Dorylaimida: Nordiidae - Brill Online Books and Journals

Nematology 17 (2015) 139-153

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Characterisation of an Iberian population of Rhyssocolpus iuventutis Andrássy, 1971 (Dorylaimida: Nordiidae), with a revised taxonomy of the genus Reyes P EÑA -S ANTIAGO 1,∗ , Pablo G UERRERO 1 , Gracia L IÉBANAS 1 , María del Carmen G ARCÍA 2 , Teresa PALOMEQUE 2 and Pedro L ORITE 2 1

Departamento de Biología Animal, Biología Vegetal y Ecología, Universidad de Jaén, Campus ‘Las Lagunillas’ s/n, Edificio B3, 23071-Jaén, Spain 2 Departamento de Biología Experimental, Universidad de Jaén, Campus ‘Las Lagunillas’ s/n, Edificio B3, 23071-Jaén, Spain Received: 9 September 2014; revised: 13 November 2014 Accepted for publication: 13 November 2014; available online: 19 December 2014

Summary – The identity and evolutionary relationships of the genus Rhyssocolpus are analysed and discussed using an integrative approach including morphological data and partial SSU-rDNA sequences. An Iberian population of R. iuventutis is characterised in detail, providing the first SEM observations of the genus. New sequences of the genera Enchodelus and Rhyssocolpus are provided for comparative purposes. Both morphological and molecular evidence support a separate status for the aforementioned two genera and Heterodorus, of which the latter and Rhyssocolpus shared a recent common ancestor, whereas Enchodelus did not, as had been traditionally assumed, occupy a close position. The Nordiidae is confirmed to be an artificial taxon. The taxonomy of Rhyssocolpus is revised and an emended diagnosis, updated list of species, key to their identification and compendium of their morphometrics are provided. Some nomenclatorial changes are also proposed: R. alleni and R. paradoxus are retained under Eudorylaimus, their original genus, whereas R. brasiliensis is transferred to Eudorylaimus as E. brasiliensis (Meyl, 1956) comb. n. Keywords – description, Enchodelus, Eudorylaimus, Eudorylaimus brasiliensis comb. n., Heterodorus, morphology, morphometrics, new combination, phylogeny, Rhyssocolpus brasiliensis, SEM, SSU-rDNA.

The genus Rhyssocolpus Andrássy, 1971, a representative of the dorylaimid family Nordiidae Jairajpuri & Siddiqi, 1964, is an interesting, rare, free-living nematode taxon that is often found in mosses, freshwater habitats or wet soils. It was originally proposed by Andrássy (1971) to accommodate the new species R. iuventutis Andrássy, 1971 along with two other known species, namely R. microdorus (Schiemer, 1965) Andrássy, 1971 and the type species, R. vulvostriatus (Stefański, 1924) Andrássy, 1971, which were transferred from Enchodelus Thorne, 1939 and Eudorylaimus Andrássy, 1959, respectively. Subsequently, several new species were added to its checklist (Ebsary, 1984; Gagarin, 1985; Andrássy, 1986, 2003; Pedram et al., 2011). The identity and the systematics of the genus have been controversial as Loof (1988) regarded it as identical to Enchodelus, an opinion that did not win general acceptance (see Jairajpuri & Ah∗ Corresponding

mad, 1992). Andrássy (2009) redefined Enchodelus and transferred a substantial proportion of its species to Heterodorus Altherr, 1952, the latter being very similar to Rhyssocolpus, although both were regarded as valid taxa. Molecular analyses have demonstrated that the evolutionary relationships between these three genera are not as close as expected and that the Nordiidae is an artificial group (Holterman et al., 2008; Pedram et al., 2011). Many species of Enchodelus – some now included in Heterodorus – were collected during several nematological surveys conducted in natural areas from the southeastern Iberian Peninsula. The morphology and taxonomy of these species were exhaustively analysed in a series of papers (Guerrero et al., 2007, 2008), although their molecular evolutionary relationships were not discussed as sequence data were not available. Abundant material of R. iuventutis was found in highland meadows from the

author, e-mail: [email protected]

© Koninklijke Brill NV, Leiden, 2015

DOI 10.1163/15685411-00002857

R. Peña-Santiago et al.

Sierra Nevada National Park (Granada province). This is the first report of the species after its original description, more than 40 years ago, and offers an ideal opportunity for its detailed morphological characterisation, including SEM observations. Molecular data of R. iuventutis, along with those of one species of Enchodelus and two species of Heterodorus, were obtained and facilitate an integrative study of the phylogeny of this interesting group of dorylaims.

