Phylogenetic position of the Paramicrolaimidae, description of a new Paramicrolaimus species and erection of a new order to accommodate the Microlaimoidea (Nematoda: Chromadorea)

Phylogenetic position of the Paramicrolaimidae, description of a new Paramicrolaimus species and... Abstract The phylogenetic position of Paramicrolaimidae Lorenzen, 1981, a rare group of marine free-living nematodes, has been the subject of debate due to the unique morphology of the buccal cavity, an unusual combination of other morphological traits, and lack of molecular sequences. Here, Paramicrolaimus hohonucola sp. nov. is described from the continental slope of New Zealand, and the position of the family Paramicrolaimidae is investigated based on analyses of the small subunit (SSU) and D2–D3 region of large subunit (LSU) rDNA genes. This is the first record of Paramicrolaimidae from the Southwest Pacific, and the deepest record of the family to date (347–1514 m depth). Observations using scanning electron microscopy revealed the presence of a transverse mouth opening formed by the fusion of the two ventrosublateral lip lobes and enlarged dorsal lip lobe, resulting in partial dorsoventral symmetry, which is an unusual feature within the phylum. Phylogenetic analyses suggest a close relationship between the Paramicrolaimidae and the family Selachinematidae Cobb, 1915 (order Chromadorida Chitwood, 1933). The Paramicrolaimidae lack cuticle punctations, a key morphological trait of all families currently classified within the Chromadorida; however, in the absence of any clear morphological affinities with other chromadorean orders, we propose that Paramicrolaimidae be placed within Chromadorida based on evidence for a close relationship with Selachinematidae as shown by SSU and LSU phylogenies. Our SSU phylogeny supports the results of previously published analyses showing a close relationship between Molgolaimus Ditlevsen, 1921 and the Microlaimidae Micoletzky, 1922. We therefore propose that Molgolaimus be removed from Desmodoroidea where it is currently classified and instead placed within Microlaimoidea. We also propose that the family Molgolaimidae Jensen, 1978 be reinstated and moved to accommodate Molgolaimus within Microlaimoidea. As in previous SSU phylogenies, our analyses provide no evidence for a close relationship between Desmodoroidea and Microlaimoidea, which currently comprise Desmodorida. Based on this molecular evidence, the clear morphological differences between Microlaimoidea and the closely related Chromadorida, and the lack of a synapomorphy linking Desmodoroidea and Microlaimoidea, we propose the order Microlaimida ord. nov. to accommodate the superfamily Microlaimoidea. D2–D3 region of large subunit (LSU) rDNA gene, Deep-Sea, Desmmodoroidea, Microlaimida order nov, Molgolaimus, Paramicrolaimus hohonucola sp. nov, small subunit (SSU) rDNA gene INTRODUCTION The placement of several marine nematode taxa remains problematic, largely because of the relatively small number of phylogenetically informative morphological traits available for higher level classification, and the frequent occurrence of convergent evolution within the phylum (Lorenzen, 1981; Filipjev, 1921; van Megen et al., 2009). Over the last two decades molecular phylogenies have provided important new insights into relationships among and within nematode orders (e.g. Bik et al., 2010). The nematode classification of De Ley & Blaxter (2002), based on a combination of a new molecular phylogeny from small subunit (SSU) rDNA and existing morphological phylogenies, and later updated by the same authors (De Ley & Blaxter, 2004), was originally based mostly on sequences of terrestrial and parasitic taxa. Meldal et al. (2007) later built on this molecular phylogeny by providing a greater representation of clades containing marine species. Their analyses, as well as the subsequent analyses of Holterman et al. (2008) and van Megen et al. (2009), indicate that the largely marine orders Chromadorida Chitwood, 1933 and Desmodorida De Coninck, 1965 are closely related and are the most basal in the class Chromadorea. These analyses also put into question the placement of the families Microlaimidae Micoletzky, 1922 and Monoposthiidae Filipjev, 1934, which together with the family Aponchiidae Gerlach, 1963 comprise the superfamily Microlaimoidea Micoletzky, 1922; at present, Microlaimoidea is classified within Desmodorida alongside Desmodoroidea Filipjev, 1922, but molecular phylogenies suggest instead that Microlaimoidea is more closely related to Chromadorida than to the rest of the Desmodorida (Meldal et al., 2007; Holterman et al., 2008). In addition, recent phylogenetic analyses suggest that the genus Molgolaimus Ditlevsen, 1921, which is currently placed in Desmodoroidea (family Desmodoridae Filipjev, 1922), in fact belongs to Microlaimoidea (Meldal et al., 2007; Leduc & Zhao, 2016). However, this new evidence is not included in recent taxonomic treatments of these orders (Tchesunov, 2014a, b), which are still largely based on the phylogeny presented by De Ley & Blaxter (2002, 2004), The classification of other marine taxa remains controversial due to the presence of unique or conflicting morphological features and absence of any molecular sequence data. An example of such a family is Paramicrolaimidae Lorenzen, 1981, which was erected to accommodate the genus Paramicrolaimus Wieser, 1954, a rare group of marine free-living nematodes. This genus is characterized by the unusual morphology of the buccal cavity with a medium to large dorsal tooth which does not project completely into the pharyngeal lumen but is instead set within, and surrounded by, the pharyngeal wall, and is always associated with a conspicuous pharyngeal gland (Lorenzen, 1981). To our knowledge, this buccal cavity structure is not found in any other nematode taxon (i.e. it is an autapomorphy of the family). This feature, and an unusual combination of other morphological traits, has resulted in the systematic position of Paramicrolaimus being changed several times since it was first described. Wieser (1954) first placed it in the family Microlaimidae because of the three distinct circles of cephalic sense organs, the deep buccal cavity provided with a dorsal tooth and the spiral amphid with 1.25 turns. Andrassy (1976) grouped Paramicrolaimus together with the genus Ohridius Gerlach & Riemann, 1973/1974, which has since been synonymized with Domorganus Goodey, 1946 within the order Plectida Gadea, 1973. Paramicrolaimus was later placed within Stilbonematinae Chitwood, 1936 (order Desmodorida) by Jensen (1978) based on the reduced buccal cavity, the slender body shape, an inferred association with bacteria and his observations of only one testis in males of Paramicrolaimus. The same author also highlighted the similarities between Paramicrolaimus and Coninckia Gerlach, 1956 (order Araeolaimida De Coninck & Schuurmans Stekhoven, 1933) in the shape of the male amphid, the arrangement of the cephalic sense organs, the structure of the pharynx and the shape of the tail. Lorenzen (1981) later showed that inferred affinities with the Stilbonematinae, which are characterized by males with only one testis, was incorrect because Paramicrolaimus in fact possesses two testes. He erected the monogeneric family Paramicrolaimidae, which he placed within Leptolaimina Lorenzen, 1981, a suborder that gathered all phylogenetically unsettled families within the order Chromadorida. De Ley & Blaxter (2002) later placed Paramicrolaimidae within the superfamily Leptolaimoidea Örley, 1880 (order Plectida) in their phylogenetic analyses based on SSU rDNA sequences; however, no molecular sequences were available for Paramicrolaimidae at the time and the position of this family was therefore presumably determined based on possible relationships with families of Leptolaimina, such as Leptolaimidae Örley, 1880 and Bastianiidae De Coninck, 1965, for which molecular sequences were available. Most recently, Holovachov (2014) acknowledged the uncertain affinities of the family Paramicrolaimidae, but noted morphological similarities with Tubolaimoididae Lorenzen, 1981 and Tarvaiidae Lorenzen, 1981 (order Plectida). The placement of Paramicrolaimidae awaits molecular analyses to clarify its relationships within Chromadorea. Paramicrolaimus currently comprises four valid species, each described from only a few individuals collected from coastal and shelf sediments (Jacob et al., 2015). During a study of the diversity of nematodes on the continental slope of New Zealand, Paramicrolaimus specimens were isolated from multiple core samples for morphological and molecular analyses. All specimens were identified as belonging to a single species, herein described as Paramicrolaimus hohonucola sp. nov. The phylogenetic relationships of the family Paramicrolaimidae were investigated based on SSU rDNA sequences representing the orders Chromadorida, Desmodorida, Araeolaimida, Plectida and Monysterida Filipjev, 1929 which contain the bulk of marine species with the class Chromadorea, and based on large subunit (LSU) rDNA sequences at the within-order level. The SSU rDNA analyses also led to a re-evaluation of the current classification of the Microlaimoidea, as previously indicated by the phylogenies of Meldal et al (2007) and Holterman et al. (2008). METHODS Sampling sites and sample processing Sediment samples were obtained from continental slope sites on Chatham Rise off the east coast of the South Island of New Zealand, from the Hikurangi Margin to the south-east of the North Island and from the Bay of Plenty off the north-east coast of the North Island (Fig. 1). Sampling was conducted using RV Tangaroa during National Institute of Water and Atmospheric Research (NIWA) voyage TAN0705 (April 2007), TAN1103 (February 2011) and TAN1701 (January 2017) to the Chatham Rise, voyage TAN1004 (April 2010) to the Hikurangi Margin and voyage TAN1206 (April 2012) to the Bay of Plenty. Figure 1. View largeDownload slide Map of central New Zealand and surrounding continental margin showing location of core samples obtained from Chatham Rise (CR, circular symbols), Hikurangi Margin (HIK, diamond symbol) and Bay of Plenty (BoP, triangular symbol); 250, 500, 1000 and 2000 m depth isobaths are shown. The RV Tangaroa sampling voyages are listed in the legend. Figure 1. View largeDownload slide Map of central New Zealand and surrounding continental margin showing location of core samples obtained from Chatham Rise (CR, circular symbols), Hikurangi Margin (HIK, diamond symbol) and Bay of Plenty (BoP, triangular symbol); 250, 500, 1000 and 2000 m depth isobaths are shown. The RV Tangaroa sampling voyages are listed in the legend. Sediment samples were collected using an Ocean Instrument MC-800A multicorer (internal diameter of core = 9.52 cm). Subsamples for the analysis of nematodes were taken using a core with an internal diameter of 26 or 29 mm to a depth of 5 cm. These cores were sliced into 0–1 and 1–5 cm sediment depth layers and preserved in 10% buffered formalin. During voyage TAN1701, the upper 5 cm layer of sediment remaining in the multicorer tube after subsampling was kept frozen. All sediment samples were rinsed on a 1 mm mesh to remove macro-infauna and on a 45 µm mesh to retain nematodes, which were extracted from the sieved sediment by the Ludox flotation method (Somerfield & Warwick, 1996). Specimens for light microscopy were transferred to distilled water, sorted using a dissecting microscope, transferred to glycerol and mounted onto permanent slides using the method of Somerfield & Warwick (1996). Frozen sediment samples (TAN1701) were thawed overnight, extracted using the Ludox flotation methods and transferred to freshwater. Specimens were sorted using a dissecting microscope, and were either transferred to a 10% formalin solution for observation using scanning electron microscopy (SEM), or transferred to lysis buffer and frozen for molecular analyses. Specimens for molecular analyses were first mounted onto a temporary slide in a drop of water to verify their identity and pictures were taken to provide image vouchers. SEM observations were conducted on specimens retrieved from frozen samples (TAN1701) and specimens previously mounted in glycerol (TAN0705 and TAN1004). Mounted specimens were gradually transferred from glycerol back to 10% formalin prior to fixation in 4% osmium tetroxide overnight. Specimens were then gradually transferred to pure ethanol using a grade ethanol series, critical point dried and mounted onto stubs before coating with gold using a sputter coater. Observations were made using a Hitachi TM3000 tabletop SEM at high vacuum mode. All measurements are in µm, and all curved structures were measured along the arc. The terminology used for describing the arrangement of morphological features such as setae follows Coomans’ (1979) typology. Type specimens are held in the NIWA Invertebrate Collection (Wellington), and the National Nematode Collection of New Zealand (NNCNZ, Auckland). Abbreviations in the text are as follows: a, body length/maximum body diameter; b, body length/pharynx length; c, body length/tail length; c′, tail length/anal body diameter; cbd, corresponding body diameter; %V, vulva distance from anterior end of body × 100/total body length. DNA extraction, sequence processing and phylogenetic inference Individual nematodes were used for DNA extraction by lysing in a buffer of 20 μL containing proteinase K (Williams et al., 1992) and DNA was extracted using the method of Zheng et al. (2002). Extracted DNA was used for PCR amplification of nearly the full length of SSU rDNA gene with two sets of primers, 1096F/1912R and 1813F/2646R (Holterman et al., 2006). Primers, D2A and D3B (Nunn, 1992) were used to amplify the D2/D3 expansion segments of the LSU rDNA gene. The PCR composition and cycling conditions for the amplification of the SSU and LSU genes, and sequencing were conducted as per Zhao et al. (2015). The sequences obtained were used to construct phylogenetic trees in Geneious 10.1.3 (http://www.geneious.com; Kearse et al. 2012). Related sequences were downloaded from GenBank and aligned with ClustalX in Geneious using the default parameter values. Phylogenetic affinities of Paramicrolaimus were investigated using the SSU and LSU rDNA genes. The SSU rDNA gene is the only locus known to resolve deep relationships among nematode taxa, whereas the LSU rDNA gene is informative at lower taxonomic levels only (De Ley et al., 2005; Bik et al., 2010). An initial phylogenetic analysis based on SSU rDNA sequences was therefore conducted using representative species (one per genus) from the orders Chromadorida, Desmodorida, Monhysterida, Araeolaimida and Plectida, which comprise the bulk of marine diversity in the class Chromadorea. Only sequences over 1200 bp were used, with the exception of Microlaimus De Man, 1880 and Gomphionema Wieser & Hopper, 1966, for which only relatively short sequences were available (399 and 680 bp, respectively). Based on the results of the first analysis, a more focused investigation of the placement of Paramicrolaimus was conducted based on Chromadorida SSU rDNA sequences, as well as Microlaimidae and Monoposthidae sequences, which have been shown to cluster either with or near the Chromadorida (Meldal et al., 2007; Holterman et al., 2008). A LSU rDNA tree was also built focusing on the Selachinematidae, the family most closely related to Parmicrolaimus hohonucola sp. nov., as identified from the SSU rDNA analyses. Sequences of the genus Richtersia Steiner, 1916, which may have affinities with the family Selachinematidae (Neira & Decraemer, 2009), were included in this analysis. Phylogenies were built in Geneious 10.1.3 (http://www.geneious.com; Kearse et al. 2012). The related sequences were aligned with ClustalW in Geneious using the default parameter values. PAUP*4.0b10 (Swofford, 2002) was used to select the best model using the Akaike information criterion. A Bayesian tree was constructed with MrBayes under the best-fit model [GTR (general time-reversible) + I (proportion of invariable sites) + G (gamma distribution)] for both SSU and LSU genes (Huelsenbeck & Ronquist, 2001). The trees were run with chain length of 1100000, burn-in length of 100000 and rooted Tobrilus gracilis (Bastian, 1865) (order Triplonchida Cobb, 1919) for SSU and Paracanthonchus miltommatus Leduc & Zhao, in press (order Chromadorida) for LSU, respectively. The Bayesian trees were viewed in Tracer 1.4 (Rambaut & Drummond, 2007) and edited in PowerPoint. The results of the phylogenetic analyses described above were tested using maximum likelihood analysis as an alternative tree-building method. These analyses were conducted in Geneious 10.1.3 with default settings and 1000 bootstrap replicates (Guindon & Gascuel, 2003). RESULTS Molecular analyses The Bayesian topology recovered in the SSU-based phylogeny shows that the Chromadorida is not monophyletic (clades A2 and B2 in Fig. 2), and together with the Microlaimoidea and Molgolaimus (clade A1), split early from other lineages, followed by the main Desmodorida clade (clade B1). The tree topology, however, did not resolve the branching order of these groups. Clade A1, which comprises the Microlaimoidea and Molgolaimus, is not grouped with the Desmodoroidea and is instead recovered as a sister taxon to the Chromadorida (85% posterior probability and 35% bootstrap value). The Araeolaimida, Monhysterida and Plectida together formed a relatively well-supported clade (clade B3 in Fig. 2; 100% posterior probability and 70% bootstrap value), but none of these individual orders was monophyletic. Figure 2. View largeDownload slide Bayesian tree of marine Chromadorean orders inferred from small subunit (SSU) sequences under the general time-reversible (GTR) + proportion of invariable sites (I) + gamma distribution (G) model. Posterior probability (left) and bootstrap values (right) are given on corresponding clades. Sequences obtained in this study are shown in bold; orders are listed on the far right and the position of the families Paramicrolaimidae, Selachinematidae, Microlaimidae and Monoposthiidae is shown. Main clades are shown by blue letters and/or numerals. The scale stands for substitutions per site. Figure 2. View largeDownload slide Bayesian tree of marine Chromadorean orders inferred from small subunit (SSU) sequences under the general time-reversible (GTR) + proportion of invariable sites (I) + gamma distribution (G) model. Posterior probability (left) and bootstrap values (right) are given on corresponding clades. Sequences obtained in this study are shown in bold; orders are listed on the far right and the position of the families Paramicrolaimidae, Selachinematidae, Microlaimidae and Monoposthiidae is shown. Main clades are shown by blue letters and/or numerals. The scale stands for substitutions per site. Our phylogenetic analyses indicate that Paramicrolaimidae constitutes a sister taxon to the family Selachinematidae (clade B2 in Fig. 2; Fig. 3). This relationship was relatively well supported in Bayesian analyses (100 and 74% posterior probability in Chromadorea- and Chromadorida-level analyses, respectively), but only weakly supported in maximum likelihood analyses (46 and 29% bootstrap values in Chromadorea- and Chromadorida-level analyses, respectively). The placement of Selachinematidae was not well resolved in our analyses of the chromadorean orders; only one of the selachinematid genera (Latronema Wieser, 1954) was placed within Chromadorida, whereas the remaining four genera formed clade B2 together with P. hohonucola sp. nov. outside of the main Chromadorida clade and all the other main clades (Fig. 2). The placement of clade B2, however, was poorly supported (73% posterior probability and 35% bootstrap value). In our analysis of SSU rDNA sequences focusing on the Chromadorida, Selachinematidae formed a monophyletic group, which together with P. hohonucola sp. nov., formed a sister group to the remaining chromadorid taxa, although there was no support for this topology (Fig. 3). Figure 3. View largeDownload slide Bayesian tree of the order Chromadorida inferred from small subunit (SSU) sequences under the general time-reversible (GTR) + proportion of invariable sites (I) + gamma distribution (G) model. Posterior probability (left) and