Zoosyst. Evol. 100 (3) 2024, 1029-1039 | DOI 10.3897/zse.100.128096 Ate BERLIN Oleaxonchium olearum gen. et sp. nov. (Nematoda, Dorylaimida) associated with an olive grove in the southern Iberian Peninsula, and new insights into the evolutionary relationships within Belondiridae Reyes Pefia-Santiago!, Miriam Garcia-Ruiz', Alba N. Ruiz-Cuenca’, Joaquin Abolafia! 1 Departamento de Biologia Animal, Biologia Vegetal y Ecologia, Universidad de Jaén, Campus “Las Lagunillas”’ s/n, 23071, Jaén, Spain https://zoobank. org/BO8F64F'7-9C 46-4F 05-A836-76A41117CE73 Corresponding author: Reyes Pefia-Santiago (rpena@ujaen.es) Academic editor: A. Schmidt-Rhaesa # Received 22 May 2024 # Accepted 25 June 2024 # Published 25 July 2024 Abstract A new belondirid, dorylaimid taxon, Oleaxonchium olearum gen. et sp. nov., collected from an olive grove in the Andalusia region of Spain, is characterised, including its morphological description, morphometrics, SEM study, and molecular (18S-, 28S-rDNA) analyses. The new genus displays a unique combination of traits that distinguishes it from its closest genera: a rectangular lip region with sclerotized margins when observed in lateral view and visibly hexagonal in face view under SEM; a comparatively long cheilostom with thickened walls at its anterior part; a short isthmus-like section separating both pharyngeal regions; a mo- no-opistho-ovarian didelphic female genital system without pars refringens vaginae, and a short and rounded tail. The new species is characterised by its 2.44—2.87 mm long body, lip region 7—7.5 um wide, odontostyle 10-10.5 um long, neck 723-973 um long, pharyngeal expansion occupying 63—72% of the total neck length, female anterior genital branch 4—-6% of body length, tripartite posterior uterus 1.9—2.6 body diameters long, with a short intermediate section bearing sclerotized elements, vulva (V = 58-61) a transverse slit, caudal region 29-35 um long (c = 74-89, c’ = 0.9-1.1), and male unknown. As derived from an integrative approach combining morphological and molecular data, the new genus is close to Metaxonchium, the polyphyly of Belondiridae is confirmed, and support is provided in favour of the monophyly of Axonchiinae. Key Words morphology, new taxa, phylogeny, SEM, taxonomy, 18S-, 28S-rDNA Introduction The free-living dorylaimid fauna of olive groves is poorly explored in spite of the fact that this culture is the Dorylaims, the members of the order Dorylaimida, are probably the most diverse nematode taxon. Being pres- ent in any continental (soil and freshwater) habitat and exploiting a wide spectrum of feeding resources, they are regarded as good bioindicators of environmental health (Pefia-Santiago 2021). With 36 valid genera and 242 valid species, the family Belondiridae Thorne, 1939 rep- resents almost 10 percent of all the dorylaims (Andrassy 2009). This family currently includes three subfamilies, one of which, Belondirinae Thorne, 1939, is less diverse and more frequent in temperate areas. most prominent and most socioeconomically important fruit tree in Europe, especially across the Mediterranean Basin (Vilar and Pereira 2018). Actually, at present, only eight species, eight genera, and six families were pre- viously found in dwelling olive orchards (unpublished data). Representatives of Belondirinae are scarce in the Iberian fauna, with four species hitherto recorded, mainly in natural areas (Jiménez-Guirado et al. 2007). A belondirid population was collected in the course of a nematological survey conducted to study the free-liv- ing fauna inhabiting olive soils in the framework of the Copyright Pena-Santiago, R. et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 1030 project Soil O-Live (EU Horizon Programme Grant No. 101091255). Its morphological and molecular study re- vealed that it represented a new generic taxon. Thus, this contribution aims to describe its morphological pattern, obtain its molecular characterization, and discuss its evo- lutionary relationships. Materials and methods Nematodes and their morphological and morphometrical study A total of 12 female specimens, found in the soils of an olive grove named “La Capilla” (Spain, Malaga