New data on two remarkable Antarctic species Amblydorylaimusisokaryon (Loof, 1975) Andrassy, 1998 and Pararhyssocolpusparadoxus (Loof, 1975), gen. n., comb. n. (Nematoda, Dorylaimida) Author Elshishka, Milka Author Lazarova, Stela Author Radoslavov, Georgi Author Hristov, Petar Author Peneva, Vlada K. text ZooKeys 2015 511 25 68 http://dx.doi.org/10.3897/zookeys.511.9793 journal article http://dx.doi.org/10.3897/zookeys.511.9793 1313-2970-511-25 89224AEDC82A4BE79C464C242CDF1B39 Taxon classification Animalia Dorylaimida Pararhyssocolpidae Pararhyssocolpus paradoxus (Loof, 1975) gen. n., comb. n. Figures 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 Eudorylaimus paradoxus Loof, 1975 Rhyssocolpus paradoxus (Loof, 1975) Andrassy , 1986 Material examined. Eighteen females, seven males and ten juveniles (J1, J3, J4) collected from three islands in Maritime Antarctic (Table 1). Measurements. See Table 5. Table 5. Morphometrics of Pararhyssocolpus paradoxus n.gen., n.comb. (adults, juveniles). All measurements, unless indicated otherwise, are in µm (and in the form: mean +/- SD (range).

Locality	King George Island		Livingston Island						Nelson Island	King George Island
Characters	KGI1		CDM		S	SDC	DM		M	KGI1

Description . Female. Habitus curved ventrad after fixation, more so in posterior body end. Cuticle smooth, when viewed under light microscope, 3-4 µm thick in postlabial region, 5-7 µm at mid-body and 4-7 µm on tail; consisting of three layers the inner one much ticker and refractive, not reaching the end of tail. Under SEM it is finely transversally striated (annules ca 0.6 µm wide). Lip region appears rounded, slightly offset by a depression, 2.3-3.4 times as broad as high, lips amalgamated, outer labial and cephalic papillae protruding above lip region contour. Under SEM inner labial papillae not elevated, close to each other and to oral aperture, outer labial and cephalic papillae below the margin of oral field. Oral aperture seems round hexagonal. Lateral pores well visible (13-14 in the pharyngeal region), the first four as two pairs at the anterior end, next more or less equally spaced. Cheilostom a truncate cone. Amphidial fovea funnel-shaped, opening at level of labial depression, its aperture about half of lip region diam. Odontostyle slender, with clear lumen, aperture subterminal, narrow (Figure 22 B) and indistinct as observed by LM in adults (Figures 18 A-C , 19B, 21F); 8-12 times longer than wide, 0.9-1.0 lip region diam. long. Odontophore simple, 1.9-2.3 times odontostyle length long. Guiding ring double, situated at 0.7-0.8 times lip region diam. from anterior end. Nerve ring located at 151-178 µm from anterior end or 32-38% of total neck length. Pharynx consisting of slender but muscular anterior section enlarging gradually and "bibulbar" ( Andrassy , 1986), basal expansion with somewhat narrower middle part, 206-231 µm long or 44-52% of total neck length (Figs 14A, 18D). Dorsal nucleus (DN) lying very close to anterior edge of pharyngeal expansion. One nucleus of anterior ventrosublateral pair of pharyngeal glands well visible, large, posterior pair of ventrosublateral nuclei slightly larger, nuclei located almost at one and the same level (pharyngeal characters presented in Table 6). Cardia conoid, measuring 28-39 x 14-19 µm , cell mass near cardia present in some specimens. The posterior end of the intestine with tongue-like projection. Prerectum short, 2-4 times, rectum 1.3-1.8 anal body diam. long. Distinct sphincter at prerectum and rectum junction. Genital system didelphic-amphidelphic, with both branches equally and well developed, anterior 450.5 +/- 21.3 (422-478) µm , posterior 463.8 +/- 37.9 (404-531) µm long, respectively. Ovaries usually large, oviduct consisting of a tubular part and well developed pars dilatata. Sphincter between oviduct and uterus moderately developed. Uterus long (anterior 220-307 µm , posterior 222.5-356 µm long, respectively), bipartite, consists of a wider proximal part followed by narrower distal part surrounded by large hyaline cells. Uteri contain sperm. Vagina extending inwards for 55-74% of body diameter, pars proximalis 35-50 x 22-30 µm , with straight walls, pars refringens (in lateral view) consisting of two massive trapezoidal separate sclerotised pieces with a combined width of 18-21 µm , pars distalis 8.5-12 µm long. Vulva a transverse slit; under SEM vulval lips spindle shaped, irregularities and ruptures of body cuticle present on both sides of vulva. Lateral vulval flaps absent. In two females uterine eggs observed, measuring 133-148 x 68.5-77 µm . Tail conical, ventrally arcuate, distal part offset, tip finger-like, sharply pointed. Three pairs of caudal pores. Table 6. Pharyngeal characters of Pararhyssocolpus paradoxus gen. n., n.comb. For abbreviations see Loof and Coomans (1970) and Andrassy (1998b) .

