Morphological and molecular characterization of Heterodera dunensis n. sp. (Nematoda: Heteroderidae) from Gran Canaria, Canary Islands

Abstract Heterodera dunensis n. sp. from the coastal dunes of Gran Canaria, Canary Islands, is described. This new species belongs to the Schachtii group of Heterodera with ambifenestrate fenestration, presence of prominent bullae, and a strong underbridge of cysts. It is characterized by vermiform second-stage juveniles having a slightly offset, dome-shaped labial region with three annuli, four lateral lines, a relatively long stylet (27-31 µm), short tail (35-45 µm), and 46 to 51% of tail as hyaline portion. Males were not found in the type population. Phylogenetic trees inferred from D2-D3 of 28S, partial ITS, and 18S of ribosomal DNA and COI of mitochondrial DNA sequences indicate a position in the ‘Schachtii clade’.

The cysts forming nematodes of the genus Heterodera Schmidt, 1871 (Nematoda: Heteroderidae) are an economically important plant-parasitic nematode (PPN) group with a worldwide distribution and a broad host range causing prominent damages to the host plants ranging from stunted and reduced growth to wilting, chlorosis, and reduced root system (Perry et al., 2018;Sikora et al., 2018). The vermiform second-stage juveniles (J2) of this PPN migrate in the root system of a host plant to feed on the vascular cylinder where they become obese sedentary females; subsequently, following fertilization and egg production, these females turn into protective cysts of more or less lemon shape, housing numerous embryonated eggs. These eggs can remain viable for years inside the cysts, until favorable environmental conditions initiate hatching of the cysts to continue further life cycle (Subbotin et al., 2010;Perry et al., 2018).
Within this genus, 85 nominal species, eight species inquirendae, and a nomen nudum have been listed in a recent update by Handoo and Subbotin (2018). Using morphological and molecular characteristics, the species of this genus have been divided into nine groups, i.e., Afenestrata, Avenae, Bifenestra, Cardiolata, Cyperi, Goettingiana, Humuli, Sacchari, and Schachtii. Morphological characterization of Heterodera species is mainly done based on vulva-slit length, vulval cone fenestration, presence or absence of bullae and underbridge in female cysts, and stylet length, lateral field differentiation, tail length, and hyaline tail length in J2 (Subbotin et al., 2010). Since the last two decades, employing molecular data such as ITS and 28S of ribosomal DNA and COI gene of mitochondrial DNA to characterize Heterodera species has been a common practice, including DNA barcoding, phylogeny, and even phylogeography (Ferris et al., 1999;Toumi et al., 2013a;Subbotin et al., 2017Subbotin et al., , 2018. Herein, we characterize Heterodera dunensis n. sp. discovered in a recent exploratory survey of PPN from Canarian coastal dunes of Gran Canaria in May 2019. The species characterization is done based on light microscopy (LM), scanning electron microscopy (SEM), and molecular information of ITS, 18S, and 28S of ribosomal DNA and COI of mitochondrial DNA.

Materials and methods
Nematode extraction and morphological analysis A sandy soil sample was collected from around the root system of Tetraena fontanesii (Webb & Berthel.) Beier & Thulin, commonly known as Sea Grape or Canarian Bean-Caper. This halophilic succulent plant was growing on a dune (GPS coordinates: 27°44′19.11″ N; 15°35′0.3″ W), about 200 m away from the Maspalomas beach of Gran Canaria. Vermiform J2 was extracted from the sand using the modified Bearmann method (Whitehead and Hemming, 1965) and stored at 4°C during the course of analysis.
For collecting female cysts, sand dried at room temperature was mixed thoroughly in water using a spoon, and after letting the sand settle, floating female cysts were picked out using a fine brush.
Morphological study of J2 was done using both heat-relaxed and fixed specimens. Individual live nematodes were heat-relaxed in a drop of water on a glass slide and examined, photographed, and measured using an Olympus BX51 DIC Microscope (Olympus Optical, Tokyo, Japan), equipped with an Olympus C5060Wz camera and a drawing tube as described in the study of Singh et al. (2018). After recording morphological information, the specimens were recovered from the slide and their genomic DNA was extracted as described in the next section. The remaining J2 juveniles were concentrated in a drop of water in a glass embryo dish, followed by adding a few drops of freshly prepared Trump's fixative [2% paraformaldehyde, 2.5% glutaraldehyde in 0.1 M Sorenson buffer (sodium phosphate buffer at pH = 7.3)]. The nematodes were then immediately heated in a microwave (700 watts) for about 4 sec, left to rest for 1 hr at room temperature and at 4°C for 24 hr, followed by gradually transferring to anhydrous glycerin to be mounted on glass slides as described in the study of Singh et al. (2018). Vulva cones of female cysts were cut in a drop of water under a stereomicroscope using a blade, and the cones were mounted in glycerolgelatin (1:1) mix on glass slides. The mounted J2 juveniles and the female vulva cones of the cysts were studied and drawn using the above-mentioned camera-and drawing tube-equipped microscope. Illustrations were improved using Adobe Photoshop CS6 Version 13.0 x 64. For SEM, specimens fixed in Trump's fixative were washed in 0.1 M phosphate buffer (pH = 7.3), and cysts were additionally sonicated for 8 min to remove any attached dirt, and dehydrated in a graded series of ethanol solutions, critical-pointdried with liquid CO 2 , mounted on stubs with carbon tabs (double conductive tapes), coated with gold of 25 nm, and photographed with a JSM-840 EM (JEOL) at 12 kV (Singh et al., 2018).