bootstrap values (right) are given on corresponding clades. Families are shown on the right-hand side. Taxa currently classified with the order Desmodorida are shown on light grey background. The scale stands for substitutions per site. Figure 3. View largeDownload slide Bayesian tree of the order Chromadorida inferred from small subunit (SSU) sequences under the general time-reversible (GTR) + proportion of invariable sites (I) + gamma distribution (G) model. Posterior probability (left) and bootstrap values (right) are given on corresponding clades. Families are shown on the right-hand side. Taxa currently classified with the order Desmodorida are shown on light grey background. The scale stands for substitutions per site. In the LSU tree of the Selachinematidae (Fig. 4), P. hohonucola sp. nov. was placed in a well-supported clade (100% posterior probability and 96% bootstrap value) comprising the bulk of Selachinematidae sequences representing the genera Choanolaimus De Man, 1880, Halichoanolaimus De Man, 1886, Bendiella Leduc, 2013, Cheironchus Cobb, 1917 and Richtersia Steiner, 1916. Figure 4. View largeDownload slide Bayesian tree inferred from D2–D3 of large subunit (LSU) sequences under the general time reversible (GTR) + proportion of invariable sites (I) + gamma distribution (G) model. Posterior probability (left) and bootstrap values (right) greater are given on corresponding clades. Sequences obtained in this study are shown in bold. The scale stands for substitutions per site. Figure 4. View largeDownload slide Bayesian tree inferred from D2–D3 of large subunit (LSU) sequences under the general time reversible (GTR) + proportion of invariable sites (I) + gamma distribution (G) model. Posterior probability (left) and bootstrap values (right) greater are given on corresponding clades. Sequences obtained in this study are shown in bold. The scale stands for substitutions per site. Taxonomic account Order Chromadorida Chitwood, 1933 Family Paramicrolaimidae Lorenzen, 1981 Synonyms: None. Diagnosis: Cuticle striated from anterior edge of amphids; no longitudinal bars. Short somatic setae present. Lip pairs usually fused into three, or in some cases two, lobes. Six inner labial papillae present; six outer labial setae located in a separate circle and slightly anterior to the four cephalic setae; cephalic setae located anterior to the amphid. Four subcephalic setae sometimes present at level of amphids. Amphidial fovea spiral with less than 2 turns. Buccal cavity narrow, tubular, asymmetrical; dorsal tooth present, set within pharyngeal tissue and only protruding slightly into buccal cavity, sometimes with lateral pointed projections, always connected to a dorsal gland; smaller ventrosublateral teeth also present. Pharynx muscular, cylindrical, with posterior bulb; muscular cardia present. Secretory-excretory system present; renette cell elongated, located at level of or posterior to cardia; excretory ampulla and duct located slightly posterior to level of nerve ring. Female reproductive system didelphic-amphidelphic with equally developed branches, ovaries reflexed antidromously. Male reproductive system diorchic with two outstretched testes. Spicules symmetrical, arcuate; gubernaculum present. Row of midventral precloacal supplements consisting of cup-shaped structures with spout-like structures pointing posteriorly. Three caudal glands and spinneret present. Remarks: The present diagnosis has been adapted from Holovachov (2014) to better correspond with our own observations, other Paramicrolaimidae species descriptions and with the current terminology of the structures described. Our observations show that the cuticle of Paramicrolaimus hohonucola sp. nov. is striated (not annulated), and that the lip region comprises two lobes, unlike other species of the genus which were described as possessing three equal lobes. In P. hohonucola sp. nov. the dorsal lobe of the lip region is enlarged and is almost equal in size and shape to the two ventrosublateral lobes, which are at least partially or completely fused together. As a result, the mouth is a straight or slightly bent transverse opening, with four inner labial papilla along its ventral side and two inner labial papilla dorsally. The subcephalic setae were described as absent in previous diagnoses, but four subcephalic setae are present in P. hohonucola sp. nov. at the level of the amphids. Males have also been described as monorchic or diorchic because only one testis was observed in the original descriptions of P. spirulifer by Jensen (1978). However, Lorenzen (1981) observed that this species is in fact diorchic like the rest of the Paramicrolaimidae. Genus Paramicrolaimus Wieser, 1954 Synonyms: None. Type species: Paramicrolaimus primus Wieser, 1954. Diagnosis: See family diagnosis. Remarks: The description of P. primus is based on one female specimen. It is therefore not adequately described and is considered species inquirenda. Valid species: P. spirulifer Wieser, 1959 P. mirus Tchesunov, 1988  = P. minus Tchesunov, 1988 in Huang & Zhang, 2005 (lapsus calami) P. damodarani Jacob, Jaleel & Vijayan, 2015 Invalid species: P. acanthus Jayasree & Warwick, 1977  = Microlaimus acanthus (Jayasree & Warwick, 1977) Kovalyev & Tchesunov, 2005 P. conothelis Lorenzen, 1973  = Microlaimus conothelis (Lorenzen, 1973) Jensen, 1978 P. lunatus Wieser & Hopper, 1967  = Microlaimus lunatus (Wieser & Hopper, 1967) Jensen, 1978 P. papillatus (Gerlach, 1956) Wieser & Hopper, 1967  = Microlaimus papillatus Gerlach, 1956 (op. Jensen, 1978) Paramicrolaimus hohonucola Verdon & Leduc sp. nov. (Figs 5–8; Table 1) Diagnosis: Paramicrolaimus hohonucola sp. nov. is characterized by body length 2150–3620 µm. Outer labial setae 8–11 µm long, cephalic setae 8–13 µm long, in two different circles; four subcephalic setae, 4–7 µm long, at level of, and adjacent to, amphids. Lip region with two equal or almost equal lobes flanking transverse mouth opening; dorsal lobe formed by enlarged dorsal lip pair and ventral lobe formed by fusion of two ventrosublateral lip pairs. Amphideal fovea 0.43–0.60 cbd. Buccal cavity with two small ventrosublateral teeth and large dorsal tooth with two lateral pointed projections (dorsosublateral teeth). Smaller ventral tooth present. Arcuate spicules 1.8–2.1 cloacal body diameter long, gubernaculum with straight apophyses pointing dorsally. Five to seven precloacal supplements consisting of cup-shaped structures with spout-like structures pointing posteriorly. Vulva located at 42–48% of body length from anterior. Short, conical tail with terminal spinneret, 2.7–3.4 cloacal body diameters long in males with ventral row of 7–8 postcloacal setae, few dorsal and lateral setae and circle of four subterminal setae; in females, tail 3.9–5.3 anal body diameters long with only one subterminal, dorsal seta. Figure 5. View largeDownload slide Paramicrolaimus hohonucola sp. nov. A, female anterior body region. B, male anterior body region. C, female cephalic region. D, male cephalic region. E, male copulatory system. F, male posterior body region. G, female posterior body region. Scale bar: A, B = 44 µm; C, D = 20 µm; E = 14 µm; F, G = 31 µm. Figure 5. View largeDownload slide Paramicrolaimus hohonucola sp. nov. A, female anterior body region. B, male anterior body region. C, female cephalic region. D, male cephalic region. E, male copulatory system. F, male posterior body region. G, female posterior body region. Scale bar: A, B = 44 µm; C, D = 20 µm; E = 14 µm; F, G = 31 µm. Figure 6. View largeDownload slide Paramicrolaimus hohonucola sp. nov. A, entire male. B, female reproductive system. Scale bar: A = 210 µm; B = 190 µm. Figure 6. View largeDownload slide Paramicrolaimus hohonucola sp. nov. A, entire male. B, female reproductive system. Scale bar: A = 210 µm; B = 190 µm. Figure 7. View largeDownload slide Paramicrolaimus hohonucola sp. nov. Light micrographs of female cephalic region. A, lateral view showing buccal armature. B, lateral view showing lips, dorsal pharyngeal gland and granules. D, dorsal tooth; Gl, dorsal pharyngeal gland; Gr, pigmented granule; L, lip; DS, dorsosublateral tooth; VS, ventrosublateral teeth. Scale bar = 18 µm. Figure 7. View largeDownload slide Paramicrolaimus hohonucola sp. nov. Light micrographs of female cephalic region. A, lateral view showing buccal armature. B, lateral view showing lips, dorsal pharyngeal gland and granules. D, dorsal tooth; Gl, dorsal pharyngeal gland; Gr, pigmented granule; L, lip; DS, dorsosublateral tooth; VS, ventrosublateral teeth. Scale bar = 18 µm. Figure 8. View largeDownload slide Paramicrolaimus hohonucola sp. nov. Scanning electron microscopy (SEM). A, male anterior body region (lateral view). B, male anterior body region (latero-ventral view). C, precloacal supplements. D, male posterior region. Arrows show position of inner labial papillae. Scale bar: A, B = 6 µm; C = 5 µm; D = 30 µm. Figure 8. View largeDownload slide Paramicrolaimus hohonucola sp. nov. Scanning electron microscopy (SEM). A, male anterior body region (lateral view). B, male anterior body region (latero-ventral view). C, precloacal supplements. D, male posterior region. Arrows show position of inner labial papillae. Scale bar: A, B = 6 µm; C = 5 µm; D = 30 µm. Table 1. Morphometrics (µm; mean, range) of Paramicrolaimus hohonucola sp. nov. Characters Holotype Paratypes Paratypes Male Males Females N - 5 6 L 3212 2544 (2152–3238) 3240 (2439–3619) a 78.3 70 (65–79) 57 (49–68) b 16.6 15 (13–16) 16 (14–18) c 27.7 29 (27–31) 24 (19–27) c′ 3.1 3.0 (2.7–3.4) 4.6 (4.0–5.3) Maximum body diameter 41 36 (28–48) 57 (49–63) Diameter at amphids 24 22.2 (19–28) 24.2 (20–26) Length of outer labial setae 11 10 (9–11) 9.5 (8–11) Length of cephalic setae 11 10 (8–12) 10.2 (9–13) Length of subcephalic setae 5 5 (4–7) 5.2 (4–7) Amphideal fovea width 10 11 (9–12) 12 (11–12) Amphideal fovea width %cbd 42 48 (43–55) 48 (46–60) Amphid distance from anterior end 17 17 (13–22) 19 (17–22) Nerve ring from the anterior end 93 82 (75–98) 100 (84–128) Nerve ring cbd 36 33 (24–43) 40 (34–48) Pharynx length 194 175 (144–207) 197 (170–222) Pharyngeal bulb length 64 55 (44–67) 64 (62–67) Pharyngeal bulb width 24 23 (17–30) 29 (26–31) Pharynx cbd at base 41 35 (28–47) 45 (39–50) Excretory pore from anterior end 122 113 (89–134) 122 (106–134) Anal/cloacal body diameter 38 30 (24–39) 30 (25–34) Tail length 116 89 (72–106) 138 (121–158) Spicule length 66 56 (45–70) - Length of gubernacular apophysis 24 20 (18–25) - Number of precloacal supplements 6 6 (5–7) - V - - 1458 (1160–1585) %V - - 45 (42–48) Vulva cbd - - 57 (49–63) Characters Holotype Paratypes Paratypes Male Males Females N - 5 6 L 3212 2544 (2152–3238) 3240 (2439–3619) a 78.3 70 (65–79) 57 (49–68) b 16.6 15 (13–16) 16 (14–18) c 27.7 29 (27–31) 24 (19–27) c′ 3.1 3.0 (2.7–3.4) 4.6 (4.0–5.3) Maximum body diameter 41 36 (28–48) 57 (49–63) Diameter at amphids 24 22.2 (19–28) 24.2 (20–26) Length of outer labial setae 11 10 (9–11) 9.5 (8–11) Length of cephalic setae 11 10 (8–12) 10.2 (9–13) Length of subcephalic setae 5 5 (4–7) 5.2 (4–7) Amphideal fovea width 10 11 (9–12) 12 (11–12) Amphideal fovea width %cbd 42 48 (43–55) 48 (46–60) Amphid distance from anterior end 17 17 (13–22) 19 (17–22) Nerve ring from the anterior end 93 82 (75–98) 100 (84–128) Nerve ring cbd 36 33 (24–43) 40 (34–48) Pharynx length 194 175 (144–207) 197 (170–222) Pharyngeal bulb length 64 55 (44–67) 64 (62–67) Pharyngeal bulb width 24 23 (17–30) 29 (26–31) Pharynx cbd at base 41 35 (28–47) 45 (39–50) Excretory pore from anterior end 122 113 (89–134) 122 (106–134) Anal/cloacal body diameter 38 30 (24–39) 30 (25–34) Tail length 116 89 (72–106) 138 (121–158) Spicule length 66 56 (45–70) - Length of gubernacular apophysis 24 20 (18–25) - Number of precloacal supplements 6 6 (5–7) - V - - 1458 (1160–1585) %V - - 45 (42–48) Vulva cbd - - 57 (49–63) a, body length/maximum body diameter; b, body length/pharynx length; c, body length/tail length; cʹ, tail length/cloacal or anal body diameter; cbd, corresponding body diameter; N, number of specimens; L, body length; V, vulva distance from anterior end of body; %V, V/total body length. View Large Table 1. Morphometrics (µm; mean, range) of Paramicrolaimus hohonucola sp. nov. Characters Holotype Paratypes Paratypes Male Males Females N - 5 6 L 3212 2544 (2152–3238) 3240 (2439–3619) a 78.3 70 (65–79) 57 (49–68) b 16.6 15 (13–16) 16 (14–18) c 27.7 29 (27–31) 24 (19–27) c′ 3.1 3.0 (2.7–3.4) 4.6 (4.0–5.3) Maximum body diameter 41 36 (28–48) 57 (49–63) Diameter at amphids 24 22.2 (19–28) 24.2 (20–26) Length of outer labial setae 11 10 (9–11) 9.5 (8–11) Length of cephalic setae 11 10 (8–12) 10.2 (9–13) Length of subcephalic setae 5 5 (4–7) 5.2 (4–7) Amphideal fovea width 10 11 (9–12) 12 (11–12) Amphideal fovea width %cbd 42 48 (43–55) 48 (46–60) Amphid distance from anterior end 17 17 (13–22) 19 (17–22) Nerve ring from the anterior end 93 82 (75–98) 100 (84–128) Nerve ring cbd 36 33 (24–43) 40 (34–48) Pharynx length 194 175 (144–207) 197 (170–222) Pharyngeal bulb length 64 55 (44–67) 64 (62–67) Pharyngeal bulb width 24 23 (17–30) 29 (26–31) Pharynx cbd at base 41 35 (28–47) 45 (39–50) Excretory pore from anterior end 122 113 (89–134) 122 (106–134) Anal/cloacal body diameter 38 30 (24–39) 30 (25–34) Tail length 116 89 (72–106) 138 (121–158) Spicule length 66 56 (45–70) - Length of gubernacular apophysis 24 20 (18–25) - Number of precloacal supplements 6 6 (5–7) - V - - 1458 (1160–1585) %V - - 45 (42–48) Vulva cbd - - 57 (49–63) Characters Holotype Paratypes Paratypes Male Males Females N - 5 6 L 3212 2544 (2152–3238) 3240 (2439–3619) a 78.3 70 (65–79) 57 (49–68) b 16.6 15 (13–16) 16 (14–18) c 27.7 29 (27–31) 24 (19–27) c′ 3.1 3.0 (2.7–3.4) 4.6 (4.0–5.3) Maximum body diameter 41 36 (28–48) 57 (49–63) Diameter at amphids 24 22.2 (19–28) 24.2 (20–26) Length of outer labial setae 11 10 (9–11) 9.5 (8–11) Length of cephalic setae 11 10 (8–12) 10.2 (9–13) Length of subcephalic setae 5 5 (4–7) 5.2 (4–7) Amphideal fovea width 10 11 (9–12) 12 (11–12) Amphideal fovea width %cbd 42 48 (43–55) 48 (46–60) Amphid distance from anterior end 17 17 (13–22) 19 (17–22) Nerve ring from the anterior end 93 82 (75–98) 100 (84–128) Nerve ring cbd 36 33 (24–43) 40 (34–48) Pharynx length 194 175 (144–207) 197 (170–222) Pharyngeal bulb length 64 55 (44–67) 64 (62–67) Pharyngeal bulb width 24 23 (17–30) 29 (26–31) Pharynx cbd at base 41 35 (28–47) 45 (39–50) Excretory pore from anterior end 122 113 (89–134) 122 (106–134) Anal/cloacal body diameter 38 30 (24–39) 30 (25–34) Tail length 116 89 (72–106) 138 (121–158) Spicule length 66 56 (45–70) - Length of gubernacular apophysis 24 20 (18–25) - Number of precloacal supplements 6 6 (5–7) - V - - 1458 (1160–1585) %V - - 45 (42–48) Vulva cbd - - 57 (49–63) a, body length/maximum body diameter; b, body length/pharynx length; c, body length/tail length; cʹ, tail length/cloacal or anal body diameter; cbd, corresponding body diameter; N, number of specimens; L, body length; V, vulva distance from anterior end of body; %V, V/total body length. View Large Material examined Holotype: male (NIWA 115460), collected in April 2010 (NIWA cruise TAN1004, station 17), Hikurangi Margin (41.6288°S, 175.8682°E), 1514 m water depth. Paratypes: One male and three female paratypes (NIWA 115461), same data as holotype. One male paratype (NNCNZ 3285), collected in April 2007 (TAN0705, station 35), Chatham Rise (43.8325°S, 176.712°E), 480 m water depth. One male paratype collected in March 2011 (NIWA cruise TAN1103, station 68), Chatham rise 43.33417°S, 178.29517°E), 347 m water depth. This specimen was used for morphological measurements, then sacrificed for SEM observations. One male and one female paratype (NIWA 115462), collected in April 2012 (NIWA cruise TAN1206, station 125), Bay of Plenty (37.4747°S, 176.7585°E), 697 m water depth. Etymology: The species name is derived from the Māori word ‘hōhonu’ which means ‘deep, deeper, profound’; and the latin suffix ‘-cola’ which means ‘dwelling in, inhabitant’. Description: Males. Brownish cylindrical body, strongly pigmented between amphids and nerve ring, and in caudal region, due to presence of brown granules. Cuticle striated from level of anterior edge of amphids to spinneret. Sparse and irregular somatic setae mainly restricted to pharyngeal and tail region, 3–4 µm long. Cephalic region slightly constricted at level of amphids. Lip region with two equal or nearly equal lobes flanking transverse mouth opening; dorsal lobe composed of dorsal lip pair, ventral lobe composed of two fused ventrosublateral lip pairs. Six-minute inner labial papillae on either side of mouth opening, only visible using SEM; two papillae on dorsal lobe and four on ventral lobe. Six outer labial setae of similar length to four cephalic setae (0.4–0.5 cbd); cephalic setae located anterior to the amphids and in separate circle to outer labial setae. Four small subcephalic setae, adjacent to, and at level of, amphids. Amphideal fovea spiral with 1.25 turns; amphideal aperture (as observed using light microscopy) oval-shaped and smaller than amphideal fovea. Buccal cavity asymmetrical, with cuticularized walls; single large dorsal tooth present, heavily cuticularized and with two lateral pointed projections (dorsosublateral teeth), set within pharyngeal tissue and protruding only slightly into buccal cavity. Dorsal gland opening into pharyngeal lumen through distal extremity of tooth. Two smaller ventrosublateral teeth present. Pharynx cylindrical and muscular, with slightly swollen anterior end surrounding buccal cavity and weak, elongated posterior bulb. Nerve ring at approximately mid-length of pharynx. Conoid and muscular cardia present, 10–13 µm long, surrounded by intestine. Secretory-excretory system present; excretory ampulla and pore situated ventrally, slightly posterior to nerve ring, renette cell located ventrally, at level or immediately posterior to cardia, 20–29 × 9–18 µm. Reproductive system with two opposed and outstreched testes, both located to left of the intestine. Sperm cells oval, 4–6 × 12–16 µm. Spicules paired, equal, arcuate, 1.8–2.1 cloacal body diameter long, with a central cuticularized projection (lamella) at proximal end. Velum and capitulum present. Tip of spicules narrow and cylindrical, forming a duct. Gubernaculum surrounding distal end of the spicules; gubernacular apophyses narrow and pointing dorsally. Five to seven precloacal supplement. Observations using SEM showed they consist of cup-shaped structures each with spout-like projection pointing posteriorly and positioned on bifid cuticularized base. Gland-like brown granules present beneath each supplement. Tail short, conical, bent ventrally, with irregularly spaced setae, row of seven ventral setae, and circle of four longer subterminal setae. Three caudal glands and spinneret present. Females. Similar to males, but with wider body. Short somatic setae sparser than in males, apparently absent in some specimens. Tail with only one subterminal dorsal seta. Reproductive system with two opposed, reflexed ovaries to left of intestine. Mature eggs 96–130 × 40–51 µm. Spermatheca not observed. Vulva located at or slightly anterior to mid-body. Proximal portion of vagina uterina surrounded by sphincter muscle. Differential diagnosis: The closest species to P. hohonucola sp. nov. is P. mirus because both species have similar a and c′ values (Table 2), and because of the similar shape and dimension of the posterior pharyngeal bulb, amphideal fovea, spicules and gubernacular apophyses. Paramicrolaimus hohonucola sp. nov. differs from P. mirus mainly by the presence of a transverse opening formed by the fusion of the two ventrosublateral lip lobes and enlarged dorsal lip lobe (vs. three lip lobes in P. mirus), and presence of caudal setae (vs. no caudal setae present in females of P. mirus). The new species also differs from P. mirus by the longer outer labial setae (8–11 µm vs. 6–8 µm in P. mirus), fewer precloacal supplements (5–7 vs. 8–10), lower values of b (13–16 vs. 16–25 in P. mirus), lower values of c (27–31 vs. 31–41 in males, and 19–27 vs. 26–31 in females) and larger body diameter at level of vulva (49–63 µm vs. 33–50 µm in P. mirus). Table 2. Morphometrics (µm) of all Paramicrolaimus species from the literature Species P. primus P. spirulifer P. mirus P. damodarani P. hohonucola sp. nov. Reference Wieser (1954) Wieser (1959) Jensen (1978) Tchesunov (1988) Huang & Zhang (2005) Jacob et al. (2015) Present study Sex Female Male Female Male Male Female Male Female Male Male Female Habitat depth (m) 250–300 0.5 28 20 64–85 95–205 347–1514 Locality Golfo de Ancud, Chile Pudget Sound, USA Øresund North, Denmark Kandalaksha Bay, Russia Yellow Sea, China Arabian Sea, India Southwestern Pacific, New Zealand L 2340 4430 4180 5170 4060 3500 3052–3600 3680–4340 1225–1310 2152–3238 2439–3619 a 44.7 147.7 139.3 136 106 105 76.3–89.5 76.3–90.7 51–52.4 65.1–79.0 48.8–67.5 b 16.8 22.1 23.3 23.5 21.7 21.8 15.9–19.8 19.4–25.4 8.8–9.14 13.0–15.6 14.4–18.3 c 22.3 55.4 52.2 30.6 40.5 26 30.5–36.7 27.6–30.6 18.8–20.3 26.6–30.6 18.9–27.3 c′ 4 2.1 3.3 4.5 2.8 4.2 2.9–3.3 4.1–4.6 - 2.7–3.4 4.0–5.3 Maximum body diameter 52 31 30 38 38 33 38–43 43–50 24–25 28–48 49–63 Diameter at amphids 22 28 26 30 24 28 22–28 23–27 20 19–28 20–26 Length of outer labial setae 9 12 12 16–17 6 6 7–8 7–8 13 9–11 8–11 Length of cephalic setae 10 14–16 14–16 18 8 8 9–10 10–11 14–15 8–12 9–13 Amphideal fovea width 12 14 13 16 12 8 11–13 11–13 11–12 9–12 11–12 Amphideal fovea height - 13 11 13 - - - - - - - Amphid % diameter 50–54 50 50 53 51 29 43–57 48–54 54–60 42–55 46–60 Amphid distance from anterior end 18 18–21 18–21 22 20–21 23 20 20 19–20 13–22 17–22 nerve ring from the anterior end 92 - - 121 70 70 85–88 82–89 - 75–98 84–128 nerve ring cbd - - - - 32 33 32–40 36–39 - 24–43 34–48 Pharynx length 139 201 179 220 153 165 172–190 145–193 140–145 144–207 170–222 Pharyngial bulb length - - - 40 40 50 50–60 50–60 38 44–67 62–67 Pharyngial bulb width - - - 17 25 25 23–30 23–30 15 17–30 26–31 Pharynx cbd at base 34 31 30 38 33 33 38–43 43–47 25 28–47 39–50 Excretory pore from anterior end 94 - - 141 98 95 94–128 113–128 - 89–134 106–134 Anal/cloacal body diameter 27 30 24 38 36 30 32–34 30–34 - 24–39 25–34 Tail length 109 80 80 171 100 125 98–105 130–148 63 72–116 121–158 Spicule length - 25 - 39 33 - 45–50 - 28–29 45–70 - Length of gubernacular apophysis - - - - 18 - 22–30 - 19 18–25 - Number of precloacal supplements - 6 - 10 9 - 8–10 - 7 5–7 - V 1083 - - - - 1575 - 1430–1780 - - 1160–1585 %V 46.3 - - - - 45 - 39–43 - - 42–48 Vulva cbd 52 - - - - 33 - 40–50 - - 49–63 Species P. primus P. spirulifer P. mirus P. damodarani P. hohonucola sp. nov. Reference Wieser (1954) Wieser (1959) Jensen (1978) Tchesunov (1988) Huang & Zhang (2005) Jacob et al. (2015) Present study Sex Female Male Female Male Male Female Male Female Male Male Female Habitat depth (m) 250–300 0.5 28 20 64–85 95–205 347–1514 Locality Golfo de Ancud, Chile Pudget Sound, USA Øresund North, Denmark Kandalaksha Bay, Russia Yellow Sea, China Arabian Sea, India Southwestern Pacific, New Zealand L 2340 4430 4180 5170 4060 3500 3052–3600 3680–4340 1225–1310 2152–3238 2439–3619 a 44.7 147.7 139.3 136 106 105 76.3–89.5 76.3–90.7 51–52.4 65.1–79.0 48.8–67.5 b 16.8 22.1 23.3 23.5 21.7 21.8 15.9–19.8 19.4–25.4 8.8–9.14 13.0–15.6 14.4–18.3 c 22.3 55.4 52.2 30.6 40.5 26 30.5–36.7 27.6–30.6 18.8–20.3 26.6–30.6 18.9–27.3 c′ 4 2.1 3.3 4.5 2.8 4.2 2.9–3.3 4.1–4.6 - 2.7–3.4 4.0–5.3 Maximum body diameter 52 31 30 38 38 33 38–43 43–50 24–25 28–48 49–63 Diameter at amphids 22 28 26 30 24 28 22–28 23–27 20 19–28 20–26 Length of outer labial setae 9 12 12 16–17 6 6 7–8 7–8 13 9–11 8–11 Length of cephalic setae 10 14–16 14–16 18 8 8 9–10 10–11 14–15 8–12 9–13 Amphideal fovea width 12 14 13 16 12 8 11–13 11–13 11–12 9–12 11–12 Amphideal fovea height - 13 11 13 - - - - - - - Amphid % diameter 50–54 50 50 53 51 29 43–57 48–54 54–60 42–55 46–60 Amphid distance from anterior end 18 18–21 18–21 22 20–21 23 20 20 19–20 13–22 17–22 nerve ring from the anterior end 92 - - 121 70 70 85–88 82–89 - 75–98 84–128 nerve ring cbd - - - - 32 33 32–40 36–39 - 24–43 34–48 Pharynx length 139 201 179 220 153 165 172–190 145–193 140–145 144–207 170–222 Pharyngial bulb length - - - 40 40 50 50–60 50–60 38 44–67 62–67 Pharyngial bulb width - - - 17 25 25 23–30 23–30 15 17–30 26–31 Pharynx cbd at base 34 31 30 38 33 33 38–43 43–47 25 28–47 39–50 Excretory pore from anterior end 94 - - 141 98 95 94–128 113–128 - 89–134 106–134 Anal/cloacal body diameter 27 30 24 38 36 30 32–34 30–34 - 24–39 25–34 Tail length 109 80 80 171 100 125 98–105 130–148 63 72–116 121–158 Spicule length - 25 - 39 33 - 45–50 - 28–29 45–70 - Length of gubernacular apophysis - - - - 18 - 22–30 - 19 18–25 - Number of precloacal supplements - 6 - 10 9 - 8–10 - 7 5–7 - V 1083 - - - - 1575 - 1430–1780 - - 1160–1585 %V 46.3 - - - - 45 - 39–43 - - 42–48 Vulva cbd 52 - - - - 33 - 40–50 - - 49–63 a, body length/maximum body diameter; b, body length/pharynx length; c, body length/tail length; cʹ, tail length/cloacal or anal body diameter; cbd, corresponding body diameter; L, body length; V, vulva distance from anterior end of body; %V, V/total body length. View Large Table 2. Morphometrics (µm) of all Paramicrolaimus species from the literature Species P. primus P. spirulifer P. mirus P. damodarani P. hohonucola sp. nov. Reference Wieser (1954) Wieser (1959) Jensen (1978) Tchesunov (1988) Huang & Zhang (2005) Jacob et al. (2015) Present study Sex Female Male Female Male Male Female Male Female Male Male Female Habitat depth (m) 250–300 0.5 28 20 64–85 95–205 347–1514 Locality Golfo de Ancud, Chile Pudget Sound, USA Øresund North, Denmark Kandalaksha Bay, Russia Yellow Sea, China Arabian Sea, India Southwestern Pacific, New Zealand L 2340 4430 4180 5170 4060 3500 3052–3600 3680–4340 1225–1310 2152–3238 2439–3619 a 44.7 147.7 139.3 136 106 105 76.3–89.5 76.3–90.7 51–52.4 65.1–79.0 48.8–67.5 b 16.8 22.1 23.3 23.5 21.7 21.8 15.9–19.8 19.4–25.4 8.8–9.14 13.0–15.6 14.4–18.3 c 22.3 55.4 52.2 30.6 40.5 26 30.5–36.7 27.6–30.6 18.8–20.3 26.6–30.6 18.9–27.3 c′ 4 2.1 3.3 4.5 2.8 4.2 2.9–3.3 4.1–4.6 - 2.7–3.4 4.0–5.3 Maximum body diameter 52 31 30 38 38 33 38–43 43–50 24–25 28–48 49–63 Diameter at amphids 22 28 26 30 24 28 22–28 23–27 20 19–28 20–26 Length of outer labial setae 9 12 12 16–17 6 6 7–8 7–8 13 9–11 8–11 Length of cephalic setae 10 14–16 14–16 18 8 8 9–10 10–11 14–15 8–12 9–13 Amphideal fovea width 12 14 13 16 12 8 11–13 11–13 11–12 9–12 11–12 Amphideal fovea height - 13 11 13 - - - - - - - Amphid % diameter 50–54 50 50 53 51 29 43–57 48–54 54–60 42–55 46–60 Amphid distance from anterior end 18 18–21 18–21 22 20–21 23 20 20 19–20 13–22 17–22 nerve ring from the anterior end 92 - - 121 70 70 85–88 82–89 - 75–98 84–128 nerve ring cbd - - - - 32 33 32–40 36–39 - 24–43 34–48 Pharynx length 139 201 179 220 153 165 172–190 145–193 140–145 144–207 170–222 Pharyngial bulb length - - - 40 40 50 50–60 50–60 38 44–67 62–67 Pharyngial bulb width - - - 17 25 25 23–30 23–30 15 17–30 26–31 Pharynx cbd at base 34 31 30 38 33 33 38–43 43–47 25 28–47 39–50 Excretory pore from anterior end 94 - - 141 98 95 94–128 113–128 - 89–134 106–134 Anal/cloacal body diameter 27 30 24 38 36 30 32–34 30–34 - 24–39 25–34 Tail length 109 80 80 171 100 125 98–105 130–148 63 72–116 121–158 Spicule length - 25 - 39 33 - 45–50 - 28–29 45–70 - Length of gubernacular apophysis - - - - 18 - 22–30 - 19 18–25 - Number of precloacal supplements - 6 - 10 9 - 8–10 - 7 5–7 - V 1083 - - - - 1575 - 1430–1780 - - 1160–1585 %V 46.3 - - - - 45 - 39–43 - - 42–48 Vulva cbd 52 - - - - 33 - 40–50 - - 49–63 Species P. primus P. spirulifer P. mirus P. damodarani P. hohonucola sp. nov. Reference Wieser (1954) Wieser (1959) Jensen (1978) Tchesunov (1988) Huang & Zhang (2005) Jacob et al. (2015) Present study Sex Female Male Female Male Male Female Male Female Male Male Female Habitat depth (m) 250–300 0.5 28 20 64–85 95–205 347–1514 Locality Golfo de Ancud, Chile Pudget Sound, USA Øresund North, Denmark Kandalaksha Bay, Russia Yellow Sea, China Arabian Sea, India Southwestern Pacific, New Zealand L 2340 4430 4180 5170 4060 3500 3052–3600 3680–4340 1225–1310 2152–3238 2439–3619 a 44.7 147.7 139.3 136 106 105 76.3–89.5 76.3–90.7 51–52.4 65.1–79.0 48.8–67.5 b 16.8 22.1 23.3 23.5 21.7 21.8 15.9–19.8 19.4–25.4 8.8–9.14 13.0–15.6 14.4–18.3 c 22.3 55.4 52.2 30.6 40.5 26 30.5–36.7 27.6–30.6 18.8–20.3 26.6–30.6 18.9–27.3 c′ 4 2.1 3.3 4.5 2.8 4.2 2.9–3.3 4.1–4.6 - 2.7–3.4 4.0–5.3 Maximum body diameter 52 31 30 38 38 33 38–43 43–50 24–25 28–48 49–63 Diameter at amphids 22 28 26 30 24 28 22–28 23–27 20 19–28 20–26 Length of outer labial setae 9 12 12 16–17 6 6 7–8 7–8 13 9–11 8–11 Length of cephalic setae 10 14–16 14–16 18 8 8 9–10 10–11 14–15 8–12 9–13 Amphideal fovea width 12 14 13 16 12 8 11–13 11–13 11–12 9–12 11–12 Amphideal fovea height - 13 11 13 - - - - - - - Amphid % diameter 50–54 50 50 53 51 29 43–57 48–54 54–60 42–55 46–60 Amphid distance from anterior end 18 18–21 18–21 22 20–21 23 20 20 19–20 13–22 17–22 nerve ring from the anterior end 92 - - 121 70 70 85–88 82–89 - 75–98 84–128 nerve ring cbd - - - - 32 33 32–40 36–39 - 24–43 34–48 Pharynx length 139 201 179 220 153 165 172–190 145–193 140–145 144–207 170–222 Pharyngial bulb length - - - 40 40 50 50–60 50–60 38 44–67 62–67 Pharyngial bulb width - - - 17 25 25 23–30 23–30 15 17–30 26–31 Pharynx cbd at base 34 31 30 38 33 33 38–43 43–47 25 28–47 39–50 Excretory pore from anterior end 94 - - 141 98 95 94–128 113–128 - 89–134 106–134 Anal/cloacal body diameter 27 30 24 38 36 30 32–34 30–34 - 24–39 25–34 Tail length 109 80 80 171 100 125 98–105 130–148 63 72–116 121–158 Spicule length - 25 - 39 33 - 45–50 - 28–29 45–70 - Length of gubernacular apophysis - - - - 18 - 22–30 - 19 18–25 - Number of precloacal supplements - 6 - 10 9 - 8–10 - 7 5–7 - V 1083 - - - - 1575 - 1430–1780 - - 1160–1585 %V 46.3 - - - - 45 - 39–43 - - 42–48 Vulva cbd 52 - - - - 33 - 40–50 - - 49–63 a, body length/maximum body diameter; b, body length/pharynx length; c, body length/tail length; cʹ, tail length/cloacal or anal body diameter; cbd, corresponding body diameter; L, body length; V, vulva distance from anterior end of body; %V, V/total body length. View Large Females of P. hohonucola sp. nov. can be differentiated from the type species of the genus, P. primus, by the absence of honeycomb-like structures on cuticle, their longer bodies (2439–3619 µm vs. 2340 µm in P. primus), higher values of a (49–68 vs. 45) and a weak and elongated posterior pharyngeal bulb (vs. well-developed bulb in P. primus). Paramicrolaimus hohonucola sp. nov. can be differentiated from P. spirulifer and P. damodarani by several features, mainly by a different body length (2439–3619 µm vs. 4180–5170 µm and 1225–1310 µm in P. spirulifer and P. damodarani, respectively), the length of their outer labial setae (8–11 µm vs. 14–18 µm and 14–15 µm in P. spirulifer and P. damodarani, respectively), lower value of a relative to P. spirulifer (49–79 vs. 136–148) and higher value of a (in males) relative to P. damodarani (65–79 vs. 51–52). Remarks: An unusual feature of the present species within the phylum is the presence of a transverse opening formed by the fusion of the two ventrosublateral lip lobes and enlarged dorsal lip lobe, resulting in apparent dorsoventral symmetry rather than triradial symmetry. A transverse mouth opening is also found in the genus Pseudonchus Cobb, 1920 (order Desmodorida); however, in this case, the transverse opening results from a reduction of the dorsal sector of the buccal cavity resulting in bilateral symmetry (De Coninck, 1942). Reduction of the dorsal sector of the buccal cavity also occurs in the genus Cheironchus Cobb, 1917 (order Chromadorida), which is characterized by a reduced dorsal mandible and two equal, large ventrosublateral mandibles displaced laterally. In P. hohonucola sp. nov., the dorsoventral symmetry is only partial because the transverse mouth opening is displaced slightly dorsally. Inequalities between dorsal and ventral regions become more pronounced in the buccal cavity, which is characterized by a large dorsal tooth with two dorsosublateral teeth and two smaller ventrosublateral teeth. Whilst the bilateral symmetry in Pseudonchus and Cheironchus may be the result of their predatory feeding habits, there is no evidence of predation in Paramicrolaimus. Paramicrolaimus hohonucola sp. nov. was found at 350–1500 m water depth, which constitutes the deepest record for the genus. Paramicrolaimus primus was found at 250–300 m (Wieser, 1953, 1954), P. damodarani at 95–205 m (Jacob et al., 2015), P. mirus at 20–90 m (Tchesunov, 1988; Huang & Zhang, 2005) and records of P. spirulifer are restricted to the sublittoral zone (Wieser, 1959; Jensen, 1978; Lorenzen, 1981). Order Microlaimida ord. nov. (Table 3) Diagnosis: Cuticle striated, annulated or smooth, rarely punctated. Anterior labial sensilla in three separate circles: six inner labial papilla, six outer labial papilla and four cephalic setae. Amphideal fovea circular, cryptocircular, unispiral or multispiral. Cheilostom with cheilorhabdia (not always visible). Buccal cavity usually with more or less pronounced dorsal tooth, smaller subventral teeth also often present. Pharynx muscular with weakly or strongly developed posterior bulb. Female reproductive system with one or two ovaries; ovaries are either outstretched or reflexed. Male reproductive system with two opposed testes or single anterior testis. Table 3. Key morphological traits characterizing the order Desmodorida (now comprising the superfamily Desmodoroidea only), Microlaimida order. nov. (comprising the Microlaimoidea and Molgolaimus) and Chromadorida Desmodorida Microlaimida order nov. Chromadorida Cuticle Striated or annulated Striated, annulated or smooth (punctations in some Microlaimus and Bolbolaimus species) Punctated (striated in Paramicrolaimidae) Cephalic capsule Present or absent Absent Absent Cephalic sensilla 6 + 6 + 4 6 + 6 + 4 6 + 6 + 4 or 6 + 10 Amphideal fovea Unispiral, multispiral or cryptocircular Circular, cryptocircular, unispiral or multispiral Unispiral, multispiral, transverse loop/slit Cheilorhabdia Present Present Present Buccal armature Typically single dorsal tooth and ≥ 2 smaller ventrosublateral teeth Typically single dorsal tooth and ≥ 2 smaller ventrosublateral teeth Typically single dorsal tooth and ≥ 2 smaller ventrosublateral teeth Pharynx Muscular with weakly or strongly developed posterior bulb Muscular with weakly or strongly developed posterior bulb Muscular with weakly or strongly developed posterior bulb Female reproductive system Two opposed and reflexed ovaries Two opposed and outstretched ovaries, two opposed and reflexed ovaries, single anterior reflexed ovary or single outstretched anterior ovary Two opposed and reflexed ovaries Male reproductive system Single anterior testis (two testes in Onepunema) Two opposed testes or single anterior testis Two opposed testes or single anterior testis Desmodorida Microlaimida order nov. Chromadorida Cuticle Striated or annulated Striated, annulated or smooth (punctations in some Microlaimus and Bolbolaimus species) Punctated (striated in Paramicrolaimidae) Cephalic capsule Present or absent Absent Absent Cephalic sensilla 6 + 6 + 4 6 + 6 + 4 6 + 6 + 4 or 6 + 10 Amphideal fovea Unispiral, multispiral or cryptocircular Circular, cryptocircular, unispiral or multispiral Unispiral, multispiral, transverse loop/slit Cheilorhabdia Present Present Present Buccal armature Typically single dorsal tooth and ≥ 2 smaller ventrosublateral teeth Typically single dorsal tooth and ≥ 2 smaller ventrosublateral teeth Typically single dorsal tooth and ≥ 2 smaller ventrosublateral teeth Pharynx Muscular with weakly or strongly developed posterior bulb Muscular with weakly or strongly developed posterior bulb Muscular with weakly or strongly developed posterior bulb Female reproductive system Two opposed and reflexed ovaries Two opposed and outstretched ovaries, two opposed and reflexed ovaries, single anterior reflexed ovary or single outstretched anterior ovary Two opposed and reflexed ovaries Male reproductive system Single anterior testis (two testes in Onepunema) Two opposed testes or single anterior testis Two opposed testes or single anterior testis Exceptions to character states are given between brackets. View Large Table 3. Key morphological traits characterizing the order Desmodorida (now comprising the superfamily Desmodoroidea only), Microlaimida order. nov. (comprising the Microlaimoidea and Molgolaimus) and Chromadorida Desmodorida Microlaimida order nov. Chromadorida Cuticle Striated or annulated Striated, annulated or smooth (punctations in some Microlaimus and Bolbolaimus species) Punctated (striated in Paramicrolaimidae) Cephalic capsule Present or absent Absent Absent Cephalic sensilla 6 + 6 + 4 6 + 6 + 4 6 + 6 + 4 or 6 + 10 Amphideal fovea Unispiral, multispiral or cryptocircular Circular, cryptocircular, unispiral or multispiral Unispiral, multispiral, transverse loop/slit Cheilorhabdia Present Present Present Buccal armature Typically single dorsal tooth and ≥ 2 smaller ventrosublateral teeth Typically single dorsal tooth and ≥ 2 smaller ventrosublateral teeth Typically single dorsal tooth and ≥ 2 smaller ventrosublateral teeth Pharynx Muscular with weakly or strongly developed posterior bulb Muscular with weakly or strongly developed posterior bulb Muscular with weakly or strongly developed posterior bulb Female reproductive system Two opposed and reflexed ovaries Two opposed and outstretched ovaries, two opposed and reflexed ovaries, single anterior reflexed ovary or single outstretched anterior ovary Two opposed and reflexed ovaries Male reproductive system Single anterior testis (two testes in Onepunema) Two opposed testes or single anterior testis Two opposed testes or single anterior testis Desmodorida Microlaimida order nov. Chromadorida Cuticle Striated or annulated Striated, annulated or smooth (punctations in some Microlaimus and Bolbolaimus species) Punctated (striated in Paramicrolaimidae) Cephalic capsule Present or absent Absent Absent Cephalic sensilla 6 + 6 + 4 6 + 6 + 4 6 + 6 + 4 or 6 + 10 Amphideal fovea Unispiral, multispiral or cryptocircular Circular, cryptocircular, unispiral or multispiral Unispiral, multispiral, transverse loop/slit Cheilorhabdia Present Present Present Buccal armature Typically single dorsal tooth and ≥ 2 smaller ventrosublateral teeth Typically single dorsal tooth and ≥ 2 smaller ventrosublateral teeth Typically single dorsal tooth and ≥ 2 smaller ventrosublateral teeth Pharynx Muscular with weakly or strongly developed posterior bulb Muscular with weakly or strongly developed posterior bulb Muscular with weakly or strongly developed posterior bulb Female reproductive system Two opposed and reflexed ovaries Two opposed and outstretched ovaries, two opposed and reflexed ovaries, single anterior reflexed ovary or single outstretched anterior ovary Two opposed and reflexed ovaries Male reproductive system Single anterior testis (two testes in Onepunema) Two opposed testes or single anterior testis Two opposed testes or single anterior testis Exceptions to character states are given between brackets. View Large Remarks: There is no synapomorphy for Microlaimida order nov. This order is created to accommodate the superfamily Microlaimoidea and genus Molgolaimus. These taxa, which were previously classified within Desmodorida, are now considered to form a distinct clade based on molecular phylogenetic analyses and reinterpretation of existing morphological data (see Discussion for detailed justification). DISCUSSION The present study provides the first molecular sequence data for the family Paramicrolaimidae, and the classification of this family is discussed below in light of molecular phylogenetic analyses. Proposed changes to the classification and composition of the Microlaimoidea, which consistently forms a clade separate from the bulk of the Desmodorida in SSU phylogenies, are explained in light of new and existing morphological and molecular evidence. Phylogenetic placement of Paramicrolaimidae Our results do not provide support for a close relationship between Paramicrolaimidae and either the family Microlaimidae, subfamily Stilbonematinae or the order Plectida as suggested by previous authors (Wieser, 1954; Jensen, 1978; Lorenzen, 1981). Instead, our phylogenetic analyses, which investigated potential relationships within marine Chromadorean orders and within the Chromadorida based on SSU rDNA sequences, indicate that Paramicrolaimidae constitutes a sister taxon to the family Selachinematidae. In our analysis of SSU rDNA sequences focusing on the