Biogeography and phylogenetic position of Enchodeloides signyensis (Loof, 1975), gen. n., comb. n. from Maritime Antarctic (Nematoda, Nordiidae)

Abstract The taxonomic position of the endemic Antarctic species Enchodeloides signyensis (Loof, 1975), gen. n., comb. n. (= Enchodelus signyensis Loof, 1975) is discussed on the basis of morphological study, including SEM, morphometric data, postembryonic observations, and sequence data of 18S rDNA and the D2-D3 expansion fragments of the large subunit rDNA. A number of characters such as the cuticle and stoma structures, including the presence of moderately developed cuticularised ring around the oral aperture, peculiarities of pharynx expansion, size and position of the posterior pair of pharyngeal nuclei, a less complex uterus, and the position of a posterior ventromedian supplement show that this species differs substantially from the other members of the genus Enchodelus. Furthermore, both the 18S and 28S rDNA-based phylogenetic trees of the Enchodelus sequences available in the GenBank formed two distinct clusters with E. signyensis being a part of a well-supported group with species of the genus Pungentus; therefore, it is proposed that its taxonomic position should be reconsidered.


Introduction
Antarctic represents unique types of habitats -polar deserts, caused by its geological history, harsh climate conditions, and remoteness. Therefore, terrestrial Antarctic biota, including nematodes, is characterised by a very high degree of endemism and low diversity (Nielsen et al. 2011). Besides, distribution of nematodes exhibits clear biogeographical patterns regarding the two Antarctic ecozones, Continental and Maritime Antarctic (Andrássy 1998a). Order Dorylaimida Pearse, 1942 is represented in Antarctic by nineteen species (12 species described from Maritime Antarctic, 7 species, from the continental part, all but one endemics; of six genera reported from this polar region, two are endemic (Elshishka et al. 2015a). Enchodelus signyensis Loof, 1975 is the only representative of the genus Enchodelus Thorne, 1939 reported from the southern hemisphere and is an endemic for the Maritime Antarctic. This species was recorded from Signy Island (Spaull 1973) as Enchodelus sp. Later Loof (1975) studied Spaull's collections from some of the islands and described this species as E. signyensis, naming it after the type locality. Subsequently, Andrássy (1998a) presented a brief description based on a female paratype specimen. Peneva et al. (2002) provided new morphological data about this species from Livingston Island, and described the males. Here new molecular and additional morphological data is presented of adults and juveniles of this species from Livingston and King George Islands, and its taxonomic position discussed.
Specimens used for SEM observations were rinsed in 0.1 M cacodylate buffer (twice for 10 min), post-fixed in 1% OsO 4 for 2 h, washed twice for 10 min in 0.1 M cacodylate buffer and dehydrated in an ethanol series (Mutafchiev et al. 2013), immersed in hexamethyldisilazane for 30 min and air dried. They were sputter coated with gold in a JEOL JFS 1200 and examined using a JEOL JSM 5510 microscope at 10 kV.
The locations of pharyngeal gland nuclei are given following Loof and Coomans (1970) and Andrássy (1998b).

DNA extraction, amplification, and sequencing
Genomic DNA was extracted from two female specimens per species using a standard nematode digestion protocol (Holterman et al. 2006). The specimens used for DNA extraction, amplification, and sequencing were from King George island (E. signyensis) and from Rila Mountain (Enchodelus sp.). For further details on the procedures used for DNA extraction, amplification, and sequencing, see Nedelchev et al. (2014). Identical sequences were obtained from both individuals of the same species and have been deposited in GenBank with the following accession numbers: for the 18S rDNA KY 881720 (E. signyensis gen. n., comb. n.) and KY766261 (Enchodelus sp.) and for D2-D3 rDNA KY881719 (E. signyensis gen. n., comb. n.) and KY766260 (Enchodelus sp.).

