4619 (2): 391–400 issn 1175-5326 (print edition)...

ZOOTAXA ISSN 1175-5326 (print edition)

ISSN 1175-5334 (online edition)

Accepted by N. Kanzaki: 20 May 2019; published: 19 Jun. 2019 391

Zootaxa 4619 (2): 391–400https://www.mapress.com/j/zt/

Copyright © 2019 Magnolia PressArticle

https://doi.org/10.11646/zootaxa.4619.2.13http://zoobank.org/urn:lsid:zoobank.org:pub:37384F80-62A3-4D83-A78A-C4C950DD9F40

Morphological and molecular characterization of Xyo pseudohystrix Travassos & Kloss, 1958 (Nematoda: Oxyuridomorpha: Hystrignathidae) from Odontotaenius disjunctus (Illiger, 1800) (Coleoptera: Passalidae) from USA and discussion on its taxonomic status

JANS MORFFE1,5, NAYLA GARCÍA1, ANDREW K. DAVIS2, KOICHI HASEGAWA3 & RAMON A. CARRENO4

1Instituto de Ecología y Sistemática, Carretera Varona 11835 e/ Oriente y Lindero, La Habana 19, CP 11900, Calabazar, Boyeros, La Habana, Cuba2Odum School of Ecology, University of Georgia, Athens GA 30602, USA3Department of Environmental Biology, College of Bioscience & Biotechnology, Chubu University, 1200 Matsumoto, Kasugai, Aichi 487–8501, Japan

4Department of Zoology, Ohio Wesleyan University, Delaware, Ohio, 43015, USA.5Corresponding author. E-mail: [email protected]

Abstract

The females of Xyo pseudohystrix Travassos & Kloss, 1958 (Nematoda: Oxyuridomorpha: Hystrignathidae) are redescribed and illustrated with the aid of SEM. New features of the cephalic end, arrangement of the cervical spines and genital tract were observed. The taxonomic status of the species is discussed on the basis of discrepancies with the generic diagnosis of Xyo Cobb, 1898. Due to the lack of proper information on the genus the status of incertae sedis is proposed. The identity of the males was confirmed by molecular studies and the morphology of the specimens previously assigned by Christie (1932) as males of Hystrignathus rigidus Leidy, 1858 correspond to the current species. New locality records are given for the states of Georgia and Ohio, USA. The phylogenetic position of the species is inferred on the basis of the D2-D3 segment of the LSU rDNA and SSU rDNA.

Key words: Xyo, redescription, SEM, LSU rDNA, SSU rDNA, phylogeny, USA

Resumen

Se redescriben e ilustran las hembras de Xyo pseudohystrix Travassos et Kloss, 1958 (Nematoda: Oxyuridomorpha: Hystrignathidae) con el empleo de Microscopía Electrónica de Barrido. Fueron observados nuevos caracteres del extremo cefálico, disposición de las espinas cervicales y sistema reproductor. La posición taxonómica de la especie es discutida sobre la base de discrepancias con la diagnosis del género Xyo Cobb, 1898 y debido a la escasez de información se propone la categoría de incertae sedis. La identidad de los machos fue confirmada mediante técnicas moleculares y, como resultado, la morfología de los ejemplares asignados a Hystrignathus rigidus Leidy, 1858 por Christie (1932) corresponde al presente taxón. Georgia y Ohio, Estados Unidos constituyen nuevos registros de localidad para la especie. La filogenia de X. pseudohystrix incertae sedis es inferida mediante el segmento D2-D3 del LSU rDNA y el SSU rDNA.

Palabras clave: Xyo, redescripción, SEM, LSU rDNA, SSU rDNA, filogenia, Estados Unidos

Introduction

The first contribution to the hystrignathid nematodes from USA was that of Leidy (1850) with the description of Hystrignathus rigidus Leidy, 1850 (Nematoda: Oxyuridomorpha: Hystrignathidae) from the passalid beetle Odon-totaenius disjunctus (Illiger, 1800) (Coleoptera: Passalidae). This is the type species of the genus Hystrignathus Leidy, 1850, which is currently the type genus of the family Hystrignathidae Travassos, 1920.