Phylogenetic analysis
The phylogenetic relationships of the new species with other related species were analyzed based on the D2-D3, ITS, 18S, and COI sequences. Phylogenetic programs implemented in Geneious Prime 2020.0.5 were used. The obtained consensus contigs were subjected to BLAST search to check for closely related species on GenBank, and all the collected sequences for each gene fragment were aligned using MUSCLE alignment of Geneious Prime 2020.0.5 using default parameters, followed by manually trimming off of the poorly aligned ends. The best nucleotide substitution model of each gene alignment (see Figures) was determined by jModelTest 2.1.10. Bayesian phylogenetic analysis (MrBayes 3.2.6) was carried out using the selected models, analyses were run under 1 × 10 6 generations (4 runs), and Markov chains were sampled every 100 generations, and 20% of the converged runs were regarded as burn-in (Huelsenbeck and Ronquist, 2001).

Description
Cyst: Body typical lemon shape to sometimes ovoid shape with protruding prominent neck and vulva. Neck regularly bent. Cysts wall light to medium brown in color with irregular zig-zag pattern on surface. Fenestration ambifenestrate. Vulva cone dome-shaped with sub terminal anus. No egg-sac observed. Vulva slit longer than fenestral length. Bullae prominent, medium brown in color, variable shape, in some cysts commonly finger-like or elongated, irregularly distributed at the periphery of vulva cone slightly above underbridge level. Underbridge furcated with central thickening, prominent in young cysts, breaks down in older cysts. Cysts containing 100-200 eggs.
J2: Body slender, tapering posteriorly. Labial region slightly offset, dome-shaped with two clear incisure under LM appearing as three lip annuli, second annule wider than the other two. En face showing oral disc fused with submedial sectors, well-separated lateral lip sectors and rectangular to square-shaped stoma opening. Lateral field with four longitudinal incisures forming three bands, outer two bands slightly wider than inner. All bands irregularly areolated, sometimes with incomplete areolation. Stylet robust, 27-31 µm long, with large rounded strongly anteriorly projecting knobs. Pharynx well-developed, ca one-third of body length with well-developed median bulb, valves and glands overlapping intestine ventrally. Nerve ring encircling isthmus. Hemizonid distinct, about two cuticular annuli long, just above secretory-excretory (SE) pore opening. SE pore at ca one-fourth of body length from anterior end. Tail 35-45 µm long, tapers gradually to a rounded terminus, hyaline region ca 50% of tail length. Phasmid opening small, roughly halfway between anus and start of hyaline tail part.
Male: Not found.

Diagnosis and relationships
Heterodera dunensis n. sp. is characterized by moderate-sized J2 of 0.43 to 0.52 mm long, lateral field with four lines, the inner band slightly smaller than the outer two bands, and all bands with irregular areolation throughout the length; a relatively long J2 stylet of 27 to 31 µm with anteriorly projected knobs, a relatively short tail of 35 to 45 µm in length, small rounded phasmids, and tail hyaline part usually ca 50% of the tail; cyst ovoid to regularly lemon-shaped, ambifenestrate, the presence of prominent finger-like bullae, and a strong underbridge.
Heterodera dunensis n. sp. is also based on D2-D3, ITS, 18S, and COI sequences clearly different from all known species, see below.