Chromadorida, Selachinematidae formed a monophyletic group, which together with P. hohonucola sp. nov., formed a sister group to the remaining chromadorid taxa, although there was no support for this topology. This finding is similar to the analyses of van Megen et al. (2009), which showed that the Selachinematidae form a distinct monophyletic clade within Chromadorida. The placement of Selachinematidae is never well supported in SSU phylogenies, and this family is not always recovered as a monophyletic group within Chromadorida (Holterman et al., 2008). However, there are strong morphological grounds for the placement of Selachinematidae within Chromadorida. The Selachinematidae are characterized by both punctated cuticle and female reproductive system with reflexed ovaries, a combination of traits not found outside Chromadorida (Lorenzen, 1981). The Paramicrolaimidae possess reflexed ovaries, but they differ from the Selachinematidae and other families currently comprising Chromadorida in having a striated cuticle. However, cuticle ornamentation sometimes varies substantially within orders. The family Comesomatidae was previously classified with Chromadorida by some authors mainly on account of the punctated cuticle (e.g. Wieser, 1954; De Coninck, 1965; Platt, 1985), but is characterized by a female reproductive system with outstretched branches, and is now placed within Areaolaimida based on molecular phylogenetic analyses (De Ley & Blaxter 2004). Therefore, in the absence of clear morphological affinities with other chromadorean orders, and despite the lack of cuticle punctations, we provisionally propose that Paramicrolaimidae be placed within Chromadorida based on our analysis of SSU and LSU sequences which indicate a close relationship with Selachinematidae. Paramicrolaimidae is the only family within the order lacking cuticle punctations, which may have been present in the ancestor and subsequently lost. The placement of both Selachinematidae and Paramicrolaimidae, however, remains equivocal due to limitations of reconstructions based on a single gene, and the classification of these families may need to be revised in the future. Composition, status and phylogenetic placement of Microlaimoidea Our molecular analyses confirm the results of previous molecular phylogenies showing that Molgolaimus, which is currently classified within Desmodoroidea, should instead be placed with Microlaimidoidea (Meldal et al., 2007; Leduc & Zhao, 2016). Molgolaimus is currently placed within the family Desmodoridae, as proposed by Lorenzen (1981) to establish the monophyly of Desmodoroidea based on the presence of only one (anterior) testis in males. Prior to this classification, Molgolaimus was originally placed with the Microlaimidae (Gerlach & Riemann, 1973/1974), presumably based on morphological similarities to the genus Microlaimus in head and amphideal fovea shape, arrangement of head sensilla and buccal cavity structure. Molgolaimus was later placed in the family Molgolaimidae Jensen, 1978 (the latter also comprising the genera Aponema Jensen, 1978 and Prodesmodora Micoletzky, 1923) mainly to reflect differences in the structure of the male and female reproductive systems between the Microlaimidae (males with one or two testes, females with outstretched ovaries) and Molgolaimidae (males with one anterior testis only, females with reflexed ovaries) (Jensen, 1978). Relationships between the Microlaimidae and Molgolaimidae were not made explicit by Jensen (1978), but their parallel treatment in the same publication implies that they were considered to be sister families within the order Desmodorida. The results of the present and previously published SSU molecular phylogenies (Meldal et al., 2007; Leduc & Zhao, 2016) provide support for the classification of Molgolaimus in a sister group to the Microlaimidae as proposed by Jensen (1978), although further work is required to clarify relationships within Microlaimoidea, which we propose should now include Molgolaimidae in addition to Microlaimidae, Monoposthiidae and Aponchiidae. There is no molecular evidence for the placement of Aponema and Prodesmodora, together with Molgolaimus within Molgolaimidae, as suggested by Jensen (1978); Aponema is currently classified with Microlaimidae based on the presence of outstretched ovaries, and Prodesmodora is classified within Desmodoridae based on the presence of reflexed ovaries (Tchesunov, 2014b). We therefore propose that the placement of these genera be left unchanged until further molecular analyses are conducted, leaving Molgolaimus as the only genus within the family Molgolaimidae. Our analyses suggest that Microlaimidae and Monoposthiidae are more closely related to Chromadorida than any other chromadorean order. The molecular evidence available to date indicates that Microlaimoidea is either (1) a monophyletic sister clade to Chromadorida (as suggested by Meldal et al. (2007) and our Chromadorea-scale analysis), (2) a paraphyletic group nested within Chromadorida (as suggested in our Chromadorida-scale analysis) or (3) a distinct clade from Chromadorida at the base of Chromadorea (Holterman et al., 2008). Difficulties in resolving the position of Microlaimoidea and other basal chromadorean taxa are likely due to the use of a single gene for phylogenetic reconstruction. Currently, the SSU rDNA gene is the only locus known to resolve deep relationships among nematode taxa; other genes such as the LSU rDNA gene are only informative at within-family level (De Ley et al., 2005; Bik et al., 2010). Obtaining a greater number of SSU rDNA sequences of microlaimoid taxa, such as representatives of Aponchiidae, may help better resolve relationships of basal Chromadorea, but finding other informative genes will probably be required in order to obtain a more stable topology. The order Desmodorida, as originally defined by De Coninck (1965), included taxa with annulated or striated cuticle and with unispiral, multispiral or loop-shaped amphideal fovea. This order originally included Desmodoroidea, Microlaimidae and Monoposthiidae, as well as other taxa which have now been moved to other orders (e.g. Ceramonematidae Cobb, 1933, and Richtersia). In the recent classification of De Ley & Blaxter (2002, 2004), Desmodorida comprises two superfamilies, Desmodoroidea and Microlaimoidea. Desmodoroidea was defined as monophyletic by Lorenzen (1981) based on the synapomorphy: only one anterior testis present. Nevertheless, two exceptions now exist: (1) Onepunema Leduc & Verschelde, 2013 possesses two testes but has been placed within the family Desmodoridae based on the presence of a cephalic capsule (a trait not found in any of the Microlaimoidea) and reflexed ovaries in females, and (2) molecular evidence indicates that Molgolaimus does not belong to Desmodoroidea despite the presence of only one anterior testis (Meldal et al., 2007; Leduc & Zhao, 2016; this study). Desmodoroidea as defined by Lorenzen (1981), and upon which the classification of De Ley & Blaxter (2004) was based, is therefore no longer monophyletic. According to Lorenzen (1981), the Microlaimoidea lacks any synapomorphy and forms the paraphyletic remains of the Desmodorida. The Microlaimoidea have several features in common with both the Desmodoroidea and Chromadorida, including the presence of cheilorhabdia, as well as arrangement of cephalic sensilla, shape of amphideal fovea and structure of the buccal cavity and pharynx (Table 3). However, two of the microlaimoid families, Microlaimidae and Aponchidae, differ from both the Desmodoroidea and Chromadorida in having outstretched ovaries. The main morphological difference between the Microlaimoidea and either the Desmodorida or Chromadorida is in the cuticle ornamentation: the Microlaimoidea have a striated or annulated cuticle whilst the Chromadorida have a punctated cuticle. There are, however, some exceptions; some microlaimid species have been described as having a punctated cuticle (e.g. Microlaimus punctulatus Gerlach, 1950, Microlaimus honestus De Man, 1922 and Bolbolaimus punctatus Cobb, 1920), and the Paramicrolaimidae, which we argue should be classified with the Chromadorida, have a striated cuticle. Cuticle punctations are relatively rare within the nematode phylum, and are only common in the order Chromadorida and family Comesomatidae. Therefore, whilst cuticle punctations (a synapomorphy) is a useful trait for inferring phylogenetic relationships of some groups, the absence of cuticle punctations, a much more widespread and common trait within the Chromadorea (i.e. a plesiomorphy), is not. Accordingly, Lorenzen (1981) did not consider the absence of cuticle punctations in Desmodoroidea and Microlaimoidea as a phylogenetically informative trait that could be used to infer a close relationship between the two superfamilies. Overall, the morphological evidence suggests that (1) there is a close relationship between Chromadorida, Desmodoroidea and Microlaimoidea based on the synapomorphy of cheilorhabdia, and (2) the Chromadorida represent a clade distinct from both the Desmodoroidea and Microlaimoidea based on the synapomorphy of cuticle punctations. However, there are no solid morphological grounds for classifying Microlaimoidea together with Desmodoroidea. Our phylogenetic analyses, as well as the phylum-wide analyses of Meldal et al. (2007) and Holterman et al. (2008), found no evidence for a sister relationship between the bulk of the Desmodoroidea and the Microlaimoidea; in SSU phylogenies, the latter always forms a distinct clade near the base of the Chromadorea and closer to Chromadorida than the rest of the Desmodorida. In light of these results, as well as lack of any morphological synapomorphy linking Desmodoroidea and Microlaimoidea, we propose to remove Microlaimoidea from the order Desmodorida, which would thus be solely comprised of Desmodoroidea, and erect the order Microlaimida order nov. to accommodate the superfamily Microlaimoidea, which we propose should include the genus Molgolaimus (see above; clade 1 in Fig. 2). This classification, which comprises the Chromadorida, Desmodorida and Microlaimida order nov. as closely related though distinct clades, is similar to the higher classification proposed by Lorenzen (1981) where the suborder Chromadorina Filipjev, 1929 comprised the superfamilies Chromadoroidea Filipjev, 1917, Desmodoroidea and Microlaimoidea. The former two superfamilies are now place within their respective orders (Chromadorida and Desmodorida, respectively), and it is therefore necessary to erect a new order to accommodate Microlaimoidea. It should be noted that although the Microlaimoidea are often recovered as a monophyletic group in SSU phylogenies (with weak or moderate support), their position is not always resolved with certainty. Future investigations based on a better representation of microlaimoid SSU sequences, or based on other sequence(s) capable of resolving deep phylogenetic relationships, may help ascertain the validity of this classification. [Version of Record, published online 21 October 2017; http://zoobank.org/urn:lsid:zoobank.org:pub:1A1C2CA3-FA39-427A-97C6-AF0D88FEFD13 ACKNOWLEDGEMENTS Funding was provided by LabexMer and NIWA’s Coasts and Oceans Centre Research Programme ‘Marine Biological Resources’ and the programme ‘Impact of Resource Use on Vulnerable Deep-Sea Communities’ (CO1X0906). We thank the participants of NIWA voyages TAN0705, TAN1004, TAN1103, TAN1206 and TAN1701 and the officers and crew of RV Tangaroa. We are grateful to Norliana Rosli (NIWA and Sultan Idris Education University) for processing the sediment samples. We also thank Janet Grieve (NIWA) and Oleksandr Holovachov (Swedish Museum of Natural History) for their advice and fruitful discussions. We thank three anonymous reviewers for providing constructive criticisms on the manuscript. REFERENCES Andrassy I . 1976 . Evolution as a basis for the systematization of nematodes . London : Pitman Publishing Ltd . Bik HM , Lambshead PJ , Thomas WK , Lunt DH . 2010 . Moving towards a complete molecular framework of the Nematoda: a focus on the Enoplida and early-branching clades . BMC Evolutionary Biology 10 : 353 . Google Scholar CrossRef Search ADS PubMed Coomans A . 1979 . A proposal for a more precise terminology of the body regions in the nematode . Annales de la Société Royale Zoologique de Belgique 108 : 115 – 117 . De Coninck LA . 1942 . Pseudonchus symmetricus De Coninck, 1942 (Nematoda-Choanolaimidae), un nématode à symétrie bilatérale secondaire de l’extrémité anthérieure . Bulletin du Musée royal d’Histoire Naturelle de Belgique, Tome 21 . De Coninck L . 1965 . Systématique des Nématodes . In: Grassé PP , ed. Traité de Zoologie Anatomie, Systématique, Biologie. Tome IV Fascicule II: Némathelminthes (Nématodes) . Paris : Masson et Cie, 1–731 De Ley P , Blaxter ML . 2002 . Systematic position and phylogeny . In: Lee DL , ed. The biology of Nematodes . London : Taylor & Francis , 1 – 30 . De Ley P , Blaxter ML . 2004 . A new system for Nematoda: combining morphological characters with molecular trees, and translating clades into ranks and taxa . Nematology Monographs & Perspectives 2 : 633 – 653 . De Ley P , De Ley IT , Morris K , Abebe E , Mundo-Ocampo M , Yoder M , Heras J , Waumann D , Rocha-Olivares A , Jay Burr AH , Baldwin JG , Thomas WK . 2005 . An integrated approach to fast and informative morphological vouchering of nematodes for applications in molecular barcoding . Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences 360 : 1945 – 1958 . Google Scholar CrossRef Search ADS PubMed Filipjev IN . 1921 . Free-living marine nematodes in the vicinity of Sevastopol . Trudy Osoboi Zoologicheskoi Laboratorii I Sevastopolskoi Biologi Stantsii Rossiysk Akademii Nauk 41 : 353 – 614 . Gerlach SA , Riemann F . 1973/1974 . The Bremerhaven checklist of aquatic nematodes. A catalogue of Nematoda Adenophorea excluding the Dorylaimida . Veroeffentlichungen des Instituts fuer Meeresforschung in Bremerhaven Suppl. 4, Part 1 (1973) and Part 2 (1974). Guindon S , Gascuel O . 2003 . A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood . Systematic Biology 52 : 696 – 704 . Google Scholar CrossRef Search ADS PubMed Holovachov O . 2014 . Chapter 7.16: order Plectida Gadea, 1973 . In: Schmidt-Raesa A , ed. Handbook of Zoology Gastrotricha, Cycloneuralia and Gnathifera. Volume 2: Nematoda . Hamburg : De Gruyter , 487 – 536 . Holterman M , Holovachov O , van den Elsen S , van Megen H , Bongers T , Bakker J , Helder J . 2008 . Small subunit ribosomal DNA-based phylogeny of basal Chromadoria (Nematoda) suggests that transitions from marine to terrestrial habitats (and vice versa) require relatively simple adaptations . Molecular Phylogenetics and Evolution 48 : 758 – 763 . Google Scholar CrossRef Search ADS PubMed Holterman M , van der Wurff A , van den Elsen S , van Megen H , Bongers T , Holovachov O , Bakker J , Helder J . 2006 . Phylum-wide analysis of SSU rDNA reveals deep phylogenetic relationships among nematodes and accelerated evolution toward crown clades . Molecular Biology and Evolution 23 : 1792 – 1800 . Google Scholar CrossRef Search ADS PubMed Huang Y , Zhang Z . 2005 . Two new species and ne new record of free-living marine nematodes from the Yellow Sea, China . Cahiers de Biologie Marine 46 : 365 – 378 . Huelsenbeck JP , Ronquist F . 2001 . MRBAYES: Bayesian inference of phylogenetic trees . Bioinformatics 17 : 754 – 755 . Google Scholar CrossRef Search ADS PubMed Jacob J , Jaleel KUA , Vijayan AK . 2015 . A new species of the rare nematode genus Paramicrolaimus Wieser, 1954 (Chromadorida: Paramicrolaimidae) from the south eastern Arabian Sea . Zootaxa 3904 : 563 – 571 . Google Scholar CrossRef Search ADS PubMed Jayasree K , Warwick RM . 1977 . Free-living nematodes of a polluted sandy beach in the Firth of Clyde, Scotland. Description of seven new species . Journal of Natural History 11 : 289 – 302 . Google Scholar CrossRef Search ADS Jensen P . 1978 . Revision of Microlaimidae, erection of Molgolaimidae fam.n and remarks on the systematic position of Paramicrolaimus (Nematoda, Desmodorida) . Zoologica Scripta 7 : 159 – 173 . Google Scholar CrossRef Search ADS Kearse M , Moir R , Wilson A , Stones-Havas S , Cheung M , Sturrock S , Buxton S , Cooper A , Markowitz S , Duran C , Thierer T , Ashton B , Meintjes P , Drummond A . 2012 . Geneious basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data . Bioinformatics 28 : 1647 – 1649 . Google Scholar CrossRef Search ADS PubMed Leduc D , Zhao ZQ . 2016 . Molecular characterisation of five nematode species (Chromadorida, Selachinematidae) from shelf and upper slope sediments off New Zealand, with description of three new species . Zootaxa 4132 : 59 – 76 . Google Scholar CrossRef Search ADS PubMed Lorenzen S . 1981 . Entwurf eines phylogenetischen Systems der freilebenden Nematoden . Veröffentlichugen de Institut für Meeresforschung in Bremerhaven 7 : 472S . Meldal BH , Debenham NJ , De Ley P , De Ley IT , Vanfleteren JR , Vierstraete AR , Bert W , Borgonie G , Moens T , Tyler PA , Austen MC , Blaxter ML , Rogers AD , Lambshead PJ . 2007 . An improved molecular phylogeny of the Nematoda with special emphasis on marine taxa . Molecular Phylogenetics and Evolution 42 : 622 – 636 . Google Scholar CrossRef Search ADS PubMed Neira C , Decraemer W . 2009 . Desmotersia levinae, a new genus and new species of free-living nematode from bathyal oxygen minimum zone sediments off Callao, Peru, with discussion on the classification of the genus Richtersia (Chromadorida: Selachinematidae) . Organisms, Diversity & Evolution 9 : 1.e1 – 1.e15 Google Scholar CrossRef Search ADS Nunn GB . 1992 . Nematode molecular evolution . PhD Thesis , University of Nottingham , UK . Platt HM . 1985 . Further observations on the Ethmolaimidae (Nematoda: Chromadorida) . Journal of Natural History 19 : 139 – 149 . Google Scholar CrossRef Search ADS Rambaut A , Drummond AJ . 2007 . Tracer v. 1.4 . Available at: http://beast.bio.ed.ac.uk/Tracer Somerfield PJ , Warwick RM . 1996 . Meiofauna in marine pollution monitoring programmes: a laboratory manual . Lowestoft : Ministry of Agriculture, Fisheries and Food . Swofford DL . 2002 . PAUP*. Phylogenetic analysis using parsimony (and other methods), version 4 . Sunderland, MA : Sinauer Associates . Tchesunov AV . 1988 . New species nematodes from the White Sea . Proceedings of the Zoological Institute, Leningrad 180 : 68 – 76 . Tchesunov AV . 2014a . Order Chromadorida Chitwood, 1933 . In: Schmidt-Raesa A , ed. Handbook of Zoology Gastrotricha, Cycloneuralia and Gnathifera. Volume 2: Nematoda . Hamburg : De Gruyter , 373 – 398 . Tchesunov AV . 2014b . Order Desmodorida De Coninck, 1965 . In: Schmidt-Raesa A , ed. Handbook of Zoology Gastrotricha, Cycloneuralia and Gnathifera. Volume 2: Nematoda . Hamburg : De Gruyter , 399 – 434 . van Megen H , van den Elsen S , Holterman M , Karssen G , Mooyman P , Bongers T , Holovachov O , Bakker J , Helder J . 2009 . A phylogenetic tree of nematodes based on about 1200 full-length small subunit ribosomal DNA sequences . Nematology 11 : 927 – 950 . Google Scholar CrossRef Search ADS Wieser W . 1953 . Free-living marine nematodes: I. Enoploidea . Acta Universita Lundensis 49 : 1–156. Wieser W . 1954 . Free-living marine nematodes: II. Chromadoroidae . Acta Universita Lundensis 50 : 1–149 Wieser W . 1959 . Free-living marine nematodes and other mall invertebrate of Puget Sound Beaches . Seattle : University of Washington Press . Williams BD , Schrank B , Huynh C , Shownkeen R , Waterston RH . 1992 . A genetic mapping system in Caenorhabditis elegans based on polymorphic sequence-tagged sites . Genetics 131 : 609 – 624 . Google Scholar PubMed Zhao Z , Li D , Davies KA , Ye W . 2015 . Schistonchus zealandicus n. sp. (Nematoda: Aphelenchoididae) associated with Ficus macrophylla in New Zealand . Nematology 17 : 53 – 66 . Google Scholar CrossRef Search ADS Zheng JW , Subbotin SA , He SS , Gu JF , Moens M . 2002 . Molecular characterisation of some Asian isolates of Bursaphelenchus xylophilus and B. mucronatus using PCR-RFLPs and sequences of ribosomal DNA . Russian Journal of Nematology 11 : 17 – 22 . © 2017 The Linnean Society of London, Zoological Journal of the Linnean Society This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Zoological Journal of the Linnean Society Oxford University Press

