Amblydorylaimus isokaryon (Loof, 1975) Andrassy , 1998

Elshishka, Milka, Lazarova, Stela, Radoslavov, Georgi, Hristov, Petar & Peneva, Vlada K., 2015, New data on two remarkable Antarctic species Amblydorylaimusisokaryon (Loof, 1975) Andrassy, 1998 and Pararhyssocolpusparadoxus (Loof, 1975), gen. n., comb. n. (Nematoda, Dorylaimida), ZooKeys 511, pp. 25-68 : 27-33

Taxon classification Animalia Dorylaimida Qudsianematidae

Amblydorylaimus isokaryon (Loof, 1975) Andrassy, 1998 Figures 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 24, 25, 26

Eudorylaimus isokaryon Loof, 1975

Material examined.

Twenty-two females, nineteen males and thirteen juveniles (J1-J4) collected from three islands from Maritime Antarctic (Table 1).

Measurements.

See Table 2.

Description.

Female. Body large, curved ventrad after fixation, especially in posterior end. Cuticle 2-4 µm thick at postlabial region, 4-6 µm at mid-body and on tail posterior to anus, three-layered, outer layer thin with fine and distinct transverse striation (especially well visible on SEM, annules 0.4-0.7 µm wide); intermediate layer also thin, refractive, especially on tail region; inner layer thicker than the others. Lateral chord occupying 20-27% of midbody diam. Lateral pores well perceptible, often conspicuous throughout entire body, 10-14 in number in neck region, dorsal pores 3-4, ventral pores along the whole body, 9-11 in neck region. Lip region angular, set off from the adjoining body by a constriction; 2-3 times as wide as high, about 23-32% of body diameter at neck base. Based on SEM photographs oral aperture dorso-ventral, vestibulum hexagonal; labial and cephalic papillae prominent, labial papillae mamilliform, surrounded by circular annules, cephalic papillae button-like, without such annules, perioral field slightly elevated. Amphidial fovea cup shaped its aperture 41-52% of lip region diam., fusus (sensillium pouch) at 29-32 µm from anterior end, small posterior pouches present which are not always discernable. Odontostyle long, weakly sclerotised, 7-9 times as long as wide, 1.2-1.4 times lip region diam., aperture occupying 1/3 to more than 1/2 of its length (30-53%), av. 2/5, depending on the position of the body (under SEM it is seen that the aperture reaches 12-14 µm which is about 2/5 of the odontostyle length), two edges of the slit do not overlap. Guiding sheath distinct, its anterior edge located at 11-13 µm (or 0.4-0.6 times lip region diam. to anterior end (in not protruded odontostyle)) and seems cuticulised stronger than the posterior edge which is located at the base of odontostyle. Odontophore rod like, 1.5-1.9 times long as odontostyle. Anterior region of pharynx enlarging gradually, basal expansion 350-450 µm, (321 µm in the specimen from King George Island), occupying 50-60% of total neck length. Anterior subventral nuclei equal in size and shape, slightly smaller than dorsal nucleus; dorsal gland nucleus 4.5-6 µm diam., first and second pair subventral gland nuclei 4-4.5 and 3-4 µm diam., respectively. Location of pharyngeal glands and their orifices is presented in Table 3. Cardia rounded conoid, ending with sharply pointed tongue, variable in size and shape. In some specimens a dorsal cellular mass present at cardia level. The posterior end of the intestine with tongue-like structure of variable length. Prerectum 1.9-3.2 (1.5 times in specimen from King George Island), rectum 1.0-1.5 times anal body diam. long. Prerectum separated from intestine by a transverse muscular ring. Sphincter between rectum and prerectum well developed. Female genital system didelphic amphidelphic, both branches equally and well developed - anterior 510.1 ± 86.5 (390-695.5) µm and posterior 527.4 ± 67.9 (430-705.5) µm long, respectively (anterior 313 and posterior 347 µm long in specimen from King George Island). Ovaries short well developed, often not reaching sphincter level. Oviduct with well-developed pars dilatata, often containing sperm. Sphincter between pars dilatata oviductus and uterus small, well developed. Uterus long, anterior 232-435 µm, posterior 226-395 µm long, without differentiation, filled with sperm. Vulva longitudinal (based on SEM observations, Fig. 12A, B). Vagina extending inwards for 51-68% of body diam.; pars proximalis 36 –44×15.5– 23 µm, pars refringens with two trapezoidal sclerotisations, with combined width of 17.5-22 µm; pars distalis 6-10 µm long. Three females each with one uterine egg, measuring 123 –135×56– 82.5 µm, in one female one egg located in pars dilatata oviductus, measuring 123 × 80 µm. Irregularities of body cuticle present around vulva, on SEM observation they appeared as additional cuticle masses. Tail short conoid, ventrally arcuate, with bluntly rounded tip, 1.3-1.7% of body length. Caudal pores two pairs, on SEM appeared papillae-like.