MORFFE Et AL.392 · Zootaxa 4619 (2) © 2019 Magnolia Press

Christie (1934) resumed studies on hystrignathids in North America and recorded H. rigidus from other states from USA. In the same hosts, the author found other females with the cervical cuticle armed with alternate rows of spines, similar to the Australian species Xyo hystrix Cobb, 1898. As the genus Xyo Cobb, 1898 had been previously synonymized with Hystrignathus by Johnston (1913), Christie (1934) assigned this species to H. hystrix. He also found male specimens and assigned them provisionally to H. rigidus. More recently, Morffe et al. (2015) rede-scribed H. rigidus using SEM studies.

Travassos & Kloss (1958) considered X. hystrix sensu Christie as different from the Australian species, mainly considering the differences in host species and geographical region. Thus, they renamed Christie´s species as X. pseudohystrix Travassos & Kloss, 1958. This arrangement was maintained by Adamson & Van Waerebeke (1992) in their revision of the Thelastomatoidea. More recently, Carreno (2018) recorded both H. rigidus and X. pseudohystrix from O. disjunctus in the state of Mississippi, USA.

In the present work, X. pseudohystrix is redescribed on the basis of new material from the southern USA, with the aid of SEM and light microscopy techniques. The taxonomic status of the species is discussed, based on new morphological features. In addition, its phylogeny is inferred with the D2-D3 domains of the LSU rDNA and the SSU rDNA.

Materials and methods

Processing of the hosts and nematodes. Specimens of Odontotaenius disjunctus (Illiger, 1800) were collected by hand from rotting logs in Athens, Georgia, USA and Ohio, USA. Beetles were maintained alive in plastic jars with moistened wood chips as a food and humidity source until arrival at the laboratory.

Hosts were killed with vapours of ethyl-ether and immediately dissected by making longitudinal incisions in both abdominal pleural membranes. Intestines were withdrawn from the bodies and dissected in Petri dishes with 0.9% NaCl or Ringer´s physiological solution. Nematodes found were killed with hot Ringer’s solution (70ºC) and fixed in TAF. Specimens for molecular studies were directly fixed in 96% ethanol. For light microscopy studies the nematodes were transferred to anhydrous glycerin via slow evaporation method (Seinhorst 1959) and mounted in the same medium. The edges of the coverslips were sealed with nail polish.

Studied material is deposited in the Colección Helmintológica de las Colecciones Zoológicas (CZACC), Insti-tuto de Ecología y Sistemática, Havana, Cuba and the Museo Nacional de Historia Natural “Prof. Eugenio de Jesús Marcano” (MNHNSD), Santo Domingo, Dominican Republic.

Morphological and morphometric studies. Measurements were taken with the aid of a calibrated eyepiece micrometer attached to a Olympus BH-2 compound microscope (Olympus, Tokyo, Japan). De Man’s indices a, b, c and V% were calculated. Variables are shown as the range followed by the mean plus standard deviation in paren-theses, and the number of measurements is also given. Micrographs were generated with an AxioCam digital camera attached to a Carl Zeiss Axioskop 2 Plus compound microscope (Carl Zeiss, Göttingen, Germany). Line drawings were made on the basis of micrographs using a Wacom Intuos Art drawing tablet with Adobe Illustrator CS6 and Adobe Photoshop CS6. Scale bars of all figures are given in micrometers.

Specimens prepared for SEM were dehydrated in an ascendant grade ethanol series and critical point dried. Dehydrated nematodes were mounted on stubs and covered with gold. Micrographs were taken with the aid of a Quanta 200 SEM (Thermo-Fisher Scientific, Hillsborogh, USA).

