A new cyst-forming nematode, Cactodera tianzhuensis n. sp. (Nematoda:Heteroderinae) from Polygonum viviparum in China with a key to the Genus Cactodera

Abstract A new cyst-forming nematode, Cactodera tianzhuensis n. sp. was isolated from the rhizosphere soil of Polygonum viviparum L. in Tianzhu county, China. Morphologically, the new species is characterized by lemon-shaped or rounded cysts that have protruding necks and vulval cones. The vulval cone of the new species appeared to be circumfenestrate without bullae and underbridge, vulval denticle present and anus distinct. Second-stage juveniles are vermiform, stylet well-developed with the rounded stylet knobs to slightly concave anteriorly. Lateral field with four incisures. Tail gradually tapering to a finely rounded terminus with a length of ca 54 (47–59) µm, outline of hyaline portion is V-shaped or U-shaped. Egg shells without visible markings or punctations. The phylogenetic analyses based on ITS-rDNA, D2-D3 of 28S-rDNA clearly revealed that the new species formed a separate clade from other Cactodera species, which further support the unique status of C. tianzhuensis n. sp. Therefore, it is described herein as a new species of the genus Cactodera.

Cactodera is a cyst-forming nematode genus of the Heteroderidae erected by Krall and Krall (1978) and the type species is cactus cyst nematode, Cactodera cacti (Filipjev and Schuurmans Stekhoven, 1941) Krall and Krall, 1978, which is distributed worldwide and mainly damaged plants of the family Cactaceae grown in glasshouse as ornamental (Skantar et al., 2019). Cactodera spp. are mainly characterized based on vulval region fenestration, bullae and underbridge absent or present in cyst, the length of stylet, tail and hyaline tail in second-stage juvenile, and the surface differentiation in eggs (Subbotin et al., 2010). However, traditional identification of cyst forming nematode based on morphology is imprecise and time-consuming to separate the related species. During the past 30 years, molecular data, including ITS-rDNA, D2-D3 region of 28S-rDNA, are more accurate tool for identification of cyst-forming nematode species. Sequence analysis of the ITS-rDNA and the D2-D3 region of 28S-rDNA of unknown species is sufficient to study the phylogenetic relationship and identify cyst-forming nematode species (Maafi et al., 2003;Subbotin et al., 2001Subbotin et al., , 2006. Up to now, the genus Cactodera contains 16 valid species and mostly parasites plants of Amaranthaceae, Cactaceae, Chenopodiae, and Polygonaceae in different regions (Escobar-Avila et al., 2020;Feng et al., 2018;Subbotin et al., 2010). To date, very little is known about the occurrence and distribution of Cactodera nematode and only three species of Cactodera have been reported in China: C. cacti (Filipjev and Schuurmans Stekhoven, 1941) Krall and Krall, 1978 parasitizing the roots of Opuntia dillenii (Pan et al., 1997) and Hylocereus undatu (Duan et al., 2012) in Fujian and Liaoning province, respectively; C. thornei (Golden and Raski, 1977) Mulvey and Golden, 1983 was found in cereal fields in Qinghai province (Peng and Vovlas, 1994); C. chenopodiae Feng et al., 2018 was described as a new cyst-forming nematode in the genus Cactodera parasitizing on Chenopodium album in Liaoning province (Feng et al., 2018).
During 2019-2020, a population of cyst-forming nematodes was found from the rhizosphere of Polygo num viviparum L. in Tianzhu county of Gansu Province, China, based on morphological, morphometric and molecular analyses. Its characters were then compared with all the related species of the genus Cactodera. This population is described herein as Cactodera tianzhuensis n. sp. due to its unique characters. To help identify the species in the genus Cactodera, a key to Cactodera species is presented.