province, Antequera municipality), were available to study. Nema- todes were extracted by centrifugation (CDFA 2015; based on Jenkins 1964) and/or with Baermann’s funnels follow- ing the protocol by Flegg (1967), somewhat modified, killed by heat, fixed in 4% formaldehyde, preserved in anhydrous glycerin according to Siddiqi’s (1964) method, mounted on permanent glass slides that were sealed with paraffin, and measured and photographed using an Eclipse 801 microscope (Nikon) equipped with differential inter- ference contrast optics, a drawing tube (camera lucida), and a DS digital camera. Morphometrics include Dema- nian indices and other measurements and ratios; some of them are presented in a separate table, while others form part of the literal description of species. Two specimens preserved in glycerin were selected for observation with a SEM, according to Abolafia (2015). The nematodes were hydrated in distilled water, dehydrated in a graded etha- nol-acetone series, critical point dried, coated with gold, and observed with a Zeiss Merlin microscope (5 kV). Molecular study For molecular analyses, single specimens were temporar- ily mounted in a drop of 1 M sodium chloride containing glass beads. This was followed by DNA extraction from single individuals, as described by Archidona- Yuste et al. (2016). The D2—D3 domains were amplified using the D2A (5'-ACAAGTACCGTGAGGGAAAGTTG-3') and D3B_ (5'-TCGGAAGGAACCAGCTACTA-3') | prim- ers (De Ley et al. 1999). The portion of 18S rRNA was amplified using primers 988F (5'-CTCAAAGATTAAG- CCATGC-3’), 1912R (5'-TTTACGGTCAGAACTAG- GG-3'), 1813F (5'-CTGCGTGAGAGGTGAAAT-3’), and 2646R (5'-GCTACCTTGTTACGACTTTT-3’) (Holterman et al. 2006). All polymerase chain reaction (PCR) assays were done according to the conditions de- scribed by Archidona-Yuste et al. (op. cit.). The amplified PCR products were purified using ExoSAP-IT (Affimet- rix, USB products) and used for direct sequencing on a DNA multicapillary sequencer (Model 3130XL genetic analyser; Applied Biosystems, Foster City, CA, USA), using the BigDye Terminator Sequencing Kit V.3.1 (Ap- plied Biosystems, Foster City, CA, USA), at the StabVida zse.pensoft.net Pena-Santiago, R. et al.: Oleaxonchium olearum gen. et sp. nov. from the Iberian Peninsula sequencing facilities (Caparica, Portugal). The newly obtained sequences were submitted to the GenBank da- tabase under the accession numbers indicated on the phy- logenetic trees. Phylogenetic analyses For phylogenetic relationships, analysis was based on 18S and 28S rDNA fragments. The obtained sequences were manually edited using Chromas 2.6.6 (Technely- sium) and aligned with other rDNA sequences available in GenBank using the ClustalW alignment tool imple- mented in MEGA7 (Kumar et al. 2016). Poorly aligned regions at extremes were removed from the alignments using MEGA7. The best-fit model of nucleotide substi- tution used for the phylogenetic analysis was statistically selected using jModelTest 2.1.10 (Darriba et al. 2012). The phylogenetic tree was generated with the Bayesian inference method using MrBayes 3.2.6 (Ronquist et al. 2012). The analysis under the general time reversible plus invariant sites plus gamma distribution (GTR + I + G) model was initiated with a random starting tree and run with the Markov chain Monte Carlo (MCMC) (Larget and Simon 1999) for 1 x 10° generations. The tree was visualised and saved with FigTree 1.4.4 (Rambaut 2018). Results Description of new taxa Oleaxonchium gen. nov. https://zoobank.org/DSB8C 148-9476-4684-AC45-255492E8F9A6 Diagnosis. Belondiridae, Belondirinae. Medium-sized nematodes, 2.44—-2.87 mm long. Cuticle dorylaimid, thick, two-layered. Body pores button-like. Lip region narrower than the adjoining body, rectangular in lateral view, with distinctly sclerotized margins, totally fused lips, and simple, pore-like cephalic papillae. Amphid cup-like, with large aperture. Cheilostom a relatively long truncate cone with thickened walls at its anterior (peri- oral) part. Odontostyle small, robust, slightly fusiform, with short aperture, up to one-third of its length. Guid- ing ring simple, refractive. Odontophore linear, lacking