Zoosyst. Evol. 100 (3) 2024, 1175-1190 | DOI 10.3897/zse.100.129009 yee BERLIN Another new ring nematode, Xenocriconemella andreae sp. nov. (Nematoda, Criconematidae), from the Iberian Peninsula Carolina Cantalapiedra-Navarrete', Ilenia Clavero-Camacho'!, Inmaculada Criado-Navarro', Rosana Salazar-Garcia!, Ana Garcia-Velazquez', Juan E. Palomares-Rius', Pablo Castillo’, Antonio Archidona- Yuste* 1 Instituto de Agricultura Sostenible, Departamento de Proteccion de Cultivos, Avenida Menén-dez Pidal s/n, 14004 Cordoba, Campus de Excelencia Internacional Agroalimentario, ceiA3, Spain https://zoobank. org/E691CFA F-0825-43 EA-8756-952C32174072 Corresponding author: Antonio Archidona- Yuste (antonio.archidona@ias.csic.es) Academic editor: A. Schmidt-Rhaesa # Received 4 June 2024 Accepted 9 July 2024 Published 23 August 2024 Abstract Nematode surveys in natural environments in the Iberian Peninsula detected three unidentified Xenocriconemella populations that closely resembled the X. macrodora-species complex, but utilization of integrative taxonomy confirmed that they comprised a new taxon described in this paper as _X. andreae sp. nov. Only females were detected in the new species (considered parthenogenetic) and delineated with a bare body (274-353 um); lip region with two annuli, continuous with body delineation; second lip annulus enclosed by the first one. Flexible and thin stylet (88.0—99.0 um), representing 30.4-47.8% of total body length. The excretory pore is positioned 2—3 annuli posterior to the level of stylet knobs, at 101.5 (87-107) um from the lip region. Female genital tract: monodelphic, prodelphic, large, and representing 34.4—52.4% of the body length; vagina slightly ventrally curved. The anus is lo- cated at (6-9) annuli from the rear end. Tail short, conoid, and blunt round terminus. Ribosomal and mitochondrial markers (D2-D3 expansion domains of 28S, ITS, partial 18S rRNA, and COI), as well as molecular phylogenetic analyses of sequences, confirmed this new taxon, and it was clearly delineated from _X. macrodora and species within the species complex (X. costaricense, X. iberica, X. paraiberica, and X. pradense). Key Words COI, description, D2-D3, integrative taxonomy, ITS, 18S, morphometry Introduction The ring nematode genus Xenocriconemella De Grisse & Loof, 1965 (De Grisse and Loof 1965) includes small ringed ectoparasite nematodes with a stylet ca. 40% of their body length. This species has become a topic of scientific attention in recent years. Particularly relevant is the novel incorporation of integrative tax- onomy studies in deciphering populations within this genus (Archidona-Yuste et al. 2024; Peraza-Padilla et al. 2024). This unlocked the long-established assump- tion that Xenocriconemella macrodora (Taylor 1936; De Grisse and Loof 1965) was the unique valid species within the genus. That is, recent integrative taxonomic studies added taxa within the genus Xenocriconemella, including the three new species described in the Iberi- an Peninsula (XY. iberica Archidona-Yuste et al. 2024, X. paraiberica Archidona-Yuste et al. 2024, and X. pra- dense Archidona-Yuste et al. 2024) and the new one from Costa Rica (X. costaricense Peraza-Padilla et al. 2024). Undoubtedly, this has allowed us to support the validity and monophyly of this genus and has also con- firmed the already reported strong association with for- est and shrub ecosystems dominated mainly by Quercus trees (Bello et al. 1986; Gomez-Barcina et al. 1989; Escuer et al. 1999). Copyright Cantalapiedra-Navarrete, C. 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. 1176 In Nematoda, and especially in plant-parasitic nem- atodes, it is quite typical that molecular differences are not manifested in variations in morphology among spe- cies (1.e., the occurrence of cryptic species complexes; Cantalapiedra-Navarrete et al. 2013; Archidona-Yuste et al. 2016, 2020; Cai et al. 2020; Clavero-Camacho et al. 2021). The integration and complementarity of different perspectives and methods of taxonomy is an imperative need for rigorous species delimitation within a cryptic complex (Proudlove and Wood 2003; Dayrat 2005; Figer and Koselj 2022). Over the last 15 years, much research has been conducted on soil nematodes in this direction (particularly in plant-parasitic species; e.g., Gutiér- rez-Gutiérrez et al. 2010; Barsi et al. 2017; Decraemer et al. 2024). The genus Xenocriconemella is a recent example where the transition from traditional to integra- tive taxonomy has unraveled a model cryptic complex of species (Archidona-Yuste et al. 2024). Indeed, molecu- lar taxonomy together with in-depth morphological and morphometrical analyses defined the X. macrodora-spe- cies complex (X. macrodora, X. iberica, X. paraiberica, and X. pradense) from nematode populations widely distributed in the Iberian Peninsula (Archidona-Yuste et al. 2024). Furthermore, several studies have revealed a wide number of cryptic species complexes within a large functional variability of plant-parasitic nematodes in the Iberian Peninsula (e.g., Gutiérrez-Gutiérrez et al. 2010; Cantalapiedra-Navarrete et al. 2013; Archidona-Yuste et al. 2016, 2020; Cai et al. 2020; Archidona-Yuste et al. 2024; Decraemer et al. 2024; among others). Although it is well known that the Iberian Peninsula stands out as one of the most biodiverse regions on the planet (Myers et al. 2000), this great diversity detected in this area could also be due to the notable scientific effort in discovering the diversity of soil nematodes in this area developed in the last few years. Here, to enhance soil