Nematode extraction and morphological characterization
Cysts, second-stage juveniles of new species were extracted from roots and soil samples of the host plant, Polygonum viviparum, in Tianzhu county, Gansu Province, China, using standard centrifugal flotation (Jenkins, 1964) and Fenwick method (Fenwick, 1940), respectively. Males were not found. For morphometric studies, second-stage juveniles were killed by gentle heating, fixed in TAF solution (formalin:triethanolamine:water = 7:2:91), and processed to ethanol-glycerin dehydration according to Seinhorst (1959) and mounted on permanent slides. For observation of vulval cones, cysts were soaked in water for several hours and dissected, the vulval cone can be bleached for 5 min in H 2 O 2 and dehydration in different gradient alcohol, the last mounted in glycerin jelly on glass slide (Subbotin et al., 2010). Light micrographs and measurements were conducted on mounted specimens using Zeiss Axio Scope A1 (Zeiss, Jena, Germany) equipped with an AxioCam 105 color camera, drawings were accomplished using a drawing tube attached to Nikon YS 100 (Nikon, Tokyo, Japan) and improved using the software Adobe illustrator CS6 x64 Version 13.0.1.

Sequence alignment and phylogenetic analysis
The newly obtained sequences for ITS-rDNA and D2-D3 of 28S-rDNA region were compared with known sequences of other related species on GenBank using BlastN homology search program. Outgroup taxa for phylogenetic analyses were selected based on the previously published studies (Cid Del Prado and Subbotin, 2014;Escobar-Avila et al., 2020;Feng et al., 2018;Soto et al., 2003;Subbotin et al., 2006Subbotin et al., , 2017. All the selected sequences were aligned by MAFFT (Standley, 2013) with default parameters and edited using Gblock (Castresana, 2000). Phyloge netic analysis of ITS-rDNA and D2-D3 of 28S-rDNA region were based on Bayesian inference (BI) using MrBayes 3.2.6 (Huelsenbeck and Ronquist, 2001). The GTR + I + G model was selected as the best-fit model of DNA evolution using MrModeltest version 2.3 (Nylander,

JOURNAL OF NEMATOLOGY
2004) according to the Akaike Information Criterion (AIC). BI analysis for each gene was initiated with a random starting tree and run with four Markov chains (three heated and one cold) for 1,000,000 generations. The Markov chains were sampled at intervals of 100 generations and the burn-in value was 25%. Two runs were performed for each analysis. After discarding burn-in samples, the remaining samples were used to generate a 50% majority rule consensus tree. Posterior probabilities (PP) were given on appropriate clades. The phylo genetic consensus trees were visualized using the software FigTree v.1.4.3 (http://tree.bio.ed.ac. uk/ software/figtree/) (Rambaut, 2016).

Eggs
Egg shells without visible markings or punctations, body of developed J2 in eggs folded about five times ( Figure 4A-C).

Male
Not found.     The new species belongs to the genus Cactodera, up to now, the genus Cactodera contains seventeen species (including C. tianzhuensis n. sp.). These species are similar in circumfenestrate fenestration, without bullae and underbridge, lateral field of J2 with four incisures. Morphologically, C. tianzhuensis n. sp. is closest to C. thornei with many overlapping morphometrics, such as cyst size, the length of J2 body, DGO, stylet, tail and hyaline tail, but differs from C. thornei in fenestral diam. (20-32 vs 31-36 μ m) for cyst and eggshell surface (smooth vs punctate). C. tianzhuensis n. sp. is similar to C. cacti, but differs from this species in the longer DGO (4.0-6.5 vs 2.9-4.4 μ m), the longer length of hyaline tail (22-29 vs 14-21 μ m) and eggshell surface (smooth vs punctate). In addition, C. tianzhuensis n. sp. can be easily distinguished from several species of Cactodera, namely C. chenopodiae, C. eremica, C. evansi, C. galinsogae, C. milleri, C. rosae and C. solani by eggshell surface smooth vs punctate.  It can be distinguished from C. radicale, C. salina and C. torreyanae by vulval denticles present vs absent.
Any other species than mentioned above of the genus Cactodera, C. tianzhuensis n. sp. can be distinguished from C. acnidae by shorter fenestral In addition, comparative important morphological and morphometric characters of C. tianzhuensis n. sp. with sixteen valid species of the genus Cactodera are listed in Table 2.

