A new ring nematode, Xenocriconemella costaricense sp. nov., (Nematoda: Criconematidae) from Costa Rica

Abstract During nematode surveys of natural vegetation in forests of La Cima de Copey de Dota, San José, San José province, Costa Rica, a Xenocriconemella species closely resembling X. macrodora and related species was found. Integrative taxonomical approaches demonstrated that it is a new species described herein as X. costaricense sp. nov. The new species is parthenogenetic (only females have been detected) and characterised by a short body (276–404 μm); lip region with two annuli, not offset, not separated from body contour; first lip annulus partially covering the second lip annulus. Stylet thin, very long (113–133 μm) and flexible, occupying 30.5–47.8% of body length. Excretory pore located from one or two annuli anterior to one or two annuli posterior to level of stylet knobs, at 42 (37–45) μm from anterior end. Female genital tract monodelphic, prodelphic, outstretched, and occupying 35–45% of body length, with vagina slightly ventrally curved (14–18 μm long). Anus located 6–11 annuli from the tail terminus. Tail conoid and bluntly rounded terminus, the last 2–3 annuli oriented dorsally. Results of molecular characterisation and phylogenetic analyses of D2-D3 expansion segments of 28S rRNA, ITS, and partial 18S rRNA, as well as cytochrome oxidase c subunit 1 gene sequences further characterised the new species and clearly separated it from X. macrodora and other related species (X. iberica, X. paraiberica, and X. pradense).


Introduction
The ring nematode genus Xenocriconemella De Grisse andLoof, 1965 (De Grisse andLoof 1965) comprises obligate ectoparasite nematodes characterised by a short body length (ca.250-400 μm), a long and flexible stylet (ca.100-110 μm and up to 40% of body length), body annuli smooth without anastomosis, vulva closed, and juveniles similar to females (Geraert 2010).Until recently, X. macrodora (Taylor, 1936) De Grisse and Loof, 1965 (Taylor 1936;De Grisse and Loof 1965) was the only nominal species within the genus.The taxonomic validity of the genus Xenocriconemella has been a scientific controversy in the nematological literature over recent decades (Loof and De Grisse 1989;Siddiqi 2000;Geraert 2010).Lübbers and Zell (1989) morphologically studied several populations of X. macrodora from Germany and asserted that German populations differed morphologically from those of the USA and several European countries in a higher number of annuli (R = 147 vs. R = 99-120), concluding that these populations belong to a new species, X. degrissei Lϋbbers andZell, 1989. Later, Ganguly et al. (2008) also studied X. macrodora populations from peach and blue pine in India and concluded that these populations belong to two new species, X. pruni Ganguly et al. 2008and X. pini Ganguly et al. 2008. However, Sturhan (2013) compared the morphometry of all three species and concluded that they all overlap with populations of X. macrodora, and the presence of a filled spermatheca described and/or figured for X. pruni and X. pini suggested the presence of males in these species (as in some X. macrodora populations).Consequently, Sturhan (2013) proposed the synonymisation of all three species with X. macrodora, but no molecular markers were provided to confirm this action.Nevertheless, recent integrative taxonomical studies on more than 25 Iberian populations of Xenocriconemella supported the validity and monophyly of this genus, as well as the description of three new species including X. iberica Archidona-Yuste et al. 2024, X. paraiberica Archidona-Yuste et al. 2024, and X. pradense Archidona-Yuste et al. 2024(Archidona-Yuste et al. 2024).This study also indicated that this genus is widespread, especially in North America and Europe, and undoubtedly has a close association with woodland forests (Archidona-Yuste et al. 2024).In Central America, the genus Xenocriconemella has been observed in Mexico (Cid del Prado 1988) and in protected areas of Costa Rica (Esquivel 2003).However, no morphological or molecular data on this nematode are available from Costa Rica.
Integrative-based taxonomy studies (using a combination of morphology and morphometry with molecular data) have proved to be accurate and useful tools for assessing populations and species boundaries within Criconematidae spp. as well as in the genus Xenocriconemella (Subbotin et al. 2005;Etongwe et al. 2020;Powers et al. 2021;Nguyen et al. 2022;Clavero-Camacho et al. 2022;Archidona-Yuste et al. 2023).A Costa Rican population of Xenocriconemella associated with forests composed of wild avocado (Persea caerulea) and Ecuador laurel (Cordia alliodora (Ruiz and Pav.) Oken were found in La Cima de Copey de Dota, San José, showing a different cytochrome c oxidase subunit 1 (COI) and ribosomal sequences to the molecular markers deposited in the GenBank for this genus.This prompted us to study this Xenocriconemella population in more detail to clarify its taxonomic position.Hence, the main objectives of this study were to (i) characterise the newly recovered Costa Rican population of Xenocriconemella morphologically and morphometrically and compare it with Xenocriconemella spp.belonging to the X. macrodora-species complex; (ii) provide molecular characterisation of this Xenocriconemella population using ribosomal (D2-D3) expansion segments of 28S rRNA, Internal Transcribed Spacer region (ITS), partial 18S rRNA), and COI markers; and (iii) study phylogenetic relationships within Criconematidae spp.and this species of the X. macrodora-species complex.