DNA extraction, gene amplification and sequencing. Genomic DNA was extracted from single individuals with the DNeasy® Blood & Tissue (Qiagen, USA) and DNAzol (Molecular Research Center Inc., Cincinnati, Ohio, USA) kits, following manufacturer´s instructions. The D2-D3 segment of the large ribosomal subunit ribosomal RNA gene (D2-D3 LSU rDNA) was amplified with the primers D2A (5´-ACA AGT ACC GTG AGG GAA AGT TG-3´) and D3B (5´-TCG GAA GGA ACC AGC TAC TA-3´) (Nunn 1992) for the specimens from Georgia and with the primers #391 (5´-AGC GGA GGA AAA GAA ACT AA-3´) and #501 (5´-TCG GAA GGA ACC AGC TAC TA-3´) (Nadler et al. 2006) for the specimens from Ohio. The small ribosomal subunit ribosomal RNA gene (SSU rDNA) in the individuals from Georgia was amplified with the primer set SSUF07_For (5´-AAA GAT TAA GCC ATG CAT G-3´) and SSUR26_Rev (5´-CAT TCT TGG CAA ATG CTT TCG-3´) (Blaxter et al. 1998). In the case of the specimen from Ohio, the SSU rDNA was amplified in two fragments using primers 47 (5´-CCC GAT TGA TTC TGT CGG C-3´) and 112 (5´-GGC TGC TGG CAC CAG ACT TGC-3´) with the overlapping primer set 135

XyO PSEUDOHyStrIX, REDESCRIPTION AND TAxONOMIC STATUS Zootaxa 4619 (2) © 2019 Magnolia Press · 393

(5´-CGG AGA GGG AGC CTG AGA AAC GGC-3´) and 136 (5´-TGA TCC TTC TGC AGG TTC ACC TAC-3´) (Carreno & Nadler 2003).

PCR reactions for the specimens from Georgia were performed in a total volume of 20 µL with the KOD Fx Neo DNA polymerase (Toyobo, Osaka, Japan). PCR cycling parameters consisted of an initial denaturation at 94ºC for 2 min followed by 35 cycles of 98ºC for 10 s, 50ºC for 30 s and 68ºC for 30 s and a final extension step of 68ºC for 5 min. The results of the PCR were checked by agarose gel electrophoresis, visualizing the DNA bands with ethidium bromide. PCR products were excised from the gel and purified with the NucleoSpin® Gel and PCR Clean Up kit (Macherey-Nagel, Düren, Germany), following the manufacturer´s protocol. Samples were submitted to Hokkaido System Science Co., Sapporo, Japan. The original PCR primers were used to sequence both strands.

PCR reactions for the specimens from Ohio were performed in a total volume of 25 µL with the Finnzymes DyNAzyme ExT polymerase (MJ Research, Watertown, Massachusetts, USA). PCR cycling parameters consisted of an initial denaturation at 94ºC for 3 min followed by 35 cycles of 94ºC for 30 s, 54ºC, 58ºC or 60ºC for 30 s (for the primer sets 391/501, 47/112 and 135/136, respectively) and 72ºC for 1 min and a final extension step of 72ºC for 7 min. PCR products were treated with the ExoSAP-IT® kit (USB Products, Affymetrix Inc., Cleveland, Ohio, USA), and sequenced using a 3730 DNA Analyzer from Applied Biosystems Inc. and BigDye® Terminator Cycle Sequencing chemistry at the Plant-Microbe Genomics Facility, Ohio State University, Columbus, Ohio.

Raw sequences were manually edited with Sequencher 3.0 and 4.1.4 (http://genecodes.com) and deposited in GenBank NCBI (http://www.ncbi.nlm.nih.gov/genbank/). The accession numbers for each taxon are provided in Table 1.

TABLE 1. GenBank accession numbers of the sequences of thelastomatoid nematodes (Oxyuridomorpha: Thelastoma-toidea) used in the present study. Newly obtained sequences in bold.

Species Country D2-D3 LSU rDNA SSU rDNAHystrignathidaeCoynema poeyi Cuba MH244508 MH577322Hystrignathus rigidus USA MH411129 MH411156Hystrignathus sp. Vietnam GQ368469 –Lepidonema magnum Cuba MH569782 MH577324Longior longior Cuba Kx427524 MH411158Longior similis Cuba Kx427528 MH411157Xyo pseudohystrix USA, Georgia MH569779 (female) MH577323

MH569780 (female) –MH569781 (male) –

USA, Ohio MK332328 (female) MK332329ThelastomatidaeCameronia multiovata Russia GQ368470 –travassosinematidaetravassossinema claudiae Japan Kx844645 Kx844644travassosinema dalei Australia HM769761 –travassosinema sp. Vietnam GQ368471 –

Phylogenetic analysis. Several sequences of thelastomatoid species (Hystrignathidae, Travassosinematidae and Thelastomatidae) were selected from GenBank for the phylogenetic analyses (accession numbers in Table 1). Cam-eronia multiovata (Thelastomatidae) and three species of travassosinema (Travassosinematidae) were used as the outgroup taxa since the monophyletic clade formed by these genera is the sister-group of Hystrignathidae, according to previous studies (Spiridonov & Guzeeva 2009; Morffe & Hasegawa 2017).