nematode records and advance the understanding of taxonomy, morphology, and molec- ular data within the genus Xenocriconemella, we carried out a further sampling campaign on the Quercus-domi- nated natural areas of the Iberian Peninsula. In this nem- atode survey, we detected several unknown populations based on the available information (that is, morphological and molecular data) of Xenocriconemella species from the USA (Powers et al. 2021), Italy (Subbotin et al. 2005), Costa Rica (Peraza-Padilla et al. 2024), and the Iberian Peninsula (Archidona-Yuste et al. 2024). The original objective of this research was therefore to explore the Cantalapiedra-Navarrete, C. et al.: Xenocriconemella andreae sp. nov. morphological-morphometrical and molecular diversity of these unresolved Xenocriconemella populations in the Iberian Peninsula genus and to compare them with the available morphometrical and molecular data by Archi- dona-Yuste et al. (2024). The key goals of this research were to (i) describe the three newly discovered Iberian Peninsula species both morphologically and morphomet- rically and compare them with other Xenocriconemella species in the X. macrodora-species complex; (11) obtain molecular information about these Xenocriconemella populations using ribosomal (D2-D3 expansion domains of 28S rRNA, ITS region, partial 18S rRNA) and COI markers; and (iii) review the phylogenetic relationships of Xenocriconemella andreae sp. nov. within Criconema- tidae spp. and the _X. macrodora-species complex. Materials and methods Nematode isolation and morphometrical characterization Continuing the nematode surveys for deciphering the molecular and morphological diversity of Xenocricone- mella isolates in the natural environments of the Iberian Peninsula started by Archidona-Yuste et al. (2024), three additional samples were collected in the autumn of 2023 containing an unidentified Xenocriconemella species (Ta- ble 1). Samples were taken from the rhizospheric soil of the selected plants and mixed to establish a single sam- ple from each sample point. The soil samples were taken from a depth of 5 to 40 cm. Afterward, nematode speci- mens were isolated from a 500 cm? soil sub-sample using the centrifugal-flotation method (Coolen 1979). Materials and methods used for light microscopy (LM) and morphometric studies followed the same protocols described by Archidona-Yuste et al. (2024) and other researchers (Seinhorst 1966; De Grisse, 1969). The fol- lowing abbreviations (i.e., measurements and ratios) are used in the morphological descriptions, data analy- ses, and figures: L (total body length); a = body length/ maximal body width; b = body length/pharyngeal length; c = body length/tail length; ¢’ = tail length/body width at anus; O = distance between stylet base and orifice of dorsal pharyngeal gland as a percentage of stylet length; R = total number of body annuli; Roes = number of an- nuli in the pharyngeal region; Rex = number of annuli between the anterior end of the body and the excretory Table 1. Host-plant species and localities of the analyzed populations of Xenocriconemella andreae sp. nov. inside the Xenocricone- mella macrodora (Taylor, 1936) De Grisse and Loof 1965 species-complex from the Iberian Peninsula in this study. Nematode species Code Host-plant species Locality, province, Country Abundance NCBI Accessions (Nem/500 D2-D3 ITS 18S Col cm! soil) Xenocriconemella andreae SINO3 Pistacia lentiscusL. — Linhd, Sintra, Portugal (type) 103 PP833567- PP833563- PP833577- ~—- PP831172- Sp. nov. PP833569 PP833564 PP833579 PP831174 Xenocriconemella andreae HUAO3 Quercus suber L. Aroche, Huelva, Spain 13 PP833570 - - PP831175- sp. nov. PP831176 Xenocriconemella andreae LEOO2 Castanea sativa Mill. Trabadelo, Leén, Spain 552 PP833571- PP833565- PP833580- PP831177 Sp. nov. PP833576 PP833566 PP833582 zse.pensoft.net Zoosyst. Evol. 100 (3) 2024, 1175-1190 pore; Rst = number of body annuli between labial disc and stylet knobs; RV = number of annuli between pos- terior end of body and vulva; Rvan = number of annul between vulva and anus; Ran = number of annuli on tail; V = (distance from anterior end to vulva/body length) x 100; VL/VB = distance between vulva and posterior end of body divided by body width at vulva; T = (dis- tance from cloacal aperture to anterior end of testis/body length) x 100 (Archidona-Yuste et al. 2023; 2024). The raw photographs were edited using Adobe Photoshop v. 22.5.2 (San Francisco, CA, USA). Molecular analyses Total genomic DNA was extracted from single nematode specimens as previously described by Archidona-Yuste et al. (2023, 2024). As in previous studies, all four molec- ular markers for each Xenocriconemella isolate were ob- tained from the same PCR tube from a single individual without any exception. Similarly to other studies, primers for ribosomal (D2- D3 expansion domains of 28S rRNA, internal transcribed pacer region (ITS) rRNA, and the partial 18S rRNA) and mitochondrial (COI) markers were the same as those spec- ified in previous papers (De Ley et al. 1999; Subbotin et al. 2001; Hu et al. 2002; Derycke et al. 2005; Holterman et al. 2006). The PCR cycling conditions were also as de- scribed in previous papers (De Ley et al. 1999; Subbotin et al. 2005; Holterman et al. 2006; Powers et al. 2021). The PCR products were treated and sequenced at the Stab Vida sequencing facility (Caparica, Portugal) (see Archi- dona-Yuste et al. 2024 for details). The sequence chro- matograms of all markers were analyzed with DNASTAR LASERGENE SeqMan v. 7.1.0. The species identity of the DNA sequences obtained