any differentiation. Pharynx entirely muscular, with both regions separated by a short, isthmus-like narrowing; basal expansion occupying ca two-thirds of the total neck length, surrounded by a conspicuous muscular sheath. Female genital system mono-opistho-ovarian, didelphic, without pars refringens vaginae, well-developed pars distalis vaginae, and transverse vulva. Caudal region short and rounded. Male unknown. Type and only species. Oleaxonchium olearum sp. nov. Etymology. The genus name is derived from Olea eu- ropaea L.., the scientific name of olive, as the type spe- cies was found associated with this typical Mediterranean fruit tree, and Axonchium, a very close taxon. Zoosyst. Evol. 100 (3) 2024, 1029-1039 Oleaxonchium olearum gen. et sp. nov. https://zoobank. org/244A BA7C-4365-4FE8-9340-F765726A 3811 Figs 1-4 Material examined. Twelve females from one location are in good condition for preservation. Morphometrics. See Table 1. Table 1. Main morphometrics of Oleaxonchium olearum gen. et sp. nov. from Spain. Measurements in um except L in mm are in the form of average + sd (range). Character n Holotype Paratypes o 119° L 2.87 2.61 + 0.12 (2.44-2.87) a 60 56.6 + 3.3 (53-64) b 2.9 3.1 + 0.3 (2.6-3.5) ¢ 84 81.8 + 5.4 (74-89) V 59 58.6 + 0.7 (58-61) Ce 1.0 1.0 + 0.1 (0.9-1.1) Lip region diameter 45 7.03 + 0.3 (7.0-7.5) Odontostyle length 10 10.1 + 0.2 (10-10.5) Odontophore length 16 15.7 + 0.4 (15-16) Neck length 973 894 + 86 (723-973) 566 + 79 (458-679) 44.5 + 1.7 (42-47) 46.2 + 1.6 (44-48) 33.6 + 0.9 (32-35) 1530 + 79 (1419-1680) 361 + 25 (326-392) 33.4 + 2.4 (29-37) 92 + 11eG'(29=35) Pharyngeal expansion length Body diameter at neck base 47 mid-body 48 anus 34 Distance vulva — anterior end Prerectum length Rectum length 35 Tail length 34 Description. Female. Very slender and medium-sized nematodes. Body cylindrical, conspicuously tapering to- wards the anterior end, much less so towards the posterior one as the tail is short and rounded. Upon fixation, habi- tus curved ventrad to a more or less open C shape. Cuti- cle smooth, two-layered, 0.5—1 um thick at anterior region, 2.54 um in mid-body, and 8—11 um on tail, consisting of a thinner outer layer and a much thicker inner layer bear- ing radial striation, especially appreciable at caudal region. Large cervical lacunae are present. Lateral chord very nar- row, 4-10 um wide, occupying up to one-fifth (9-21%) of midbody diameter, bearing abundant elliptical gland bodies, more perceptible at posterior body region. Lateral pores but- ton-like, when observed with SEM. Lip region almost rect- angular in lateral view, visibly narrower than the adjoining body, 1.8—2.1 times wider than high, and ca one-sixth (15- 17%) of body diameter at neck base, its anterior and lateral margins sclerotized and somewhat refractive, with hardly visible labial and cephalic papillae. SEM observations: lip region almost octogonal when observed in face view, with totally fused lips; its margin slightly elevated, marking off a somewhat sunken area; oral field comparatively small as inner labial papillae are almost equidistant between the oral aperture and the marging, far from the outer labial papillae; labial papillae button-like, delimited by a small circular in- cisure, with a distinct pore at their center, cephallic papillae unusually simple, small pore-like. Amphid fovea cup-like, its aperture 6.5—7.5 um long, almost equal (0.9-1.1 times) to 1031 the lip region width. Cheilostom a truncate cone 11.5—12 um long, 1.6—1.7 times as long as the lip region width, with thin walls that distinctly thicken at its anterior (perioral) end. Odontostyle slightly fusiform, 5—7 times as long as wide, longer (1.3—1.5 times) than lip region width, 0.35—0.42% of body length, its aperture 2—3 um or less than one-third (20- 30%) of total length. Guiding ring simple, distinct. Odonto- phore rod-like, 1.4—1.6 times the odontostyle, bearing very weak thickenings at its base. Pharynx very long, consisting of a slender anterior region, a very short narrower (isth- mus-like) intermediate section, and a large, strongly muscu- lar posterior expansion 18—38 times as long as wide, 10—15 times longer