Locality	King George Island				Livingston Island
Characters	KGI1				CDM		DM
1
11
12
2
21
22
1
2
1
2

Males. General morphology similar to that of female, except for the genital system. Arrangement of pharyngeal gland nuclei presented at Table 6. Genital system diorchic , testes opposed, anterior 318-474 µm (n=3) and posterior 278-436 µm (n=2) long, respectively. Spicules dorylaimid, stout, 1.7-2.6 cloacal body diam. long. Lateral guiding piece with triangular distal part, 19-24 µm long. Sperm oval, measuring 5 -9x3- 4 µm . Ventromedian supplements contiguous, 24-28 in number, preceded by one adcloacal pair of papillae located at 9-16 µm distance from cloacal opening, out of spicules range; a series of well developed subventral spaced papillae ( Jairajpuri and Ahmad 1992 ) in number 11-18 observed. Post-cloacal papilla present. Tail compared to that in female with narrower finger like tip. Three pairs of caudal pores. Juveniles. Comparison of length of functional and replacement odontostyle and body length yielded in identification of three juvenile stages (second stage juvenile not found). The tail in J1 elongated, sigmoid, in J3 tail elongate with long hyaline extension, ventrally arcuate, sometimes slightly sigmoid, sharply tipped; in J4 ventrally arcuate with gradually tapering distal part, c' decreases during successive stages to females (Table 5). Figure 14. Pararhyssocolpus paradoxus (Loof, 1975), gen. n., comb. n. A, B, D-FFemale : A Pharyngeal region (KGI) B Anterior region (KGI) D Posterior genital branch (LI, DM) E Posterior end (KGI) F Tongue like projection (KGI) Male (LI, DM): C Pharyngeal expansion. Scale bar: 50 µm ( A-F ). Figure 15. Pararhyssocolpus paradoxus (Loof, 1975) gen. n., comb. n. Female: A-C Vulval region (KGI) D-F Tail ends (D, E KGI; F LI, DM). Scale bar: 50 µm ( A-F ). Figure 16. Pararhyssocolpus paradoxus (Loof, 1975), gen. n., comb. n. Male: A Anterior region (LI, DM) B Tail end (KGI) C Lateral guiding piece (KGI). Scale bar: 50 µm ( A-C ). Figure 17. Pararhyssocolpus paradoxus (Loof, 1975), gen. n., comb. n. Juveniles (KGI): A-E Lip region of A J1 B, C J3 D, E J4 F-J Tail end of F J1 G, H J3 I, J J4. Scale bar: 50 µm ( A-J ). Figure 18. Pararhyssocolpus paradoxus (Loof, 1975), gen. n., comb. n. Female: A-C Anterior region (A KGI; B LI, DM; C NI) D Pharyngeal expansion, cardia (KGI) E Amphidial fovea (KGI) F Entire body (KGI) G Sphincter between uterus and pars dilatata oviductus (KGI) H Anterior genital branch (KGI) I, J Vulval region (KGI) K Irregularities around vulva (KGI). Scale bars: 10 µm ( A-C , G, I-K ); 50 µm (D, H); 6 µm (E); 200 µm (F). Figure 19. Pararhyssocolpus paradoxus (Loof, 1975), gen. n., comb. n. Male: A Pharyngeal region (LI, CDM) B, C Anterior end (B KGI; C LI, DM) D Entire body (KGI) E Sperm (KGI) F Posterior testis (KGI) G, I Spicules (KGI) H Lateral guiding piece (KGI) J Ventromedian supplements (KGI) K Subventral papillae marked by arrows (KGI). Scale bars: 50 µm (A, F); 10 µm (B, C, E, G, H); 200 µm (D); 20 µm ( I-K ). Figure 20. Pararhyssocolpus paradoxus (Loof, 1975), comb. n. A-CFemale : A-C Tail ends (A KGI; B LI, DM; C NI). D-FMale : D Posterior end (KGI) E, F Tail ends (E CDM, LI; F KGI). Scale bars: 10 µm ( A-C , E, F); 50 µm (D). Figure 21. Pararhyssocolpus paradoxus (Loof, 1975), comb. n. A-E , G-IJuveniles (KGI): A-E Lip region of A J1 B, C J3 D, E J4 G-I Tail end of G, H J3 I J4. F, JFemale: (KGI): F Lip region J Tail end. Scale bars: 6 µm (A); 10 µm ( B-J ). Figure 22. SEM