in this study was confirmed by the basic local alignment search tool (BLAST) at the National Center for Biotechnology Information (NCBI) (Altschul et al. 1990). The accomplished sequences were delivered to the GenBank database under accession num- bers specified on the phylogenetic trees and in Table 1. Species delineation analyses We used two independent approaches to species delin- eation to resolve the species boundaries within the X. macrodora species complex, counting morphometric and molecular data. First, we conducted a principal component analysis (PCA) to delimit species using morphometric data (Leg- endre and Legendre 2012). We established the species delimitation amongst the new Xenocriconemella popu- lations found in the Iberian Peninsula and other species recently described within this genus, and we further eval- uated the relationships between these new isolates and those previously designated within Xenocriconemella. PCA was constructed upon the following morphological 1177 characters: L, stylet length, R, Rst, Roes, Rex, RV, Rvan, Ran, and the ratios a, b, c, V, VL/VB (Archidona- Yuste et al. 2024). For this analysis, we chose 25 _X. macrodora s.1. populations previously characterized and recorded from numerous countries (Archidona- Yuste et al. 2024), as well as 28 Iberian populations previously studied under an in- tegrative taxonomical approach, including 13 belonging to X. iberica, 12 belonging to _X. paraiberica, 3 belonging to X. pradense, and | belonging to _X. costaricense from Costa Rica (Archidona-Yuste et al. 2024; Peraza-Padilla et al. 2024). After data standardization (Zuur et al. 2010), the diagnostic character-data set was tested for collinear- ity using the values of the variance inflation factor (VIF) as recommended by Montgomery et al. (2012). PCA was carried out by means of the PCA function supplied in the software package ‘FactoMineR’ (Lé et al. 2008). All data analyses were performed with R version 4.2.2 (R Core Team 2022; https://www.R-project.org). Species delimitation with molecular data and to com- pute intra- and inter-species disparity was performed by the P ID liberal and Rosenberg’s PAB value using the species delimitation plugin implemented in the software Geneious Prime v2022.1.1. (Geneious, Auckland, New Zealand) (Masters et al. 2011). Genetic distance was calcu- lated based on intra- and interspecies molecular variations established by determining the ratio between the average genetic distance between specimens within a species and the average genetic distance between specimens belonging to sister species; if the ratio is less than 0.10, the probabil- ity of species identification is high (Masters et al. 2011). The P ID (liberal) value (Ross et al. 2008) means the likeli- hood that a correct species identification would be carried out using the closest genetic distance or placement on a tree (falling within or being sister to a monophyletic spe- cies clade). Taxa with a P ID (liberal) > 0.93 were consid- ered to be satisfactorily demarcated (Hamilton et al. 2014). Rosenberg’s P,,, means the likelihood that the monophyly of a group of sequences is the result of random branching; significant values were <0.05 (Rosenberg 2007). Phylogenetic analyses Methods and software programs for aligning, sequence edi- tion, and phylogenetic analyses were performed following the same procedures already specified in previous papers, including outgroup selection and tree visualization (Hall 1999; Ronquist and Huelsenbeck 2003; Darriba et al. 2012; Tan et al. 2015; Rambaut, 2018; Katoh et al. 2019; Eton- gwe et al. 2020; Nguyen et al. 2022; Archidona-Yuste et al. 2024). The best-fit models for each marker were: the transi- tional model with invariable sites and a gamma-shaped dis- tribution (TIM3 + I+ G) for the D2-D3 expansion domains of 28S rRNA; the general time-reversible model with in- variable sites and a gamma-shaped distribution (GTR + I + G) for ITS and the partial 18S rRNA gene; and the 3-pa- rameter model with invariable sites and a gamma-shaped distribution (TPM3uf + I + G) for the COI gene. zse.pensoft.net 1178 Results Low to moderate densities (312, 13, -552 nematodes/500 cm? of soil) of the currently characterized isolates of Xe- nocriconemella were determined in the soil samples col- lected from the rhizosphere of mastic tree, cork oak, and chestnut Linho, Sintra region, Portugal, Aroche, Huelva province, Spain, and Trabadelo, Leon province, Spain, respectively. Comprehensive morphological, morpho- metric, and molecular data about this species are supplied below, confirming its identification as a new taxon within the Xenocriconemella macrodora-species complex. Species delineation using morphometry Our PCA results showed a wide intraspecific variation amongst the specimens of Xenocriconemella spp., espe- cially for X. iberica and X. paraiberica, based on the wide morphometric variation in the following features: R, Rv, Roes, Rst, Rex, Stylet, V, and (VL/VB), confirming that previously described by Archidona-Yuste et al. (2024) (Fig. 1). As anticipated, we confirmed the high morpho- logical variation displayed by X. macrodora (Archido- na-Yuste et al. 2024). PCA clearly distinguished between almost all the individuals of X. andreae sp. nov., X. pra- dense, and_X. costaricense, as well as those identified with- in_X. iberica and X. paraiberica. However, this spatial sep- aration occurred to a lower degree between X. pradense and X. iberica, where some individuals were found close to each other (Fig. 1). This species segregation was mostly observed along the first and second