majority rule consensus tree as inferred from ITS rRNA sequence alignment under the transversion model with invariable sites and a gamma-shaped distribution (TrN + I + G) model.Posterior probabilities of more than 0.70 are given for appropriate clades.Newly obtained sequences in this study are shown in bold.The scale bar indicates expected changes per site.

Fig. 7 .
Fig. 7. Phylogenetic relationships among Cryptaphelenchus spp.using the Bayesian 50% majority rule consensus tree as inferred from 18S rRNA sequence alignment under the transversion model with a gamma-shaped distribution (TrN + G).Posterior probabilities of more than 0.70 are given for appropriate clades.Newly obtained sequences in this study are shown in bold.The scale bar indicates expected changes per site.

Table 1 .
Morphometrics of Cryptaphelenchus curvatum n. sp.All measurements are in μm and in the form: mean ± s.d.(range).

Table 2 .
Morphometrics of Cryptaphelenchus tumidus n. sp.All measurements are in μm and in the form: mean ± s.d.(range).