Multiple sequence alignments were made using the Muscle algorithm with the default parameters as imple-mented in MEGA6 (Tamura et al. 2013). Poorly aligned regions and gaps were automatically removed with trimAl (Capella-Gutiérrez et al. 2009). Phylogenetic analyses were performed to the both D2-D3 LSU rDNA and SSU rDNA as well as a concatenated dataset of both genes. MEGA6 was also used to identify the optimal model of evolution for the dataset (K2+G for the SSU rDNA and GTR+G for the LSU rDNA and the concatenated dataset)


following the Akaike Information Criterion (AIC) and to construct phylogenetic trees based on the Maximum Like-lihood (ML) method. Nodal support was inferred by bootstrap analysis using 1,000 iterations. Bayesian Inference analysis (BI) was performed with MrBayes v3.2.6 (Ronquist et al. 2012), with 3×106 generations, sampling every 100 generations and discarding the first 25% of the sample runs as burn-in. The convergence statistics of the BI process stationarity and the number of burn-in trees were checked using Tracer v1.5 (Rambaut et al. 2003).

Systematics

Family Hystrignathidae Travassos, 1920

Xyo Cobb, 1898

Xyo pseudohystrix Travassos & Kloss, 1958 incertae sedisFig. 1 A–I, Fig. 2 A–G

Material examined. Vouchers: 15♀♀, USA, Georgia, Athens; in Odontotaenius disjunctus; 15/V/2016; A. K. Davis coll.; CZACC 11.7135–11.7149. 4♀♀, same data as the latter; MNHNSD 05.0022–05.0025.

redescription. Female. Body comparatively robust, widening gradually posterior to head, reaching its maxi-mum width at level of the vulva, then narrowing gradually towards tail. Sub-cuticular striae present. Cervical cuticle armed with alternate rows of spines, from the base of the annulated region to ca. one body-width posterior to the basal bulb. First row with 20 minute spines, number that increases to ca. 28 in the last rows. Next to the first row the spines become larger and then start to diminish their size at ca. the midpoint of the spiny region, being shorter, finer and more scattered. Lateral alae absent. Lateral gap present in the spiny region; from the level of its second third to its end, the separation of the spines is ca. 14 µm. Head bearing eight paired, rounded and flattened cephalic papillae, their diameter of ca. 3 µm. Cephalic papillae with a central depression, its diameter of ca. 2 µm. Oral opening surrounded by a cuticular, prominent annular lip, not in contact with the cephalic papillae. Amphids lateral, their opening triradiate and located at a small protuberance at level of the external edges of the cephalic papillae. Short, finely annulated region (ca. 10 µm in length) with ca. 3 annuli extending from the base of the head. Stoma with triradiate lumen, surrounded by an oesophageal collar. Oesophagus consisting of a muscular, sub-cylindrical procorpus, well differentiated from the cylindrical isthmus. Basal bulb rounded, valve-plate well-developed. In-testine simple, sub-rectilinear, its fore region dilated. Rectum short. Anus a crescent-like ventromedian transverse slit (ca. 28 µm in length). Nerve ring encircling procorpus at ca. its midpoint. Excretory pore ventral, located at ca. two body-widths posterior to the basal bulb. Vulva a median transverse slit ca. 40 µm in length, its lips barely prominent, located near the midbody. Vagina muscular, forwardly directed. Genital tract didelphic-amphidelphic, both ovaries reflexed. Distal end of the anterior ovary reflexed at the level of the excretory pore, distal flexure ca. two body-widths long. Distal end of the posterior ovary reflexed at ca. 2.5 body-widths anterior to the level of the anus, distal flexure ca. five body-widths long, its distal end reaching the level of the vulva. Oocytes in single rows. Fusiform spermatheca present in the posterior uterus, near the level of the basal end of the anterior ovary. Sperma-theca absent in the anterior branch of the genital tract. Eggs ellipsoidal, smooth-shelled and numerous. Tail conical and attenuate, ending in a sharp tip.