dimensions (Dim 1 and Dim2; 38.57 and 21.9% of the total variance, respectively). Dim1 was largely dominated by R, Rv, Roes, Rst, Rex, sty- let length, and VL/VB (Fig. 1). On the other hand, Dim2 was mainly dominated by the Rvan and c ratio, thereby relating this dimension to the posterior part of the nema- tode. Except in specific cases, the delimitation of species boundaries provided by the PCA occurs through a linear combination of multiple diagnostic characters (Archido- na-Yuste et al. 2016, 2024). Thus, we detected that species separations were mainly based on a combination of the fol- lowing diagnostic characters: R, Roes, Rst, Rex, Rv, Rvan, c ratio, and stylet length. More explicitly, individuals with higher values in R, Rv, Roes, Rst, Rex, and Rv and longer stylet length were located on the right (i.e., X. pradense and X. costaricense), and those with lower values for these characters were located on the left side along Dim] (..e., X. paraiberica and X. andreae sp. nov.). Likewise, speci- mens with higher values of Rvan and c ratio (that is, longer length in the posterior part of the body) were located at the top (i.e., X. costaricense and X. andreae sp. nov.), and those with lower values for these characters were located at the bottom side along the Dim? (1.e., X. pradense). Ul- timately, we could conclude that Roes, Rst, Rex, and Rv were the most useful morphometrics for separating species within this cryptic complex in the genus Xenocricone- mella. However, most of the specimens of X. iberica and zse.pensoft.net Cantalapiedra-Navarrete, C. et al.: Xenocriconemella andreae sp. nov. X. paraiberica were located overlying each other, given their similar values for traits associated with Dim 1 and Dim2 (Fig. 1). Thus, we confirmed that both species are strictly related morphologically. Additionally, we found an analogous arrangement for individuals (i.e., mean values of isolates) of X. macrodora as described by Archidona-Yuste et al. (2024). Definitively, our data confirmed the idea that these already described species (1.¢e., X. iberica, X. parai- berica, and X. pradense) encompass a model complex of cryptic species (1.e., the X. macrodora species complex). However, PCA allowed us to separate the new taxa _X. an- dreae sp. nov. and the already described species (X. costar- icense) from this species cryptic complex. Species delineation using ribosomal and mitochondrial DNA Species delineation using ribosomal and mitochondri- al markers proved that X. andreae sp. nov., X. iberica, X. paraiberica, X. pradense, and X. costaricense were un- doubtedly distinguished among them, as were X. macro- dora from the USA and Italy. The ratio between intra- and inter-species molecular variation for the D2—D3 expansion domains of 28S rRNA and the ITS region of all four Iberi- an Peninsula species was very low (0.01—0.08). In contrast, COI variation was higher in_X. macrodora (0.33), followed by X. iberica (0.18). X. paraiberica (0.18), X. andreae sp. nov. (0.15), X. pradense (0.09), and_X. costaricense (0.03), confirming that COI is more diversified in the USA than in the Iberian Peninsula and Costa Rica populations (Table 2). However, for all five species, the D2-D3 expansion do- mains of 28S rRNA and ITS genes undoubtedly displayed intra- and inter-species molecular variation (Table 2), sig- nifying that the likelihood of species separation with these loci was high (Ross et al. 2008). Similarly, the P ID (liberal) values for all six species and loci were > 0.93 (the probabil- ity for P ID (liberal) to be considered adequately delimited in the species delimitation is P > 0.93), signifying that the six Xenocriconemella species can be adequately separated (Ross et al. 2008; Hamilton et al. 2014). The P ID (liberal) value (Ross et al. 2008) reveals the likelihood that a precise species identification would be completed using BLAST, the closest genetic distance, or placement on a tree. Species with a P ID (liberal) > 0.93 were considered to be ade- quately delimited (Hamilton et al. 2014). Additionally, all clades were well-supported (PP = 1.00) in the phylogenetic trees for the three loci, and Rosenberg’s PAB values also supported the monophyly (Rosenberg’s significant values = P <0.05) of the six species distinctly (Rosenberg 2007). Ribosomal and mitochondrial DNA characterization Xenocriconemella andreae sp. nov. was molecularly characterized by the sequences of three ribosomal genes, D2-D3 expansion domains of 28S rRNA, ITS rRNA, Zoosyst. Evol. 100 (3) 2024, 1175-1190 X. costaricense |@|x. andreae sp.nov. mx. Dim2 (21.9%) Dim3 (10.8%) i=) -2 2.5 0.0 2.5 Dim2 (21.9%) iberica , Macrodora | Dim3 (10.8%) 1179 X, paraiberica bq] X, pradense 2 Dim1 (38.5%) ‘ a ~? SAN AN ) ~) 0.72 ls. a ae N b Tal 0.58 3 c|)h6£ a V 6 J 0.43 © Stylet I < RE 3 2 Rst _ uae d Roes _ 5 ~ Rex a : Rv _ 0.14 3 O06 25 52 75 Contributions (%) Figure 1. Principal component of the Xenocriconemella macrodora species complex. Projections of species on the plane of dimensions 1 and 2, 1 and 3, and 2 and 3. Correlation plot between dimensions and qualities of representation of the morphometric characters (“square cosine” (cos2)). Barplot showing the standardized contribution (%) of mor- phometric variables for the three dimensions retained by the PCA (only dimensions with sum of squares (SS) loadings > 1 were extracted). A reference soil (red) line is also shown on the barplot. This reference line corresponds to the expected value if the contribution were uniform. For a given dimension, any row or column with a contribution above the reference line could be considered important in contributing to the dimension. and partial 18S rRNA, and the mitochondrial gene COI. The amplification of these regions yielded single frag- ments of approximately 800, 800, 1600, and 400 bp, respectively, based on gel electrophoresis. Ten D2-D3 expansion domains of 28S rRNA sequences from 676 to 714 bp (PP833567—PP833576), four ITS rRNA se- quences from 677 to 830 bp (PP833563—PP833566), six 18S rRNA sequences from 1681 to 1708 bp (PP833577- PP833582), and six COI sequences from 368 to 385 bp (PP831172—PP831177) were generated for this new spe- cies. Intraspecific sequence variations in ribosomal and mitochondrial markers were low in D2-D3 expansion do- mains of 28S rRNA (99.5—100.0%, 1-3 bp and 0 indel), in the ITS region (99. 