Phylogenetic position of the Paramicrolaimidae, description of a new Paramicrolaimus species and erection of a new order to accommodate the Microlaimoidea (Nematoda: Chromadorea)

Loading next page...
 
/lp/ou_press/phylogenetic-position-of-the-paramicrolaimidae-description-of-a-new-L70CQBE0HN
Publisher
Oxford University Press
Copyright
© 2017 The Linnean Society of London, Zoological Journal of the Linnean Society
ISSN
0024-4082
eISSN
1096-3642
D.O.I.
10.1093/zoolinnean/zlx072
Publisher site
See Article on Publisher Site

Abstract

Abstract The phylogenetic position of Paramicrolaimidae Lorenzen, 1981, a rare group of marine free-living nematodes, has been the subject of debate due to the unique morphology of the buccal cavity, an unusual combination of other morphological traits, and lack of molecular sequences. Here, Paramicrolaimus hohonucola sp. nov. is described from the continental slope of New Zealand, and the position of the family Paramicrolaimidae is investigated based on analyses of the small subunit (SSU) and D2–D3 region of large subunit (LSU) rDNA genes. This is the first record of Paramicrolaimidae from the Southwest Pacific, and the deepest record of the family to date (347–1514 m depth). Observations using scanning electron microscopy revealed the presence of a transverse mouth opening formed by the fusion of the two ventrosublateral lip lobes and enlarged dorsal lip lobe, resulting in partial dorsoventral symmetry, which is an unusual feature within the phylum. Phylogenetic analyses suggest a close relationship between the Paramicrolaimidae and the family Selachinematidae Cobb, 1915 (order Chromadorida Chitwood, 1933). The Paramicrolaimidae lack cuticle punctations, a key morphological trait of all families currently classified within the Chromadorida; however, in the absence of any clear morphological affinities with other chromadorean orders, we propose that Paramicrolaimidae be placed within Chromadorida based on evidence for a close relationship with Selachinematidae as shown by SSU and LSU phylogenies. Our SSU phylogeny supports the results of previously published analyses showing a close relationship between Molgolaimus Ditlevsen, 1921 and the Microlaimidae Micoletzky, 1922. We therefore propose that Molgolaimus be removed from Desmodoroidea where it is currently classified and instead placed within Microlaimoidea. We also propose that the family Molgolaimidae Jensen, 1978 be reinstated and moved to accommodate Molgolaimus within Microlaimoidea. As in previous SSU phylogenies, our analyses provide no evidence for a close relationship between Desmodoroidea and Microlaimoidea, which currently comprise Desmodorida. Based on this molecular evidence, the clear morphological differences between Microlaimoidea and the closely related Chromadorida, and the lack of a synapomorphy linking Desmodoroidea and Microlaimoidea, we propose the order Microlaimida ord. nov. to accommodate the superfamily Microlaimoidea. D2–D3 region of large subunit (LSU) rDNA gene, Deep-Sea, Desmmodoroidea, Microlaimida order nov, Molgolaimus, Paramicrolaimus hohonucola sp. nov, small subunit (SSU) rDNA gene INTRODUCTION The placement of several marine nematode taxa remains problematic, largely because of the relatively small number of phylogenetically informative morphological traits available for higher level classification, and the frequent occurrence of convergent evolution within the phylum (Lorenzen, 1981; Filipjev, 1921; van Megen et al., 2009). Over the last two decades molecular phylogenies have provided important new insights into relationships among and within nematode orders (e.g. Bik et al., 2010). The nematode classification of De Ley & Blaxter (2002), based on a combination of a new molecular phylogeny from small subunit (SSU) rDNA and existing morphological phylogenies, and later updated by the same authors (De Ley & Blaxter, 2004), was originally based mostly on sequences of terrestrial and parasitic taxa. Meldal et al. (2007) later built on this molecular phylogeny by providing a greater representation of clades containing marine species. Their analyses, as well as the subsequent analyses of Holterman et al. (2008) and van Megen et al. (2009), indicate that the largely marine orders Chromadorida Chitwood, 1933 and Desmodorida De Coninck, 1965 are closely related and are the most basal in the class Chromadorea. These analyses also put into question the placement of the families Microlaimidae Micoletzky, 1922 and Monoposthiidae Filipjev, 1934, which together with the family Aponchiidae Gerlach, 1963 comprise the superfamily Microlaimoidea Micoletzky, 1922; at present, Microlaimoidea is classified within Desmodorida alongside Desmodoroidea Filipjev, 1922, but molecular phylogenies suggest instead that Microlaimoidea is more closely related to Chromadorida than to the rest of the Desmodorida (Meldal et al., 2007; Holterman et al., 2008). In addition, recent phylogenetic analyses suggest that the genus Molgolaimus Ditlevsen, 1921, which is currently placed in Desmodoroidea (family Desmodoridae Filipjev, 1922), in fact belongs to Microlaimoidea (Meldal et al., 2007; Leduc & Zhao, 2016). However, this new evidence is not included in recent taxonomic treatments of these orders (Tchesunov, 2014a, b), which are still largely based on the phylogeny presented by De Ley & Blaxter (2002, 2004), The classification of other marine taxa remains controversial due to the presence of unique or conflicting morphological features and absence of any molecular sequence data. An example of such a family is Paramicrolaimidae Lorenzen, 1981, which was erected to accommodate the genus Paramicrolaimus Wieser, 1954, a rare group of marine free-living nematodes. This genus is characterized by the unusual morphology of the buccal cavity with a medium to large dorsal tooth which does not project completely into the pharyngeal lumen but is instead set within, and surrounded by, the pharyngeal wall, and is always associated with a conspicuous pharyngeal gland (Lorenzen, 1981). To our knowledge, this buccal cavity structure is not found in any other nematode taxon (i.e. it is an autapomorphy of the family). This feature, and an unusual combination of other morphological traits, has resulted in the systematic position of Paramicrolaimus being changed several times since it was first described. Wieser (1954) first placed it in the family Microlaimidae because of the three distinct circles of cephalic sense organs, the deep buccal cavity provided with a dorsal tooth and the spiral amphid with 1.25 turns. Andrassy (1976) grouped Paramicrolaimus together with the genus Ohridius Gerlach & Riemann, 1973/1974, which has since been synonymized with Domorganus Goodey, 1946 within the order Plectida Gadea, 1973. Paramicrolaimus was later placed within Stilbonematinae Chitwood, 1936 (order Desmodorida) by Jensen (1978) based on the reduced buccal cavity, the slender body shape, an inferred association with bacteria and his observations of only one testis in males of Paramicrolaimus. The same author also highlighted the similarities between Paramicrolaimus and Coninckia Gerlach, 1956 (order Araeolaimida De Coninck & Schuurmans Stekhoven, 1933) in the shape of the male amphid, the arrangement of the cephalic sense organs, the structure of the pharynx and the shape of the tail. Lorenzen (1981) later showed that inferred affinities with the Stilbonematinae, which are characterized by males with only one testis, was incorrect because Paramicrolaimus in fact possesses two testes. He erected the monogeneric family Paramicrolaimidae, which he placed within Leptolaimina Lorenzen, 1981, a suborder that gathered all phylogenetically unsettled families within the order Chromadorida. De Ley & Blaxter (2002) later placed Paramicrolaimidae within the superfamily Leptolaimoidea Örley, 1880 (order Plectida) in their phylogenetic analyses based on SSU rDNA sequences; however, no molecular sequences were available for Paramicrolaimidae at the time and the position of this family was therefore presumably determined based on possible relationships with families of Leptolaimina, such as Leptolaimidae Örley, 1880 and Bastianiidae De Coninck, 1965, for which molecular sequences were available. Most recently, Holovachov (2014) acknowledged the uncertain affinities of the family Paramicrolaimidae, but noted morphological similarities with Tubolaimoididae Lorenzen, 1981 and Tarvaiidae Lorenzen, 1981 (order Plectida). The placement of Paramicrolaimidae awaits molecular analyses to clarify its relationships within Chromadorea. Paramicrolaimus currently comprises four valid species, each described from only a few individuals collected from coastal and shelf sediments (Jacob et al., 2015). During a study of the diversity of nematodes on the continental slope of New Zealand, Paramicrolaimus specimens were isolated from multiple core samples for morphological and molecular analyses. All specimens were identified as belonging to a single species, herein described as Paramicrolaimus hohonucola sp. nov. The phylogenetic relationships of the family Paramicrolaimidae were investigated based on SSU rDNA sequences representing the orders Chromadorida, Desmodorida, Araeolaimida, Plectida and Monysterida Filipjev, 1929 which contain the bulk of marine species with the class Chromadorea, and based on large subunit (LSU) rDNA sequences at the within-order level. The SSU rDNA analyses also led to a re-evaluation of the current classification of the Microlaimoidea, as previously indicated by the phylogenies of Meldal et al (2007) and Holterman et al. (2008). METHODS Sampling sites and sample processing Sediment samples were obtained from continental slope sites on Chatham Rise off the east coast of the South Island of New Zealand, from the Hikurangi Margin to the south-east of the North Island and from the Bay of Plenty off the north-east coast of the North Island (Fig. 1). Sampling was conducted using RV Tangaroa during National Institute of Water and Atmospheric Research (NIWA) voyage TAN0705 (April 2007), TAN1103 (February 2011) and TAN1701 (January 2017) to the Chatham Rise, voyage TAN1004 (April 2010) to the Hikurangi Margin and voyage TAN1206 (April 2012) to the Bay of Plenty. Figure 1. View largeDownload slide Map of central New Zealand and surrounding continental margin showing location of core samples obtained from Chatham Rise (CR, circular symbols), Hikurangi Margin (HIK, diamond symbol) and Bay of Plenty (BoP, triangular symbol); 250, 500, 1000 and 2000 m depth isobaths are shown. The RV Tangaroa sampling voyages are listed in the legend. Figure 1. View largeDownload slide Map of central New Zealand and surrounding continental margin showing location of core samples obtained from Chatham Rise (CR, circular symbols), Hikurangi Margin (HIK, diamond symbol) and Bay of Plenty (BoP, triangular symbol); 250, 500, 1000 and 2000 m depth isobaths are shown. The RV Tangaroa sampling voyages are listed in the legend. Sediment samples were collected using an Ocean Instrument MC-800A multicorer (internal diameter of core = 9.52 cm). Subsamples for the analysis of nematodes were taken using a core with an internal diameter of 26 or 29 mm to a depth of 5 cm. These cores were sliced into 0–1 and 1–5 cm sediment depth layers and preserved in 10% buffered formalin. During voyage TAN1701, the upper 5 cm layer of sediment remaining in the multicorer tube after subsampling was kept frozen. All sediment samples were rinsed on a 1 mm mesh to remove macro-infauna and on a 45 µm mesh to retain nematodes, which were extracted from the sieved sediment by the Ludox flotation method (Somerfield & Warwick, 1996). Specimens for light microscopy were transferred to distilled water, sorted using a dissecting microscope, transferred to glycerol and mounted onto permanent slides using the method of Somerfield & Warwick (1996). Frozen sediment samples (TAN1701) were thawed overnight, extracted using the Ludox flotation methods and transferred to freshwater. Specimens were sorted using a dissecting microscope, and were either transferred to a 10% formalin solution for observation using scanning electron microscopy (SEM), or transferred to lysis buffer and frozen for molecular analyses. Specimens for molecular analyses were first mounted onto a temporary slide in a drop of water to verify their identity and pictures were taken to provide image vouchers. SEM observations were conducted on specimens retrieved from frozen samples (TAN1701) and specimens previously mounted in glycerol (TAN0705 and TAN1004). Mounted specimens were gradually transferred from glycerol back to 10% formalin prior to fixation in 4% osmium tetroxide overnight. Specimens were then gradually transferred to pure ethanol using a grade ethanol series, critical point dried and mounted onto stubs before coating with gold using a sputter coater. Observations were made using a Hitachi TM3000 tabletop SEM at high vacuum mode. All measurements are in µm, and all curved structures were measured along the arc. The terminology used for describing the arrangement of morphological features such as setae follows Coomans’ (1979) typology. Type specimens are held in the NIWA Invertebrate Collection (Wellington), and the National Nematode Collection of New Zealand (NNCNZ, Auckland). Abbreviations in the text are as follows: a, body length/maximum body diameter; b, body length/pharynx length; c, body length/tail length; c′, tail length/anal body diameter; cbd, corresponding body diameter; %V, vulva distance from anterior end of body × 100/total body length. DNA extraction, sequence processing and phylogenetic inference Individual nematodes were used for DNA extraction by lysing in a buffer of 20 μL containing proteinase K (Williams et al., 1992) and DNA was extracted using the method of Zheng et al. (2002). Extracted DNA was used for PCR amplification of nearly the full length of SSU rDNA gene with two sets of primers, 1096F/1912R and 1813F/2646R (Holterman et al., 2006). Primers, D2A and D3B (Nunn, 1992) were used to amplify the D2/D3 expansion segments of the LSU rDNA gene. The PCR composition and cycling conditions for the amplification of the SSU and LSU genes, and sequencing were conducted as per Zhao et al. (2015). The sequences obtained were used to construct phylogenetic trees in Geneious 10.1.3 (http://www.geneious.com; Kearse et al. 2012). Related sequences were downloaded from GenBank and aligned with ClustalX in Geneious using the default parameter values. Phylogenetic affinities of Paramicrolaimus were investigated using the SSU and LSU rDNA genes. The SSU rDNA gene is the only locus known to resolve deep relationships among nematode taxa, whereas the LSU rDNA gene is informative at lower taxonomic levels only (De Ley et al., 2005; Bik et al., 2010). An initial phylogenetic analysis based on SSU rDNA sequences was therefore conducted using representative species (one per genus) from the orders Chromadorida, Desmodorida, Monhysterida, Araeolaimida and Plectida, which comprise the bulk of marine diversity in the class Chromadorea. Only sequences over 1200 bp were used, with the exception of Microlaimus De Man, 1880 and Gomphionema Wieser & Hopper, 1966, for which only relatively short sequences were available (399 and 680 bp, respectively). Based on the results of the first analysis, a more focused investigation of the placement of Paramicrolaimus was conducted based on Chromadorida SSU rDNA sequences, as well as Microlaimidae and Monoposthidae sequences, which have been shown to cluster either with or near the Chromadorida (Meldal et al., 2007; Holterman et al., 2008). A LSU rDNA tree was also built focusing on the Selachinematidae, the family most closely related to Parmicrolaimus hohonucola sp. nov., as identified from the SSU rDNA analyses. Sequences of the genus Richtersia Steiner, 1916, which may have affinities with the family Selachinematidae (Neira & Decraemer, 2009), were included in this analysis. Phylogenies were built in Geneious 10.1.3 (http://www.geneious.com; Kearse et al. 2012). The related sequences were aligned with ClustalW in Geneious using the default parameter values. PAUP*4.0b10 (Swofford, 2002) was used to select the best model using the Akaike information criterion. A Bayesian tree was constructed with MrBayes under the best-fit model [GTR (general time-reversible) + I (proportion of invariable sites) + G (gamma distribution)] for both SSU and LSU genes (Huelsenbeck & Ronquist, 2001). The trees were run with chain length of 1100000, burn-in length of 100000 and rooted Tobrilus gracilis (Bastian, 1865) (order Triplonchida Cobb, 1919) for SSU and Paracanthonchus miltommatus Leduc & Zhao, in press (order Chromadorida) for LSU, respectively. The Bayesian trees were viewed in Tracer 1.4 (Rambaut & Drummond, 2007) and edited in PowerPoint. The results of the phylogenetic analyses described above were tested using maximum likelihood analysis as an alternative tree-building method. These analyses were conducted in Geneious 10.1.3 with default settings and 1000 bootstrap replicates (Guindon & Gascuel, 2003). RESULTS Molecular analyses The Bayesian topology recovered in the SSU-based phylogeny shows that the Chromadorida is not monophyletic (clades A2 and B2 in Fig. 2), and together with the Microlaimoidea and Molgolaimus (clade A1), split early from other lineages, followed by the main Desmodorida clade (clade B1). The tree topology, however, did not resolve the branching order of these groups. Clade A1, which comprises the Microlaimoidea and Molgolaimus, is not grouped with the Desmodoroidea and is instead recovered as a sister taxon to the Chromadorida (85% posterior probability and 35% bootstrap value). The Araeolaimida, Monhysterida and Plectida together formed a relatively well-supported clade (clade B3 in Fig. 2; 100% posterior probability and 70% bootstrap value), but none of these individual orders was monophyletic. Figure 2. View largeDownload slide Bayesian tree of marine Chromadorean orders inferred from small subunit (SSU) sequences under the general time-reversible (GTR) + proportion of invariable sites (I) + gamma distribution (G) model. Posterior probability (left) and bootstrap values (right) are given on corresponding clades. Sequences obtained in this study are shown in bold; orders are listed on the far right and the position of the families Paramicrolaimidae, Selachinematidae, Microlaimidae and Monoposthiidae is shown. Main clades are shown by blue letters and/or numerals. The scale stands for substitutions per site. Figure 2. View largeDownload slide Bayesian tree of marine Chromadorean orders inferred from small subunit (SSU) sequences under the general time-reversible (GTR) + proportion of invariable sites (I) + gamma distribution (G) model. Posterior probability (left) and bootstrap values (right) are given on corresponding clades. Sequences obtained in this study are shown in bold; orders are listed on the far right and the position of the families Paramicrolaimidae, Selachinematidae, Microlaimidae and Monoposthiidae is shown. Main clades are shown by blue letters and/or numerals. The scale stands for substitutions per site. Our phylogenetic analyses indicate that Paramicrolaimidae constitutes a sister taxon to the family Selachinematidae (clade B2 in Fig. 2; Fig. 3). This relationship was relatively well supported in Bayesian analyses (100 and 74% posterior probability in Chromadorea- and Chromadorida-level analyses, respectively), but only weakly supported in maximum likelihood analyses (46 and 29% bootstrap values in Chromadorea- and Chromadorida-level analyses, respectively). The placement of Selachinematidae was not well resolved in our analyses of the chromadorean orders; only one of the selachinematid genera (Latronema Wieser, 1954) was placed within Chromadorida, whereas the remaining four genera formed clade B2 together with P. hohonucola sp. nov. outside of the main Chromadorida clade and all the other main clades (Fig. 2). The placement of clade B2, however, was poorly supported (73% posterior probability and 35% bootstrap value). In our analysis of SSU rDNA sequences focusing on the Chromadorida, Selachinematidae formed a monophyletic group, which together with P. hohonucola sp. nov., formed a sister group to the remaining chromadorid taxa, although there was no support for this topology (Fig. 3). Figure 3. View largeDownload slide Bayesian tree of the order Chromadorida inferred from small subunit (SSU) sequences under the general time-reversible (GTR) + proportion of invariable sites (I) + gamma distribution (G) model. Posterior probability (left) and bootstrap values (right) are given on corresponding clades. Families are shown on