Sequences and phylogenetic analyses
The 18S and D2-D3 28S rDNA sequences were compared with those of other nematode species available at the GenBank sequence database using BLASTN similarity search tool. The sequences revealing the highest similarity were used for sequence and phylogenetic analyses (Meldal et al. 2007;Holterman et al. 2008;Pedram et al. 2009Pedram et al. , 2011aPedram et al. 2011b;Pedram et al. 2015, etc.). Bayesian Inference (BI) algorithm implemented in MrBayes 3.2.5 was used for reconstruction of phylogenetic relationships (Huelsenbeck and Ronquist 2001;Ronquist et al. 2012). For further details on phylogeny analyses and tree visualisation, see Lazarova et al. (2016). Based on previous studies (Holterman et al. 2006;Elshishka et al. 2015a) Aporcelaimellus spp. were selected as an outgroup for both phylogenies. The estimates of evolutionary divergences between sequences/species within and between groups (numbers of base differences and p-distances) were performed with MEGA7 (Kumar et al. 2016). The analyses involved nine nucleotide sequences with 790 and 1666 positions in total for D2-D3 and 18S rDNA, respectively. Loof, 1975 Figs 1-6 Material examined. Twenty-eight females and twenty-one juveniles (J1-J4) from Livingston and King George Islands (Table 1).

Enchodelus signyensis
Description. Measurements. See Table 2-4. Female. Habitus curved ventrally after fixation, adopting a C-shape. Cuticle consisting of four layers with different refraction, the outer two layers thinner, the second outer with stronger refraction, the inner layers thicker, especially at tail region. Cuticle 2-3 µm thick at postlabial region at the level of the guiding ring, 2-4 µm at mid-body and 4-6 µm on tail; outer layer with very fine transverse striations, innermost layer coarsely striated (Figs 1, 2). Lip region 4-5 µm high angular (following terminology adopted by Peña-Santiago (2006)), offset from the adjoining body by a constriction; about 3 times as wide as high. Based on SEM photographs (Fig. 3), perioral area high, disc-like structure with apparently four elevations surrounding oral aperture, oral aperture appearing cross-like in shape in frontal view. Labial and cephalic papillae prominent; labial papillae button-like, each surrounded by a small ring, their openings pore-like. Inner labial papillae located at distinct elevations; separated from each other, and far from oral aperture and outer labial papillae; divided from the outer labial and cephalic papillae by a circular striation (Fig. 3). Cephalic papillae button-like; outer labial and cephalic papillae below the margin of oral field. Six radial striations beginning from the oral field interrupted by inner and ending at outer labial papillae. Amphidial fovea cup-shaped, its aperture approximately half of lip region diameter, its margin curved; under SEM, the amphidial aperture with an operculum, however the presence of this structure should be confirmed with further studies. Cheilostom a truncate cone with weakly developed walls, its anteriormost part representing a moderately cuticularised perioral ring, appearing as small perioral refractive dots. Odontostyle short and slender, straight, 18-20 times as long as wide, 1.2-1.6 times lip region diameter, aperture 14-16% of its length, 1.2-1.7% of body length. Odontophore 1.2-1.6 times as long as odontostyle, with small swellings at its base. Guiding ring double, located at 0.8-1.0 times lip region diameter from anterior end. Anterior region of pharynx enlarging gradually; pharyngeal expansion 112.5-134 µm, occupying 37-45% of total pharynx length. Location of pharyngeal gland nuclei and their orifices is presented in Table 3. Distance DO-DN 14-19 µm, nuclei of dorsal and second ventrosublateral glands clearly visible, nuclei of first ventrosublateral glands in most specimens indistinct, located slightly behind the middle of the distance DN-S 2 N (n = 1). Nuclei of dorsal glands 3.5-5 µm diameter, first and second pair ventrosublateral 1 µm and 2-3 µm, respectively. Excretory pore opposite the nerve ring with slightly cuticularised canal clearly visible at 100-112 µm from the anterior end. Cardia rounded conoid. Prerectum 1.7-4.8, rectum 0.9-1.4 times anal body diameter long. Tail bluntly conoid, 2-3% of body length, with numerous saccate bodies. Hyaline part 4-8 µm wide or 12-25% of tail length. Two pairs of caudal pores present. Both branches of female genital system equally and well-developed (in specimens of Livingston Island shorter: anterior 236.2 ± 23.3 (186-275) µm and posterior 208.2 ± 34.4 (143-259) µm long, in specimens from King George Island anterior 298.3 ± 31.9 (245-330) µm and posterior 323.1 ± 46.4 (243-361) µm long). Ovaries short, rarely reaching sphincter level; oviduct with well-developed pars dilatata. Sphincter well developed. Uteri tubular, thick walled, surrounded by hyaline cells along almost the whole length, anterior uterus 104-152 µm long, posterior 105-156 µm long, 2-3 times correspond-  (Loof, 1975), gen. n., comb. n. (= Enchodelus signyensis Loof, 1975).   (Loof, 1975), gen. n., comb. n. (= Enchodelus signyensis Loof, 1975). Female: A-D Tail ends (A specimen from King George Island; B, C, D specimens from Livingston Island) E-G Tail ends with saccate bodies (E specimen from King George Island F, G specimens from Livingston Island). Scale bar: 10 µm.