1—100.0%, 0-6 bp and 0-3 indels), zse.pensoft.net 1180 Cantalapiedra-Navarrete, C. et al.: Xenocriconemella andreae sp. nov. Table 2. Parameters evaluating Xenocriconemella macrodora species-complex delimitation based on two rRNA genes (D2-D3 ex- pansion segments of the 28S rRNA, ITS) and one mtDNA barcoding locus, COI, for six Xenocriconemella species of the complex. Species Gene Intra/Inter 2 P ID (Liberal) » Clade Support ° Rosenberg’s P,,, 4 Xenocriconemella macrodora D2-D3 - - - ITS - - - - COl 0.33 0.97 (0.94, 0.99) © 1.00 1.2 x 10-3! Xenocriconemella andreae sp. nov. D2-D3 0.08 0.98 (0.93, 1.0) 1.00 5.6 x 10” ITS 0.04 0.97 (0.86, 1.0) 1.00 1.1 x 10-3 COl 0.15 0.96 (0.86, 1.0) 1.00 1.1 x 10° Xenocriconemella iberica D2-D3 0.04 0.98 (0.87, 1.0) 1.00 9.7x 105 ITS 0.02 0.98 (0.87, 1.0) 1.00 4.1 x 10-3 COl 0.18 0.98 (0.95, 1.0) 1.00 1.1 x 10° Xenocriconemella paraiberica D2-D3 0.05 0.97 (0.87, 1.0) 1.00 1.1 x 10° ITS 0.03 0.97 (0.86, 1.0) 1.00 1.1 x 103 Col 0.18 0.98 (0.95, 1.0) 1.00 4.9 x 107 Xenocriconemella pradense D2-D3 0.03 0.98 (0.87, 1.0) 1.00 0.04 ITS 0.01 0.98 (0.87, 1.0) 1.00 4.1 x 10-3 Col 0.09 0.98 (0.93, 1.0) 1.00 0.00 Xenocriconemella costaricense D2-D3 0.04 0.97 (0.86, 1.0) 1.00 1.1 x 10° ITS 0.02 0.97 (0.82, 1.0) 1.00 4.9 x 10-3 COl 0.03 0.97 (0.86, 1.0) 1.00 4.9 x 107 * Intra-species variation relative to inter-species variation. > The P ID (liberal) value represents the probability (with the 95% confidence interval) for making a correct identification of an unknown specimen of the focal species using DNA barcoding (closest genetic distance). P ID (liberal) values > 0.93 were considered to be significantly delimited (Hamilton et al. 2014). Numbers in bold represent significant values. ‘ Clade support: posterior probabilities from Bayesian trees. ‘ Rosenberg’s P,,, value is the probability that the monophyly of a group of sequences is the result of random branching; Rosenberg’s significant values = P < 0.05. ° Significant results are indicated in bold. (-) Not obtained or performed because only a single sequence of D2-D3 or ITS for this species is available in NCBI. in 18S rRNA (99.8—100.0%, 0-3 bp and 0 indel), and in COI (97.8—100.0%, 0-8 bp and 0-1 indel). D2-D3 ex- pansion domains of 28S rRNA of X. andreae sp. nov. (PP833567—PP833576) were 95.0—94.6% similar (differ- ing by 33-41 bp, 24 indels) to X. paraiberica from Spain (OR880152—OR880200), 93.3-93.5% similar (differing by 33-44 bp, 0 indels) to X. costaricense from Costa Rica (PP209388—PP209391 ), 92.3—92.7% similar (differing by 49-50 bp, 3 indels) to X. iberica from Spain and Portugal (OR880112—OR880149), 91.0-91.3% similar (differing by 58 bp, | indel) to_X. pradense from Spain (OR880203- OR880217), and 91.4-91.2% similar (differing by 47- A8 bp, 1 indel) to X. macrodora from Italy (AY780960). ITS of X. andreae sp. nov. (PP833563—PP833566) was 82.7% similar (differing by 138 bp, 51 indels) to _X. co- staricense from Costa Rica (PP209397, PP209398), 80.9— 81.1% similar (differing by 165-167 bp, 102—103 indels) to X. paraiberica from Spain (OR878338—OR878349), 80.8% similar (differing by 161 bp, 49 indels) to X. pra- dense (OR878350), 79.5% similar (differing by 173-174 bp, 76—77 indels) to X. iberica (OR878332—OR878336), and 78.2—78.6% similar (differing by 64—65 bp, 33-34 in- dels) to _X. macrodora from USA (JQ708139), but with a low coverage (50-59%). Partial 18S rRNA of _X. andreae sp. nov. (PP833577—PP833582) was 98.8% similar (dif- fering by 20 bp, 5 indels) to_X. paraiberica (OR878358), 98.6-98.7% similar (differing by 23—24 bp, 3-4 indels) to X. macrodora (MF094906, MF094973, MF095001), 98.2—98.5% similar (differing by 25-30 bp, 3 indels) to X. costaricense (PP209396), 97.9-98.1% similar (differ- ing by 32-35 bp, 5 indels) to X. pradense (OR878360— OR878361), and 97.8% similar (differing by 37 bp, 5 in- dels) to. X. iberica (OR878356). Finally, COI of X. andreae sp. nov. (PP831172—PP831177) was 92.5-93.4% similar (differing by 23—27 bp, 0-1 indel) to XY. iberica from Spain and Portugal (OR885936—OR885976), 90.3—92.0% sim- zse.pensoft.net ilar (differing by 28-36 bp, 0-1 indel) to X. paraiberi- ca from Spain (OR885983—OR886017), 89.1% similar (differing by 41-36 bp, 1 indel) to_X. costaricense from Costa Rica (PP210897—PP210900), 89.0-91.7% similar (differing by 37-46 bp, 1 indel) to X. macrodora from USA (MF770894—MF770950, MN711386—MN711444), and 89.8—90.2% similar (differing by 31—32 bp, | indel) to _X. pradense from Spain (OR886020—OR886029). Phylogenetic analysis Phylogenetic analysis among Xenocriconemella_ spe- cies, based on the D2—D3 expansion domains of 28S, ITS, the partial 18S rRNA, and the partial COI mtDNA gene sequences, was carried out using BI (Figs 2, 3, 4, 5, respectively). The phylogenetic trees created with the ribosomal and mitochondrial DNA markers includ- ed 78, 