A new aphelench nematode, Basilaphelenchus brevistylus n. sp. (Aphelenchoididae: Tylaphelenchinae) from Pinus massoniana in China

Abstract Basilaphelenchus brevistylus n. sp. was isolated from masson pine (Pinus massoniana) in Guangdong province, China. The new species is characterized by an offset lip region, short stylet (female stylet 4.5-5.5 μm and male stylet 4-5 μm long) with three elongate posteriorly directed knobs, posteriorly located metacorpal valve and lateral field composed of three lines. The female has an elongate postuterine sac and a short conical tail, uniformly narrowing toward a sharp tip, or tapering to a slightly offset mucronate tip in a few individuals. The male has a conical tail with a sharp terminal mucro, three pairs of caudal papillae, and small arcuate spicules with a bluntly rounded condylus and small pointed rostrum. B. brevistylus n. sp. can be distinguished from all described Basilaphelenchus nematodes by numerous morphological and morphometrical traits, especially the tail morphology of both sexes and stylet length. In addition, molecular phylogenetic trees inferred from rRNA small subunit and D2-D3 expansion domains of large subunit revealed that this nematode belongs to the Basilaphelenchus, and is clearly different from all the other Basilaphelsenchus species.

The family Aphelenchoididae Skarbilovich, 1947, with over 400 species, is a large group of aphelench nematodes (Hunt, 2008). Ecologically, they include phytoparasites, mycetophagous species, and predators. Many species are reported to be asso ciates or parasites of insects (Hunt, 1993). Six subfamilies within the Aphelenchoididae were listed by Hunt (2008), whereas seven subfamilies were proposed on the basis of the classification for the Aphelenchoididae given by Kanzaki (2014). The difference between the two taxonomy systems is that the latter placed Anomyctus (Allen, 1940) in a separate subfamily, the Anomyctinae (Goodey, 1960). By the year of 2014, one new subfamily Tylaphelenchinae  belonging to the Aphelenchoididae was established . Currently four genera Tylaphelenchus (Rühm, 1956), Pseudaphelenchus (Kanzaki et al., 2009), Albiziaphelenchus (Bajaj, 2012), and Basilaphelenchus  comprise the subfamily Tylaphelenchinae. Morphologically, they all have at least one tylenchid-like character, such as small spherical median bulb, tylenchid-type bursa, and elongate posteriorly directed stylet knobs (Mirzaie Fouladvand et al., 2019a). Phylogenetically, although molecular data are unavailable for the two genera Tylaphelenchus and Albiziaphelenchus, recent phylogenetic analysis based on rRNA small subunit (SSU) and D2-D3 expansion domains of large subunit (LSU D2-D3) confirmed that Pseudaphelenchus and Basilaphelenchus form a monophyly of the Tylaphelenchinae (Aliramaji et al., 2020;Kanzaki, 2021;Mirzaie Fouladvand et al., 2019a, b;Pedram et al., 2018).
In a survey of aphelench nematodes from pine wood in China, an unknown species of aphelenchoidid was extracted from a dead Pinus massoniana Lamb. in Xingning city, Guangdong Province, China. Intensive morphological and molecular studies of the nematode revealed that it is a new species of the genus Basilaphelenchus. The new species is described and illustrated herein as Basilaphelenchus brevistylus n. sp. Phylogenetic analysis based on SSU and LSU D2-D3 was performed to investigate the relationships of the new species with other species of Tylaphelenchinae.

Nematode extraction and morphological observations
Decaying wood and its bark samples were collected from a standing dead Pinus massoniana in Xingning city, Guangdong province in the south of China during early June 2020. The nematodes were extracted from samples by the Baermann funnel method (Feng, 2001), killed by gentle heat, fixed in DESS solution (Yoder et al., 2006), and processed by the glycerinethanol method for permanent slides (Seinhorst, 1959). Specimens were measured and photographed with the aid of a Nikon ECLIPSE Ni microscope equipped with a Nikon Digital Sight Camera and exclusive NIS-Elements BR software (Nikon, Tokyo, Japan).