Materials and methods M ATERIAL EXAMINED Nematodes were collected in natural habitats from the Sierra Nevada National Park (province of Granada, Spain) in the summer of 2010, extracted from soil samples using the methods of Baermann (1917) and Flegg (1967), relaxed and killed by heat, fixed in 4% formaldehyde, processed to anhydrous glycerin following Siddiqi’s (1964) protocol and mounted on permanent glass slides to enable handling. Several specimens of three Iberian species of the Nordiidae, collected from the Sierra Mágina Natural Park and previously characterised by Guerrero et al. (2007, 2008), namely Enchodelus saxifragae Popovici, 1995, Heterodorus morgensis (Loof, 1988) Andrássy, 2009 and H. brevidentatus (Thorne, 1939) Andrássy, 2009, were also included in this study.

DNA EXTRACTION , PCR AND SEQUENCING DNA extraction was performed following the protocol of Subbotin et al. (2006) with some modifications. Each nematode was cut in several pieces under a binocular microscope and the fragments transferred to a tube containing 32 μl DNA/RNA-free water. Next, 6 μl of proteinase K (10 mg ml−1 ), 10 μl β-mercaptoethanol (98%) and 5 μl 10× PCR buffer were added. Tubes were incubated at 65°C for 45 min and then at 95°C for 15 min. After incubation, the tubes were centrifuged for 2 min at 12 000 g and stored at −20°C until use. A 600 bp fragment of the small subunit unit (SSU) rDNA was amplified by PCR using the primers 18S-965 (5 -GGCGATCAGATACCGCCCTAGTT-3 ) and 18S1573 (5 -TACAAAGGGCAGGGACGTAAT-3 ) (Mullin et al., 2005). The amplification was done for two specimens of each of the four nordiid species analysed. PCR was performed using 5 μl of extracted DNA, 2 μl 10× PCR buffer, 0.25 μl MgCl2 (50 mM), 0.25 μl dNTP (10 mM), 2 μl of each primer (10 nM), 0.3 μl Taq polymerase (5 U μl−1 ) and 13.2 μl of water. The PCR program was as follows: denaturation at 92°C for 2 min, followed by 40 cycles of denaturation at 92°C for 30 s, annealing at 56°C for 60 s, and extension at 72°C for 1 min. A final extension was performed at 72°C for 5 min. The amplified fragments were analysed by electrophoresis in agarose gels. Purified fragments were inserted into the pGEMT-Easy vector (Promega). Recombinant plasmids were sequenced on both strands by the dideoxy sequencing method. The sequences were deposited in the EMBL database (accession nos HG797023-HG797026).

L IGHT MICROSCOPY Nematodes were observed and measured using a Nikon Eclipse 80i light microscope. Morphometrics included de Man’s indices and most of the usual measurements. Some of the best preserved specimens were photographed with a Nikon DS digital camera. Photographs were edited with Adobe® Photoshop® CS. S CANNING ELECTRON MICROSCOPY (SEM) After examination and identification, a few specimens were recycled for observation under SEM following the protocol of Abolafia & Peña-Santiago (2005). Nematodes were hydrated in distilled water, dehydrated in a graded ethanol series (25, 30, 50, 70, 95, 100%) and acetone (100%), critical point-dried, coated with gold, and observed with a JEOL JSM-5800 microscope. 140

P HYLOGENETIC ANALYSES The partial 18S sequence from R. iuventutis was used to do a homology search in GenBank using the BLAST program with the megablast (highly similar sequences) option. Sequences with high levels of homology, including all available sequences of nordiid taxa, were selected. This dataset was used for phylogenetic analyses. Multiple-sequence alignments were initially performed using CLUSTALW (Larkin et al., 2007) and edited by hand. The best-fit nucleotide substitution models were evaluated using MEGA 5.0. The program lists models with decreasing BIC (Bayesian Information Criterion) values, along with log likelihood as well as AIC (Akaike Information Criterion) values for each model. The models with the lowest BIC scores were considered to be better for describing the substitution pattern (Tamura et al., Nematology

Rhyssocolpus iuventutis and taxonomy of the genus

2011). Using this criterion, the best model was K2 + G + I (Kimura 2-parameter using a discrete Gamma distribution and assuming that a certain fraction of sites are evolutionarily invariable). However, the other two criteria have also been frequently used in nematode phylogenetic studies (Mullin et al., 2005; Pedram et al., 2011). According to these other two criteria, the best model was GTR + G + I (General Time Reversible). In order to avoid possible bias due to selected model, both models were used to create phylogenetic trees. Phylogenetic relationships among the sequences were explored by the maximum-likelihood (ML) method using MEGA 5.0 (Tamura et al., 2011). Bootstrap (BS) values for each clade were assessed from 1000 replicates in all cases and assigned to clades in the form: ‘BS value calculated using the model K2 + G + I/BS value calculated using the model GTR + G + I’. Three nematode 18S sequences were used as outgroups: Aporcelaimus americanus Thorne & Swanger, 1936 (AY146509), Aporcelaimus cf. eurydorys (Ditlevsen, 1911) Thorne & Swanger, 1936 (AY911906), and Sectonema barbatoides Heyns, 1965 (AY284814). These sequences have a high homology with the selected sequences in the first dataset although they belong to the family Aporcelaimidae Heyns, 1965, which is quite close to the Nordiidae (van Megen et al., 2009). For an external group, a Solididens vulgaris (Thorne, 1930) Heyns, 1968 18S sequence (AY552977) was selected as this species belongs to the suborder Nylgolaimina Ahmad & Jairajpuri, 1979, unlike all the other species which belong to the suborder Dorylaimina Pearse, 1936.