61, 94, and 171 sequences, and their alignment had 702, 728, 1694, and 360 characters, respectively. The Bayesian 50% majority rule consensus tree inferred from the D2-D3 expansion domains of the 28S rRNA alignment is given in Fig. 2. For this ribosomal mark- er, all six species belonging to the genus Xenocricone- mella clustered together in a well-supported clade (PP = 1.00), clearly separated from all other genera within Criconematidae (Fig. 2). The Xenocriconemella clade was subdivided into four subclades; one of them (PP = 1.00) comprises all the sequences for XY. andreae sp. nov. (PP833567—PP833576), followed by another one (PP = 0.96) including X. paraiberica (OR880152—OR880202) and X. costaricense (PP209388—PP209391), the third one (PP = 0.99) comprises X. pradense (OR880203- OR880218) and the single sequence for X. macrodora from Italy (AY780960), and the fourth subclades include X. iberica (OR880107—OR880151). Zoosyst. Evol. 100 (3) 2024, 1175-1190 28S Criconematidae spp. 1.00 0.90 1.00 1.00 1.00 1181 OR880152 Xenocriconemella paraiberica 0.89 OR880153 Xenocriconemella paraiberica “"HOR880154 Xenocriconemella paraiberica 1.00 |fOR880157 Xenocriconemella paraiberica OR880159 Xenocriconemella paraiberica OR880202 Xenocriconemella paraiberica PP209388 Xenocriconemella costaricense 1.00 -PP209389 Xenocriconemella costaricense PP209390 Xenocriconemella costaricense PP209391 Xenocriconemella costaricense OR880203 Xenocriconemella pradense OR880204 Xenocriconemella pradense 1.00 }OR880206 Xenocriconemella pradense OR880208 Xenocriconemella pradense OR880211 Xenocriconemella pradense OR880209 Xenocriconemella pradense AY780960 Xenocriconemella macrodora Italy 0.99;0R880116 Xenocriconemella iberica OR880148 Xenocriconemelia iberica 4.09 {OR880107 Xenocriconemella iberica OR880109 Xenocriconemella iberica OR880111 Xenocriconemella iberica OR880112 Xenocriconemella iberica 0.99 1.00 MK253536 Discocriconemella hengsungica ON877368 Criconema pseudoannuliferum MN783701 Criconema annuliferum MH828126 Criconema demani MW938539 Ogma decalineatus 1.00;ON705078 Criconema plesioannuliferum AY780952 Criconema sp. Vovias IPP ON705071 Criconema paraannuliferum MF683234 Criconema silvum 1.00 MG029572 Hemicriconemoides parataiwanensis MG029571 Hemicriconemoides paracamelliae MW291449 Hemicriconemoides chitwoodi KP192481 Hemicriconemoides strictathecatus MH444624 Hemicriconemoides fujianensis KF856514 Hemicriconemoides ortonwilliamsi MN720099 Hemicriconemoides brachyurus MW938526 Hemicriconemoides rosae KF856529 Hemicriconemoides pseudobrachyurus 0.999MZ262320 Criconema mutabile 1.00} "FN433864 Criconema sp. Livingston ‘AY780953 Criconema sp. Crozzoli IZA 1.00-AY780954 Criconema mutabile 1.00 1.00 4.00 1.00 1.00 1.00 1.00 M2Z265065 Paratylenchus parastraeleni MN088372 Paratylenchus bukowinensis M2Z265080 Paratylenchus enigmaticus 0.05 1.00 1.00 0.70 1.00 1.00 MZ220549 Mesocriconema onoense MW938536 Mesocriconema ornatum MN888460 Mesocriconema antipolitanum MN720094 Mesocriconema curvatum -MN720087 Mesocriconema nebraskense FN433856 Mesocriconema xenoplax OR336053 Mesocriconema basili MN738724 Criconemoides parainformis MN738726 Criconemoides geraerti MK253537 Discocriconemella sinensis MN888465 Criconemoides informis MN628431 Criconemoides amorphus MW938513 Ogma hechuanensis MW938523 Nothocriconemoides hangzhouensis 1.00 rpOR880219 Criconemella rosmarini OR880220 Criconemella rosmarini MZ262311 Discocriconemella limitanea JQ231186 Criconemoides obtusicaudatus MN888467 Criconemoides parvus MH444643 Criconemoides myungsugae Figure 2. Phylogenetic relationships of Xenocriconemella andreae sp. nov. with Criconematidae spp. Bayesian 50% majority rule consensus tree as inferred from D2 and D3 expansion domains of 28S rRNA sequence alignment under the TIM3 + I+ G model (—InL = 8665.6142; AIC = 17655.228420; freqA = 0.1888; freqC = 0.2391; freqG = 0.3304; freqT = 0.2418; R(a) = 0.4795; R(b) = 1.6113; R(c) = 1.0000; R(d) = 0.4795; R(e) = 4.0922; R(f) = 1.0000; Pin- va = 0.4170; and Shape = 0.9390). Posterior probabilities greater than 0.70 are given for appropriate classes. The newly obtained sequences in this study are shown in bold, and colored boxes indicate the clade association of the new species. Scale bar: expected changes per site. In the ITS region tree (Fig. 3), phylogenetic rela- tionships showed all Xenocriconemella species, except for X. macrodora from the USA (JQ708139), within a well-supported clade (PP = 1.00), undoubtedly separated from all other genera of Criconematidae. This clade was subdivided into two subclades, one of them (PP = 1.00), comprising all the sequences for X. andreae sp. nov. (PP833563—PP833566), X. paraiberica (OR878338- zse.pensoft.net 1182 PP833563 Xenocriconemella andreae sp. nov. 1.00)PP833565 Xenocriconemella andreae sp. nov. PP833564 Xenocriconemella andreae sp. nov. __ §PP833566 Xenocriconemella andreae sp. nov. OR878338 Xenocriconemelia paraiberica ITS Criconematidae spp. 1.00 Cantalapiedra-Navarrete, C. et al.: Xenocriconemella andreae sp. nov. 1.00] OR878342 Xenocriconemelia paraiberica OR878344 Xenocriconemella paraiberica OR878349 Xenocriconemella paraiberica 1.00 ,PP209397 Xenocriconemella costaricense 1.00 PP209398 Xenocriconemella costaricense OR878350 Xenocriconemella pradense 1.00 ]OR878352 Xenocriconemella pradense OR878354 Xenocriconemella pradense 0.78 1.00 OR878355 Xenocriconemella pradense OR878335 Xenocriconemella iberica 1.00J]OR878337 Xenocriconemella iberica 0.79 OR878333 Xenocriconemella iberica OR878332 Xenocriconemella iberica MK253544 Discocriconemella hengsungica 1.00 JQ708132 Criconema mutabile FN435300 Criconema sp. Livingston4 1.00 JQ708139 Xenocriconemella macrodora USA 1.00 jON705082 Criconema paraannuliferum 1.00 ON705083 Criconema paraannuliferum 1.00 0.74 ON705113 Criconema plesioannuliferum 1.00'ON705112 Criconema plesioannuliferum 0.99 1.00 ;ON877377 Criconema pseudoannuliferum y ON877376 Criconema pseudoannuliferum. 