than body diameter at neck base, which occu- pies ca two-thirds (63-72%) of the total neck length, and appears enveloped by a strong spiral muscular sheath with almost strait bands; gland nuclei obscure in the specimens examined. Nerve ring situated at 147-178 um, or 17-22% of the total neck length from the anterior end. Pharyngo-in- testinal junction consisting of a cylindrical, tongue-like, 20— 27 x 6.5—11 um cardia, almost totally surrounded by intesti- nal tissue. Genital system mono-opistho-ovarian, didelphic: anterior branch reduced, 102—165 um long or 4-6% of body length, consisting of a tube-like uterus, a narrowing, proba- bly a very weak sphincter, and a distal cell mass 20-24 um long, certainly representing a vestigial oviduct and/or ova- ry; posterior branch well-developed, 190-303 um long or 811% of body length, with a variably large ovary 56-146 um long, often not reaching the sphincter, bearing oocytes first in several rows and then in a single row, oviduct 72—116 um long or 1.6—2.6 body diameters that consists of a slender distal region made of prismatic cells and a proximal pars dilatata with visible lumen inside, sphincter present but not especially distinct between oviduct and uterus, uterus 87— 122 um long or 1.9—2.6 body diameters (its length however should be taken with caution as it always appears convolut- ed), tripartite as it consists of a longer proximal region with wider lumen, a shorter intermediate section that bears refrac- tive elements (Z-like structure), and a dilate, almost spheri- cal, distal part. Vagina 22—27 um long, extending inwards to often more than one-half (48-60%) of body diameter: pars proximalis 11-16 x 8-17 um and encircled by moderately developed musculature, pars distalis 9-12 um long. Vulva a transverse slit ca 10 um long. Prerectum 9.6—12.2, rec- tum 0.9—1.1 anal body diameters long. Anus a visibly arched transverse slit ca 4 um long. Caudal region short and round- ed, its inner core reaching 61—77% of tail length, caudal pores two pairs, sublateral, at the middle of tail. Male. Unknown. Molecular characterization. After sequencing and ed- iting, ten sequences of type specimens were obtained for phylogenetic analyses. Six 18S rDNA sequences, which were 1653, 1696, 1705, and 1709 (three sequences) bp length (acc. PP956610—PP956615, respectively), present- ed 1651 bp in common, and a Blast search showed that they were 98.97% similar to those of Axonchium sp. (acc. MG921264 and O0Q946544). Four 28S rDNA sequenc- es, which were 728, 739, 742, and 745 bp length (acc. PP956616—PP956619, respectively), presented 720 bp in common, anda Blast search showed that they were 93.34% zse.pensoft.net 1032 Pena-Santiago, R. et al.: Oleaxonchium olearum gen. et sp. nov. from the Iberian Peninsula Figure 1. Oleaxonchium olearum gen. et sp. nov. (female). A. Entire; B-D. Anterior body region in lateral median (B, C) and lateral surface (D) views; E. Neck region; F. Isthmus-like narrowing between both pharyngeal sections; G. Vagina; H. Genital system; I. Oviduct-uterus junction; J. Pharyngo-intestinal junction; K, L. Caudal region; M. Detail of uterine Z-like sructure Scale bars: 200 um (A); 5 um (B—-D, G, M); 100 um (E); 10 um (F, I-L). 50 um (Hi). zse.pensoft.net Zoosyst. Evol. 100 (3) 2024, 1029-1039 Figure 2. Oleaxonchium olearum gen. et sp. nov. (female, general morphology, LM). A. Neck region; B. Entire; C, D, F, I. Anterior body region in lateral median (C, D, F) and lateral surface (I) views; E. Isthmus-like narrowing between both pharyngeal sections; G. Posterior body region; H. Pharyngo-intestinal junction; J. Gland-like bodies at the lateral chord. Scale bars: 100 um (A); 200 um (B); 5 um (C, D, F, 1); 50 um (G); 10 um (E, H, J). zse.pensoft.net 1034 Pena-Santiago, R. et al.: Oleaxonchium olearum gen. et sp. nov. from the Iberian Peninsula Figure 3. Oleaxonchium olearum gen. et sp. nov. (female, genital system, and caudal region, LM). A. Genital system; B. Anterior genital branch; C. Oviduct-uterus junction; D, E. Vagina; F, G. Rectum and caudal region; H. Detail of uterine Z-like structure. Scale bars: 50 um (A, B); 10 um (C, F, G); 5 um (D, E, H). similar to those of