Results Rhyssocolpus iuventutis Andrássy, 1971 (Figs 1-4) M ATERIAL EXAMINED

D ESCRIPTION Adult Moderately slender nematodes of medium size, 1.311.66 mm long. Body cylindrical, tapering towards both extremities, but more so towards posterior end due to conical tail. Habitus curved ventrad upon fixation, C-shaped in female, G-shaped in male. Cuticle two-layered, 1.5-2.5 μm thick in anterior region, 2.0-5.0 μm at mid-body and 3.05.0 μm on tail, outer layer bearing fine transverse striation, almost indistinguishable under LM. Lateral chord occupying 7-27% of mid-body diam., two lateral cervical pores present, arranged close together at ca one lip region diam. posterior to amphidial aperture. Lip region weakly angular, truncate anteriad, nearly continuous with adjacent body, 2.8-4.2 times as broad as high and onefifth to one-third (20-30%) of body diam. at neck base. With SEM, lips amalgamated, oral field circular, separated from remaining lip region by an annular incisure and containing six inner labial papillae, perioral area apparently consisting of four liplets (one dorsal, one ventral and two lateral) covering oral aperture, papillae button-like, each surrounded by a small, ring-like annulus. Amphidial fovea stirrup-shaped, its aperture occupying 54-70% of lip region diam. Cheilostom a truncate cone, with slightly thickened walls. Odontostyle short, attenuated, 7-10 times as long as wide and 0.7-0.8 lip region diam. long, aperture very short, difficult to observe. Guiding ring double, with fixed ring at 6.0-7.0 μm or 0.5-0.7 lip region diam. from anterior end. Odontophore rod-like, 1.8-2.3 odontostyle lengths long. Pharynx distinctly muscular, enlarging very gradually, basal expansion occupying up to onethird (27-33%) of total neck length, pharyngeal gland nuclei located as follows: DN = 76-81, AS indistinct, PS1 = 43-61, PS2 = 45-62. Cardia short, rounded, (5-10) × (813) μm. Caudal region conical, regularly curved ventrad, with more or less acute tip, its inner core not reaching tail end to form a well defined hyaline portion occupying onefourth to one-half (23-47%) of tail length. Female

See Table 1.

Genital system didelphic-amphidelphic, both branches equally and well developed. Ovaries large, anterior 103236 μm, posterior 108-297 μm long, often reaching and surpassing sphincter level, oocytes first in two or more rows, then in single row. Oviduct 73-145 μm long or 1.2-2.9 times corresponding body diam., consisting of a slender part with prismatic cells and a moderately developed pars dilatata often containing sperm cells. Weak sphincter present at oviduct-uterus junction. Uterus

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An Iberian population of 31 females and 11 males was studied, the nematodes being in variable, but often acceptable, states of preservation. M ORPHOMETRICS


Fig. 1. Rhyssocolpus iuventutis Andrássy, 1971 (female, LM). A: Entire; B-D: Anterior region in lateral median view; E: Anterior genital branch; F: Lip region in lateral surface view; G-I: Vagina and vulva, showing cuticular irregularities in vulval area; J: Posterior body region; K-M: Caudal region. (Scale bars: A = 200 μm; B, G-I, K-M = 10 μm; C, D, F = 5 μm; E = 50 μm; J = 20 μm.)

bipartite, i.e., consisting of a wider and shorter proximal region with visible lumen and a narrower and longer distal portion with narrow lumen, its length 138-228 μm or 2.1-3.7 corresponding body diam. long. Vagina extending 142

inwards for ca one-half (40-60%) of body diam., pars proximalis (22-31) × (12-17) μm in size, with slightly sigmoid walls surrounded by weak musculature, pars refringens with two refractive triangular pieces, each Nematology


Fig. 2. Rhyssocolpus iuventutis Andrássy, 1971 (male, LM). A: Entire; B-D: Pharyngeal expansion, showing location of DN (B) and S2 N (C, D); E: Neck region; F: Posterior body region; G: Caudal region; H, I: Spicules. (Scale bars: A = 200 μm; B-D = 20 μm; E, F = 50 μm; G-I = 10 μm.)