Male. General morphology similar to that of the female, except for the genital system. In one specimen the odontostyle aperture ventral. Arrangement of pharyngeal gland nuclei and their orifices is presented at Table 3. Genital system diorchic, with opposite testes: anterior 376.4 ± 28.9 (339-418) µm and posterior 361.9 ± 56.8 (279-442) µm long (n=6), (anterior 237 and posterior 255 µm in a specimen from King George Island), respectively. Spicules dorylaimoid, strongly curved ventrad and robust, their length about 1.7-2.3 times cloacal body diam. Ventromedian supplements preceded by one adcloacal pair of papillae, 12-16 in number, regularly spaced, with small cuticular folds between them, adcloacal pair located at 29-37.5 µm apart from cloacal opening (26 µm in specimen from King George Island). Sperm spindle shaped, measuring 11 –13×3– 4 µm. Lateral guiding pieces, cylindrical with bifurcate end, measuring 24 –31×3– 5 µm. Tail short conoid, ventrally arcuate, with obtusely rounded tip, two pairs of caudal pores.

Juveniles. Morphometrics obtained from juvenile specimens, and the relationship between the lengths of their functional and replacement odontostyles and body lengths, identified four juvenile stages (Figure 13). Tail in J1-J3 elongated conoid, ventrally arcuate with rounded terminus, in J4 as in females, c’ decreasing during successive stages to female (Table 4, Figs 6, 10).

Sequence and phylogenetic analyses.

The BLAST search using D2-D3 region sequence of Amblydorylaimus isokaryon showed highest similarity (96%) to Aporcelaimellus salicinus Álvarez-Ortega, Subbotin & Peña-Santiago, 2013 (JX094341-42), while the 18S rDNA sequence was closest (99% similarity, 4-10 nucleotide differences of about 1700 bp) to several Aporcelaimellus spp., Allodorylaimus andrassyi (Meyl, 1955) Andrássy, 1986 and four sequences acquired during environmental studies of arable soil ( Griffiths et al. 2006) and trembling aspen rhizosphere ( Lesaulnier et al. 2008). Since the 18S rDNA phylogram based on bigger dataset (Figure 24) did not show clearly the evolutionary relationships of Amblydorylaimus isokaryon a smaller dataset with the closest sequences was analysed (Figure 25). Although the very low 18S rDNA resolution this analysis yielded a tree with Amblydorylaimus isokaryon being a part of well-resolved group of species assigned to three families: Dorylaimidae De Man, 1876 ( Labronema vulvapapillatum (Meyl, 1954) Loof & Grootaert, 1981 and Mesodorylaimus centrocercus (de Man, 1880), Geraert, 1966, Qudsianematidae ( Ecumenicus spp.) and Aporcelaimidae (includes mainly Aporcelaimellus spp. and Allodorylaimus andrassyi which probably is misidentified). Further, in the 28S rDNA-based phylogenetic tree Amblydorylaimus isokaryon appeared a sister species of Aporcelaimellus salicinus , again being a part of a well-supported group of several Aporcelaimellus spp. and Allodorylaimus andrassyi (Figure 26).

Discussion.

The main morphological characters of the studied populations are very similar, only the specimen from King George Island differs by its shorter body, pharynx, pharyngeal expansion, anterior and posterior female genital branches, prerectum and tail (Table 2). Our materials generally agree well with the type specimens (Loof, 1975), although some differences occurred: the present specimens have broader lip region (length of odontostyle 1.2-1.3 vs 1.5 times longer than lip region diam., longer uterine eggs (123-135 vs 117-122 µm), and somewhat longer distance adcloa cal pair of papillae - cloaca (29-37.5 vs 26-29 µm) ( Andrássy 1998a). Loof (1975) described the vulva as longitudinal but according to Andrássy (1998a) it is more or less a roundish pore, although he may not have observed females in ventral position. Our SEM studies confirm Loof’s observations. Andrássy (1998a) reported that spermatozoids have an atypical shape for dorylaimids being rounded or potato-like, however our observations showed that their shape is spindle-like, similar to the drawings by Loof (1975). Further, the presence of a tongue-like projection between the intestine and prerectum not mentioned in the original description was observed. None of the above mentioned authors reported the cuticular irregularities around the vulva documented here both by LM and SEM.