Nematode population and morphological characterisation
In winter 2023, a nematological survey was conducted in a forest area in La Cima de Copey de Dota, San José, Costa Rica, and in one of them, low population densities of a Xenocriconemella species were detected.Soil samples for nematode analysis were collected with a shovel from randomly selected trees and mixed to constitute a soil sample from each sampling site; samples came from the upper 5-40 cm of soil.Subsequently, nematodes were extracted from a 500 cm 3 subsample of soil by centrifugal flotation and modification of Cobb´s decanting and sieving methods (Flegg 1967;Coolen 1979).
Specimens for light microscopy (LM) and morphometric studies were killed at 70-75°C and fixed in an aqueous solution of 4% formaldehyde + 1% glycerin, dehydrated using an alcoholsaturated chamber, and processed to pure glycerin using Seinhorst's method (Seinhorst 1966) as modified by De Grisse (1969).Light micrographs and measurements of the nematode population, including important diagnostic characteristics were conducted using a Leica DM6 compound microscope with a Leica DFC7000 T digital camera (Leica, Wetzlar, Germany).Measurements and ratios included: L, (total body length); a = body length/ maximal body width; b = body length/pharyngeal length; c = body length/tail length; c' = tail length/body width at anus; O = distance between stylet base and orifice of dorsal pharyngeal gland as percentage of stylet length; R = total number of body annuli; Roes = number of annuli in pharyngeal region; Rex = number of annuli between anterior end of body and excretory pore; Rst = number of body annuli between labial disc and stylet knobs; RV = number of annuli between posterior end of body and vulva; Rvan = number of annuli between vulva and anus; Ran = number of annuli on tail; V = (distance from anterior end to vulva/body length) × 100; VL/VB = distance between vulva and posterior end of body divided by body width at vulva; T = (distance from cloacal aperture to anterior end of testis/body length) × 100 (Archidona-Yuste et al. 2023;2024).The raw photographs were edited using Adobe Photoshop SC6 v 24.7.2 (San Francisco, CA, USA).

DNA extraction and molecular characterisation
To avoid mistakes in case of mixed populations in the sample, single specimens were temporarily mounted in a drop of 1 M NaCl containing glass beads (to avoid nematode crushing/damaging specimens) to ensure specimens conformed with the target population.Genomic DNA extraction from single specimens was conducted as described by Archidona-Yuste et al. (2023).Briefly, an individual nematode was cut using a scalpel in a drop of polymerase chain reaction (PCR) buffer (ThermoPol®, Biolabs, New England, USA) (20 μL), and 2 μL proteinase K (600 μg/mL) was added.Tubes were frozen at -80°C (15 min) and then incubated at 65°C (1 h) and 95°C (10 min) consecutively.Tubes were centrifuged (1 min, 16,000 × g) and kept at À20°C until use in PCR; more importantly, all three molecular markers for the population of Xenocriconemella were extracted from the same single individual in each PCR tube without any exception.

