Description of Heterodera microulae sp. n. (Nematoda: Heteroderinae) from China – a new cyst nematode in the Goettingiana group

Abstract A new cyst-forming nematode, Heterodera microulae sp. n., was isolated from the roots and rhizosphere soil of Microula sikkimensis in China. Morphologically, the new species is characterized by lemon-shaped body with an extruded neck and obtuse vulval cone. The vulval cone of the new species appeared to be ambifenestrate without bullae and a weak underbridge. The second-stage juveniles have a longer body length with four lateral lines, strong stylets with rounded and flat stylet knobs, tail with a comparatively longer hyaline area, and a sharp terminus. The phylogenetic analyses based on ITS-rDNA, D2-D3 of 28S rDNA, and COI sequences revealed that the new species formed a separate clade from other Heterodera species in Goettingiana group, which further support the unique status of H. microulae sp. n. Therefore, it is described herein as a new species of genus Heterodera; additionally, the present study provided the first record of Goettingiana group in Gansu Province, China.

Due to overlapping morphological characters and phenotypic plasticity, it is difficult to distinguish closely related Heterodera species; therefore, sequence-based diagnosis is gaining more reliability for precise and accurate identification of cystforming nematodes (Peng et al., 2003). The internal transcribed spacer region of the ribosomal DNA (ITS-rDNA), the D2 and D3 expansion fragments of the 28S ribosomal DNA genes (D2-D3 of 28S-rDNA), and mitochondrial DNA (COI gene) units are good candidate genes for molecular taxonomic and phylogenetic studies (Subbotin et al., 2001;Subbotin et al., 2006;Madani et al., 2004;Vovlas et al., 2017). Based on morphomolecular characterizations, Handoo and Subbotin (2018) divided Heterodera into nine distinct groups such as Afenestrata, Avenae, Bifenestra, Cardiolata, Cyperi, Goettingiana, Humuli, Sacchari, and Schachtii group. Sequence analysis of these groups is significant to study the phylogenetic relationship and identifying the Heterodera species.
During 2018 and 2019, a population of cyst nematode was collected from the rhizosphere of Microula sikkimensis in Tianzhu county of Gansu Province, China. Considering the economic value of the cyst nematode, morphomolecular studies were performed; the preliminary studies indicated that the population belongs to Goettingiana group of Heterodera. The species characters were then compared with all the related species and concluded that this population possess unique characters and it is described herein as Heterodera microulae sp. n.

Isolation and morphological observation of nematodes
The nematodes were extracted from root and soil samples of Microula sikkimensis in Tianzhu county, Gansu Province, China. Cysts and white females were collected using sieving-decanting method, while second-stage juveniles (J2s) were recovered from hatched eggs and kept in water suspension until further use (Hooper, 1970;Golden, 1990). 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) as modified by De Grisse (1969) and mounted on permanent slides. Vulval cones were mostly mounted in glycerin jelly. Measurements were made on mounted specimens using a Nikon Eclipse E100 Microscope (Nikon, Tokyo, Japan). Light micrographs and illustrations were produced using a Zeiss Axio Scope A1 microscope (Zeiss, Jena, Germany) equipped with an AxioCam 105 color camera and Nikon YS 100 with a drawing tube (Nikon, Tokyo, Japan), respectively.

Sequence alignment and phylogenetic analysis
The newly obtained sequences for each gene (ITS-rDNA, D2-D3 region of 28S-rDNA, and COI gene) were compared with known sequences of Heterodera using BLASTn homology search program. Outgroup taxa for phylogenetic analyses were selected based on the previously published studies (Subbotin et al., 2001;Maafi et al., 2003;Mundo-Ocampo et al., 2008;Kang et al., 2016;Madani et al., 2018;Vovlas et al., 2017). The selected sequences were aligned by MAFFT (Kazutaka and Standley, 2013) with default parameters and edited using Gblock (Castresana, 2000). Phylogenetic analyses were based on Bayesian inference (BI) using MrBayes 3.1.2 (Huelsenbeck and Ronquist, 2001). The GTR + I + G model was selected as the best-fit model of DNA evolution for both 28S D2-D3, ITS, and COI regions using MrModeltest version 2.3 (Nylander, 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 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 phylogenetic consensus trees were visualized using FigTree v.1.4.3 software (http://tree. bio.ed.ac.uk/software/figtree/) (Rambaut, 2016). The species in Goettingiana group and their localities, hosts, and GenBank accession numbers used in this study were presented in Table S1.

Description Cyst
It is lemon-shaped with an obtuse vulval cone, neck extruding, and cuticle thick with an irregular zig-zag pattern. The color was white to pale to medium brown; remnants of the subcrystalline layer were rarely present. The egg sac was usually absent (Figs. 1G, 3B, C). The vulval cone was ambifenestrate-like waning crescent moon and separated by a well-developed vulval bridge. The anus area was distinct, bullae were absent (Figs. 1F, 3D, E). The vulval slit was longer than fenestral width (39.00 vs 37.75 µm); the underbridge was weak and often lost during cone preparation.

Female
The female was lemon-shaped, pearl white, or pale yellow in color. It was rarely rounded with a protruding   neck and vulva, the subcrystalline layer was present, and the egg sac absent (Figs. 2A, B, 3A). There was a labial region with two annuli. Labial sclerotization was weak, the stylet was strong, and basal knobs were rounded and anteriorly flattened. The excretory pore was indistinct, median bulb was rounded and massive, and other parts of the pharynx were not clearly discernable. There was vulval slit in a cleft on the cone terminus ( Fig. 2C, D).