the right-hand side. Taxa currently classified with the order Desmodorida are shown on light grey background. The scale stands for substitutions per site. Figure 3. View largeDownload slide Bayesian tree of the order Chromadorida inferred from small subunit (SSU) sequences under the general time-reversible (GTR) + proportion of invariable sites (I) + gamma distribution (G) model. Posterior probability (left) and bootstrap values (right) are given on corresponding clades. Families are shown on the right-hand side. Taxa currently classified with the order Desmodorida are shown on light grey background. The scale stands for substitutions per site. In the LSU tree of the Selachinematidae (Fig. 4), P. hohonucola sp. nov. was placed in a well-supported clade (100% posterior probability and 96% bootstrap value) comprising the bulk of Selachinematidae sequences representing the genera Choanolaimus De Man, 1880, Halichoanolaimus De Man, 1886, Bendiella Leduc, 2013, Cheironchus Cobb, 1917 and Richtersia Steiner, 1916. Figure 4. View largeDownload slide Bayesian tree inferred from D2–D3 of large subunit (LSU) sequences under the general time reversible (GTR) + proportion of invariable sites (I) + gamma distribution (G) model. Posterior probability (left) and bootstrap values (right) greater are given on corresponding clades. Sequences obtained in this study are shown in bold. The scale stands for substitutions per site. Figure 4. View largeDownload slide Bayesian tree inferred from D2–D3 of large subunit (LSU) sequences under the general time reversible (GTR) + proportion of invariable sites (I) + gamma distribution (G) model. Posterior probability (left) and bootstrap values (right) greater are given on corresponding clades. Sequences obtained in this study are shown in bold. The scale stands for substitutions per site. Taxonomic account Order Chromadorida Chitwood, 1933 Family Paramicrolaimidae Lorenzen, 1981 Synonyms: None. Diagnosis: Cuticle striated from anterior edge of amphids; no longitudinal bars. Short somatic setae present. Lip pairs usually fused into three, or in some cases two, lobes. Six inner labial papillae present; six outer labial setae located in a separate circle and slightly anterior to the four cephalic setae; cephalic setae located anterior to the amphid. Four subcephalic setae sometimes present at level of amphids. Amphidial fovea spiral with less than 2 turns. Buccal cavity narrow, tubular, asymmetrical; dorsal tooth present, set within pharyngeal tissue and only protruding slightly into buccal cavity, sometimes with lateral pointed projections, always connected to a dorsal gland; smaller ventrosublateral teeth also present. Pharynx muscular, cylindrical, with posterior bulb; muscular cardia present. Secretory-excretory system present; renette cell elongated, located at level of or posterior to cardia; excretory ampulla and duct located slightly posterior to level of nerve ring. Female reproductive system didelphic-amphidelphic with equally developed branches, ovaries reflexed antidromously. Male reproductive system diorchic with two outstretched testes. Spicules symmetrical, arcuate; gubernaculum present. Row of midventral precloacal supplements consisting of cup-shaped structures with spout-like structures pointing posteriorly. Three caudal glands and spinneret present. Remarks: The present diagnosis has been adapted from Holovachov (2014) to better correspond with our own observations, other Paramicrolaimidae species descriptions and with the current terminology of the structures described. Our observations show that the cuticle of Paramicrolaimus hohonucola sp. nov. is striated (not annulated), and that the lip region comprises two lobes, unlike other species of the genus which were described as possessing three equal lobes. In P. hohonucola sp. nov. the dorsal lobe of the lip region is enlarged and is almost equal in size and shape to the two ventrosublateral lobes, which are at least partially or completely fused together. As a result, the mouth is a straight or slightly bent transverse opening, with four inner labial papilla along its ventral side and two inner labial papilla dorsally. The subcephalic setae were described as absent in previous diagnoses, but four subcephalic setae are present in P. hohonucola sp. nov. at the level of the amphids. Males have also been described as monorchic or diorchic because only one testis was observed in the original descriptions of P. spirulifer by Jensen (1978). However, Lorenzen (1981) observed that this species is in fact diorchic like the rest of the Paramicrolaimidae. Genus Paramicrolaimus Wieser, 1954 Synonyms: None. Type species: Paramicrolaimus primus Wieser, 1954. Diagnosis: See family diagnosis. Remarks: The description of P. primus is based on one female specimen. It is therefore not adequately described and is considered species inquirenda. Valid species: P. spirulifer Wieser, 1959 P. mirus Tchesunov, 1988  = P. minus Tchesunov, 1988 in Huang & Zhang, 2005 (lapsus calami) P. damodarani Jacob, Jaleel & Vijayan, 2015 Invalid species: P. acanthus Jayasree & Warwick, 1977  = Microlaimus acanthus (Jayasree & Warwick, 1977) Kovalyev & Tchesunov, 2005 P. conothelis Lorenzen, 1973  = Microlaimus conothelis (Lorenzen, 1973) Jensen, 1978 P. lunatus Wieser & Hopper, 1967  = Microlaimus lunatus (Wieser & Hopper, 1967) Jensen, 1978 P. papillatus (Gerlach, 1956) Wieser & Hopper, 1967  = Microlaimus papillatus Gerlach, 1956 (op. Jensen, 1978) Paramicrolaimus hohonucola Verdon & Leduc sp. nov. (Figs 5–8; Table 1) Diagnosis: Paramicrolaimus hohonucola sp. nov. is characterized by body length 2150–3620 µm. Outer labial setae 8–11 µm long, cephalic setae 8–13 µm long, in two different circles; four subcephalic setae, 4–7 µm long, at level of, and adjacent to, amphids. Lip region with two equal or almost equal lobes flanking transverse mouth opening; dorsal lobe formed by enlarged dorsal lip pair and ventral lobe formed by fusion of two ventrosublateral lip pairs. Amphideal fovea 0.43–0.60 cbd. Buccal cavity with two small ventrosublateral teeth and large dorsal tooth with two lateral pointed projections (dorsosublateral teeth). Smaller ventral tooth present. Arcuate spicules 1.8–2.1 cloacal body diameter long, gubernaculum with straight apophyses pointing dorsally. Five to seven precloacal supplements consisting of cup-shaped structures with spout-like structures pointing posteriorly. Vulva located at 42–48% of body length from anterior. Short, conical tail with terminal spinneret, 2.7–3.4 cloacal body diameters long in males with ventral row of 7–8 postcloacal setae, few dorsal and lateral setae and circle of four subterminal setae; in females, tail 3.9–5.3 anal body diameters long with only one subterminal, dorsal seta. Figure 5. View largeDownload slide Paramicrolaimus hohonucola sp. nov. A, female anterior body region. B, male anterior body region. C, female cephalic region. D, male cephalic region. E, male copulatory system. F, male posterior body region. G, female posterior body region. Scale bar: A, B = 44 µm; C, D = 20 µm; E = 14 µm; F, G = 31 µm. Figure 5. View largeDownload slide Paramicrolaimus hohonucola sp. nov. A, female anterior body region. B, male anterior body region. C, female cephalic region. D, male cephalic region. E, male copulatory system. F, male posterior body region. G, female posterior body region. Scale bar: A, B = 44 µm; C, D = 20 µm; E = 14 µm; F, G = 31 µm. Figure 6. View largeDownload slide Paramicrolaimus hohonucola sp. nov. A, entire male. B, female reproductive system. Scale bar: A = 210 µm; B = 190 µm. Figure 6. View largeDownload slide Paramicrolaimus hohonucola sp. nov. A, entire male. B, female reproductive system. Scale bar: A = 210 µm; B = 190 µm. Figure 7. View largeDownload slide Paramicrolaimus hohonucola sp. nov. Light micrographs of female cephalic region. A, lateral view showing buccal armature. B, lateral view showing lips, dorsal pharyngeal gland and granules. D, dorsal tooth; Gl, dorsal pharyngeal gland; Gr, pigmented granule; L, lip; DS, dorsosublateral tooth; VS, ventrosublateral teeth. Scale bar = 18 µm. Figure 7. View largeDownload slide Paramicrolaimus hohonucola sp. nov. Light micrographs of female cephalic region. A, lateral view showing buccal armature. B, lateral view showing lips, dorsal pharyngeal gland and granules. D, dorsal tooth; Gl, dorsal pharyngeal gland; Gr, pigmented granule; L, lip; DS, dorsosublateral tooth; VS, ventrosublateral teeth. Scale bar = 18 µm. Figure 8. View largeDownload slide Paramicrolaimus hohonucola sp. nov. Scanning electron microscopy (SEM). A, male anterior body region (lateral view). B, male anterior body region (latero-ventral view). C, precloacal supplements. D, male posterior region. Arrows show position of inner labial papillae. Scale bar: A, B = 6 µm; C = 5 µm; D = 30 µm. Figure 8. View largeDownload slide Paramicrolaimus hohonucola sp. nov. Scanning electron microscopy (SEM). A, male anterior body region (lateral view). B, male anterior body region (latero-ventral view). C, precloacal supplements. D, male posterior region. Arrows show position of inner labial papillae. Scale bar: A, B = 6 µm; C = 5 µm; D = 30 µm. Table 1. Morphometrics (µm; mean, range) of Paramicrolaimus hohonucola sp. nov. Characters Holotype Paratypes Paratypes Male Males Females N - 5 6 L 3212 2544 (2152–3238) 3240 (2439–3619) a 78.3 70 (65–79) 57 (49–68) b 16.6 15 (13–16) 16 (14–18) c 27.7 29 (27–31) 24 (19–27) c′ 3.1 3.0 (2.7–3.4) 4.6 (4.0–5.3) Maximum body diameter 41 36 (28–48) 57 (49–63) Diameter at amphids 24 22.2 (19–28) 24.2 (20–26) Length of outer labial setae 11 10 (9–11) 9.5 (8–11) Length of cephalic setae 11 10 (8–12) 10.2 (9–13) Length of subcephalic setae 5 5 (4–7) 5.2 (4–7) Amphideal fovea width 10 11 (9–12) 12 (11–12) Amphideal fovea width %cbd 42 48 (43–55) 48 (46–60) Amphid distance from anterior end 17 17 (13–22) 19 (17–22) Nerve ring from the anterior end 93 82 (75–98) 100 (84–128) Nerve ring cbd 36 33 (24–43) 40 (34–48) Pharynx length 194 175 (144–207) 197 (170–222) Pharyngeal bulb length 64 55 (44–67) 64 (62–67) Pharyngeal bulb width 24 23 (17–30) 29 (26–31) Pharynx cbd at base 41 35 (28–47) 45 (39–50) Excretory pore from anterior end 122 113 (89–134) 122 (106–134) Anal/cloacal body diameter 38 30 (24–39) 30 (25–34) Tail length 116 89 (72–106) 138 (121–158) Spicule length 66 56 (45–70) - Length of gubernacular apophysis 24 20 (18–25) - Number of precloacal supplements 6 6 (5–7) - V - - 1458 (1160–1585) %V - - 45 (42–48) Vulva cbd - - 57 (49–63) Characters Holotype Paratypes Paratypes Male Males Females N - 5 6 L 3212 2544 (2152–3238) 3240 (2439–3619) a 78.3 70 (65–79) 57 (49–68) b 16.6 15 (13–16) 16 (14–18) c 27.7 29 (27–31) 24 (19–27) c′ 3.1 3.0 (2.7–3.4) 4.6 (4.0–5.3) Maximum body diameter 41 36 (28–48) 57 (49–63) Diameter at amphids 24 22.2 (19–28) 24.2 (20–26) Length of outer labial setae 11 10 (9–11) 9.5 (8–11) Length of cephalic setae 11 10 (8–12) 10.2 (9–13) Length of subcephalic setae 5 5 (4–7) 5.2 (4–7) Amphideal fovea width 10 11 (9–12) 12 (11–12) Amphideal fovea width %cbd 42 48 (43–55) 48 (46–60) Amphid distance from anterior end 17 17 (13–22) 19 (17–22) Nerve ring from the anterior end 93 82 (75–98) 100 (84–128) Nerve ring cbd 36 33 (24–43) 40 (34–48) Pharynx length 194 175 (144–207) 197 (170–222) Pharyngeal bulb length 64 55 (44–67) 64 (62–67) Pharyngeal bulb width 24 23 (17–30) 29 (26–31) Pharynx cbd at base 41 35 (28–47) 45 (39–50) Excretory pore from anterior end 122 113 (89–134) 122 (106–134) Anal/cloacal body diameter 38 30 (24–39) 30 (25–34) Tail length 116 89 (72–106) 138 (121–158) Spicule length 66 56 (45–70) - Length of gubernacular apophysis 24 20 (18–25) - Number of precloacal supplements 6 6 (5–7) - V - - 1458 (1160–1585) %V - - 45 (42–48) Vulva cbd - - 57 (49–63) a, body length/maximum body diameter; b, body length/pharynx length; c, body length/tail length; cʹ, tail length/cloacal or anal body diameter; cbd, corresponding body diameter; N, number of specimens; L, body length; V, vulva distance from anterior end of body; %V, V/total body length. View Large Table 1. Morphometrics (µm; mean, range) of Paramicrolaimus hohonucola sp. nov. Characters Holotype Paratypes Paratypes Male Males Females N - 5 6 L 3212 2544 (2152–3238) 3240 (2439–3619) a 78.3 70 (65–79) 57 (49–68) b 16.6 15 (13–16) 16 (14–18) c 27.7 29 (27–31) 24 (19–27) c′ 3.1 3.0 (2.7–3.4) 4.6 (4.0–5.3) Maximum body diameter 41 36 (28–48) 57 (49–63) Diameter at amphids 24 22.2 (19–28) 24.2 (20–26) Length of outer labial setae 11 10 (9–11) 9.5 (8–11) Length of cephalic setae 11 10 (8–12) 10.2 (9–13) Length of subcephalic setae 5 5 (4–7) 5.2 (4–7) Amphideal fovea width 10 11 (9–12) 12 (11–12) Amphideal fovea width %cbd 42 48 (43–55) 48 (46–60) Amphid distance from anterior end 17 17 (13–22) 19 (17–22) Nerve ring from the anterior end 93 82 (75–98) 100 (84–128) Nerve ring cbd 36 33 (24–43) 40 (34–48) Pharynx length 194 175 (144–207) 197 (170–222) Pharyngeal bulb length 64 55 (44–67) 64 (62–67) Pharyngeal bulb width 24 23 (17–30) 29 (26–31) Pharynx cbd at base 41 35 (28–47) 45 (39–50) Excretory pore from anterior end 122 113 (89–134) 122 (106–134) Anal/cloacal body diameter 38 30 (24–39) 30 (25–34) Tail length 116 89 (72–106) 138 (121–158) Spicule length 66 56 (45–70) - Length of gubernacular apophysis 24 20 (18–25) - Number of precloacal supplements 6 6 (5–7) - V - - 1458 (1160–1585) %V - - 45 (42–48) Vulva cbd - - 57 (49–63) Characters Holotype Paratypes Paratypes Male Males Females N - 5 6 L 3212 2544 (2152–3238) 3240 (2439–3619) a 78.3 70 (65–79) 57 (49–68) b 16.6 15 (13–16) 16 (14–18) c 27.7 29 (27–31) 24 (19–27) c′ 3.1 3.0 (2.7–3.4) 4.6 (4.0–5.3) Maximum body diameter 41 36 (28–48) 57 (49–63) Diameter at amphids 24 22.2 (19–28) 24.2 (20–26) Length of outer labial setae 11 10 (9–11) 9.5 (8–11) Length of cephalic setae 11 10 (8–12) 10.2 (9–13) Length of subcephalic setae 5 5 (4–7) 5.2 (4–7) Amphideal fovea width 10 11 (9–12) 12 (11–12) Amphideal fovea width %cbd 42 48 (43–55) 48 (46–60) Amphid distance from anterior end 17 17 (13–22) 19 (17–22) Nerve ring from the anterior end 93 82 (75–98) 100 (84–128) Nerve ring cbd 36 33 (24–43) 40 (34–48) Pharynx length 194 175 (144–207) 197 (170–222) Pharyngeal bulb length 64 55 (44–67) 64 (62–67) Pharyngeal bulb width 24 23 (17–30) 29 (26–31) Pharynx cbd at base 41 35 (28–47) 45 (39–50) Excretory pore from anterior end 122 113 (89–134) 122 (106–134) Anal/cloacal body diameter 38 30 (24–39) 30 (25–34) Tail length 116 89 (72–106) 138 (121–158) Spicule length 66 56 (45–70) - Length of gubernacular apophysis 24 20 (18–25) - Number of precloacal supplements 6 6 (5–7) - V - - 1458 (1160–1585) %V - - 45 (42–48) Vulva cbd - - 57 (49–63) a, body length/maximum body diameter; b, body length/pharynx length; c, body length/tail length; cʹ, tail length/cloacal or anal body diameter; cbd, corresponding body diameter; N, number of specimens; L, body length; V, vulva distance from anterior end of body; %V, V/total body length. View Large Material examined Holotype: male (NIWA 115460), collected in April 2010 (NIWA cruise TAN1004, station 17), Hikurangi Margin (41.6288°S, 175.8682°E), 1514 m water depth. Paratypes: One male and three female paratypes (NIWA 115461), same data as holotype. One male paratype (NNCNZ 3285), collected in April 2007 (TAN0705, station 35), Chatham Rise (43.8325°S, 176.712°E), 480 m water depth. One male paratype collected in March 2011 (NIWA cruise TAN1103, station 68), Chatham rise 43.33417°S, 178.29517°E), 347 m water depth. This specimen was used for morphological measurements, then sacrificed for SEM observations. One male and one female paratype (NIWA 115462), collected in April 2012 (NIWA cruise TAN1206, station 125), Bay of Plenty (37.4747°S, 176.7585°E), 697 m water depth. Etymology: The species name is derived from the Māori word ‘hōhonu’ which means ‘deep, deeper, profound’; and the latin suffix ‘-cola’ which means ‘dwelling in, inhabitant’. Description: Males. Brownish cylindrical body, strongly pigmented between amphids and nerve ring, and in caudal region, due to presence of brown granules. Cuticle striated from level of anterior edge of amphids to spinneret. Sparse and irregular somatic setae mainly restricted to pharyngeal and tail region, 3–4 µm long. Cephalic region slightly constricted at level of amphids. Lip region with two equal or nearly equal lobes flanking transverse mouth opening; dorsal lobe composed of dorsal lip pair, ventral lobe composed of two fused ventrosublateral lip pairs. Six-minute inner labial papillae on either side of mouth opening, only visible using SEM; two papillae on dorsal lobe and four on ventral lobe. Six outer labial setae of similar length to four cephalic setae (0.4–0.5 cbd); cephalic setae located anterior to the amphids and in separate circle to outer labial setae. Four small subcephalic setae, adjacent to, and at level of, amphids. Amphideal fovea spiral with 1.25 turns; amphideal aperture (as observed using light microscopy) oval-shaped and smaller than amphideal fovea. Buccal cavity asymmetrical, with cuticularized walls; single large dorsal tooth present, heavily cuticularized and with two lateral pointed projections (dorsosublateral teeth), set within pharyngeal tissue and protruding only slightly into buccal cavity. Dorsal gland opening into pharyngeal lumen through distal extremity of tooth. Two smaller ventrosublateral teeth present. Pharynx cylindrical and muscular, with slightly swollen anterior end surrounding buccal cavity and weak, elongated posterior bulb. Nerve ring at approximately mid-length of pharynx. Conoid and muscular cardia present, 10–13 µm long, surrounded by intestine. Secretory-excretory system present; excretory ampulla and pore situated ventrally, slightly posterior to nerve ring, renette cell located ventrally, at level or immediately posterior to cardia, 20–29 × 9–18 µm. Reproductive system with two opposed and outstreched testes, both located to left of the intestine. Sperm cells oval, 4–6 × 12–16 µm. Spicules paired, equal, arcuate, 1.8–2.1 cloacal body diameter long, with a central cuticularized projection (lamella) at proximal end. Velum and capitulum present. Tip of spicules narrow and cylindrical, forming a duct. Gubernaculum surrounding distal end of the spicules; gubernacular apophyses narrow and pointing dorsally. Five to seven precloacal supplement. Observations using SEM showed they consist of cup-shaped structures each with spout-like projection pointing posteriorly and positioned on bifid cuticularized base. Gland-like brown granules present beneath each supplement. Tail short, conical, bent ventrally, with irregularly spaced setae, row of seven ventral setae, and circle of four longer subterminal setae. Three caudal glands and spinneret present. Females. Similar to males, but with wider body. Short somatic setae sparser than in males, apparently absent in some specimens. Tail with only one subterminal dorsal seta. Reproductive system with two opposed, reflexed ovaries to left of intestine. Mature eggs 96–130 × 40–51 µm. Spermatheca not observed. Vulva located at or slightly anterior to mid-body. Proximal portion of vagina uterina surrounded by sphincter muscle. Differential diagnosis: The closest species to P. hohonucola sp. nov. is P. mirus because both species have similar a and c′ values (Table 2), and because of the similar shape and dimension of the posterior pharyngeal bulb, amphideal fovea, spicules and gubernacular apophyses. Paramicrolaimus hohonucola sp. nov. differs from P. mirus mainly by the presence of a transverse opening formed by the fusion of the two ventrosublateral lip lobes and enlarged dorsal lip lobe (vs. three lip lobes in P. mirus), and presence of caudal setae (vs. no caudal setae present in females of P. mirus). The new species also differs from P. mirus by the longer outer labial setae (8–11 µm vs. 6–8 µm in P. mirus), fewer precloacal supplements (5–7 vs. 8–10), lower values of b (13–16 vs. 16–25 in P. mirus), lower values of c (27–31 vs. 31–41 in males, and 19–27 vs. 26–31 in females) and larger body diameter at level of vulva (49–63 µm vs. 33–50 µm in P. mirus). Table 2. Morphometrics (µm) of all Paramicrolaimus species from the literature Species P. primus P. spirulifer P. mirus P. damodarani P. hohonucola sp. nov. Reference Wieser (1954) Wieser (1959) Jensen (1978) Tchesunov (1988) Huang & Zhang (2005) Jacob et al. (2015) Present study Sex Female Male Female Male Male Female Male Female Male Male Female Habitat depth (m) 250–300 0.5 28 20 64–85 95–205 347–1514 Locality Golfo de Ancud, Chile Pudget Sound, USA Øresund North, Denmark Kandalaksha Bay, Russia Yellow Sea, China Arabian Sea, India Southwestern Pacific, New Zealand L 2340 4430 