measuring 2-5 μm long, and with a combined width of 1520 μm, pars distalis 1.5-2.5 μm long. Vulva a transverse slit ca 10 μm long. Abundant and very distinct cuticular irregularities (folds, wrinkles, holes, etc.) in perivulval area forming a strip or band around entire body. Prerectum 1.2-2.1 anal body diam. long, usually with transverse, septum-like structures. Rectum 0.7-1.3 anal body diam. long. Male Prerectum 2.4-3.3 corresponding body diam. long. Genital system diorchic, testes opposed. In addition to adcloacal pair situated at 14-17 μm from cloacal aperture, a series of 6-8 irregularly spaced, ventromedian supplements, posteriormost of which lying at 47-72 μm from adVol. 17(2), 2015

cloacal pair and outside range of spicules. Spicules strong, curved ventrad, 4.6-5.4 times as long as wide. Lateral guiding pieces 10-25 μm long, ca four times as long as wide. D IAGNOSIS Rhyssocolpus iuventutis is characterised by its 1.301.66 mm long body, lip region nearly continuous and 1113 μm broad, odontostyle 7-10 μm long with very small aperture, odontophore rod-like, guiding ring double, neck 232-307 μm long, pharyngeal expansion 67-98 μm long or occupying 24-33% of total neck length, female genital system amphidelphic, pars refringens vaginae with two well developed separate triangular pieces, V = 45-51, 143


Fig. 3. Rhyssocolpus iuventutis Andrássy, 1971 (male, SEM). A: Anterior region in sublateral view; B: Lip region in face view; C: Caudal region in subventral view; D: Posterior body region; E: Caudal region in ventral view. (Scale bars: A, B = 5 μm; C, E = 20 μm; D = 50 μm.)

very abundant and distinct cuticular irregularities near vulva, tail conical and curved ventrad (31-52 μm, c = 3055, c = 1.0-1.5), spicules 50-60 μm long and 5-8 spaced ventromedian supplements. 144

D ISTRIBUTION South-eastern Iberian Peninsula, province of Granada, Sierra Nevada National Park, where the specimens were Nematology


Fig. 4. Rhyssocolpus iuventutis Andrássy, 1971 (female, SEM). A: Anterior region in lateral view; B: Same in sublateral view; C: Lip region in face view; D-F: Vulval region; G-I: Caudal region. (Scale bars: A-C = 5 μm; D-F = 20 μm; G-I = 10 μm.) Vol. 17(2), 2015

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Table 1. Morphometrics of Rhyssocolpus iuventutis Andrássy, 1971 from the Iberian Peninsula. All measurements are in μm (except for L in mm) and in the form: mean ± s.d. (range). Character n L a b c c V Lip region diam. Odontostyle length Odontophore length Guiding ring from anterior end Neck length Pharyngeal expansion length Body diam. at neck base mid-body anus/cloaca Prerectum length Rectum length Tail length Spicule length Ventromedian supplements

Females

Males

31 1.54 ± 0.07 (1.41-1.66) 24.4 ± 2.0 (21-28) 5.7 ± 0.3 (4.8-6.2) 35.7 ± 3.2 (30-44) 1.3 ± 0.1 (1.0-1.5) 47.7 ± 1.2 (45-51) 12.4 ± 0.4 (11.5-13.0) 9.3 ± 0.3 (8.5-10.0) 19.1 ± 1.1 (16-21) 7.0 ± 0.3 (6.5-7.5) 272 ± 11 (246-307) 83 ± 5.8 (70-98) 48 ± 3 (43-54) 64 ± 5.7 (52-74) 35 ± 1.9 (30-39) 59 ± 8.2 (43-75) 37 ± 4.6 (24-47) 44 ± 3.7 (35-52) – –

11 1.47 ± 0.1 (1.31-1.59) 27.5 ± 2.0 (24-30) 5.7 ± 0.3 (4.9-5.9) 32.4 ± 1.8 (28-35) 1.3 ± 0.06 (1.2-1.4) – 11.9 ± 0.4 (11-13) 9.3 ± 0.3 (9-10) 18.9 ± 1.2 (17-21) 6.9 ± 0.4 (6.0-7.5) 262 ± 10 (248-279) 74 ± 3.7 (67-79) 44 ± 3.3 (40-52) 54 ± 4.5 (47-63) 35 ± 2.7 (30-38) 107 ± 11 (88-125) 46 ± 5.3 (38-53) 45 ± 3.1 (41-50) 57 ± 2.0 (54-60) 6-8

collected near a rivulet leading to the ‘Laguna de las Yeguas’, associated with highland meadows, locally termed ‘borreguiles’ (plant series Genisto baeticae-Festuceto clementei sigmetum), latitude 37.16612°N, longitude 3.585743°E. R EMARKS This is the first record of the species since its original description, which was based on material from northern Hungary (Andrássy, 1971) and new details are herein provided for its better characterisation, especially those referring to the morphology of the lip region and the nature of the female genital system. The Iberian specimens perfectly fit the type population in their general morphology and morphometrics with the exception of their less slender body (a = 21-28 vs 27-32 in females; 24-30 vs 30-34 in males), longer tail (35-52 vs 31-35 μm; c = 30-44 vs 43-55) and longer spicules (54-60 vs 50-54 μm). These differences are regarded as geographical variation since other key features are identical. Compared with other Rhyssocolpus species, R. iuventutis is very similar to R. aquilonius Andrássy, 2003, R. arcticus Ebsary, 1984, R. vinciguerrae Pedram, Pourjam, 146

