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 Author Morffe, Jans Author García, Nayla Author Davis, Andrew K. Author Hasegawa, Koichi Author Carreno, Ramon A. text Zootaxa 2019 2019-06-19 4619 2 391 400 journal article 26456 10.11646/zootaxa.4619.2.13 0436e7e3-cd42-420d-a6e9-6b69afb0a911 1175-5326 3249571 37384F80-62A3-4D83-A78A-C4C950DD9F40 Xyo Cobb, 1898 Xyo pseudohystrix Travassos & Kloss, 1958 incertae sedis Fig. 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 maximum 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. Intestine 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. Spermatheca 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 Discussion Taxonomic 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 differences 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 cervical 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 hystrignathids from O. disjunctus . A single male specimen was found in the present study. From light microscopical observations 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. pseudohystrix (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 current 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 statement 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 Noxubee 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 Georgia (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 morphology, 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. 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. pseudohystrix 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 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 (reconstructed 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. 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). Cameronia 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.