0.94 0.907 or 08133 Criconema sphagni JQ708135 Criconema sphagni MF683235 Ogma decalineatus 1.00 jMF683236 Criconema silvum MF683237 Criconema silvum 0.99 1.00 pa KF856546 Hemicriconemoides minutus 1.00°7 KF856547 Hemicriconemoides wessoni . KF856553 Hemicriconemoides brachyurus KF8&56556 Hemicriconemoides macrodorus 0.92 EF126179 Hemicriconemoides kanayaensis 1.00 JQ708131 Criconema arkaense JQ708130 Criconema arkaense JQ708129 Criconema arkaense 0.96 JQ708128 Criconema arkaense JQ708136 Criconema petasum 1.00 MF094994 Lobocriconema thornei 1.00 0.95 1.00 0.85 1.00 MF095014 Lobocriconema incrassatum MF683239 Neobakernema variabile FN433849 Mesocriconema xenoplax KY574863 Mesocriconema nebraskense 0.98 -OM904066 Discocriconemella limitanea 1.00 ;MZ361700 Mesocriconema onoense KC937032 Criconemoides brevistylus 4.00 JQ231189 Criconemoides obtusicaudatus : 1.007-—-MN738718 Criconemoides geraerti 1.00 0.97 MZ820665 Discocriconemella parasinensis 4.00 MN738717 Criconemoides parainformis - MZ015576 Discocriconemella sinensis MN876030 Nothocriconemoides hangzhouensis 1.00 MN738720 Criconemoides rotundicaudatus OM904057 Criconemoides myungsugae M2Z265015 Paratylenchus baldaccil MW798336 Paratylenchus baldaccii 0.2 Figure 3. Phylogenetic relationships of Xenocriconemella andreae sp. nov. with Criconematidae spp. Bayesian 50% majority rule consensus tree as inferred from ITS rRNA sequence alignment under the GTR + I + G model (-InL = 11773.3526; AIC = 23086.70524; freqA = 0.2179; freqC = 0.2568; freqG = 0.2584; freqT = 0.2669; R(a) = 1.3037; R(b) = 2.5717; R(c) = 1.7862; R(d) = 06486; R(e) = 2.8826; R(f) = 1.0000; Pinva = 0.1410; and Shape = 0.8680). Posterior probabilities greater than 0.70 are given for appropriate classes. The newly obtained sequences in this study are shown in bold, and colored boxes indicate the clade association of the new species. Scale bar: expected changes per site. OR878349), and_X. costaricense (PP209397—PP209398), and the second one (PP = 1.00), comprising sequences from_X. pradense and_X. iberica. Xenocriconemella mac- rodora from the USA (JQ708139) clustered with a low support (PP = 0.79) from the rest of Xenocriconemella spp. (and also with Discocriconemella hengsungica, MK253544), establishing a well-supported subclade with Criconema mutabile (JQ708132) and Criconema sp. 4 Livingston (FN435300). In 18S rRNA phylogeny (Fig. 4), all the sequenc- es included in the genus Xenocriconemella clustered in a well-supported clade (PP = 1.00), situated at the top of the tree (Fig. 4). This clade was subdivided into six zse.pensoft.net subclades, each one separating a different Xenocricone- mella species, including X. costaricense (PP209392- PP209396), X. macrodora (MF095001, MF094973, MF094906, and JF972482), X. iberica (OR878356— OR878357), X. pradense (OR878360—OR878361), X. paraiberica (OR878358—OR878359), X. andreae sp. nov. (PP833577—PP833582), and X. macrodora from Portugal (MT229843) (Fig. 4). Lastly, using COI gene sequences, the phylogenetic po- sition of X. andreae sp. nov. (PP831172—PP831177) and all other Xenocriconemella species was shown in Fig. 5. The phylogenetic position of X. andreae sp. nov. was well separated from other species of the genus (PP = 1.00), Zoosyst. Evol. 100 (3) 2024, 1175-1190 18S Criconematidae spp. 1.00 1 0.95 1.00 1.00 0.99 0.7371 0.94 1.00 1.00 rPP209392 Xenocriconemella costaricense 0.92 PP209396 Xenocriconemella costaricense 1.00 | FPP209393 Xenocriconemella costaricense PP209394 Xenocriconemella costaricense 0.99 PP209395 Xenocriconemella costaricense -r MF095001 Xenocriconemella macrodora USA 0.95 J*ME094973 Xenocriconemella macrodora USA ; MF094906 Xenocriconemella macrodora USA ae -"JF972482 Xenocriconemella macrodora USA OR878356 Xenocriconemella iberica 1.00 OR878357 Xenocriconemella iberica 0.97 OR878360 Xenocriconemella pradense ; 1.00*OR878361 Xenocriconemella pradense 0.97 OR878358 Xenocriconemella paraiberica OR878359 Xenocriconemella paraiberica PP833577 Xenocriconemella andreae sp. nov. PP833581 Xenocriconemella andreae sp. nov. PP833579 Xenocriconemella andreae sp. nov. PP Xenocriconemella andreae sp. nov. PP833578 Xenocriconemella andreae sp. nov. ____- *PP833582 Xenocriconemella andreae sp. nov. =M1229843 Xenocriconemella macrodora Portuga AJ966480 Criconema sp. PDL 2005 | ME094935 Criconema sphagni ME094936 Criconema sphagni MF094941 Criconema sphagni MF094942 Criconema sphagni 1.00 FMF094982 Criconema sphagni ME094968 Criconema sphagni ME094970 Criconema sphagni JE972462 Criconema sphagni JE972463 Criconema sphagni JF972464 Criconema ee 1 oc? 72 MF094910 Criconema sp. N1247 MF094919 Criconema sp. N1399 FJ489519 Criconema permistum MF094960 Crossonema fimbriatum MF094934 Crossonema menzeli MF094933 Ogma seymouri EU669918 Ogma cobbi ON877385 Criconema pseudoannuliferum 1.00 &FON877384 Criconema pseudoannuliferum ON877383 Criconema pseudoannuliferum ME094927 Criconema petasum 1.00 RFMFE094958 Criconema petasum - MFQ94959 Criconema petasum : 1.00TQN705042 Criconema paraannuliferum —=+ON705040 Criconema paraannuliferum ON705041 Criconema paraannuliferum ON705048 Criconema plesioannuliferum ON705049 Criconema plesioannuliferum ON705050 Criconema plesioannuliferum 0.941. fKX344495 Criconema longulum