Robbins, Ye & Peña-Santiago, 2011 and R. vulvostriatus. It can be distinguished from R. arcticus and R. vulvostriatus by the absence vs presence of well developed cuticular flaps in the vulval region (but see below the morphological comparative analysis under the next section); from R. aquilonius by its more anterior vulva (V = 4551 vs 52-54), larger spicules (54-60 vs 41 μm) and fewer (5-8 vs 11) ventromedian supplements; and from R. vinciguerrae by its larger general size (female body = 1.411.66 vs always under 1.4 mm long), absence vs presence of a dorsal cell mass between the cardia and the end of the anterior ovary/testis, transverse vs longitudinal vulval aperture, comparatively shorter female tail (c = 1.0-1.5 vs 1.4-1.9) with more acute vs finely rounded tip, and larger spicules (54-60 vs 43-51 μm). The evolutionary relationships of R. iuventutis, as revealed by a phylogenetic tree derived from partial 18S rDNA analysis (Fig. 5), shows it is most closely related to R. vinciguerrae, the only other species of the genus sequenced so far. Both Rhyssocolpus species form part of a moderately supported clade that also includes representatives of Heterodorus, along with several members of the Qudsianematidae and one sequence of Longidorella parva, another member of the Nordiidae. Nematology


Fig. 5. Maximum-likelihood tree generated with the K2 + G + I model from the SSU rDNA sequence alignment. The scale bar represents the number of substitutions per site units. In clades the first value corresponds to the bootstrap value calculated using the K2 + G + I model and the second value corresponds to the bootstrap value calculated using the GTR + G + I model. Newly obtained sequences are indicated in boldface.

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Taxonomy of the genus Rhyssocolpus E VIDENCE DERIVED FROM MORPHOLOGICAL COMPARATIVE ANALYSIS

When describing Enchodelus morgensis Loof, 1988, Loof compared and discussed several diagnostic characters of the genera Enchodelus and Rhyssocolpus, concluding that the latter was identical to the former, and transferred most Rhyssocolpus species to Enchodelus. Nevertheless, Loof’s actions were not followed by other authors (Jairajpuri & Ahmad, 1992; Andrássy, 2009). Loof (1988) noted that two relevant features, namely the odontostyle length and the nature of the cuticle in the vulval area, were different in both genera, but considered that these differences had “no more than specific value”. The conical-tailed members of Enchodelus, i.e., those comparable to Rhyssocolpus species, were recently separated from rounded-tailed forms by Andrássy (2009, 2011), who grouped them under the (resurrected) genus Heterodorus – until then a junior synonym of Enchodelus – and retained the rounded-tailed species under the (true) genus Enchodelus. Consequently, Rhyssocolpus is herein compared with Heterodorus. Three features might be relevant to separate Rhyssocolpus from Heterodorus using a morphological approach: odontostyle length, uterus morphology, and the presence/absence of cuticular irregularities around the vulval area. Rhyssocolpus representatives have a comparatively small odontostyle, up to 13 μm long, often only up to 10 μm long and always shorter than the lip region diam., whereas Heterodorus species have a larger odontostyle, never less than 10 μm long and very often easily surpassing this value (reaching up to 70 μm), and almost always longer than, only very exceptionally equal to (never shorter than), the lip region diam. The uterus is bipartite in both genera, but the distal part close to the sphincter bears some differentiation (refractive granules in its lumen, hyaline cells surrounding it, etc.) in many members of Heterodorus such as H. arcuatus (Thorne, 1939) Andrássy, 2009, H. brevidentatus, H. geraldi (Winiszweska, 1987) Andrássy, 2009, H. magnificus Altherr, 1952 (the type species), H. morgensis, H. porosus Guerrero, Liébanas & Peña-Santiago, 2007 and H. veletensis Guerrero, Liébanas & Peña-Santiago, 2007, which have recently been studied in some detail (cf., Guerrero & Peña-Santiago, 2007; Guerrero et al., 2007). This type of uterine differentiation has not been reported in Rhyssocolpus forms. The presence of very strong cuticular irregularities in the vulval area is a remarkably common feature of Rhyssocolpus 148