Metaxonchium giennense (acc. 0Q473053—0Q473054), 91.14% to those of Metaxon- chium magnum (OQ099691, 0Q099693), and 88.86% to that of Axonchium sp. (ON927906). Diagnosis. The new species is characterised by its 2.44—2.87 mm long body, lip region 7—7.5 um wide, odontostyle 10-10.5 um long, neck 723-973 um long, pharyngeal expansion occupying 63-72% of the to- tal neck length, female genital system mono-opist- ho-ovarian, didelphic, with anterior branch occupying 46% of body length, posterior uterus 87-122 um long or 1.9-2.6 body diameters and tripartite, with a short intermediate section bearing refractive elements, vul- va (V = 58-61) a transverse slit, caudal region short zse.pensoft.net and rounded (29-35 um, c = 74-89, c’ = 0.9-1.1), and male unknown. Type locality and habitat. Spain, Malaga province, Antequera municipality, “La Capilla” farm (37.198283, -4.543868, elevation 491 m), where the new species was collected in an olive grove with intensive tillage. Type material. Female holotype and nine female para- types were deposited in the nematode collection of the University of Jaén, Spain. Two female paratypes were de- posited with the United States Department of Agriculture Nematode Collection (USDANC) at Beltsville, MD, USA. Etymology. The species epithet is the Latin term olearum = belonging to or corresponding to olives, as type material was found in an olive grove. Zoosyst. Evol. 100 (3) 2024, 1029-1039 Figure 4. Oleaxonchium olearum gen. et sp. nov. (female, SEM). A-C. Lip region, in face view; D. Lateral pores; E. Anterior body region, lateral view; F. Anterior body region, ventral view; G. Caudal region, sublateral view; H. Vulva, ventral view. Scale bars: 1 um (A-C); 5 um (D); 2 um (E, F); 4 um (G, H). Evolutionary relationships of the new taxon As derived from morphological data The general appearance of the new genus is very simi- lar to that found in representatives of the Axonchium-like pattern, including comparatively short and slightly fusi- form odontostyle, simple odontophore, both pharyngeal sections distinctly separated, very long pharyngeal ex- pansion, mono-opistho-ovarian, didelphic female genital system, and a short and rounded tail (cf. Coomans and Nair 1975). Some of these traits should be interpreted as relevant apomorphies (fusiform odontostyle, very long pharyngeal expansion, mono-opistho-ovarian female genital system), if not autapomorphies (separation of both pharyngeal sections) within the family Belondiri- dae (cf. Pefia-Santiago and Abolafia 2023). Nevertheless, Oleaxonchium gen. nov. 1s easily distinguishable from other Axonchium-like genera in the unique aspect of its lip region (rectangular in lateral view, plane and almost octagonal in face view, and with sclerotized margins vs. typically cap-like and without sclerotized margins), which is herein interpreted as an outstanding autapomor- phy. The very small cephalic papillae (simple pores) ob- served in the type species, as well as its button-like lat- eral pores, represent apomorphic (if not autapomorphic) features too. Besides, the cheilostom, comparatively long and with thickened walls at its anterior, perioral part, 1s an apomorphic condition, never recorded in other genera that bear shorter and thin-walled cheilostoms. In having a short, narrower (isthmus-like) section be- tween both pharyngeal sections, the new genus is close to Metaxonchium Coomans & Nair, 1975, but, leaving aside the lip region, both taxa differ in the shape of the vagina, which lacks (vs. bears) pars refringens and presents very well-developed (vs. very short) pars distalis, respective- ly. In the absence of pars refringens vaginae, Oleaxon- chium gen. nov. also resembles the genera Axonchium Cobb, 1920, and Syncheilaxonchium Coomans & Nair, 1975, differing from them in the nature of their pharynx: a Slender anterior region separated from the posterior ex- pansion by a short isthmus-like section vs. a well-muscu- lar anterior region separated from the posterior one by a marked constriction (abutting). As derived from molecular analyses The results of the analyses of six 18S rDNA and four 28S rDNA sequences (see above) are presented in the trees