Male. See Discussiontaxonomic remarks. Christie (1934) found the second species from O. disjunctus with the females having the

cervical cuticle with alternate rows of spines. The author mentioned that the first row of spines have 32 elements as in the Australian X. hystrix described by Cobb (1898). Following the criteria of Johnston (1913) who considered Xyo as a junior synonym of Hystrignathus, Christie (1934) assigned this species to X. hystrix sensu Cobb.

Travassos & Kloss (1958) considered X. hystrix sensu Christie as a different species, based mainly in the differ-ences of host species and geographical region and renamed it as X. pseudohystrix. Kloss (1962) did not follow this and synonymized the species with X. hystrix sensu Cobb. Hunt (1982) regarded both X. hystrix sensu Cobb and X. pseudohystrix as species inquirendae based on the lack of elements for a specific determination in the description of both species. However, Adamson & Van Waerebeke (1992) followed the criteria of Travassos & Kloss (1958) and kept X. pseudohystrix as a valid species. Carreno (2018) in his record of the species for Mississippi, USA, also


considered the species as X. pseudohystrix, but remarked on the need of a proper revision of the taxon. One of the main features for the definition of the genus Xyo is the presence of alternate rows of spines in the cer-

vical cuticle, with the first row having 32 elements (Adamson & Van Waerebeke 1992). This feature was observed by Christie (1934) in X. pseudohystrix under light microscopy and by Hunt (1982) in X. xiphacanthus Hunt, 1982 with the aid of SEM. However, in the current study, the SEM images show that the specimens of X. pseudohystrix present 20 elements in the first row of spines. This number increases to only ca. 28 spines at the level of the last rows.

Christie (1934) found male specimens that he provisionally assigned to H. rigidus. The decision of the author was based on the lack of morphological elements to properly assign these males to one of the both species of hystri-gnathids from O. disjunctus. A single male specimen was found in the present study. From light microscopical ob-servations it coincided with Christie´s description. The specimen was used for DNA studies and a partial sequence of the D2-D3 LSU rDNA was obtained. The sequence matches with the ones from three females of X. pseudohys-trix (two from Georgia and one from Ohio). The latter points to a misidentification of Christie´s male specimens. Still, the collection of more male hystrignathid individuals from O. disjunctus is needed in order to assure a better morphological characterization of X. pseudohystrix based on light microscopy and SEM, as well as to describe the males of H. rigidus.

The aforementioned differences with Christie´s description and the generic diagnosis of Xyo found in the cur-rent study make the correct placement of X. pseudohystrix within the genus difficult. This is further complicated by the scarce available data on the male morphology. With the available information it is not recommended to establish a new genus in order to accommodate the species. Thus, we propose the status of incertae sedis for X. pseudohystrix with different criteria than those of Hunt (1982) who considered it as species inquirenda for the following reasons:

1. The presence of 20 elements in the first row of alternate spines of X. pseudohystrix is not consistent with the generic diagnosis of Xyo. Assuming that X. hystrix sensu Cobb presents 32 elements in the first row of spines, the presence of 20 elements in X. pseudohystrix make them different species. This disagrees with Hunt´s state-ment since according to the International Code of Zoological Nomenclature (ICZN) a species inquirenda is “…a species of doubtful identity needing further investigation”.

2. The lack of a proper description and type material of X. hystrix sensu Cobb make it necessary to collect new material in order to perform a proper redescription of the species as well as to confirm the features of the generic diagnosis, more preferably with the aid of SEM and molecular techniques. Such studies will clarify the identity of X. hystrix sensu Cobb itself as well as the rest of the species among the genus, including X. pseudohystrix. So far, with the existing data on the genus Xyo we can assure the position of X. pseudohystrix. This fits better with the status of incertae sedis: defined by the ICZN as “…of uncertain taxonomic position”.