species and has not been described with comparable development in Heterodorus forms, although it is known to occur in isolated species of several genera such as Eudorylaimus and Mesodorylaimus Andrássy, 1959. In conclusion, from a morphological perspective, Heterodorus and Rhyssocolpus show relevant differences that allow their differentiation into two groups. E VIDENCE DERIVED FROM MOLECULAR ANALYSIS The results obtained from partial 18S rDNA analysis (Fig. 5), including new sequences of four species (see Material and methods section), show that representatives of the Nordiidae are split into three major subgroups with variable clade support. The first group consists of sequences of Pungentus Thorne & Swanger, 1936 plus one sequence of Californidorus Robbins & Weiner, 1978. The second includes sequences of Enchodelus as well as several sequences of four non-nordiid genera: Prodorylaimus Andrássy, 1959 in Dorylaimidae de Man, 1876, and Crassolabium Yeates, 1967, Eudorylaimus and Epidorylaimus Andrássy, 1986 in Qudsianematidae. The third group is formed by sequences of the members of Heterodorus and Rhyssocolpus, along with other species from the Qudsianematidae and Nordiidae (see above). These results generally agree with those published by Holterman et al. (2008) and Pedram et al. (2011), and confirm that the Nordiidae is not a natural (monophyletic) taxon and that the relationships between dorylaimid families are still far from being well established. The evolutionary relationships between the members of the third clade are not well resolved in the tree and hence serious uncertainties persist concerning the systematics of its members, especially due to the inclusion of the qudsianematid taxa and because the sequences of Heterodorus species do not group together. Thus, as yet there is insufficient molecular evidence to elucidate the nature of the relationship between Heterodorus and Rhyssocolpus (cf., Pedram et al., 2011), although it is relevant that the two sequences of Rhyssocolpus are the closest to each other within the clade. I NTEGRATIVE APPROACH Both morphological and molecular (with currently available data) evidence support the conclusion that Rhyssocolpus species form a natural group. On the one hand, the morphological differences between Rhyssocolpus and Heterodorus are small but certainly significant, and can be interpreted under a cladistic approach. The very short and attenuate odontostyle along with the presence of Nematology


strong cuticular irregularities at vulval level are considered apomorphic conditions of their respective characters, and synapomorphies which define the Rhyssocolpus pattern. The differentiations observed in the distal part of the uterus in members of Heterodorus are equally regarded as an apomorphic state characterising the Heterodorus pattern. On the other hand, in spite of available molecular data not satisfactorily defining the evolutionary relationships of Rhyssocolpus and Heterodorus, the two available Rhyssocolpus sequences lie close together in the tree. Thus, contrary to Loof’s (1988) opinion, and following the opinions of other authors (Jairajpuri & Ahmad, 1992; Vinciguerra, 2006; Andrássy, 2009), Enchodelus, Heterodorus and Rhyssocolpus are considered as valid genera, with Heterodorus and Rhyssocolpus probably sharing a common recent ancestor and being distinctly separated from Enchodelus. A revised taxonomic characterisation of Rhyssocolpus therefore follows.

Genus Rhyssocolpus Andrássy, 1971 D IAGNOSIS ( EMENDED ) Nordiidae. Small- to medium-sized nematodes, 0.701.98 mm long. Cuticle dorylaimoid. Lip region continuous or offset by depression, exceptionally by constriction. Odontostyle small and attenuate, never longer and usually shorter than lip region diam., with narrow lumen and minute aperture. Guiding ring double. Odontophore rod-like. Pharynx dorylaimoid, with gradual enlargement and pharyngeal expansion occupying up to two-fifths (2740%) of total neck length; S1 N (AS) very reduced. Female genital system didelphic-amphidelphic, uterus bipartite, pars refringens vaginae present, vulval aperture a transverse or longitudinal slit. Strong cuticular irregularities (wrinkles, folds, etc., and occasionally flaps) present near vulval area. Caudal region conical, regularly curved ventrad in both sexes. Spicules dorylaimoid, 37-60 μm long. Male ventromedian supplements 2-11, spaced, with hiatus. R EMARKS Rhyssocolpus is a rather homogeneous dorylaimid taxon from a morphological perspective, its species being separated by means of small and subtle differences (see below). It seems to be a genuine representative of the Holarctic nematode fauna and is widely spread in Eurasia and North America (see geographical distribution in Table 2). Vol. 17(2), 2015

T YPE SPECIES R. vulvostriatus (Stefański, 1924) Andrássy, 1971 = Dorylaimus vulvostriatus Stefański, 1924 = Eudorylaimus vulvostriatus (Stefański, 1924) Andrássy, 1959 = Enchodelus vulvostriatus (Stefański, 1924) Loof, 1988 = Dorylaimus gracilis apud Micoletzky (1925) nec de Man (1884) [syn. by Andrássy (1971)] OTHER SPECIES R. aquilonius Andrássy, 2003 R. arcticus Ebsary, 1984 = Enchodelus ebsaryi Loof, 1988 R. fluviatilis Gagarin, 1985 R. iuventutis Andrássy, 1971 = Enchodelus iuventutis (Andrássy, 1971) Loof, 1988 R. microdorus (Schiemer, 1965) Andrássy, 1971 = Enchodelus microdorus Schiemer, 1965 R. repis (Brzeski, 1992) Vinciguerra, 2006 = Enchodelus repis Brzeski, 1992 R. vinciguerrae Pedram, Pourjam, Robbins, Ye & PeñaSantiago, 2011 S PECIES TRANSFERRED TO , OR RETAINED UNDER , OTHER GENERA