of Fig. 5 (18S) and Fig. 6 (28S). In both trees, the sequences of the new taxon form maximally sup- ported clades, confirming their identity. Nevertheless, zse.pensoft.net 1036 Pena-Santiago, R. et al.: Oleaxonchium olearum gen. et sp. nov. from the Iberian Peninsula 7100 100 100 100 18S rDNA MZ496941 Mononchus truncatus AY297821 Mononchus aquaticus DQ418791 Romanomermis culicivorax 0.02 100 MN296088 Neometadorylaimus coomansi MN296089 Neometadorylaimus coomansi MG921289 Tylencholaimus cynodonti 1400 MG921287 Tylencholaimus cynodonti 100 MG921280 Tylencholaimus cynodonti MG921272 Tylencholaimus ibericus MG921273 Tylencholaimus ibericus 100 AY552978 Tylencholaimellus affinis AY284837 Tylencholaimellus striatus AJ966472 Allodorylaimus sp. 100- ~KY942068 Allodorylaimus sp. Figg} KY942067 Allodorylaimus sp. KY119448 Allodorylaimus sp. og AY284794 Eudorylaimus minutus 100 AY146525 Longidorella parva 100) AY 284789 Longidorella sp. 100 AY284790 Longidorella sp. AY284804 Microdorylaimus miser i007 £U477379 Heterodorus veletensis me HG797025 Heterodorus brevidentatus HG797024 Heterodorus morgensis 100 HG797023 Rhyssocolpus juventutis HQ637471 Rhyssocolpus vinciguerrae 100 AY284805 Microdorylaimus modestus ji FJ042952 Enchodelus babakicus oo HM851184 Enchodelus longispiculus ld HQ404367 Enchodelus sardashtensis 100} =AY911988 Enchodelus hopedorus FJ042953 Enchodelus macrodorus HG797026 Enchodelus saxifragae AY284802 Epidorylaimus lugdunensis AJ966484 Eudorylaimus carteri AY593946 Prodorylaimus mas AY284795 Crassolabium circuliferum KY881720 Enchodeloides signyensis AY284788 Pungentus silvestris AY919274 Pungentus pungens AJ966501 Pungenius sp. AY283155 Californidorus sp. 100 74 100 KT258982 Belondira bagongshanensis coor =AY146482 Belondira cf. apitica 100 AY911924 Belondira cf. apitica 100 EU880009 Belondira sp 100 KT258983 Belondira bagongshanensis 100 MG921265 Belondira sp. MG921266 Belondira sp. AY284821 Dorylaimellus montenegricus AY911968 Dorylaimellus parvulus AY911972 Dorylaimellus tenuidens 56 AY552969 Dorylaimellus virginianus AY 284831 Leptonchus granulosus 66 KJ636399 Proleptonchus weischeri —— AJ966510 Tylencholaimus sp. 100 100 100 BO 56 KU992903 Tylencholaimus helanensis AB661628 Tylencholaimus cf. parvus EF207253 Tylencholaimus mirabilis 89] 100 AY284828 Discolaimus major MH915669 Moshajia idiofora AY284801 Allodorylaimus andrassyi AY284812 Aporcelaimellus cf. paraobtusicaudatus 8. AY911895 Aporcelaimellus krygeri 100 KF717497 Calcaridorylaimus castaneae LC457654 Calcaridorylaimus signatus AJ966489 Mesodorylaimus japonicus 79) MG921244 Cailcaridorylaimus sp. AJ966490 Mesodorylaimus cf. nigritulus 7§ AY284783 Ecumenicus monohystera KY119890 Ecumenicus monohystera KJ636386 Laimydorus sp. 67- MN605663 Aporcelaimellus nigeriensis 99 SOAY 284779 Chrysonema attenuatur MK089266 Lenonchium zanjaniensis DQ141212 Aporcelaimellus obtusicaudatus OM269524 Aporcelaimellus obtusicaudatus OP048823 Aporcelaimelius obtusicaudatus °¢ ~=MK796433 Aporcelaimellus obtusicaudatus AY911896 Aporcelaimelius obscuraides KM092519 Amblydorylaimus isokaryon 9¢ AY911892 Aporcelaimellus cf. capitatus AY911893 Metaporcelaimus conoidus AY593948 Aporcella simplex ] AY593950 Dorylaimoides limnophilus 4 24 AY284830 Dorylaimoides micoletzkyi sq |e KU662325 Dorylaimoides sp. 100 0Q920987 Paraxonchium carmenae AY284809 Paraxonchium laetificans EF207248 Mesodorylaimus centrocercus 00 AY284785 Sylphodorylaimus sylphoides AY284823 Oxydirus oxycephaloides AY284777 Dorylaimus stagnalis AY284824 Oxydirus oxycephalus 53) (0 AY284825 Oxydirus oxycephalus op AY593949 Oxydirus nethus EF207251 Oxydirus nethus AY284776 Dorylaimus stagnalis 100 GU446710 Paractinolaimus decraemerae 100 if §=AY284826 Paractinolaimus macrolaimus AY993978 Paractinolaimus macrolaimus GU178030 Paractinolaimus sahandi AY552972 Labronema ferox AY284807 Labronema vulvapapillatum 100 109 MK894247 Labronema montanum | MK894248 Labronema montanum CS os OP793643 Talanema ibericum OP793644 Talanema ibericum OP793645 Talanema ibericum °F =MT645226 Talanema bagrii MT645227 Talanema baqrii 100 100 KJ636342 Aquatides aquaticus — AY552963 Aquatides christei AY593944 Clavicaudoides clavicaudatus