Juveniles. Based on morphometrics of juvenile specimens and the relationships between the lengths of their functional and replacement odontostyles and body lengths, four juvenile stages were identified (Figs 4-7). Habitus in first juvenile stage slightly ventrally curved, lip region flat, continuous with the body, genital primordium 11-12 µm long, tail conical elongated with long central peg (Figs 4-6). Tail in J2 and J3 conoid elongated in J4 bluntly conoid as in females with numerous saccate bodies on tail, c' decreasing during the successive stages to J4 and females.
Enchodeloides gen. n. http://zoobank.org/0AFC0BD5-CA16-4A19-9165-16CD7EE71176 Diagnosis. Nordiidae. Nematodes of medium size. Cuticle dorylaimoid, consisting of four layers, outer layer finely, inner layer coarsely transversally striated. Lip region angular; stoma entrance surrounded by a moderately developed cuticularised ring, appearing as small perioral refractive dots. Amphidial fovea cup-shaped, its aperture about half of lip region diameter, curved. Odontostyle short and slender, straight. Odontophore with small swellings. Guiding ring double. Anterior region of pharynx enlarging gradually into pharyngeal expansion. Posterior pair of pharyngeal nuclei smaller than dorsal nucleus, located posteriorly in pharyngeal expansion. Cardia rounded conoid. Female genital system amphidelphic. Uterus not differentiated. Vagina moderately sclerotised. Vulva a transverse slit. Males rare. Spicula stout ventrally curved. Lateral guiding pieces present. Sperm cells spindle-shaped. Supplements 2 to 4 in number preceded by an ad-cloacal pair of papillae, starting far behind the level of the spicules. Tail bluntly conoid, with numerous saccate bodies on tail. First juvenile stage with elongate conical tail with long central peg.