D2-D3 of 28S rDNA
Three D2-D3 sequences (MT508987-MT508989) of 987-1039 bp were pro duced without intraspecific sequence variation. The closest available sequence on GenBank was MK292129 of H. glycines with 95.9% similarity (43 out of 1039 bp differences). The D2-D3 alignment of 750 bp long consisted of 75 Heterodera sequences of 33 species and a Cryphodera sinensis sequence (JX566455) as the outgroup. The resulting D2-D3 tree revealed an      Hyaline tail length 20.6 ± 1.9 (16-23) Note: All measurements are except percentage and ratio in μ m and in the form: mean ± sd (range).

18S of rDNA
Three partial 18S sequences (MT509422-MT509424) of 806-845 bp were genera ted without intraspecific sequence variation. The sequences were closest to EU306357 of H. koreana with 98.9% similarity (9 out of 845 bp differences). The resulting phylogenetic tree revealed an unresolved position of several species, including H. dunensis n. sp., and is, therefore, not provided.

Type material
Holotype J2 and seven J2 paratypes in two slides, two cyst vulval cones, and two whole cysts in separate slides were deposited at the National Plant Protection Organization, Wageningen Nematode Collection, Wageningen, The Netherlands (WaNeCo). Six paratype J2 and three cyst vulval cones in two slides were submitted to the Ghent University Museum, Zoology Collections, Belgium. Additionally, five J2 paratypes and two cyst vulval cones were also deposited at the UGent Nematode Collection (slide UGnem-189-190) of the Nematology Research Unit, Department of Biology, Ghent University, Belgium.

Discussion
Heterodera dunensis n. sp. is easily distinguishable from other Heterodera species and from all other members of Schachtii group by both morphology and molecular data (D2-D3, ITS, 18S, and COI sequences). The obtained phylogenetic trees revealed a consistent phylogenetic position of the new species always forming a clade together with other members of the Schachtii group. The ITS tree provided slightly better-resolved phylogenetic relationships among different Heterodera species compared to the D2-D3 and the COI trees, while the 18S tree had an inferior resolution.
Heterodera dunensis n. sp. was present in a Canarian dune sample with a moderately large population of only J2 and cysts together with very few saprophytic nematodes and no other PPN species. A resampling in search of males from the type location and an attempt to culture the species at NPPO, Wageningen green house did not succeed. Coastal regions are a relatively common habitat for Heterodera spp. To our knowledge, nine Heterodera spp. have been reported from similar habitats around the world, namely Heterodera arenaria Cooper, 1955, parasitizing on marram grass (Ammophila arenaria) on mobile sand dunes from several coastal sites in the United Kingdom and the Netherlands (Robinson et al., 1996;Brzeski, 1998); Heterodera aucklandica Wouts and Sturhan, 1995 associated with meadow rice grass (Microlaena stipoides) in Auckland, New Zealand (Wouts and Sturhan, 1995); Heterodera hordecalis Andersson, 1975 on marram grass in the Netherlands (Van der Putten and Van der Stoel, 2006); Heterodera leuceilyma Di Edwardo and Perry, 1964 from the coastal regions of Florida parasitizing on St Augustine grass (Stenotaphrum secundatum) (Di Edwardo and Perry, 1964); Heterodera litoralis Wouts and Sturhan, 1996 associated with a succulent plant, beaded glasswort (Sarcocornia quingueflora) in South Island, New Zealand (Wouts and Sturhan, 1996); H. mediterranea associated with a woody plant, lentisc (Pistacia lentiscus) on The Adriatic coast of Southern Italy (Vovlas et al., 1981); Heterodera pratensis Gäbler, Sturhan, Subbotin and Rumpenhorst, 2000 in a pasture near the coast of the Baltic Sea at Lindhöft, Germany (Gäbler et al., 2000); Heterodera riparia (Kazachenko, 1993) Subbotin, Sturhan, Rumpenhorst and Moens, 2003 associated with couch grass, Elymus repens (L.) from along the coast of Olga Bay, the coast of Okhot Sea, Kamchatka region of Russia (Subbotin et al., 2003), and Heterodera salixophila Kirjanova, 1969 parasitizing roots of the willow tree, Salix purpurae from the shores of Kurdish Bay, Baltic Sea, Kaliningrad region of Russia (Kirjanova, 1969).
This new species was found associated with Tetraena fontanesii (Sea Grape or Canarian Bean-Caper), a succulent plant with a limited distribution, in Canary Islands and some parts of West Africa. To the best of our knowledge, no report of an association of PPN with this host has been made before. Sampling in similar habitats should reveal if H. dunensis n. sp. is endemic for the Canarian islands and to what extent it is host-specific.