Phylogenetic analyses
D2-D3 expansion domains of the 28S, ITS, 18S rRNA, and COI mtDNA sequences of the recovered unidentified Xenocriconemella species were obtained in this study.These sequences and other sequences of Criconematidae spp.from GenBank were used for phylogenetic analyses.The selection of outgroup taxa for each dataset was based on previously published studies (Etongwe et al. 2020;Nguyen et al. 2022;Archidona-Yuste et al. 2024).Multiple sequence alignments of the different genes were completed using the FFT-NS-2 algorithm of MAFFT V.7.450 (Katoh et al. 2019).The BioEdit program V.7.2.5 (Hall 1999) was used for sequence alignment visualisation and manually edited and trimmed of the poorly aligned positions using a light filtering strategy (up to 20% of alignment positions), which has little impact on tree accuracy and may save computation time, as suggested by Tan et al. (2015), because methods for automated filtering of multiple sequence alignments frequently worsen single-gene phylogenetic inference (Tan et al. 2015).
Phylogenetic analyses of the sequence datasets were based on Bayesian inference (BI) using MrBayes 3.1.2(Ronquist and Huelsenbeck 2003).The best-fit model of DNA evolution was achieved using JModelTest V.2.1.7 (Darriba et al. 2012) with the Akaike information criterion (AIC).The best-fit model, base frequency, proportion of invariable sites, and gamma distribution shape parameters and substitution rates in the AIC were then used in MrBayes for phylogenetic analyses.The general time-reversible model with invariable sites and a gamma-shaped distribution (GTR + I + G) for the D2-D3 expansion segments of 28S rRNA and the partial 18S rRNA gene, the TPM2uf model with a gammashaped distribution (TPM2uf + G) for the ITS region, and the Hasegawa, Kishino, and Yano model with invariable sites and a gamma-shaped distribution (HKY + I + G) for the COI gene were run with four chains for 4 × 10 6 generations.A combined analysis of the three ribosomal genes was not undertaken because some sequences were not available for all species.The sampling for Markov chains was conducted at intervals of 100 generations.For each analysis, two runs were conducted.After discarding burn-in samples of 30% and evaluating convergence, the remaining samples were retained for more in-depth analyses.The topologies were used to generate a 50% majority-rule consensus tree.For each appropriate clade, posterior probabilities (PP) were given.FigTree software version v.1.4.4 (Rambaut 2018) was used for visualising trees from all analyses.

Results
The presently studied population of Xenocriconemella was detected at low density (31 nematodes per 500 cm 3 of soil) in one of two soil samples collected from a forest area in La Cima de Copey de Dota, San José province, Costa Rica.Detailed morphological, morphometrical, and molecular information about this species is provided below, confirming its identity as a new species of Xenocriconemella described herein.

Not found.
Juveniles Body similar to females, including tail shape, but shorter.Edge of body annuli without appendages, marked with delicate irregular punctations.

Etymology
The species epithet refers to the country where the species was detected, Costa Rica.

Type host and locality
The new species was recovered from a rainforest in the rhizosphere of wild avocado (Persea caerulea) and Ecuador laurel (Cordia alliodora (Ruiz and Pav.) Oken from a forest in La Cima de Copey de Dota, San José, San José Province, Costa Rica (coordinates 9°4 2'41.2"N, 83°56'59.2"W).

Type material
Holotype female and 19 female paratypes were deposited at Laboratorio de Nematología, Escuela de Ciencias Agrarias, Universidad Nacional, Heredia, Costa Rica; two females are at Institute for Sustainable Agriculture (IAS) of Spanish National Research Council (CSIC), Córdoba, Spain; and two females are at the USDA Nematode Collection (T-8028p).
The phylogenetic position of X. costaricense sp.nov. in the ITS region tree is shown in Figure 4. Phylogenetic relationships observed for this region were very similar to D2-D3 expansion segments of the 28S rRNA gene.In this analysis, X. costaricense sp.nov (PP209392-PP209393) and X. paraiberica clustered together, although clearly separated, in a well-supported subclade (PP = 1.00), whereas X. iberica and X. pradense clustered with another well-supported subclade (PP = 1.00).For this region X. macrodora from USA (JQ708139) clustered separately from the rest of Xenocriconemella spp.accessions, forming a well-supported subclade with Criconema mutabile (JQ708132) and Criconema sp. 4 Livingston (FN435300).
In 18S rRNA phylogeny, all accessions belonging to the Xenocriconemella genus clustered together in a well-supported clade (PP = 1.00), occupying the top of the tree.However, the phylogenetic relationships between the different Xenocriconemella spp.were not well resolved, as many of the internal clades were poorly supported.
Finally, the phylogenetic position of X. costaricense sp.nov.(PP210897-PP210900) and the other species, using COI gene sequences, are shown in Figure 6.The position of X. costaricense sp.nov.was well-supported (PP = 1.00), clustering alone in a basal position of the tree.Accessions from X. macrodora from USA appeared together in a well-supported (PP = 1.00) subclade.On the contrary, relationships of Xenocriconemella spp.from the Iberian Peninsula-X.pradense, X, iberica, and X. paraiberica-were not well resolved for this gene (Figure 6).