Second-stage juvenile
The body was straight or slightly curved ventrally after heat treatment (Fig. 4A). The lip region was offset and rounded, measuring 3.90 to 5.50 (4.63) µm in height and 9.65 to 12.75 (11.01)-µm wide. The cephalic framework was strongly sclerotized (Figs. 1B, 4C). The stylet was strong; knobs were well developed, rounded and flat, or slightly concave anteriorly (Figs. 1C, 4C). The dorsal esophageal gland orifice measured from 5.32 to 6.32 (5.61) µm posterior to the stylet knob. Median bulb was rounded with a strong valvular apparatus. The pharyngeal glands were well developed, overlapping the intestine dorsoventrally (Figs. 1A, 4B). The hemizoind was distinct from one to three annuli long (Fig. 4H), the excretory pore was situated 102.46 to 130.79 (114.40) µm from the anterior end, and one to two annules were posterior to the hemizonid (Fig. 4G). There was a lateral field with four incisures (Figs. 1D, 4G). The dorsal gland nucleus and subventral gland nuclei were distinct (Fig. 4E, F). Genital primordium situated at 59 to 62% of body length behind the anterior end, with two distinct nucleate cells (Fig. 4I). The tail was conoid, gradually tapering to a finely rounded terminus. The hyaline portion was irregularly annulated occupying 50% of tail length. Phasmid was absent (Figs. 1E, 4D).

Eggs
Body hyaline without any markings was presented; juveniles folded six times (Fig. 3F).

Male
The male was not found.   Note: All measurements are in μ m, and in the form: mean ± standard (range).

Type host and locality
Heterodera microulae sp. n. was collected from the roots and rhizosphere soil of Microula sikkimensis Hemsl. (Boraginaceae, Tubiflorae, Metachlamydeae) in Tianzhu county of Gansu Province, China. The geographical position is N 37°11′46″; E 102°47′6″. This site was located in continental highland with the vegetation type of meadow grassland and the soil is composed of chernozems. The climatic parameters of the site include 450 mm of average rainfall and an approximate −2 air temperature.

Etymology
The species is named after the host of its isolation.

Diagnosis and relationships
Heterodera microulae sp. n. is characterized by having lemon-shaped cysts that have protruding necks and obtuse vulval cones. The cysts are 414 to 543-µm long and 305 to 456-µm wide having ambifenestrate vulval cone and bullae are absent. Females are white in color with a subcrystalline layer. Second-stage juveniles are straight or slightly curved ventrally with four incisures in the lateral field. The juveniles are 506 to 628-µm long having strong stylets with welldeveloped rounded stylet knobs, genital primordium situated at 59 to 62% of body length, and tail 49 to 61-µm long with a hyaline portion of 24 to 33 µm. Eggs are hyaline without any markings; juveniles inside the eggs form sixfold.

JOURNAL OF NEMATOLOGY
Additionally, comparative morphological and morphometric characters of H. microulae sp. n. with other valid species of Goettingiana group are listed in Table 2.

Molecular characterization and phylogenetic relationships
The H. microulae sp. n. sequences of D2-D3 region of 28 S (734 bp), ITS (993 bp), and COI (415 bp) gene were obtained and submitted to the GenBank.

Discussion
Taxonomy of Heterodera species has been revised extensively in the past; Baldwin and Mundo-Ocampo (1991) placed 23 Heterodera species into Goettingiana group. However, Sturhan (1998) and Subbotin et al. (2001) used J2's lateral field characters and host preferences to separate Heterodera species into different groups (such as Bifenestra, Cyperi, and Humuli groups). The key morphological characters of the Goettingiana group include lemon-shaped cysts having a protruding neck, ambifenestration, and absence of bullae (small bullae occasionally present); some species may have an egg sac, vulval slit length > 35 µm, a thin vulval bridge, fenestral length (30-45 µm), and a weak underbridge. There were second-stage juveniles with body length > 400 µm, stylet length > 20 µm, tail length > 45 µm, hyaline tail portion > 20 µm, and lateral field with four lines (Subbotin and Turhan, 2004). The new species also belong to the Goettingiana group and morphologically very close to H. urticae; however, morphometrics of J2s body lengths, DGO and excretory pore position, fenestral length, vulval slit length, and cyst width can be used to differentiate both species.
Phylogenetically, it is evident that H. microulae sp. n. is a member of Goettingiana group. In our analyses, it is also noted that Heterodera sp. DP-2010 (HM560791, HM560855, and HM560856) and H. goettingiana (HM370423 and HM370425) from Qinghai, China, formed a well-supported molecular clade with the H. microulae sp. n. Moreover, the nucleotide differences of these sequences with our new species sequences are also very low (2-4-bp difference for ITS and 0-1 bp for 28S). Previously, Escobar-Avila et al. (2018) indicated that the sequences of H. goettingiana (HM370423 and HM370425) from Qinghai, China, might be a case of misidentification. Based on our phylogenetic and  Toumi et al. (2013) and later corrected to Heterodera carotae by Madani et al. (2018).
Heterodera microulae sp. n. is isolated from Microula sikkimensis, it is a biennial herbaceous plant that grows in forests, meadows, and forest edges at altitudes of 2,200 to 4,700 m, and it is widely distributed in South and East Asian countries (Pi et al., 2014). H. microulae sp. n. was found in Gansu and Qinghai Provinces, but we speculate that it is likely to be found in some localities that are characterized by low temperature, high rainfall, and high altitude.
The present study described a new species found in the rhizosphere of M. sikkimensis; further research is needed to understand the distribution and biology of the new species. In addition, plenty of leguminous crops (pea, kidney bean, pole bean, etc.) are growing in the same locality. Therefore, host-suitability tests of H. microulae sp. n. are an open research field to investigate the damage potential of this species.