This species was originally described as Eudorylaimus isokaryon by Loof (1975); later Andrássy (1998a) established a new genus, Amblydorylaimus to accommodate it on the basis of several morphological characters (amphidial fovea and odontostyle shape, equally sized mid-pharyngeal nuclei, atypical sperm shape, nipper-like adspicular pieces and unusual location of adcloacal pair of supplements). He described and illustrated Amblydorylaimus isokaryon having a specific shape of odontostyle - resembling garden shears; the aperture appeared small. He suggested that this unusual shape was not caused by fixation artefacts as "other organellum of cuticular origin is clearly visible, without any deformation" and "all other Eudorylaimus species collected by Spaull (Loof, 1975) in his study trip do possess normally shaped, well preserved dorylaimid spear". Andrássy (1998a) suggested that it would be necessary to know if living specimens possessed this shape of odontostyle. We examined living specimens of this species, and did not find the peculiarities of the odontostyle shape observed by this author. In the original description, Loof (1975) did not mention this special feature of odontostyle and noted that the odontostyle aperture occupied one-third of its length. In our specimens the odontostyle is weakly sclerotised, regular with usual dorylaimid shape; the length of aperture longer, occupying 1/3-1/2 of the odontostyle. In earlier prepared slides, the odontostyle showed some irregularities similar to those described by Andrássy (1998a). The same author ( 1998a, 2009a) considered this genus as a member of family Qudsianematidae , but noted that it significantly differs from every genus of this family with its characteristic morphology. Molecular data based on 28S rDNA, however showed that this genus is a member of family Aporcelaimidae and not family Qudsianematidae . This conclusion is supported by our morphological evidences: large aperture of odontostyle (reaching almost ½ of odontostyle length), oral opening a dorso-ventral slit, cuticle thick with refractive layer, not fixed guiding ring etc. which confirm Amblydorylaimus fits better to the family Aporcelaimidae . Based on morphology and molecular data (28S) Amblydorylaimus is closely related to genus Aporcelaimellus Heyns, 1965 from which it can be differentiated by its longer and not robust odontostyle with shorter aperture (av. 2/5 vs more than 1/2 of odontostyle length), and not overlapping vs overlapping edges, lip region with radial vs bilateral symmetry ( Álvarez-Ortega and Peña-Santiago 2013), vulva longitudinal vs transverse (except Aporcelaimellus macropunctatus (Heyns, 1967) Jimenez-Guirado, 1994 distinguished by its longitudinal vulva), position of adcloacal pair of papillae in males (more distant from cloacal opening vs very close) and lateral guiding pieces bifurcate vs simple. Recently, Andrássy (2009b) proposed a new genus close to Aporcelaimellus and Amblydorylaimus , the genus Aporcelinus Andrássy, 2009. The latter genus differs from the genus Amblydorylaimus by the structure of cardia (with a small dorsal lobe), transverse vulva, eggshell wrinkled, ventromedian supplements small, irregularly spaced, without precloacal space, location of adcloacal pair and shape of tail (conoid tail with sharply pointed terminus). Vinciguerra et al. (2014) believed that the taxonomic position of Aporcelinus is ambiguous; they noted that this genus could also be assigned to family Qudsianematidae on the basis of its morphological features (odontostyle aperture length, simple guiding ring and thickness of cuticle, composed of two layers). Related to the cuticle structure, it should be mentioned that genus Aporcelinus has three layered cuticle with inner refractive layer, well visible on several photomicrographs (Figs 4E, 8 A–C) presented by Vinciguerra et al. (2014). Further, the location of adcloacal pair of male ventromedian papillae (comparatively far from cloaca opening) in Amblydorylaimus isokaryon shows some similarity to Crassolabium persicum Jabbari, Niknam, Vinciguerra, Moslehi, Abolafia & Peña-Santiago, 2012, but the latter species differs from it by the odontostyle structure (weakly sclerotised vs quite robust), not differentiated vs bipartite uterus, structure of pars distalis (without differentiation vs with two small sclerotisations close to the pars refringens in Crassolabium persicum ) ( Jabbari et al. 2012).

On the basis of morphological and molecular data, we propose the genus Amblydorylaimus to be transferred from family Qudsianematidae to the family Aporcelaimidae . It is worth mentioning that the latter family obviously is non monophyletic and we propose this taxonomic change on the base of the close relationships with the genus Aporcelaimellus now regarded as a member of family Aporcelaimidae .

Diagnosis (emended).

Amblydorylaimus .

Aporcelaimidae .

Aporcelaiminae. Body large, about 3 mm. Cuticle three-layered, outer layer thin with fine but distinct transverse striation. Lip region angular, offset from adjacent body by a constriction. Oral aperture dorso-ventral, hexagonal. Amphidial fovea caliciform with small posterior pouches. Odontostyle long, weakly sclerotised. Guiding sheath distinct, anterior and posterior edges moderately cuticularised. Odontophore rod like. Pharynx expanded in its posterior half. Nuclei distinct, dorsal nucleus fairly posterior in position, first subventral pair large and equal in size, posterior pair rather far from the end of pharyngeal expansion. Prerectum sharply separated from mid-intestine. Female genital system didelphic amphidelphic. Ovaries very short, uterus long without differentiation. Vulva longitudinal, cuticular irregularities present around it. Pars refringens vaginae well developed. The posterior end of the intestine with tongue-like structure. Sperm spindle shaped. Spicula dorylaimid, lateral guiding piece distally bifurcate. Ventromedian supplements numerous, regularly spaced, preceded by one adcloacal pair of papillae comparatively far from cloacal aperture. Tail similar in both sexes, short conoid, ventrally arcuate, with bluntly rounded tip. Tail in J1-J3 conoid elongated, in J4 as in female.

Distribution.

Amblydorylaimus is an endemic genus of the maritime Antarctic. It has been reported from several islands (Intercurrence, Elephant, Galindez, Livingston and King George) ( Loof 1975; Maslen 1979; Peneva et al. 2009; Kito 2009). The present finding from Nelson Island represents a new geographical record.