Discussion
Initially, Mulvey (1972) divided several cyst-forming nematodes into five groups based on cyst characteristics. Four species (namely, H. betulae Hirschmann and Riggs, 1969;H. cacti Filipjev and Schuurmans Stekhoven, 1941;H. estonica Kirjanova andKrall, 1963 andH. weissi Steiner, 1949) were characterized by cysts with circumfenestrate vulval cone and posterior protuberance in group two. Subsequently, Krall and Krall (1978) proposed the genus Cactodera from the type species name and several Heterodera species added in this genus. The key morphological characters of the genus Cactodera include cysts lemon-shaped to spherical with posterior protuberance, fenestra circumfenestrate, absence of bullae and underbridge, vulval denticles usually present and anus without fenestration, second-stage juveniles have strong stylet, lateral field with four lines and phasmid opening punctiform. Eggshells punctate or smooth (Subbotin et al., 2010). These characteristics clearly indicate that the new species belongs to the genus Cactodera. Morphologically and morphometrically, C. tianzhuensis n. sp. is most similar to C. thornei in having a longer body (average length > 500 μ m) and longer tail (average length > 54 μ m) that can be differentiated from other Cactodera spp. However,  (1977) Note: All measurements are in μ m, and in the form: mean ± standard (Range).
the lower and upper morphometric data of the ranges may overlap with other related species, Cactodera spp. will be identified more accurately based on morphological, morphometric, and molecular data. In our molecular phylogenetic studies, C. tianzhuensis n. sp. formed a single clade with Cactodera species and showed closely related to C. estonica and Cactodera sp. (original identified in GenBank as C. estonica; unpublished). However, sequence divergence (ranged from 6 to 8 bp for 28 S; 30 to 38 bp for ITS) and morphological characteristics can easily distinguish these species. Presently, out of seventeen valid species, six Cactodera species are not represented in GenBank database (i.e., C. acnidae (Schuster and Brezina, 1979) Wouts, 1985, C. amaranthi (Stoyanov, 1979 Krall and Krall, 1978, C. eremica Baldwin and Bell, 1985, C. evansi Cid Del Prado and Rowe, 2000, C. radicale Chizhov, Udalova and Nasonova, 2008, C. thornei (Golden and Raski, 1977 Mulvey and Golden, 1983). Thus, sequences information with the genus Cactodera is still limited in molecular data and need to be completed in more studies.
C. tianzhuensis n. sp. is isolated from Polygonum viviparum L. in Tianzhu county, this habitat located in continental highland with the vegetation type of meadow grassland and the soil is composed of chernozems. The previous studies reported only three species of cyst-forming nematodes (two Heterodera species and one Globodera species) from this habitat. Li et al., 2020 described a new Heterodera species found in the rhizosphere of Microula sikkimensis and named Heterodera microulae  and several scholars reported Heterodera avenae Wollenweber, 1924 associated with meadow grass (Kobresia myoscuroides, Kobresia humilis and Achnatherum inebrains) (Li et al., 2015;Zhang et al., 2019) and Globodera artemisiae parasitizing on Chinese herbal medicine (Artemisia argyi) (Han et al., 2020). To our best knowledge, there is no report of the genus Cactodera damage plants in this habitat and this is first species described of Cactodera species in this habitat, the fourth Cactodera species in China. Though few studies on the host-suitability of several species of Cactodera spp. have been evaluated, barley (Hordeum vulgare L.) is known as being a host for C. galinsogae and C. rosae (Cid Del Prado and Miranda, 2008), and recently described C. solani on tomato (Solanum lycopersicum) was reported (Escobar-Avila et al., 2020). In addition, Graney and Bird (1990) performed a host range test of C. milleri including 34 plant species and indicated this species can reproduce on quinoa (Chenopodium quinoa). Moreover, three species of Cactodera (namely, Figure 6: Molecular phylogenetic tree of C. tianzhuensis n. sp. (highlighted in bold) inferred from 28 S D2/D3 extension region under GTR + I + G model. The posterior probability values exceeding 50% are given on appropriate clades. *Originally identified as C. estonica in the GenBank. **Originally identified as C. rosae in the GenBank. ***Originally identified as C. estonica in the GenBank.
C. chenopodiae, C. torreyanae, C. solani) were shown to be endoparasitic to semi-endoparasitic in sessile habit, a characteristic that the juveniles penetrate with anterior body into the host roots and the posterior body protruding from the surface of the roots (Cid Del Prado and Subbotin, 2014;Escobar-Avila et al., 2020;Feng et al., 2018). Therefore, the biology, hostsuitability, and distribution of Cactodera species (including C. tianzhuensis n. sp.) should further studies to explore.