The specimens from Athens, Georgia and central Ohio (the latter locality including a single specimen used only for DNA studies) constitute new state records for the species. In the previous studies of Christie (1934), the taxon was recorded for the states of Illinois, Louisiana, Maryland and Virginia. The latest record of the species is the Nox-ubee National Wildlife Refuge, Starkville, state of Mississippi (Carreno 2018). The individuals from Georgia (Table 2) agree in most of the morphometrics with the population from Mississippi. However, the specimens from Georgia are more robust (a = 17.75–23.39 vs. 22.00–24.00) and their isthmus is slightly shorter (38–48 µm vs. 50 µm).

DNA studies. Four partial sequences of the D2-D3 LSU rDNA were obtained, three from specimens from Geor-gia (two females and one male) and one from an individual from Ohio. All of these sequences are identical. Besides, two sequences of the SSU rDNA were obtained from two specimens from Georgia and Ohio, respectively. These are also identical.

ML and BI analyses were performed for the D2-D3 LSU rDNA, the SSU rDNA and a concatenated dataset of both markers. The trees of the D2-D3 LSU rDNA and the concatenated datasets were the ones with the highest values of bootstrap support. Since the topology of both ML and BI trees was identical only the former are shown (Fig. 3).

For both, the LSU rDNA and the concatenated dataset the results are contradictory in the context of morphol-ogy, since X. pseudohystrix incertae sedis present spines in the cervical cuticle and since the female genital tract is didelphic-amphidelphic as in H. rigidus, Hystrignathus sp. and Lepidonema magnum Morffe & García, 2010.


FIgURE 1. Xyo pseudohystrix Travassos & Kloss, 1958 incertae sedis (Nematoda: Oxyuridomorpha: Hystrignathidae). Female. A. Oesophageal region, lateral view. B. Tail, lateral view. C. Cephalic end, optical section. D. Cephalic end, external view (re-constructed from SEM images). E. Cephalic end, en face view (reconstructed from SEM images). F. Spines at the midpoint of the spiny region (reconstructed from SEM images). G. Spines at the end of the spiny region (reconstructed from SEM images). H. Genital tract, lateral view. I. Habitus, lateral view.


FIgURE 2. Xyo pseudohystrix Travassos & Kloss, 1958 incertae sedis (Nematoda: Oxyuridomorpha: Hystrignathidae). Female. SEM images. A. Habitus, lateral view. B. Cervical region, lateral view. C. Tail, ventral view. D. Cephalic end, en face view. E. Cephalic end, lateral view. F. Spines at the midpoint of the spiny region. G. Spines at the end of the spiny region. Scale bars: A. 500 µm. B. 300 µm. C. 200 µm. D. 30 µm. E, G. 50 µm. F. 20 µm.


In both trees H. rigidus and L. magnum cluster together in a well-supported clade and Hystrignathus sp. is basal to it in the analysis of the LSU rDNA. With respect to morphology, such an arrangement could be attributed to the aforementioned presence of cervical spines and a didelphic-amphidelphic genital tract. However, X. pseudohys-trix incertae sedis forms a monophyletic clade with two species of Longior Travassos & Kloss, 1958. The latter genus has an unarmed cervical cuticle and the genital tract monodelphic-prodelphic vs. armed cervical cuticle and didelphic-amphidelphic genital tract. The procorpus is cylindrical and very elongated vs. sub-cylindrical and not elongated in Xyo.

FIgURE 3. Maximum likelihood (ML) tree inferred from: A. D2-D3 LSU rDNA and B. concatenated dataset of the D2-D3 LSU rDNA and the SSU rDNA for several species of the superfamily Thelastomatoidea (Nematoda: Oxyuridomorpha). Cam-eronia multiovata (Thelastomatidae) and three species of travassosinema (Travassosinematidae) were used as outgroup taxa. Values at the nodes correspond to bootstrap resampling (≥70)/posterior probability (≥0.90).


There is not a morphological justification to such topology. The analysis includes all the hystrignathid genera with sequences available in GenBank which are only a minimal sample of all the genera among the family. This makes the phylogeny of the family Hystrignathidae far from complete. The inclusion of more molecular data and a better characterization of the morphology of many taxa (including the morphology of the males) could lead to a better and more logical arrangement of Xyo among Hystrignathidae. Therefore, the results discussed here must be considered provisional and can change if new information is added.