Phylogenetic analysis
The sequences of B. brevistylus n. sp. were compared with aphelench nematode sequences in GenBank using the BLAST homology search program. The close-related and published sequences of aphelench nematodes were chosen for phylogenetic analyses. Outgroup taxa for each dataset were selected according to previous phylogenetic study for aphelench nematodes (Aliramaji et al., 2020). DNA sequences were aligned by ClustalW implemented in the program MEGA6.0 (Tamura et al., 2013) using default parameters. Models of base substitution were evaluated using Modeltest3.7 (Posada and Crandall, 1998) combined with PAUP4.0 (Swofford, 1998). The Akaike-supported model, the base frequencies, the proportion of invariable sites, the gamma distribution shape parameters, and substitution rates were used in our phylogenetic analyses. Bayesian analysis for SSU and LSU D2-D3 under the GTR + I + G model was employed to confirm the tree topology using MrBayes 3.2 (Huelsenbeck and Ronquist, 2001) running four chains for 1 × 10 6 generations and setting the 'burnin' at 2,500. The MCMC (Markov Chain Monte Carlo) method was used within a Bayesian framework to estimate the posterior probabilities of the phylogenetic trees (Larget and Simon, 1999) and generate a 50% majority rule consensus tree. TreeView1.6 was used to display and edit the trees (Page, 1996).

Measurements
Measurements of the new species are given in Table 1.

Description Female
Small size. Body slender and slightly ventrally curved when heat-relaxed; annules fine. Lateral fields with three incisures. Lip region raised, 1.5 to 2.5 times wider than high, offset from body, separated from body by a clear constriction; vestibule well sclerotized, X-shaped in lateral view. Stylet short, 4.5 to 5.5 μ m long, with three elongate and posteriorly directed knobs, stylet cone comprising ca. 30% of total stylet. Procorpus cylindrical, ca. three to four stylet lengths. Metacorpus (median bulb) small, spherical, its width 66.5 ± 3.2 (59.1-78.3)% corresponding body diam., with glandular anterior part and muscular posteior part. Valve of median bulb weak, but discernible, situating posteriorly, at 60.7 to 72.0% of

Etymology
The specific epithet is derived from the shorter stylet of the new species compared with the other Basilaphelenchus species.
In addition, the SSU phylogenetic analysis revealed that B. brevistylus n. sp. has a close relationship to Pseudaphelenchus spp., however, B. brevistylus n. sp. can be easily distinguished from Pseudaphelenchus spp. by the absence or presence of bursa (male tail without bursa vs male tail with long bursa), the different stylet shape (stylet with three elongate posteriorly directed knobs vs stylet bipartite with small  Posterior probability values exceeding 50% are given on appropriate clades. and conspicuous basal knobs) and a shorter body of female (less than 500 μ m vs generally more than 500 μ m).

Molecular profiles and phylogenetic status
The 628-bp LSU D2-D3 and 1597-bp near full-length SSU were sequenced. The molecular phylogenetic status of B. brevistylus n. sp. is presented in Figures 4  and 5, and the two phylogenetic trees reconstructed based on sequences of LSU D2-D3 and SSU both confirm that the new species was within the Basila phelenchus clade. In Figure 4, the phylogenetic tree is based on LSU D2-D3 from a multiple alignment of 1142 total characters, all Basilaphelenchus species reside within a 78% supported monophyletic clade. In the clade, B. brevistylus n. sp. is closely related to B. persicus with a 100% support, and they are clearly distinguished from each other. And the Basilaphelenchus clade is sister to the Pseuda phelenchus clade, forming a monophyletic clade of Tylaphelenchinae with a 66% support. In Figure 5, the phylogenetic tree is based on SSU from a multiple alignment of 2,734 total characters, B. brevistylus n. sp. is also closely related to B. persicus with a 100% support and clearly distinguished from it, but these two species and other Basilaphelenchus species do not form a monophyletic clade. Similar with the tree inferred from LSU D2-D3, all Basilaphelenchus and Pseudaphelenchus species form a monophyly of the subfamily Tylaphelenchinae, with a 100% support.