micrographs. Pararhyssocolpus paradoxus (Loof, 1975), comb. n. A-H , JFemale: A-D Lip region (A Sublateral view (NI); B Lateral view (NI); C, D (LI) In face view) E Vulval region (NI) F Cuticle striations (NI) G, H Vulval region, irregularities around vulva, lateral body pores marked by arrows (NI) J Tail end (LI) I, K-M Male (LI): I Posterior end, lateral view K Tail end L Cloaca M Ventromedian supplements and subventral papillae (marked by arrows). Scale bars: 5 µm (A, B, D); 10 µm (C, G, H, J, L, M); 50 µm (I); 20 µm (K); 2 µm (E); 1 µm (F). Figure 23. Pararhyssocolpus paradoxus (Loof, 1975), comb. n. Scatter plot of the functional (●) and replacement odontostyle (▲) in relation to the body length of the juvenile stages and adults: females (▲) and males (▲). Figure 24. Hypothesis of the phylogenetic relationships of Amblydorylaimus isokaryon (Loof, 1975) and Pararhyssocolpus paradoxus (Loof, 1975), gen. n. comb. n. based on 18S rDNA (61 sequences) inferred from a Bayesian analysis using GTR+G model and Coomansus gerlachei (de Man, 1904) for rooting the tree. Posterior probabilities higher than 0.8 are presented. Figure 25. Hypothesis of the phylogenetic relationships of Amblydorylaimus isokaryon (Loof, 1975) and Pararhyssocolpus paradoxus (Loof, 1975), gen. n. comb. n. based on 18S rDNA of closest species (17 sequences) inferred from a Bayesian analysis using GTR+G model and midpoint rooting of the tree. Posterior probabilities higher than 0.8 are presented. Species coloured according the classification of Andrassy (2009a) and Pena-Santiago and Alvarez-Ortega (2014) : dark blue - fam. Aporcelaimidae , light blue - fam. Actinolaimidae , green - fam. Dorylaimidae , red - fam. Qudsianematidae . Figure 26. Hypothesis of the phylogenetic relationships of Amblydorylaimus isokaryon (Loof, 1975) and Pararhyssocolpus paradoxus (Loof, 1975), gen. n. comb. n. based on 28S rDNA D2-D3 inferred from a Bayesian analysis using GTR+G model and midpoint rooting of the tree. Posterior probabilities higher than 0.8 are presented. Species coloured according the classification of Andrassy (2009a) and Pena-Santiago and Alvarez-Ortega (2014) : dark blue - fam. Aporcelaimidae , light blue - fam. Actinolaimidae , dark green - fam. Dorylaimidae , light green - fam. Thornenematidae , red - fam. Qudsianematidae , yellow - fam. Swangeriidae , violet - fam. Belondiridae . *For abbreviations of localities see Table 1 Sequence and phylogenetic analyses. The BLAST search using D2-D3 region sequence of Pararhyssocolpus paradoxus gen. n., comb. n. showed highest similarity (93%) to the sequences of several Opisthodorylaimus sylphoides (Williams, 1959) Carbonell & Coomans, 1985 clones and Prodorylaimus sp. (AY593008-10, EF207241, Holterman et al. 2008 ). The 18S rDNA sequence showed 99% similarity to several dorylaimid species belonging to different families including Amblydorylaimus isokaryon , and various Aporcelaimellus spp. The hypothesis testing using closely and more distantly related 18S rDNA sequences (Figure 24) revealed distant relationship of Pararhyssocolpus paradoxus gen. n., comb. n. to the only available sequences of Rhyssocolpus Andrassy , 1971 ( Rhyssocolpus vinciguerrae Pedram, Pourjam, Robbins, Ye, Pena-Santiago , 2011, Figure 4) (fam. Nordiidae ) and Eudorylaimus Andrassy , 1959 (two Eudorylaimus spp.) (fam. Qudsianematidae ). The ambiguous position of both Pararhyssocolpus paradoxus gen. n., comb. n. and Amblydorylaimus isokaryon could be a result of the low resolution