R. alleni (Brzeski, 1962) Andrássy, 1986, retained under Eudorylaimus = Eudorylaimus alleni Brzeski, 1962 R. brasiliensis (Meyl, 1956) Andrássy, 1986, transferred to Eudorylaimus (see below) = Dorylaimus brasiliensis Meyl, 1956 = Prodorylaimus brasiliensis (Meyl, 1956) Andrássy, 1959 R. paradoxus (Loof, 1975) Andrássy, 1986, retained under Eudorylaimus = Eudorylaimus paradoxus Loof, 1975 N OTES ON SOME SPECIES Rhyssocolpus alleni: Loof (1988) suggested that this species “probably . . . does not belong to the Enchodelus complex either”, and several of its diagnostic features support its retention under Eudorylaimus: large size (body length 2.5-2.7 mm, see Brzeski (1962) and Loof (1971)), odontostyle 21-23 μm long, pharyngeal expansion occupying about one-half of total neck length, and absence of strong cuticular irregularities in the vulval area. 149

150 12∗ – – – – – – – 9 –

25 0.81-1.12 20-29 4.0-5.1 20-27 1.5-2.2 50-57 10-11 15 0.85-1.07 24-29 4.2-4.9 19-25 1.5-1.7 – –

11 1.04-1.37 21-29 4.6-5.3 26-34 1.4-1.9 48-55 11-13 8 1.13-1.35 25-33 4.7-5.6 24-28 1.5-1.7 – 11-12 14∗ – – – – – – 53-54 – 12-14 50 –

repis

vinciguerrae

1.37-1.68 1.40-1.83 1.71-1.98 1.74-1.88 2.40 1.43-1.62 1.19-1.87 1.25-1.44 1.33-1.48 1.40

1.42-1.65 1.30-1.45 1.41-1.66 1.31-1.59

17-23 19-29 22-33 32-34 31 23-25 20-31 25-27 27-28 26

27-32 30-34 21-28 24-30

5.0-6.7 5.5-6.0 5.7-6.9 5.4-6.6 5.5 6.0-7.2 5.1-8.1 4.7-5.5 5.4-7.2 5.9

5.7-6.6 5.6-6.8 4.8-6.2 4.9-5.9

21-38 32-36 33-41 35-46 50 39-55 30-53 43-45 44-50 49

– – – – – – – – – –

–

50 – 47-51 – 53 48-52

7-8 7.5 –

– 11-12∗ 51-53 10.5∗ – – 7-9 7-9

9-10 – 8-10 9-10

43-55 1.0-1.1 47-51 12-13 42-50 1.0 – – 30-44 1.0-1.5 45-51 11-13 28-35 1.2-1.4 – 11-13 –

90-128 –

50∗ 59∗

30-33 – 70-79 67-79

31-35 – 35-52 41-50

248∗ 279∗ – – – – – – – 237∗

33% – – – – – – – – –

227-265 31-35% 232-275 –

196-236 27-35% 197-229 –

37 – – – – – – – – –

35-50 44-55

31-51 38-46

207, 214 30% 43, 38∗ ∗ 202-205 30-35% 36∗ ∗ – – 203

232-250∗ – 246-307 248-279

– – – – – – – 58-60 –

– 43-51

– 37-41

36 – –

– 50-54 – 54-60

–

– 50-58

7 – – – – 9-12 –

8-11

– 6-8

– 2-6

6 – 5

– 5-7 – 6-8

–

– 7-11

13 14 4 15

Denmark Italy

12

10, 11

9

8

Switzerland Italy

?

Poland

Iran

Korea

6 7

5

Spain

Austria Italy

4

3

2

Hungary

Russia

Canada

Ref. 1

Abbreviations: LRD = lip region diam.; Odont. = odontostyle; Ph.exp. = pharyngeal expansion; Ve.sup = ventral supplements; Distrib. = geographical distribution. References: 1: Andrássy (2003); 2: Ebsary (1984); 3: Gagarin (1985); 4: Andrássy (1971); 5: Present paper; 6: Schiemer (1965); 7: Vinciguerra & De Francisci (1973); 8: Brzeski (1992); 9: Pedram et al. (2011); 10: Stefański (1924); 11: Thorne & Swanger (1936); 12: Micoletzky (1925); 13: Altherr (1952); 14: Zullini (1970); 15: Zullini (1971). ∗ Values calculated from other measurements and/or illustrations.