4180 5170 4060 3500 3052–3600 3680–4340 1225–1310 2152–3238 2439–3619 a 44.7 147.7 139.3 136 106 105 76.3–89.5 76.3–90.7 51–52.4 65.1–79.0 48.8–67.5 b 16.8 22.1 23.3 23.5 21.7 21.8 15.9–19.8 19.4–25.4 8.8–9.14 13.0–15.6 14.4–18.3 c 22.3 55.4 52.2 30.6 40.5 26 30.5–36.7 27.6–30.6 18.8–20.3 26.6–30.6 18.9–27.3 c′ 4 2.1 3.3 4.5 2.8 4.2 2.9–3.3 4.1–4.6 - 2.7–3.4 4.0–5.3 Maximum body diameter 52 31 30 38 38 33 38–43 43–50 24–25 28–48 49–63 Diameter at amphids 22 28 26 30 24 28 22–28 23–27 20 19–28 20–26 Length of outer labial setae 9 12 12 16–17 6 6 7–8 7–8 13 9–11 8–11 Length of cephalic setae 10 14–16 14–16 18 8 8 9–10 10–11 14–15 8–12 9–13 Amphideal fovea width 12 14 13 16 12 8 11–13 11–13 11–12 9–12 11–12 Amphideal fovea height - 13 11 13 - - - - - - - Amphid % diameter 50–54 50 50 53 51 29 43–57 48–54 54–60 42–55 46–60 Amphid distance from anterior end 18 18–21 18–21 22 20–21 23 20 20 19–20 13–22 17–22 nerve ring from the anterior end 92 - - 121 70 70 85–88 82–89 - 75–98 84–128 nerve ring cbd - - - - 32 33 32–40 36–39 - 24–43 34–48 Pharynx length 139 201 179 220 153 165 172–190 145–193 140–145 144–207 170–222 Pharyngial bulb length - - - 40 40 50 50–60 50–60 38 44–67 62–67 Pharyngial bulb width - - - 17 25 25 23–30 23–30 15 17–30 26–31 Pharynx cbd at base 34 31 30 38 33 33 38–43 43–47 25 28–47 39–50 Excretory pore from anterior end 94 - - 141 98 95 94–128 113–128 - 89–134 106–134 Anal/cloacal body diameter 27 30 24 38 36 30 32–34 30–34 - 24–39 25–34 Tail length 109 80 80 171 100 125 98–105 130–148 63 72–116 121–158 Spicule length - 25 - 39 33 - 45–50 - 28–29 45–70 - Length of gubernacular apophysis - - - - 18 - 22–30 - 19 18–25 - Number of precloacal supplements - 6 - 10 9 - 8–10 - 7 5–7 - V 1083 - - - - 1575 - 1430–1780 - - 1160–1585 %V 46.3 - - - - 45 - 39–43 - - 42–48 Vulva cbd 52 - - - - 33 - 40–50 - - 49–63 Species P. primus P. spirulifer P. mirus P. damodarani P. hohonucola sp. nov. Reference Wieser (1954) Wieser (1959) Jensen (1978) Tchesunov (1988) Huang & Zhang (2005) Jacob et al. (2015) Present study Sex Female Male Female Male Male Female Male Female Male Male Female Habitat depth (m) 250–300 0.5 28 20 64–85 95–205 347–1514 Locality Golfo de Ancud, Chile Pudget Sound, USA Øresund North, Denmark Kandalaksha Bay, Russia Yellow Sea, China Arabian Sea, India Southwestern Pacific, New Zealand L 2340 4430 4180 5170 4060 3500 3052–3600 3680–4340 1225–1310 2152–3238 2439–3619 a 44.7 147.7 139.3 136 106 105 76.3–89.5 76.3–90.7 51–52.4 65.1–79.0 48.8–67.5 b 16.8 22.1 23.3 23.5 21.7 21.8 15.9–19.8 19.4–25.4 8.8–9.14 13.0–15.6 14.4–18.3 c 22.3 55.4 52.2 30.6 40.5 26 30.5–36.7 27.6–30.6 18.8–20.3 26.6–30.6 18.9–27.3 c′ 4 2.1 3.3 4.5 2.8 4.2 2.9–3.3 4.1–4.6 - 2.7–3.4 4.0–5.3 Maximum body diameter 52 31 30 38 38 33 38–43 43–50 24–25 28–48 49–63 Diameter at amphids 22 28 26 30 24 28 22–28 23–27 20 19–28 20–26 Length of outer labial setae 9 12 12 16–17 6 6 7–8 7–8 13 9–11 8–11 Length of cephalic setae 10 14–16 14–16 18 8 8 9–10 10–11 14–15 8–12 9–13 Amphideal fovea width 12 14 13 16 12 8 11–13 11–13 11–12 9–12 11–12 Amphideal fovea height - 13 11 13 - - - - - - - Amphid % diameter 50–54 50 50 53 51 29 43–57 48–54 54–60 42–55 46–60 Amphid distance from anterior end 18 18–21 18–21 22 20–21 23 20 20 19–20 13–22 17–22 nerve ring from the anterior end 92 - - 121 70 70 85–88 82–89 - 75–98 84–128 nerve ring cbd - - - - 32 33 32–40 36–39 - 24–43 34–48 Pharynx length 139 201 179 220 153 165 172–190 145–193 140–145 144–207 170–222 Pharyngial bulb length - - - 40 40 50 50–60 50–60 38 44–67 62–67 Pharyngial bulb width - - - 17 25 25 23–30 23–30 15 17–30 26–31 Pharynx cbd at base 34 31 30 38 33 33 38–43 43–47 25 28–47 39–50 Excretory pore from anterior end 94 - - 141 98 95 94–128 113–128 - 89–134 106–134 Anal/cloacal body diameter 27 30 24 38 36 30 32–34 30–34 - 24–39 25–34 Tail length 109 80 80 171 100 125 98–105 130–148 63 72–116 121–158 Spicule length - 25 - 39 33 - 45–50 - 28–29 45–70 - Length of gubernacular apophysis - - - - 18 - 22–30 - 19 18–25 - Number of precloacal supplements - 6 - 10 9 - 8–10 - 7 5–7 - V 1083 - - - - 1575 - 1430–1780 - - 1160–1585 %V 46.3 - - - - 45 - 39–43 - - 42–48 Vulva cbd 52 - - - - 33 - 40–50 - - 49–63 a, body length/maximum body diameter; b, body length/pharynx length; c, body length/tail length; cʹ, tail length/cloacal or anal body diameter; cbd, corresponding body diameter; L, body length; V, vulva distance from anterior end of body; %V, V/total body length. View Large Table 2. Morphometrics (µm) of all Paramicrolaimus species from the literature Species P. primus P. spirulifer P. mirus P. damodarani P. hohonucola sp. nov. Reference Wieser (1954) Wieser (1959) Jensen (1978) Tchesunov (1988) Huang & Zhang (2005) Jacob et al. (2015) Present study Sex Female Male Female Male Male Female Male Female Male Male Female Habitat depth (m) 250–300 0.5 28 20 64–85 95–205 347–1514 Locality Golfo de Ancud, Chile Pudget Sound, USA Øresund North, Denmark Kandalaksha Bay, Russia Yellow Sea, China Arabian Sea, India Southwestern Pacific, New Zealand L 2340 4430 4180 5170 4060 3500 3052–3600 3680–4340 1225–1310 2152–3238 2439–3619 a 44.7 147.7 139.3 136 106 105 76.3–89.5 76.3–90.7 51–52.4 65.1–79.0 48.8–67.5 b 16.8 22.1 23.3 23.5 21.7 21.8 15.9–19.8 19.4–25.4 8.8–9.14 13.0–15.6 14.4–18.3 c 22.3 55.4 52.2 30.6 40.5 26 30.5–36.7 27.6–30.6 18.8–20.3 26.6–30.6 18.9–27.3 c′ 4 2.1 3.3 4.5 2.8 4.2 2.9–3.3 4.1–4.6 - 2.7–3.4 4.0–5.3 Maximum body diameter 52 31 30 38 38 33 38–43 43–50 24–25 28–48 49–63 Diameter at amphids 22 28 26 30 24 28 22–28 23–27 20 19–28 20–26 Length of outer labial setae 9 12 12 16–17 6 6 7–8 7–8 13 9–11 8–11 Length of cephalic setae 10 14–16 14–16 18 8 8 9–10 10–11 14–15 8–12 9–13 Amphideal fovea width 12 14 13 16 12 8 11–13 11–13 11–12 9–12 11–12 Amphideal fovea height - 13 11 13 - - - - - - - Amphid % diameter 50–54 50 50 53 51 29 43–57 48–54 54–60 42–55 46–60 Amphid distance from anterior end 18 18–21 18–21 22 20–21 23 20 20 19–20 13–22 17–22 nerve ring from the anterior end 92 - - 121 70 70 85–88 82–89 - 75–98 84–128 nerve ring cbd - - - - 32 33 32–40 36–39 - 24–43 34–48 Pharynx length 139 201 179 220 153 165 172–190 145–193 140–145 144–207 170–222 Pharyngial bulb length - - - 40 40 50 50–60 50–60 38 44–67 62–67 Pharyngial bulb width - - - 17 25 25 23–30 23–30 15 17–30 26–31 Pharynx cbd at base 34 31 30 38 33 33 38–43 43–47 25 28–47 39–50 Excretory pore from anterior end 94 - - 141 98 95 94–128 113–128 - 89–134 106–134 Anal/cloacal body diameter 27 30 24 38 36 30 32–34 30–34 - 24–39 25–34 Tail length 109 80 80 171 100 125 98–105 130–148 63 72–116 121–158 Spicule length - 25 - 39 33 - 45–50 - 28–29 45–70 - Length of gubernacular apophysis - - - - 18 - 22–30 - 19 18–25 - Number of precloacal supplements - 6 - 10 9 - 8–10 - 7 5–7 - V 1083 - - - - 1575 - 1430–1780 - - 1160–1585 %V 46.3 - - - - 45 - 39–43 - - 42–48 Vulva cbd 52 - - - - 33 - 40–50 - - 49–63 Species P. primus P. spirulifer P. mirus P. damodarani P. hohonucola sp. nov. Reference Wieser (1954) Wieser (1959) Jensen (1978) Tchesunov (1988) Huang & Zhang (2005) Jacob et al. (2015) Present study Sex Female Male Female Male Male Female Male Female Male Male Female Habitat depth (m) 250–300 0.5 28 20 64–85 95–205 347–1514 Locality Golfo de Ancud, Chile Pudget Sound, USA Øresund North, Denmark Kandalaksha Bay, Russia Yellow Sea, China Arabian Sea, India Southwestern Pacific, New Zealand L 2340 4430 4180 5170 4060 3500 3052–3600 3680–4340 1225–1310 2152–3238 2439–3619 a 44.7 147.7 139.3 136 106 105 76.3–89.5 76.3–90.7 51–52.4 65.1–79.0 48.8–67.5 b 16.8 22.1 23.3 23.5 21.7 21.8 15.9–19.8 19.4–25.4 8.8–9.14 13.0–15.6 14.4–18.3 c 22.3 55.4 52.2 30.6 40.5 26 30.5–36.7 27.6–30.6 18.8–20.3 26.6–30.6 18.9–27.3 c′ 4 2.1 3.3 4.5 2.8 4.2 2.9–3.3 4.1–4.6 - 2.7–3.4 4.0–5.3 Maximum body diameter 52 31 30 38 38 33 38–43 43–50 24–25 28–48 49–63 Diameter at amphids 22 28 26 30 24 28 22–28 23–27 20 19–28 20–26 Length of outer labial setae 9 12 12 16–17 6 6 7–8 7–8 13 9–11 8–11 Length of cephalic setae 10 14–16 14–16 18 8 8 9–10 10–11 14–15 8–12 9–13 Amphideal fovea width 12 14 13 16 12 8 11–13 11–13 11–12 9–12 11–12 Amphideal fovea height - 13 11 13 - - - - - - - Amphid % diameter 50–54 50 50 53 51 29 43–57 48–54 54–60 42–55 46–60 Amphid distance from anterior end 18 18–21 18–21 22 20–21 23 20 20 19–20 13–22 17–22 nerve ring from the anterior end 92 - - 121 70 70 85–88 82–89 - 75–98 84–128 nerve ring cbd - - - - 32 33 32–40 36–39 - 24–43 34–48 Pharynx length 139 201 179 220 153 165 172–190 145–193 140–145 144–207 170–222 Pharyngial bulb length - - - 40 40 50 50–60 50–60 38 44–67 62–67 Pharyngial bulb width - - - 17 25 25 23–30 23–30 15 17–30 26–31 Pharynx cbd at base 34 31 30 38 33 33 38–43 43–47 25 28–47 39–50 Excretory pore from anterior end 94 - - 141 98 95 94–128 113–128 - 89–134 106–134 Anal/cloacal body diameter 27 30 24 38 36 30 32–34 30–34 - 24–39 25–34 Tail length 109 80 80 171 100 125 98–105 130–148 63 72–116 121–158 Spicule length - 25 - 39 33 - 45–50 - 28–29 45–70 - Length of gubernacular apophysis - - - - 18 - 22–30 - 19 18–25 - Number of precloacal supplements - 6 - 10 9 - 8–10 - 7 5–7 - V 1083 - - - - 1575 - 1430–1780 - - 1160–1585 %V 46.3 - - - - 45 - 39–43 - - 42–48 Vulva cbd 52 - - - - 33 - 40–50 - - 49–63 a, body length/maximum body diameter; b, body length/pharynx length; c, body length/tail length; cʹ, tail length/cloacal or anal body diameter; cbd, corresponding body diameter; L, body length; V, vulva distance from anterior end of body; %V, V/total body length. View Large Females of P. hohonucola sp. nov. can be differentiated from the type species of the genus, P. primus, by the absence of honeycomb-like structures on cuticle, their longer bodies (2439–3619 µm vs. 2340 µm in P. primus), higher values of a (49–68 vs. 45) and a weak and elongated posterior pharyngeal bulb (vs. well-developed bulb in P. primus). Paramicrolaimus hohonucola sp. nov. can be differentiated from P. spirulifer and P. damodarani by several features, mainly by a different body length (2439–3619 µm vs. 4180–5170 µm and 1225–1310 µm in P. spirulifer and P. damodarani, respectively), the length of their outer labial setae (8–11 µm vs. 14–18 µm and 14–15 µm in P. spirulifer and P. damodarani, respectively), lower value of a relative to P. spirulifer (49–79 vs. 136–148) and higher value of a (in males) relative to P. damodarani (65–79 vs. 51–52). Remarks: An unusual feature of the present species within the phylum is the presence of a transverse opening formed by the fusion of the two ventrosublateral lip lobes and enlarged dorsal lip lobe, resulting in apparent dorsoventral symmetry rather than triradial symmetry. A transverse mouth opening is also found in the genus Pseudonchus Cobb, 1920 (order Desmodorida); however, in this case, the transverse opening results from a reduction of the dorsal sector of the buccal cavity resulting in bilateral symmetry (De Coninck, 1942). Reduction of the dorsal sector of the buccal cavity also occurs in the genus Cheironchus Cobb, 1917 (order Chromadorida), which is characterized by a reduced dorsal mandible and two equal, large ventrosublateral mandibles displaced laterally. In P. hohonucola sp. nov., the dorsoventral symmetry is only partial because the transverse mouth opening is displaced slightly dorsally. Inequalities between dorsal and ventral regions become more pronounced in the buccal cavity, which is characterized by a large dorsal tooth with two dorsosublateral teeth and two smaller ventrosublateral teeth. Whilst the bilateral symmetry in Pseudonchus and Cheironchus may be the result of their predatory feeding habits, there is no evidence of predation in Paramicrolaimus. Paramicrolaimus hohonucola sp. nov. was found at 350–1500 m water depth, which constitutes the deepest record for the genus. Paramicrolaimus primus was found at 250–300 m (Wieser, 1953, 1954), P. damodarani at 95–205 m (Jacob et al., 2015), P. mirus at 20–90 m (Tchesunov, 1988; Huang & Zhang, 2005) and records of P. spirulifer are restricted to the sublittoral zone (Wieser, 1959; Jensen, 1978; Lorenzen, 1981). Order Microlaimida ord. nov. (Table 3) Diagnosis: Cuticle striated, annulated or smooth, rarely punctated. Anterior labial sensilla in three separate circles: six inner labial papilla, six outer labial papilla and four cephalic setae. Amphideal fovea circular, cryptocircular, unispiral or multispiral. Cheilostom with cheilorhabdia (not always visible). Buccal cavity usually with more or less pronounced dorsal tooth, smaller subventral teeth also often present. Pharynx muscular with weakly or strongly developed posterior bulb. Female reproductive system with one or two ovaries; ovaries are either outstretched or reflexed. Male reproductive system with two opposed testes or single anterior testis. Table 3. Key morphological traits characterizing the order Desmodorida (now comprising the superfamily Desmodoroidea only), Microlaimida order. nov. (comprising the Microlaimoidea and Molgolaimus) and Chromadorida Desmodorida Microlaimida order nov. Chromadorida Cuticle Striated or annulated Striated, annulated or smooth (punctations in some Microlaimus and Bolbolaimus species) Punctated (striated in Paramicrolaimidae) Cephalic capsule Present or absent Absent Absent Cephalic sensilla 6 + 6 + 4 6 + 6 + 4 6 + 6 + 4 or 6 + 10 Amphideal fovea Unispiral, multispiral or cryptocircular Circular, cryptocircular, unispiral or multispiral Unispiral, multispiral, transverse loop/slit Cheilorhabdia Present Present Present Buccal armature Typically single dorsal tooth and ≥ 2 smaller ventrosublateral teeth Typically single dorsal tooth and ≥ 2 smaller ventrosublateral teeth Typically single dorsal tooth and ≥ 2 smaller ventrosublateral teeth Pharynx Muscular with weakly or strongly developed posterior bulb Muscular with weakly or strongly developed posterior bulb Muscular with weakly or strongly developed posterior bulb Female reproductive system Two opposed and reflexed ovaries Two opposed and outstretched ovaries, two opposed and reflexed ovaries, single anterior reflexed ovary or single outstretched anterior ovary Two opposed and reflexed ovaries Male reproductive system Single anterior testis (two testes in Onepunema) Two opposed testes or single anterior testis Two opposed testes or single anterior testis Desmodorida Microlaimida order nov. Chromadorida Cuticle Striated or annulated Striated, annulated or smooth (punctations in some Microlaimus and Bolbolaimus species) Punctated (striated in Paramicrolaimidae) Cephalic capsule Present or absent Absent Absent Cephalic sensilla 6 + 6 + 4 6 + 6 + 4 6 + 6 + 4 or 6 + 10 Amphideal fovea Unispiral, multispiral or cryptocircular Circular, cryptocircular, unispiral or multispiral Unispiral, multispiral, transverse loop/slit Cheilorhabdia Present Present Present Buccal armature Typically single dorsal tooth and ≥ 2 smaller ventrosublateral teeth Typically single dorsal tooth and ≥ 2 smaller ventrosublateral teeth Typically single dorsal tooth and ≥ 2 smaller ventrosublateral teeth Pharynx Muscular with weakly or strongly developed posterior bulb Muscular with weakly or strongly developed posterior bulb Muscular with weakly or strongly developed posterior bulb Female reproductive system Two opposed and reflexed ovaries Two opposed and outstretched ovaries, two opposed and reflexed ovaries, single anterior reflexed ovary or single outstretched anterior ovary Two opposed and reflexed ovaries Male reproductive system Single anterior testis (two testes in Onepunema) Two opposed testes or single anterior testis Two opposed testes or single anterior testis Exceptions to character states are given between brackets. View Large Table 3. Key morphological traits characterizing the order Desmodorida (now comprising the superfamily Desmodoroidea only), Microlaimida order. nov. (comprising the Microlaimoidea and Molgolaimus) and Chromadorida Desmodorida Microlaimida order nov. Chromadorida Cuticle Striated or annulated Striated, annulated or smooth (punctations in some Microlaimus and Bolbolaimus species) Punctated (striated in Paramicrolaimidae) Cephalic capsule Present or absent Absent Absent Cephalic sensilla 6 + 6 + 4 6 + 6 + 4 6 + 6 + 4 or 6 + 10 Amphideal fovea Unispiral, multispiral or cryptocircular Circular, cryptocircular, unispiral or multispiral Unispiral, multispiral, transverse loop/slit Cheilorhabdia Present Present Present Buccal armature Typically single dorsal tooth and ≥ 2 smaller ventrosublateral teeth Typically single dorsal tooth and ≥ 2 smaller ventrosublateral teeth Typically single dorsal tooth and ≥ 2 smaller ventrosublateral teeth Pharynx Muscular with weakly or strongly developed posterior bulb Muscular with weakly or strongly developed posterior bulb Muscular with weakly or strongly developed posterior bulb Female reproductive system Two opposed and reflexed ovaries Two opposed and outstretched ovaries, two opposed and reflexed ovaries, single anterior reflexed ovary or single outstretched anterior ovary Two opposed and reflexed ovaries Male reproductive system Single anterior testis (two testes in Onepunema) Two opposed testes or single anterior testis Two opposed testes or single anterior testis Desmodorida Microlaimida order nov. Chromadorida Cuticle Striated or annulated Striated, annulated or smooth (punctations in some Microlaimus and Bolbolaimus species) Punctated (striated in Paramicrolaimidae) Cephalic capsule Present or absent Absent Absent Cephalic sensilla 6 + 6 + 4 6 + 6 + 4 6 + 6 + 4 or 6 + 10 Amphideal fovea Unispiral, multispiral or cryptocircular Circular, cryptocircular, unispiral or multispiral Unispiral, multispiral, transverse loop/slit Cheilorhabdia Present Present Present Buccal armature Typically single dorsal tooth and ≥ 2 smaller ventrosublateral teeth Typically single dorsal tooth and ≥ 2 smaller ventrosublateral teeth Typically single dorsal tooth and ≥ 2 smaller ventrosublateral teeth Pharynx Muscular with weakly or strongly developed posterior bulb Muscular with weakly or strongly developed posterior bulb Muscular with weakly or strongly developed posterior bulb Female reproductive system Two opposed and reflexed ovaries Two opposed and outstretched ovaries, two opposed and reflexed ovaries, single anterior reflexed ovary or single outstretched anterior ovary Two opposed and reflexed ovaries Male reproductive system Single anterior testis (two testes in Onepunema) Two opposed testes or single anterior testis Two opposed testes or single anterior testis Exceptions to character states are given between brackets. View Large Remarks: There is no synapomorphy for Microlaimida order nov. This order is created to accommodate the superfamily Microlaimoidea and genus Molgolaimus. These taxa, which were previously classified within Desmodorida, are now considered to form a distinct clade based on molecular phylogenetic analyses and reinterpretation of existing morphological data (see Discussion for detailed justification). DISCUSSION The present study provides the first molecular sequence data for the family Paramicrolaimidae, and the classification of this family is discussed below in light of molecular phylogenetic analyses. Proposed changes to the classification and composition of the Microlaimoidea, which consistently forms a clade separate from the bulk of the Desmodorida in SSU phylogenies, are explained in light of new and existing morphological and molecular evidence. Phylogenetic placement of Paramicrolaimidae Our results do not provide support for a close relationship between Paramicrolaimidae and either the family Microlaimidae, subfamily Stilbonematinae or the order Plectida as suggested by previous authors (Wieser, 1954; Jensen, 1978; Lorenzen, 1981). Instead, our phylogenetic analyses, which investigated potential relationships within marine Chromadorean orders and within the Chromadorida based on SSU rDNA sequences, indicate that Paramicrolaimidae constitutes a sister taxon to the family Selachinematidae. In our analysis of SSU rDNA sequences focusing on the Chromadorida, Selachinematidae formed a monophyletic group, which together with P. hohonucola sp. nov., formed a sister group to the remaining chromadorid taxa, although there was no support for this topology. This finding is similar to the analyses of van Megen et al. (2009), which showed that the Selachinematidae form a distinct monophyletic clade within Chromadorida. The placement of Selachinematidae is never well supported in SSU phylogenies, and this family is not always recovered as a monophyletic group within Chromadorida (Holterman et al., 2008). However, there are strong morphological grounds for the placement of Selachinematidae within Chromadorida. The Selachinematidae are characterized by both punctated cuticle and female reproductive system