TABLE 2. Morphometrics of Xyo pseudohystrix Travassos & Kloss, 1958 incertae sedis (Nematoda: Oxyuridomorpha: Hystrignathidae) females from Athens, Georgia, USA. All the measurements are given in micrometers unless otherwise indicated.

Charactera 17.75–23.39 (20.45 ± 1.72, n = 18)b 6.45–8.07 (7.05 ± 0.41, n = 15)c 9.26–11.03 (10.20 ± 0.48, n = 18)V% 51.92–56.10 (53.86 ± 1.24, n = 18)Total length (mm) 3.075–3.900 (3.589 ± 0.246, n = 18)Maximum width 140–210 (177 ± 19, n = 19)Stoma length 50–73 (60 ± 5, n = 19)Procorpus length 350–460 (395 ± 28, n = 15)Isthmus length 38–48 (43 ± 3, n = 18)Basal bulb diameter 63–80 (70 ± 4, n = 19)Oesophagus length 450–570 (511 ± 32, n = 15)Nerve ring-anterior end 250–320 (279 ± 21, n = 15)Excretory pore-anterior end 730–960 (888 ± 50, n = 19)Vulva-anterior end (mm) 1.725–2.075 (1.931 ± 0.102, n = 18)Tail length 290–400 (353 ± 31, n = 19)Eggs 90–103×33–45 (98 ± 4×37 ± 4, n = 13)

Acknowledgements

We thank George Keeney for provision of O. disjunctus from Ohio. We are very grateful to Jullien Cillis (Royal Bel-gian Institute of Natural Sciences) for his help with the SEM pictures. We thank MSc. Eduardo Furrazola (Instituto de Ecología y Sistemática) for his help with the micrographs. The Belgian Development Cooperation, through the Belgian Focal Point of the Global Taxonomy Initiative (GTI; 2013 calls) supported access by the senior author to SEM at the Royal Belgian Institute of Natural Sciences. Access to molecular techniques at Chubu University was made possible with funds from the Japanese Society for the Promotion of Science (JSPS), Long Term Fellowship (ID No. L16566). We thank the Plant-Microbe Genomics Facility at The Ohio State University for their contribu-tion to this publication. This research was also supported by the project “Colecciones Zoológicas, su conservación y manejo III”, Ministerio de Ciencia, Tecnología y Medio Ambiente, Cuba, and the Research Institute for Biological Function, Chubu University.

Literature cited

Adamson, M.L. & Van Waerebeke, D. (1992) Revision of the Thelastomatoidea, Oxyurida of invertebrate hosts III. Hystri-gnathidae. Systematic Parasitology, 22, 111–130.

https://doi.org/10.1007/BF00009604Blaxter, M.L., De Ley, P., Garey, J.R., Liu, L.x., Scheldeman, P., Vierstraete, A., Vanfleteren, J.R., Mackey, L.Y., Dorris, M.,

Frisse, L.M., Vida, J.T. & Thomas, W.K. (1998) A molecular evolutionary framework for the phylum Nematoda. Nature, 392, 71–75.

https://doi.org/10.1038/32160Capella-Gutiérrez, S., Silla-Martínez, J.M. & Gabaldón, T. (2009) trimAl: a tool for automated alignment trimming in large-


scale phylogenetic analyses. Bioinformatics, 25, 1972–1973. https://doi.org/10.1093/bioinformatics/btp348Carreno, R.A. & Nadler, S.A. (2003) Phylogenetic analysis of the Metastrongyloidea (Nematoda: Strongylida) inferred from

ribosomal RNA gene sequences. Journal of Parasitology, 89, 965–973. https://doi.org/10.1645/GE-76RCarreno, R.A. (2018) Pinworms (Nematoda: Thelastomatoidea) from Insects Collected in Mississippi, USA, with description

of a new species of Protrellus Cobb, 1920 from the Cockroach Ischnoptera deropeltiformis Brunner von Wattenwyl, 1865 (Blattaria: Ectobiidae). Zootaxa, 4531 (4), 567–577.