Relationships. The new genus resembles members of the subfamily Pungentinae Siddiqi, 1969, especially the genera Enchodelus, Pungentella Andrássy, 2009, Pungentus and Stenodorylaimus Álvarez-Ortega & Peña-Santiago, 2011. It differs from Enchodelus by having lip region with six radial striae starting from inner and ending at outer labial papillae vs absent (seen under SEM), four vs three layered cuticle, two vs one thicker inner layer at tail region (under light microscopy), cheilostom thin walled vs thick walled, a moderately developed cuticularised ring around the oral ap-  (Loof, 1975), gen. n., comb. n. (= Enchodelus signyensis Loof, 1975) based on 18S rDNA inferred from a Bayesian analysis (GTR+G model) and two Aporcelaimellus species used as an outgroup. * Thonus is currently considered a synonym of Crassolabium (Peña-Santiago and Ciobanu, 2008). erture vs absent; less developed vs well developed basal swellings; a pharynx enlargement gradually expanding vs abruptly expanding into basal expansion (Fig. 4G, H), the posterior pair of pharyngeal nuclei generally smaller than dorsal nucleus vs as Figure 9. Phylogenetic relationships of Enchodeloides signyensis (Loof, 1975), gen. n., comb. n. (= Enchodelus signyensis Loof, 1975) based on 28S rDNA D2-D3 inferred from a Bayesian analysis (GTR+G model) and two Aporcelaimellus species used as an outgroup. * Thonus is currently considered a synonym of Crassolabium (Peña-Santiago & Ciobanu, 2008). large as dorsal nucleus (Andrássy 2009), except for E. macrodorus Thorne, 1939 (Guerrero andPeña-Santiago 2007) and located more posteriorly, more than 89% vs 83-88% of the pharyngeal expansion (Loof and Coomans 1970); less complex uterus vs tripartite (bipartite in E. distinctus Ahmad &Jairajpuri, 1980 andE. ponorensis Popovici, 1995); posteriormost ventromedian supplement located at a considerable distance from the adcloacal pair and outside of the spicule range vs posteriormost one or two ventromedian supplements rather close to the adcloacal pair and inside the spicule range, 2-4 vs 7-16 in number, and finally, all representatives of the genus Enchodelus have been reported only from the northern hemisphere. Enchodeloides gen. n. differs from Pungentella by having transversally striated cuticle vs smooth; a longer odontostyle (much longer vs equal to or slightly longer than lip region diam.) with a smaller aperture (up to one-sixth vs one-fourth to one-third its length); a moderately developed cuticularised ring vs four small platelets around the oral aperture and the guiding ring double vs simple. From Pungentus it differs in having a moderately developed cuticularised ring vs four distinct circumoral platelets around the oral aperture; a straight vs arcuate odontostyle; shorter odontostyle (1.2-1.6 times vs 2-3 times lip region diameter (Andrássy 2009a); the first pair of ventrosublateral pharyngeal gland nuclei indistinct, difficult to observe vs well developed; a long distance DO-DN (5-6% vs 2-4% (Loof and Coomans 1970)); ventromedian supplements located at a considerable distance from the adcloacal pair and outside of the spicule range vs posteriormost 1-4 supplements lying within the spicule range, and with vs without hiatus. From the genus Stenodorylaimus it differs by having a shorter body (L=1.2-1.9 vs 3.7-5.1 mm), and a slender vs more robust odontostyle (1.2-1.7 vs 0.51-0.87% of body length); a longer pharynx (b-ratio up to 6 vs more than 7); saccate bodies present vs absent; the first pair of ventrosublateral pharyngeal gland nuclei indistinct, difficult to observe vs well developed; ventromedian supplements spaced vs irregularly spaced, 2-4 vs 14-19 in number, and with vs without hiatus.
Consequently, the new combination Enchodeloides signyensis (Loof, 1975) is proposed to accommodate the only nordiid species occurring in Maritime Antarctic.

Distribution
Enchodeloides signyensis is a widespread endemic for the Maritime Antarctic, occurring in several islands: Signy (Loof 1975;Maslen 1981;Caldwell 1981), Coronation, Elephant, Galindez, Blaiklock (Loof 1975), Alamode (Loof 1975;Maslen and Convey 2006), Dream (Shishida and Ohyama 1989), Charcot (Convey et al. 2000;Maslen and Convey 2006), Livingston (Peneva et al. 2002(Peneva et al. , 2004Elshishka et al. 2015b), Alexander (Maslen and Convey 2006), and King George Islands (Russell et al. 2014). It has been recorded from various microhabitats, different moss and algae communities, and in association with species of higher plants, reported from Maritime Antarctic (D. antarctica and C. quitensis) ( Table 6). Data from previous records and the present study show that E. signyensis is associated with different type of microhabitats. Like other terrestrial nematodes in extreme polar conditions, a majority of which colonise all microhabitats, this species does not show specific biotope preferences. According to Chernov et al. (2011) the major life strategy of organisms inhabiting extreme environments is the development of tolerance and plasticity, not specialisation and competitiveness, which is typical of other biomes.