of the SSU rDNA, non-monophyly of these four families and/or probably incorrect species identifications. The majority of the nematode sequences belonging to the superfamily Dorylaimoidea de Man, 1876 available at the GenBank have no morphological and metrical data and their identification is questionable. In an additional analysis using the most closely related sequences performed in order to clarify the possible evolutionary relationships of Pararhyssocolpus paradoxus gen. n., comb. n. (Figure 25): it clustered into the same clade with Amblydorylaimus isokaryon and some other species of the families Qudsianematidae , Dorylaimidae and Aporcelaimidae . Further, in the 28S rDNA-based phylogenetic tree Pararhyssocolpus paradoxus gen. n., comb. n. grouped with species belonging to different families (Figure 26) and no close relationships to any of them were revealed. Discussion. The specimens examined generally agree well with data reported for this species, although some differences occurred: lip region offset by slight depression vs deep depression; vulva transverse vs "probably pore-like rather than transverse", smaller DN-DO distance (0.5-1 vs 1.6-3.4%) ( Loof 1975 ). Further, the distinct sphincter at prerectum/rectum junction, tongue-like structure at the posterior end of intestine and subventral papillae in male were not mentioned in the original description. Originally this species was attributed to family Qudsianematidae . Loof (1975) placed it in Eudorylaimus , because of widened near the middle pharynx and numerous ventromedian supplements. Nevertheless, he reported that it showed several characters close to Rhyssocolpus (shape of lip region, short odontostyle, and wrinkled cuticle near vulva, although he regarded the last one a not generic rank character). Subsequently Andrassy (1986) included it in family Nordiidae (genus Rhyssocolpus ) ignoring the characters in which this species differs from the other members of genus Rhyssocolpus e.g. the greater number of contiguous ventromedian supplements and specific shape of pharyngeal expansion. Again, Loof (1988) reported that many features of this species (numerous and contiguous supplements, pharyngeal expansion at about half pharynx length, DN lying at about 60% of pharynx, distinct first pair of ventrosublateral pharyngeal glands) conflicted with the diagnosis of Rhyssocolpus and continued to regard this Antarctic species as a member of Eudorylaimus ( Qudsianematidae ). Very recently, Pena-Santiago et al. (2015) provided a revised taxonomy of the genus Rhyssocolpus and proposed Rhyssocolpus paradoxus be retained under Eudorylaimus . However, it differs from the latter genus by the arrangement of ventromedian supplements in males (contiguous vs spaced ), double vs single guiding ring, slender vs wider odontostyle and specific shape of pharyngeal expansion. Recent molecular studies ( Holterman et al. 2008 ; Pedram et al. 2011 ; Pena-Santiago et al. 2015 ) as well as our molecular data inferred from the analysis of 18S and D2-D3 expansion segments of the 28S rDNA, showed that this genus could not be assigned to any known Dorylaimoidea family. With considering the differences discussed above, as well as molecular data, the herein studied species cannot be regarded either as a member of the genus Rhyssocolpus or the genus Eudorylaimus and their attributed families, consequently a new genus Pararhyssocolpus gen. n., and a new family Pararhyssocolpidae fam. n. are proposed to accommodate this species.