Discussion
In China, the genus Basilaphelenchus has not been reported to date. The finding of Basilaphelenchus brevistylus n. sp. expands the geographic distribution of this genus. The Basilaphelenchus is a relatively new genus within the family Aphelenchoididae. It was established in 2018 . Since then, six Basilaphelenchus have been reported. Five of the six were found in Iran (Aliramaji et al., 2020;Golhasan et al., 2021;Mirzaie Fouladvand et al., 2019a, b;Pedram et al., 2018), and the remaining one was described in Japan more recently (Kanzaki, 2021). Besides, Tylaphelenchus grosmannae (Rühm, 1965), originating from Chile, was transferred to the genus Basilaphelenchus as B. grosmannae due to typological similarities . Therefore, B. brevistylus n. sp. is the eighth Basilaphelen chus species. So far, all Basilaphelenchus species were found in wood of trees, including Araucaria araucana, Fagus orientalis, and several unidentified trees (Golhasan et al., 2021;Kanzaki, 2021). In this study, B. brevistylus n. sp. was isolated from Pinusm massoniana, which is the first report of the genus from pine tree.
Currently, little is known about the biology of the genus Basilaphelenchus. However, a mycetophagus habit for this genus has been suggested as all Basilaphelenchus species were found in dead wood and rotten material, and multiple species, including B. persicus, B. pedrami, B. hyrcanus, and B. gorganensis, had been successfully multiplied on fungi (Golhasan et al., 2021;Kanzaki, 2021). Although we did not try to culture B. brevistylus n. sp. in fungi, the new species was also extracted from decaying wood. We therefore agree with the mycophagy hypothesis for this genus. In addition, it has also been proposed that this genus may be associated with wood borer and bark beetle insects because all Basilaphelenchus species were from wood and bark samples (Golhasan et al., 2021). However, so far only B. grosmannae was discovered to be carried by a bark beetle Hylurgonotus brunneus (Rühm, 1965). Insect associations of the other seven Basilaphlenchus species including B. brevistylus n. sp. have not been demonstrated. Interestingly, we noted that all Basilaphelenchus species stylets have an unique shape (with three elongate and posteriorly directed knobs) and are very short (no more than 10 μ m). It has been found that stylet shape and length of several aphelenchoidid species are related to their biological characters. For example, Bursaphelenchus sinensis showed morphological differences between a mycophagous and predaceous form (Kanzaki et al., 2019); the parasitic generation of Bursaphelenchus sexdentati has a smaller stylet than the free-living generation (Vosilite, 1990). Therefore, it would be valuable to further investigate potential insect carriers of Basilaphelenchus nematodes and the possible stylet modifications indicative of a specific insectnematode relationship.
Given that the small body sizes and morphological similarity with Aphelenchoides, it is possible that the Basilaphelenchus nematodes were overlooked during nematode surveys, and molecular techniques are of great assistance to confirm the status of Basilaphelenchus spp. (Kanzaki, 2021). In this study, our molecular phylogenetic analyses based on two rDNA markers, LSU D2-D3 and SSU, both place B. brevistylus n. sp. in a highly supported clade with B. persicus, and B. brevistylus n. sp. is clearly distinguished from all the other Basilaphelenchus species, which is in line with the result of morphological identification, confirming this nematode is a new Basilaphelenchus species. Interestingly, the paraphyly of the genus Basilaphelenchus had been indicated according to several studies based on phylogenetic analyses inferred from SSU and LSU D2-D3, and Basilaphelenchus and Pseudaphelenchus always formed a Tylaphelenchinae monophyly (Aliramaji et al., 2020;Kanzaki, 2021;Mirzaie Fouladvand et al., 2019a, b). In our study, Basilaphelenchus spp. are closely related to Pseudaphelenchus spp. but the exact nature of that relationship is not clear. Both LSU D2-D3 and SSU provide weak to moderate support for a sister genus relationship and the monophyly of Basilaphelenchus. To date we have not identified a consistent morphological or host-range character that define the relevant clades. Similar unresolved relationships have been reported in other Tylenchina, e.g. Rotylenchus (Cantalapiedra-Navarretea et al., 2013), Filenchus (Qing et al., 2017), Mesocriconema, and Criconemoides (Powers et al., 2017). We believe additional genetic markers and additional taxa will improve our understanding of the relationships in this often overlooked genus of fungal feeding nematodes.