Discussion
The recent integrative taxonomic study on numerous X. macrodora populations from the Iberian Peninsula defined a new species complex within X. macrodora including at least four species, viz.X. iberica, X. macrodora, X. paraiberica, and X. pradense (Archidona-Yuste et al. 2024).The main objective of this study was to identify and describe, morphologically and molecularly, a new population of Xenocriconemella detected in a natural environment in a forest of Dota, San José province, Costa Rica, as well as clarify the phylogenetic relationships within the genus Xenocriconemella.All results confirmed that the unknown Xenocriconemella population is morphologically and morphometrically close to the X. macrodora-species complex, except for differences in body size, stylet length, and other minor morphometric differences.Nevertheless, all molecular markers clearly separated the new Costa Rican population from all other species, confirming that the new population is a new valid species of the genus Xenocriconemella.These data provide clear evidence that the worldwide biodiversity within this genus may be higher than previously understood, as recently suggested by Archidona-Yuste et al. (2024).Interestingly, although more studies are needed to confirm this hypothesis, our results suggest that additional taxa can be included within the widely reported populations of X. macrodora s.l. in those regions where  the species complex has been reported (Archidona-Yuste et al.

2024).
Again, ribosomal and mitochondrial markers (D2-D3 expansion domains of the 28S rRNA and ITS rRNA, and the mtDNA gene COI) are important tools for accurate identification of Xenocriconemella and remain essential for accurate diagnosis of ring nematodes (Subbotin et al. 2005;Etongwe et al. 2020;Powers et al. 2021;Nguyen et al. 2022;Archidona-Yuste et al. 2024).However, the low nucleotide variability found in partial 18S rRNA makes it difficult to identify individuals at the species level, but this molecular marker clearly separated the genus from other genera of the family Criconematidae.Phylogenetic analyses based on ribosomal genes resulted in a general consensus of species' phylogenetic positions for the majority of species and was congruent with those given by previous phylogenetic analyses (Subbotin et al. 2005;Etongwe et al. 2020;Nguyen et al. 2022;Archidona-Yuste et al. 2024).In particular, in all three ribosomal gene trees, X. costaricense sp.nov.clustered with all morphologically related species, including X. iberica, X. macrodora, X. paraiberica, and X. pradense (Figures 3-5).
In summary, the present study confirms the usefulness of an integrative approach based on the combination of morphometric and morphological traits and genotyping rRNA and mtDNA markers for correct discrimination among Xenocriconemella species, suggesting the need for continuing nematode surveys in natural environments to complete the unexplored worldwide biodiversity of this genus.

Table 1 .
Morphometrics of Xenocriconemella costaricense sp.nov.from the rhizosphere of wild avocado (Persea caerulea) and Ecuador laurel (Cordia alliodora (Ruiz & Pav.) Oken in La Cima de Dota, San José Province, Costa Rica : All measurements are in μm and in the form: mean ± s.d.(range).Abbreviations: a, body length/maximal body width; b, body length/pharyngeal length; c, body length/tail length; c', tail length/body width at anus; L, total body length; n, number; O, distance between stylet base and orifice of dorsal pharyngeal gland as percentage of stylet length; R, total number of body annuli; Roes, number of annuli in pharyngeal region; Rex, number of annuli between anterior end of body and excretory pore; Rst, number of body annuli between labial disc and stylet knobs; RV, number of annuli between posterior end of body and vulva; Rvan, number of annuli between vulva and anus; Ran, number of annuli on tail; V, (distance from anterior end to vulva/body length) × 100; VL/VB, distance between vulva and posterior end of body divided by body width at vulva. Note