with reflexed ovaries, a combination of traits not found outside Chromadorida (Lorenzen, 1981). The Paramicrolaimidae possess reflexed ovaries, but they differ from the Selachinematidae and other families currently comprising Chromadorida in having a striated cuticle. However, cuticle ornamentation sometimes varies substantially within orders. The family Comesomatidae was previously classified with Chromadorida by some authors mainly on account of the punctated cuticle (e.g. Wieser, 1954; De Coninck, 1965; Platt, 1985), but is characterized by a female reproductive system with outstretched branches, and is now placed within Areaolaimida based on molecular phylogenetic analyses (De Ley & Blaxter 2004). Therefore, in the absence of clear morphological affinities with other chromadorean orders, and despite the lack of cuticle punctations, we provisionally propose that Paramicrolaimidae be placed within Chromadorida based on our analysis of SSU and LSU sequences which indicate a close relationship with Selachinematidae. Paramicrolaimidae is the only family within the order lacking cuticle punctations, which may have been present in the ancestor and subsequently lost. The placement of both Selachinematidae and Paramicrolaimidae, however, remains equivocal due to limitations of reconstructions based on a single gene, and the classification of these families may need to be revised in the future. Composition, status and phylogenetic placement of Microlaimoidea Our molecular analyses confirm the results of previous molecular phylogenies showing that Molgolaimus, which is currently classified within Desmodoroidea, should instead be placed with Microlaimidoidea (Meldal et al., 2007; Leduc & Zhao, 2016). Molgolaimus is currently placed within the family Desmodoridae, as proposed by Lorenzen (1981) to establish the monophyly of Desmodoroidea based on the presence of only one (anterior) testis in males. Prior to this classification, Molgolaimus was originally placed with the Microlaimidae (Gerlach & Riemann, 1973/1974), presumably based on morphological similarities to the genus Microlaimus in head and amphideal fovea shape, arrangement of head sensilla and buccal cavity structure. Molgolaimus was later placed in the family Molgolaimidae Jensen, 1978 (the latter also comprising the genera Aponema Jensen, 1978 and Prodesmodora Micoletzky, 1923) mainly to reflect differences in the structure of the male and female reproductive systems between the Microlaimidae (males with one or two testes, females with outstretched ovaries) and Molgolaimidae (males with one anterior testis only, females with reflexed ovaries) (Jensen, 1978). Relationships between the Microlaimidae and Molgolaimidae were not made explicit by Jensen (1978), but their parallel treatment in the same publication implies that they were considered to be sister families within the order Desmodorida. The results of the present and previously published SSU molecular phylogenies (Meldal et al., 2007; Leduc & Zhao, 2016) provide support for the classification of Molgolaimus in a sister group to the Microlaimidae as proposed by Jensen (1978), although further work is required to clarify relationships within Microlaimoidea, which we propose should now include Molgolaimidae in addition to Microlaimidae, Monoposthiidae and Aponchiidae. There is no molecular evidence for the placement of Aponema and Prodesmodora, together with Molgolaimus within Molgolaimidae, as suggested by Jensen (1978); Aponema is currently classified with Microlaimidae based on the presence of outstretched ovaries, and Prodesmodora is classified within Desmodoridae based on the presence of reflexed ovaries (Tchesunov, 2014b). We therefore propose that the placement of these genera be left unchanged until further molecular analyses are conducted, leaving Molgolaimus as the only genus within the family Molgolaimidae. Our analyses suggest that Microlaimidae and Monoposthiidae are more closely related to Chromadorida than any other chromadorean order. The molecular evidence available to date indicates that Microlaimoidea is either (1) a monophyletic sister clade to Chromadorida (as suggested by Meldal et al. (2007) and our Chromadorea-scale analysis), (2) a paraphyletic group nested within Chromadorida (as suggested in our Chromadorida-scale analysis) or (3) a distinct clade from Chromadorida at the base of Chromadorea (Holterman et al., 2008). Difficulties in resolving the position of Microlaimoidea and other basal chromadorean taxa are likely due to the use of a single gene for phylogenetic reconstruction. Currently, the SSU rDNA gene is the only locus known to resolve deep relationships among nematode taxa; other genes such as the LSU rDNA gene are only informative at within-family level (De Ley et al., 2005; Bik et al., 2010). Obtaining a greater number of SSU rDNA sequences of microlaimoid taxa, such as representatives of Aponchiidae, may help better resolve relationships of basal Chromadorea, but finding other informative genes will probably be required in order to obtain a more stable topology. The order Desmodorida, as originally defined by De Coninck (1965), included taxa with annulated or striated cuticle and with unispiral, multispiral or loop-shaped amphideal fovea. This order originally included Desmodoroidea, Microlaimidae and Monoposthiidae, as well as other taxa which have now been moved to other orders (e.g. Ceramonematidae Cobb, 1933, and Richtersia). In the recent classification of De Ley & Blaxter (2002, 2004), Desmodorida comprises two superfamilies, Desmodoroidea and Microlaimoidea. Desmodoroidea was defined as monophyletic by Lorenzen (1981) based on the synapomorphy: only one anterior testis present. Nevertheless, two exceptions now exist: (1) Onepunema Leduc & Verschelde, 2013 possesses two testes but has been placed within the family Desmodoridae based on the presence of a cephalic capsule (a trait not found in any of the Microlaimoidea) and reflexed ovaries in females, and (2) molecular evidence indicates that Molgolaimus does not belong to Desmodoroidea despite the presence of only one anterior testis (Meldal et al., 2007; Leduc & Zhao, 2016; this study). Desmodoroidea as defined by Lorenzen (1981), and upon which the classification of De Ley & Blaxter (2004) was based, is therefore no longer monophyletic. According to Lorenzen (1981), the Microlaimoidea lacks any synapomorphy and forms the paraphyletic remains of the Desmodorida. The Microlaimoidea have several features in common with both the Desmodoroidea and Chromadorida, including the presence of cheilorhabdia, as well as arrangement of cephalic sensilla, shape of amphideal fovea and structure of the buccal cavity and pharynx (Table 3). However, two of the microlaimoid families, Microlaimidae and Aponchidae, differ from both the Desmodoroidea and Chromadorida in having outstretched ovaries. The main morphological difference between the Microlaimoidea and either the Desmodorida or Chromadorida is in the cuticle ornamentation: the Microlaimoidea have a striated or annulated cuticle whilst the Chromadorida have a punctated cuticle. There are, however, some exceptions; some microlaimid species have been described as having a punctated cuticle (e.g. Microlaimus punctulatus Gerlach, 1950, Microlaimus honestus De Man, 1922 and Bolbolaimus punctatus Cobb, 1920), and the Paramicrolaimidae, which we argue should be classified with the Chromadorida, have a striated cuticle. Cuticle punctations are relatively rare within the nematode phylum, and are only common in the order Chromadorida and family Comesomatidae. Therefore, whilst cuticle punctations (a synapomorphy) is a useful trait for inferring phylogenetic relationships of some groups, the absence of cuticle punctations, a much more widespread and common trait within the Chromadorea (i.e. a plesiomorphy), is not. Accordingly, Lorenzen (1981) did not consider the absence of cuticle punctations in Desmodoroidea and Microlaimoidea as a phylogenetically informative trait that could be used to infer a close relationship between the two superfamilies. Overall, the morphological evidence suggests that (1) there is a close relationship between Chromadorida, Desmodoroidea and Microlaimoidea based on the synapomorphy of cheilorhabdia, and (2) the Chromadorida represent a clade distinct from both the Desmodoroidea and Microlaimoidea based on the synapomorphy of cuticle punctations. However, there are no solid morphological grounds for classifying Microlaimoidea together with Desmodoroidea. Our phylogenetic analyses, as well as the phylum-wide analyses of Meldal et al. (2007) and Holterman et al. (2008), found no evidence for a sister relationship between the bulk of the Desmodoroidea and the Microlaimoidea; in SSU phylogenies, the latter always forms a distinct clade near the base of the Chromadorea and closer to Chromadorida than the rest of the Desmodorida. In light of these results, as well as lack of any morphological synapomorphy linking Desmodoroidea and Microlaimoidea, we propose to remove Microlaimoidea from the order Desmodorida, which would thus be solely comprised of Desmodoroidea, and erect the order Microlaimida order nov. to accommodate the superfamily Microlaimoidea, which we propose should include the genus Molgolaimus (see above; clade 1 in Fig. 2). This classification, which comprises the Chromadorida, Desmodorida and Microlaimida order nov. as closely related though distinct clades, is similar to the higher classification proposed by Lorenzen (1981) where the suborder Chromadorina Filipjev, 1929 comprised the superfamilies Chromadoroidea Filipjev, 1917, Desmodoroidea and Microlaimoidea. The former two superfamilies are now place within their respective orders (Chromadorida and Desmodorida, respectively), and it is therefore necessary to erect a new order to accommodate Microlaimoidea. It should be noted that although the Microlaimoidea are often recovered as a monophyletic group in SSU phylogenies (with weak or moderate support), their position is not always resolved with certainty. Future investigations based on a better representation of microlaimoid SSU sequences, or based on other sequence(s) capable of resolving deep phylogenetic relationships, may help ascertain the validity of this classification. [Version of Record, published online 21 October 2017; http://zoobank.org/urn:lsid:zoobank.org:pub:1A1C2CA3-FA39-427A-97C6-AF0D88FEFD13 ACKNOWLEDGEMENTS Funding was provided by LabexMer and NIWA’s Coasts and Oceans Centre Research Programme ‘Marine Biological Resources’ and the programme ‘Impact of Resource Use on Vulnerable Deep-Sea Communities’ (CO1X0906). We thank the participants of NIWA voyages TAN0705, TAN1004, TAN1103, TAN1206 and TAN1701 and the officers and crew of RV Tangaroa. We are grateful to Norliana Rosli (NIWA and Sultan Idris Education University) for processing the sediment samples. We also thank Janet Grieve (NIWA) and Oleksandr Holovachov (Swedish Museum of Natural History) for their advice and fruitful discussions. We thank three anonymous reviewers for providing constructive criticisms on the manuscript. REFERENCES Andrassy I . 1976 . Evolution as a basis for the systematization of nematodes . London : Pitman Publishing Ltd . Bik HM , Lambshead PJ , Thomas WK , Lunt DH . 2010 . Moving towards a complete molecular framework of the Nematoda: a focus on the Enoplida and early-branching clades . BMC Evolutionary Biology 10 : 353 . Google Scholar CrossRef Search ADS PubMed Coomans A . 1979 . A proposal for a more precise terminology of the body regions in the nematode . Annales de la Société Royale Zoologique de Belgique 108 : 115 – 117 . De Coninck LA . 1942 . Pseudonchus symmetricus De Coninck, 1942 (Nematoda-Choanolaimidae), un nématode à symétrie bilatérale secondaire de l’extrémité anthérieure . Bulletin du Musée royal d’Histoire Naturelle de Belgique, Tome 21 . De Coninck L . 1965 . Systématique des Nématodes . In: Grassé PP , ed. Traité de Zoologie Anatomie, Systématique, Biologie. Tome IV Fascicule II: Némathelminthes (Nématodes) . Paris : Masson et Cie, 1–731 De Ley P , Blaxter ML . 2002 . Systematic position and phylogeny . In: Lee DL , ed. The biology of Nematodes . London : Taylor & Francis , 1 – 30 . De Ley P , Blaxter ML . 2004 . A new system for Nematoda: combining morphological characters with molecular trees, and translating clades into ranks and taxa . Nematology Monographs & Perspectives 2 : 633 – 653 . De Ley P , De Ley IT , Morris K , Abebe E , Mundo-Ocampo M , Yoder M , Heras J , Waumann D , Rocha-Olivares A , Jay Burr AH , Baldwin JG , Thomas WK . 2005 . An integrated approach to fast and informative morphological vouchering of nematodes for applications in molecular barcoding . Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences 360 : 1945 – 1958 . Google Scholar CrossRef Search ADS PubMed Filipjev IN . 1921 . Free-living marine nematodes in the vicinity of Sevastopol . Trudy Osoboi Zoologicheskoi Laboratorii I Sevastopolskoi Biologi Stantsii Rossiysk Akademii Nauk 41 : 353 – 614 . Gerlach SA , Riemann F . 1973/1974 . The Bremerhaven checklist of aquatic nematodes. A catalogue of Nematoda Adenophorea excluding the Dorylaimida . Veroeffentlichungen des Instituts fuer Meeresforschung in Bremerhaven Suppl. 4, Part 1 (1973) and Part 2 (1974). Guindon S , Gascuel O . 2003 . A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood . Systematic Biology 52 : 696 – 704 . Google Scholar CrossRef Search ADS PubMed Holovachov O . 2014 . Chapter 7.16: order Plectida Gadea, 1973 . In: Schmidt-Raesa A , ed. Handbook of Zoology Gastrotricha, Cycloneuralia and Gnathifera. Volume 2: Nematoda . Hamburg : De Gruyter , 487 – 536 . Holterman M , Holovachov O , van den Elsen S , van Megen H , Bongers T , Bakker J , Helder J . 2008 . Small subunit ribosomal DNA-based phylogeny of basal Chromadoria (Nematoda) suggests that transitions from marine to terrestrial habitats (and vice versa) require relatively simple adaptations . Molecular Phylogenetics and Evolution 48 : 758 – 763 . Google Scholar CrossRef Search ADS PubMed Holterman M , van der Wurff A , van den Elsen S , van Megen H , Bongers T , Holovachov O , Bakker J , Helder J . 2006 . Phylum-wide analysis of SSU rDNA reveals deep phylogenetic relationships among nematodes and accelerated evolution toward crown clades . Molecular Biology and Evolution 23 : 1792 – 1800 . Google Scholar CrossRef Search ADS PubMed Huang Y , Zhang Z . 2005 . Two new species and ne new record of free-living marine nematodes from the Yellow Sea, China . Cahiers de Biologie Marine 46 : 365 – 378 . Huelsenbeck JP , Ronquist F . 2001 . MRBAYES: Bayesian inference of phylogenetic trees . Bioinformatics 17 : 754 – 755 . Google Scholar CrossRef Search ADS PubMed Jacob J , Jaleel KUA , Vijayan AK . 2015 . A new species of the rare nematode genus Paramicrolaimus Wieser, 1954 (Chromadorida: Paramicrolaimidae) from the south eastern Arabian Sea . Zootaxa 3904 : 563 – 571 . Google Scholar CrossRef Search ADS PubMed Jayasree K , Warwick RM . 1977 . Free-living nematodes of a polluted sandy beach in the Firth of Clyde, Scotland. Description of seven new species . Journal of Natural History 11 : 289 – 302 . Google Scholar CrossRef Search ADS Jensen P . 1978 . Revision of Microlaimidae, erection of Molgolaimidae fam.n and remarks on the systematic position of Paramicrolaimus (Nematoda, Desmodorida) . Zoologica Scripta 7 : 159 – 173 . Google Scholar CrossRef Search ADS Kearse M , Moir R , Wilson A , Stones-Havas S , Cheung M , Sturrock S , Buxton S , Cooper A , Markowitz S , Duran C , Thierer T , Ashton B , Meintjes P , Drummond A . 2012 . Geneious basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data . Bioinformatics 28 : 1647 – 1649 . Google Scholar CrossRef Search ADS PubMed Leduc D , Zhao ZQ . 2016 . Molecular characterisation of five nematode species (Chromadorida, Selachinematidae) from shelf and upper slope sediments off New Zealand, with description of three new species . Zootaxa 4132 : 59 – 76 . Google Scholar CrossRef Search ADS PubMed Lorenzen S . 1981 . Entwurf eines phylogenetischen Systems der freilebenden Nematoden . Veröffentlichugen de Institut für Meeresforschung in Bremerhaven 7 : 472S . Meldal BH , Debenham NJ , De Ley P , De Ley IT , Vanfleteren JR , Vierstraete AR , Bert W , Borgonie G , Moens T , Tyler PA , Austen MC , Blaxter ML , Rogers AD , Lambshead PJ . 2007 . An improved molecular phylogeny of the Nematoda with special emphasis on marine taxa . Molecular Phylogenetics and Evolution 42 : 622 – 636 . Google Scholar CrossRef Search ADS PubMed Neira C , Decraemer W . 2009 . Desmotersia levinae, a new genus and new species of free-living nematode from bathyal oxygen minimum zone sediments off Callao, Peru, with discussion on the classification of the genus Richtersia (Chromadorida: Selachinematidae) . Organisms, Diversity & Evolution 9 : 1.e1 – 1.e15 Google Scholar CrossRef Search ADS Nunn GB . 1992 . Nematode molecular evolution . PhD Thesis , University of Nottingham , UK . Platt HM . 1985 . Further observations on the Ethmolaimidae (Nematoda: Chromadorida) . Journal of Natural History 19 : 139 – 149 . Google Scholar CrossRef Search ADS Rambaut A , Drummond AJ . 2007 . Tracer v. 1.4 . Available at: http://beast.bio.ed.ac.uk/Tracer Somerfield PJ , Warwick RM . 1996 . Meiofauna in marine pollution monitoring programmes: a laboratory manual . Lowestoft : Ministry of Agriculture, Fisheries and Food . Swofford DL . 2002 . PAUP*. Phylogenetic analysis using parsimony (and other methods), version 4 . Sunderland, MA : Sinauer Associates . Tchesunov AV . 1988 . New species nematodes from the White Sea . Proceedings of the Zoological Institute, Leningrad 180 : 68 – 76 . Tchesunov AV . 2014a . Order Chromadorida Chitwood, 1933 . In: Schmidt-Raesa A , ed. Handbook of Zoology Gastrotricha, Cycloneuralia and Gnathifera. Volume 2: Nematoda . Hamburg : De Gruyter , 373 – 398 . Tchesunov AV . 2014b . Order Desmodorida De Coninck, 1965 . In: Schmidt-Raesa A , ed. Handbook of Zoology Gastrotricha, Cycloneuralia and Gnathifera. Volume 2: Nematoda . Hamburg : De Gruyter , 399 – 434 . van Megen H , van den Elsen S , Holterman M , Karssen G , Mooyman P , Bongers T , Holovachov O , Bakker J , Helder J . 2009 . A phylogenetic tree of nematodes based on about 1200 full-length small subunit ribosomal DNA sequences . Nematology 11 : 927 – 950 . Google Scholar CrossRef Search ADS Wieser W . 1953 . Free-living marine nematodes: I. Enoploidea . Acta Universita Lundensis 49 : 1–156. Wieser W . 1954 . Free-living marine nematodes: II. Chromadoroidae . Acta Universita Lundensis 50 : 1–149 Wieser W . 1959 . Free-living marine nematodes and other mall invertebrate of Puget Sound Beaches . Seattle : University of Washington Press . Williams BD , Schrank B , Huynh C , Shownkeen R , Waterston RH . 1992 . A genetic mapping system in Caenorhabditis elegans based on polymorphic sequence-tagged sites . Genetics 131 : 609 – 624 . Google Scholar PubMed Zhao Z , Li D , Davies KA , Ye W . 2015 . Schistonchus zealandicus n. sp. (Nematoda: Aphelenchoididae) associated with Ficus macrophylla in New Zealand . Nematology 17 : 53 – 66 . Google Scholar CrossRef Search ADS Zheng JW , Subbotin SA , He SS , Gu JF , Moens M . 2002 . Molecular characterisation of some Asian isolates of Bursaphelenchus xylophilus and B. mucronatus using PCR-RFLPs and sequences of ribosomal DNA . Russian Journal of Nematology 11 : 17 – 22 . © 2017 The Linnean Society of London, Zoological Journal of the Linnean Society This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

Journal

Zoological Journal of the Linnean SocietyOxford University Press

Published: Oct 21, 2017

There are no references for this article.

You’re reading a free preview. Subscribe to read the entire article.


DeepDyve is your
personal research library

It’s your single place to instantly
discover and read the research
that matters to you.

Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.

All for just $49/month

Explore the DeepDyve Library

Search

Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly

Organize

Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.

Access

Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.

Your journals are on DeepDyve

Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.

All the latest content is available, no embargo periods.

See the journals in your area

DeepDyve

Freelancer

DeepDyve

Pro

Price

FREE

$49/month
$360/year

Save searches from
Google Scholar,
PubMed

Create lists to
organize your research

Export lists, citations

Read DeepDyve articles

Abstract access only

Unlimited access to over
18 million full-text articles

Print

20 pages / month

PDF Discount

20% off