17 15 4 3 5 206 4 3

2 0.95, 0.92 25, 24 4.6, 4.3 22, 24 2 0.91-0.92 21-23 4.5 21-23 0.69 23 3.4 19

microdorus

vulvostriatus

8-10 9-10

31 20 31 11

283∗ 268∗

11-13 242-304∗ 35-40% 46-56∗

iuventutis

1.49-1.62 22-24 4.9-6.7 29-33 1.4-1.5 50-51 41 579

8-12 –

3

28-37 5.9-7.9 31-37 1.3-1.6 46-52 11-13 36-43 6.0-7.0 29-37 1.1-1.6 – –

fluviatilis

1.4-2.2 1.7-2.0

25 22

arcticus

Table 2. Compendium of morphometrics of species belonging to Rhyssocolpus Andrássy, 1971. All measurements are in μm except for L (mm). Species n/sex L a b c c V LRD Odont. Neck Ph.exp. Tail Spicule Ve.sup. Distrib. aquilonius 5 1.23-1.40 21-24 4.6-4.9 27-32 1.3-1.5 52-54 12-13 7-9 265-285 32-38% 40-48 – – Alaska 1.50 27 5.5 35 1.2 – – – – – 44 52 11


Nematology


Rhyssocolpus arcticus and R. iuventutis: the separation of these two species is problematic. The presence and absence, respectively, of vulval flaps is the only relevant morphological difference, whereas the ranges of their morphometrics widely overlap. Rhyssocolpus brasiliensis: the true identity of this species remains obscure, but the available information suggests that it is not compatible with the Rhyssocolpus pattern: odontostyle longer (1.3 times) than lip region diam. and comparatively stronger, pharyngeal expansion occupying ca 45% of total neck length, and 1314 contiguous ventromedian supplements. It is provisionally transferred to Eudorylaimus as E. brasiliensis (Meyl, 1956) comb. n. Rhyssocolpus microdorus and R. repis: the separation of these two species is also problematic. The absence and presence, respectively, of vulval flaps is the only relevant morphological difference, whereas the ranges of their morphometrics widely overlap. Rhyssocolpus fluviatilis: the original description of this species is not very detailed and some doubts persist as to the identity as the lip region is distinctly angular and offset by a constriction (see Gagarin’s Figure 3.3) and the odontostyle is as long as the lip region diam. – two features that do not fit the pattern of the genus. Nevertheless, other characters (attenuate odontostyle, guiding ring double and the presence of coarse cuticle irregularities in the vulval area) support its classification under this genus. Rhyssocolpus paradoxus: as Loof (1988) pointed out, this species does not fit either the Enchodelus pattern, or the Rhyssocolpus pattern because of its larger general size (body length 2.37-3.13 mm), odontostyle 28-35 μm long, pharyngeal expansion occupying more than twofifths of total neck length, absence of strong cuticle irregularities near the vulval area, and higher number (2227) of contiguous ventromedian supplements.

Key to species 1 Lip region distinctly angular and offset by constriction; male unknown . . . . . . . . . . . . . . . . . . . . . . . . . . . . fluviatilis Lip region rounded or weakly angular and continuous or offset by depression; male known . . . . . . . . . . . . . . . 2 2 Smaller general size, body length up to 1.1 mm long, spicules up to 41 μm long. . . . . . . . . . . . . . . . . . . . . . . . .3 Larger general size, body length over 1.0 mm long, spicules 43 μm long or more . . . . . . . . . . . . . . . . . . . . . . 4 3 Vulval flaps absent . . . . . . . . . . . . . . . . . . . . . . microdorus Vol. 17(2), 2015

Vulval flaps present . . . . . . . . . . . . . . . . . . . . . . . . . . . repis 4 Vulval flaps present . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Vulval flaps absent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 5 Tail longer (c = 1.3-1.6), strongly curved ventrad, hook-like; most posterior ventromedian supplement anterior to anterior end of spicules, far from adcloacal pair and close to second-most posterior ventromedian supplement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . arcticus Tail shorter, about as long as anal body diam., weakly curved ventrad, not hook-like; most posterior ventromedian supplement anterior to anterior end of spicules, mid-way between adcloacal pair and second-most posterior ventromedian supplement . . . . . . . . . vulvostriatus 6 Male bearing 11 ventromedian supplements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . aquilonius Male bearing 5-8 ventromedian supplements . . . . . . . . 7 7 Larger general size, body 1.31-1.66 mm long; vulva transverse; spicules 54-60 μm long . . . . . . . . . iuventutis Smaller general size, body 1.04-1.37 mm long; vulva longitudinal; spicules 43-51 μm long . . . . vinciguerrae A compendium of morphometrics for valid species is presented in Table 2.

Acknowledgements The authors especially thank the financial support received from the projects entitled Fauna Ibérica: Nematoda, Dorylaimoidea (excepto Longidoridae) (Spanish Ministry of Science and Innovation, ref. CGL200766786-C08-08; co-financed FEDER) and Revision de la familia Nordiidae (Consejería de Innovación, Ciencia y Empresa; Andalusian Regional Government, Spain; ref. RNM-475; co-financed FEDER). SEM pictures were obtained with the assistance of technical staff and the equipment of ‘Scientific and Technical Instrumentation Center’, University of Jaén.

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