AY552974 Nygolaimus cf. parvus AY552976 Paravulvus hartingit 56 87 100 89 89 100 100) 100 Figure 5. Bayesian inference tree from the newly sequenced Oleaxonchium olearum gen. et sp. nov. based on sequences of the 18S rDNA region. Bayesian posterior probabilities (%) are given for each clade. The scale bar shows the number of substitutions per site. zse.pensoft.net Zoosyst. Evol. 100 (3) 2024, 1029-1039 98 1037 JX445112 Longidorus magnus JX445106 Longidorus baeticus JX445109 Longidorus vineacola JX445117 Longidorus intermedius KY881719 Enchodeloides signyensis 100; 100 EJ 59 100 100 ‘96 a7 100 97] 99 93 100 66 95 81 100 28S rDNA 100 AY593054 Enchodelus macrodorus AY593053 Pungentus silvestris MH346474 Pungentus engadinensis MN855359 Pungentus sufiyanensis KR184124 Enchodorus dolichurus KX691911 Enchodorus yeatsi KP963965 Heterodorus brevidentatus KR184127 Heterodorus youbertghostai KP204547 Rhyssocolpus vinciguerrae HM235514 Dorydorella bryophila AY593045 Longidorella sp. MH884066 Dorydorella sp. HM235515 Longidorella penetrans AY593042 Longidorella cf. macramphis AY593046 Microdorylaimus miser AY593047 Eudorylaimus minutus AY593048 Eudorylaimus minutus AY593049 Microdorylaimus modestus AY593035 Epidorylaimus lugdunensis AY593036 Epidorylaimus lugdunensis AY593037 Eudorylaimus sp. 100, 100 100 100 100 KT258984 Belondira bagongshanensis KT258985 Belondira bagongshanensis MF363124 Belondira coomansi MG921267 Belondira sp. MG921268 Belondira s KX151719 Aporcella charidemiensis KX151720 Aporcella charidemiensis 7] MW243335 Kochinema farodai JQ778273 Aporcella simplex MN262455 Aporcella simplex MH727512 Aporcella simplex JQ778274 Aporcella simplex MN296487 Neometadorylaimus coomansi ‘100 MG921305 Tylencholaimus ibericus KU992905 Tylencholaimus helanensis AY593027 Tylencholaimus mirabilis EF207243 Tylencholaimus cf, teres AY593023 Carcharodiscus banaticus AY593024 Carcharodiscus banaticus EF 207238 Discolaimoides symmetricus MT776559 Discolaimoides symmetricus 5 AY593025 Discolaimus major EF 207239 Discolaimus cf. major AY593026 Discolaimus major MT776558 Discolaimus major KY750844 Discolaimus sp. AY593029 Chrysonema attenuatum JQ927438 Metaporcelaimus capitatus JQ927440 Metaporcelaimus ovogranulosus AY593031 Sectonema barbatoides KU589226 Sectonema ciliatum 4 100 AY593016 Allodorylaimus andrassyi AY593015 Allodorylaimus andrassyi MKO007554 Alfodorylaimus sp. MH727518 Aporcelinus mediterraneus 100 MH619735 Aporcelinus amazonicus MK007553 Aporcelinus floridensis MH184520 Aporcelinus infundibulicaudatus MH619728 Aporcelinus insularis MF 134400 Aporcelinus jiaonanensis MK007552 Aporcelinus paolae MH619727 Aporcelinus elongicaudatus AY593018 Aporcelaimellus obtusicaudatus JX428789 Aporcelaimellus waenga JX094340 Aporcelaimellus obtusicaudatus KM406917 Aporcelaimellus pycnus AY593022 Axonchium propinquum KY492387 Makatinus crassiformis KY492387 Makatinus macrostylus KY492386 Makatinus crassiformis KM092520 Amblydorylaimus isokaryon 100 MH004440 Crassolabium costaricense MH004441 Crassolabium costaricense 0Q920984 Paraxonchium carmenae AY593001 Paraxonchium laetificans AY593002 Paraxonchium laetificans AY593003 Dorylaimoides limnophilus AY593004 Dorylaimoides micoletzkyi AY593013 Ecumenicus monohystera AY593014 Ecumenicus sp. AY593005 Mesodorylaimus sp. AY593006 Mesodorylaimus sp. AY593007 Eudorylaimus centrocercus MK894244 Labronema montanum MK894246 Labronema montanum AY593008 Syiphodorylaimus syiphoides AY593010 Syiphodorylaimus syiphoides AY593011 Oxydirus nethus AY593012 Oxydirus oxycephalus AY592994 Dorylaimus stagnalis GU446711 Paractinolaimus decraemerae AY592998 Paractinolaimus macrolaimus KM067903 Paractinolaimus morus GU178031 Paractinolaimus sahandi ON054911 Trachactinolaimus nanjingensis ON054912 Trachactinolaimus nanjingensis ON054913 Trachactinolaimus nanjingensis ON133539 Westindicus sp. AY592996 Labronema vulvapapillatum AY592997 Labronema vulvapapillatum JN242245 Nevadanema nevadense EF207241 Prodorylaimus sp. KM092522 Pararhyssocolpus paradoxus MG921241 Aetholaimus rotundicauda EF207234 Clavicaudoides clavicaudatus EF207236 Clavicaudoides trophurus EF207237 Clavicaudoides trophurus AY593061 Nygolaimus