https://doi.org/10.11646/zootaxa.4531.4.7Christie, J.R. (1934) The nematode genera Hystrignathus Leidy, Lepidonema Cobb and Artigasia, n.g. (Thelastomatidae). Pro-

ceedings of the Helminthological Society of Washington, 1, 43–48. Cobb, N.A. (1898) Extract from a manuscript report on the parasites of stock. Agricultural Gazette of New South Wales, 9,

296–321.Hunt, D.J. (1982) Hystrignathus ferox n.sp. and Xyo xiphacanthus n.sp. (Oxyurida: Hystrignathidae) with additional data on

Carlosia tijucana Travassos & Kloss, 1957. Systematic Parasitology, 4, 59–68. https://doi.org/10.1007/BF00012229Johnston, T.H. (1913) Notes on some entozoa. Proceedings of the royal Society of Queensland, 24, 63–91.Kloss, G.R. (1962) Alguns parasitos do intestino de Coleopteros da familia Passalidae. Papéis Avulsos do Departamento de

Zoologia, São Paulo, 15, 163–171.Leidy, J. (1850) Description of some nematoid Entozoa infesting insects. Proceedings of the Academy of Natural Sciences,

Philadelphia, 5, 100–102.Morffe, J., García, N. & Davis, A.K. (2015) Redescription of the females of Hystrignathus rigidus Leidy, 1850 (Nematoda:

Hystrignathidae), parasites of Odontotaenius disjunctus (Coleoptera: Passalidae) from eastern USA. Zootaxa, 3941 (1), 131–136.

https://doi.org/10.11646/zootaxa.3941.1.8Morffe, J. & Hasegawa, K. (2017) Morphological and molecular characterization of travassosinema claudiae n. sp. (Oxyurido-

morpha: Travassosinematidae) from the Japanese millipede Parafontaria laminata (Attems, 1909) (Polydesmida: xysto-desmidae). Zootaxa, 4282 (1), 166–178.

https://doi.org/10.11646/zootaxa.4282.1.10Nadler, S.A., De Ley, P., Mundo-Ocampo, M., Smythe, A.B., Stock, S.P., Bumbarger, D., Adams, B.J., De Ley, I.T., Holovachov,

O. & Baldwin, J.G. (2006) Phylogeny of Cephalobina (Nematoda): molecular evidence for recurrent evolution of probolae and incongruence with traditional classifications. Molecular Phylogenetics and Evolution, 40, 696–711.

https://doi.org/10.1016/j.ympev.2006.04.005Nunn, G.B. (1992) Nematode molecular evolution. I. University of Nottingham, Nottingham, 187 pp.Rambaut, A., Suchard, M.A., xie, W. & Drummond, A.J. (2003) tracer. Version 1.6. Available from: http://beast.bio.ed.ac.

uk/Tracer/ (accessed 12 October 2016)Ronquist, F., Teslenko, M., van der Mark, P., Ayres, D.L., Aaron, D., Höhna, S., Larget, B., Liu, L., Suchard, M.A. & Huelsen-

beck, J.P. (2012) MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic Biology, 61, 539–542.

https://doi.org/10.1093/sysbio/sys029Seinhorst, J.W. (1959) A rapid method for the transfer of nematodes from fixative to anhydrous glycerin. Nematologica, 4,

67–69. https://doi.org/10.1163/187529259x00381Spiridonov, S.E. & Guzeeva, E.A. (2009) Phylogeny of nematodes of the superfamily Thelastomatoidea (Oxyurida) inferred

from LSU rDNA sequence. russian Journal of Nematology, 17, 127–134.Tamura, K., Stecher, G., Peterson, D., Filipski, A. & Kumar, S. (2013) MEGA6: Molecular Evolutionary Genetics Analysis ver-

sion 6.0. Molecular Biology and Evolution, 30, 2725–2729. https://doi.org/10.1093/molbev/mst197Travassos, L. & Kloss, G.R. (1958) Sobre a fauna de nematodeos dos coleopteros Passalidae da Estaçao Biologica de Boracéia.

Arquivos de Zoologia, Sao Paulo, 11, 23–57.

http://beast.bio.ed.ac.uk/Tracer/

http://beast.bio.ed.ac.uk/Tracer/

4619 (2): 391–400 issn 1175-5326 (print edition)...

Documents