cf. brachyuris 100 98 78) 100 100 100 100 109 100 jit a 100 100 100 96 100 AY593063 Mononchus tunbridgensis 100 EF417153 Romanomermis culicivorax 0.07 AY593064 Mononchus truncatus Figure 6. Bayesian inference tree from the newly sequenced Oleaxonchium olearum gen. et sp. nov. based on sequences of the 28S rDNA region. Bayesian posterior probabilities (%) are given for each clade. The scale bar shows the number of substitutions per site. zse.pensoft.net 1038 other aspects of its evolutionary relationships significant- ly differ when the trees are compared. The 18S tree provides very unsatisfactory branching resolution. On the one hand, the Oleaxonchium gen. nov. clade forms part of a very lowly (55%) supported clade, where it appears as the sister group of an also lowly (56%) supported subclade that includes sequences of sev- eral Metaxonchium species. On the other hand, sequenc- es of Axonchium-like genera (Axonchium, Axonchoides, and Syncheilaxonchium) and M. giennensis (all of them highlighted in green in the tree) form part of a maximally supported clade with sequences of the genera Belondira and Dorylaimellus. The 28S tree offers better resolution as all Axonchi- um-like sequences form a maximally supported clade. Nevertheless, some uncertainties persist about the in- ternal relationships within this clade, mainly due to the apparent polyphyly of Metaxonchium, whose sequences form part of three different subclades, namely (new taxon + M. giennense), (Axonchoides smokyensis + (M. steno- spiculum + M. toroense)), and (M. magnum + Syncheilax- onchium nairi). Besides, the Axonchium-like clade is in- cluded in a much larger clade, with Be/ondira sequences not forming part of it. An integrative approach and general discussion The singularity of the lip region of Oleaxonchium gen. nov., unique within Belondiridae and probably in Dory- laimida, and a recognisable autopomorphy, supports its separation from its closest relatives. The peculiar shape of its body pores and cephalic papillae also represent re- markable differences with other axonchiid genera. Mo- lecular analyses based on both 18S and 28S genes coin- cide when indicating that there is a closer evolutionary relationship between the new taxon and Metaxonchium representatives, but the results significantly differ in the nature and robutness of this relationship when the respec- tive molecular trees are compared. Anyway, Metaxon- chium is a rather homogeneous genus with a recognis- able morphological pattern (Pefia-Santiago and Abolafia 2023), easily distinguishable from Oleaxonchium gen. nov. (see above). Present results confirm that the internal evolutionary relationships of Belondiridae remain unsatisfactorily re- solved, with morphological and molecular data not al- ways in agreement. First, the polyphyly of the family is once again noted in both 18S and 28S trees, as Oxydirus (subfamily Swangeriinae) sequences appear in a separate clade, far from those including other representatives of the group. Second, morphological features and 28S anal- ysis support the validity of Axonchiinae Thorne, 1964, as a monophyletic taxon that should be reinstaurated (cf. Andrassy 2009). Third, relationships between Belondira (subfamily Belondirinae) and axonchiid genera remain obscure, as 18S and 28S analyses did not provide com- parable evidence. Once more, further research should be accomplished to clarify these issues. zse.pensoft.net Pena-Santiago, R. et al.: Oleaxonchium olearum gen. et sp. nov. from the Iberian Peninsula Acknowledgments This contribution derives from the project Soil O-Live. This project has received funding from the European Union’s Horizon Europe research and innovation pro- gramme under grant agreement No. 101091255 (Soil Deal for Europe - HORIZON-MISS-2021-SOIL-02-03). The authors thank Dr. Pablo Castillo (IAS, Cordoba, Spain) for his collaboration in molecular analyses and are grateful for the SEM pictures obtained with the assis- tance of technical staff (Amparo Martinez-Morales) and equipment belonging to the Centro de Instrumentacion Cientifico-Técnica (CICT) of the University of Jaén. A.N. 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