KX344496 Criconema acriculum MF094954 oon octangularis MF094952 Ogma decalineatus KX344497 Criconema loofi : MF 90 Discocriconemella hengsungica M116022 Criconema crotaloides AY 284622 Hemicriconemoides pseudobrachyurus KJ934166 Hemicriconemoides wessoni 7 MG029559 Hemicriconemoides kanayaensis 0.83¢MF094914 Criconema mutabile 1.00 | *MF094998 Criconema mutabile 1.00 FMF094925 Criconema sp. N2460 MF094931 Criconema sp. N2686 4.00 FME094899 Criconema permistum : MF094900 Criconema permistum 0.97 *MF094920 Criconema permistum MH444636 Hemicriconemoides parasinensis 1.00 0.99 0.95 0.82-— H 0.97, 00 MG029554 Hemicriconemoides paracamelliae MH444615 Hemicriconemoides chitwoodi ~-h=MG029556 Hemicriconemoides parataiwanensis MH444626 Hemicriconemoides fujianensis MZ041014 Criconemoides myungsugae MF095024 Criconemoides annulatus | MN738716 Criconemoides rotundicaudatus MF094921 Mesocriconema sphaerocephalum 1.00 MF094896 Mesocriconema xenoplax AY284625 Mesocriconema xenoplax MF094891 Mesocriconema curvatum MF094965 Mesocriconema rusticum 1.00 MF 93 Mesocriconema ornatum —=IVIF094909 Mesocriconema onoense MF795592 Discocriconemella limitanea MK546401 Lobocriconema iranense _ MF094994 Lobocriconema thornei MN738713 Criconemoides geraerti MF094902 Criconemoides informis | MK253543 Discocriconemella sinensis - MG701279 Hemicycliophora subbotini MF094908 Bakernema inaequale 0.97 1.00 1.00 1.00 ¢ KJ636364 Tylenchocriconema alleni KF668498 Paratylenchus shenzhenensis 0.02 1183 Figure 4. Phylogenetic relationships of Xenocriconemella andreae sp. nov. with Criconematidae spp. Bayesian 50% majority rule consensus tree as inferred from 18S rRNA sequence alignment under the GTR + I+ G model (— InL = 7859.2560; AIC = 16114.51198; freqA = 0.2451; freqC = 0.2388; freqG = 0.2784; freqT = 0.2378; R(a) = 1.4017; R(b) = 2.0249; R(c) = 1.0058; R(d) = 0.6630; R(e) = 5.7196; R(f) = 1.0000; Pinva = 0.6610; and Shape = 0.5740). Pos- terior probabilities greater than 0.70 are given for appropriate classes. The newly obtained sequences in this study are shown in bold, and colored boxes indicate the clade association of the new species. Scale bar: expected changes per site. zse.pensoft.net 1184 Cantalapiedra-Navarrete, C. et al.: Xenocriconemella andreae sp. nov. MN711400 Xenocriconemella macrodora N1210 1.00 FMN711443 Xenocriconemella macrodora P201089 MN711442 Xenocriconemella macrodora P201088 MN711399 Xenocriconemella macrodora N1209 Col MN711444 Xenocriconemella macrodora P201090 MN711425 Xenocriconemella macrodora N3474 5 = Het yaee senearooneme recor eeret 4 ‘enocriconemella macrodora Xenocriconemella spp. MF770950 Xenacriconemella macrodora N3483 - o.94f77MN711410 Xenocriconemella macrodora N3132 aos MN711411 Xenocriconemella macrodora N3142 : ME770941 Xenocriconemella macrodora N3141 MN711406 Xenocriconemelia macrodora N3105 MN711408 Xenocriconemella macrodora N3116 MN711419 Xenocriconemella macrodora N342T MN711390 Xenocriconemella macrodora N274 MN711395 Xenocriconemella macrodora N971 MN711396 Xenocriconemella macrodora N972 MN711424 Xenocriconemella macrodora N3444 MN?711423 Xenocriconemella macrodora N3443 MN?11422 Xenocriconemella macrodora N3442 MN711421 Xenocriconemella macrodora N3441 MN711394 Xenocriconemella macrodora N937 MN711441 Xenocriconemella macrodora N9434 MN711405 Xenocriconemelia macrodora N3018 MN711418 Xenocriconemelia macrodora N3386 MN711427 Xenocriconemella macrodora N3479 MN711426 Xenocriconemella macrodora N3478 MN711417 Xenocriconemella macrodora N3380 MIN711404 Xenocriconemella macrodora N2871 MIN711403 Xenocriconemella macrodora N2854 MN711402 Xenocriconemelia macrodora N2843 MN711401 Xenocriconemella macrodora N2837 MN711420 Xenocriconemella macrodora N3440 MN711409 Xenocriconemella macrodora N3120 MN711386 Xenocriconemella macrodora N14. MN711391 Xenocriconemella macrodora N602 MN?711392 Xenocriconemelia macrodora N604 = MN711398 Xenocriconemella macrodora N1128 MN711437 Xenocriconemella macrodora N8226 MN711414 Xenocriconemella macrodora N3228 MN711412 Xenocriconemella macrodora N3184 MN7?11434 Xenocriconemella macrodora N3653 MN711433 Xenocriconemella macrodora N3649 MN711432 Xenocriconemella macrodora N3644 MN711407 Xenocriconemella macrodora N3115. 4,00 [~MN711393 Xenocriconemella macrodora N772 = MN711415 Xenocriconemella macrodora N3252 0.97 MN711430 Xenocriconemella macrodora N3497 0.917 MN7 11387 Xenocriconemella macrodora N256 MN711388 Xenocriconemella macrodora N259 MN711416 Xenocriconemella macrodora N3338 MN711440 Xenocriconemella macrodora N9348 =f MN711439 Xenocriconemella macrodora N8417 MN711438 Xenocriconemella macrodora N8402 0.98 MN711431 Xenocriconemella macrodora N3582 1.00 -—MN711413 Xenocriconemella macrodora N3217 | MN711435 Xenocriconemella macrodora NS605 MN711436 Xenocriconemella macrodora N5853 MN711389 Xenocriconemella macrodora N270 MF770894 Xenocriconemella macrodora N1212 MF770895 Xenocriconemella macrodora N1213 MN711397 Xenocriconemella macrodora N1111 OR886015 Xenocriconemella paraiberica ‘OR886018 Xenocriconemella paraiberica 0.88 OR886007 Xenocriconemealia paraiberica —F+-OR886008 Xenocriconemelia paraiberica OR886006 Xenocriconemella paraiberica OR886010 Xenocriconemella paraiberica OR886009 Xenocriconemella paraiberica 0.99 FOR886014 Xenocnconemella paraiberica OR886013 Xenocriconemella paraiberica ‘OR886012 Xenocriconemella paraiberica ‘OR886011 Xenocriconemella paraiberica 0.99," OR885996 Xenocriconemella paraiberica OR885995 Xenocriconemella paraiberica OR885984 Xenocriconemella paraiberica = OR885983 Xenocriconemella paraiberica OR885985 Xenocriconemelia paraiberica ‘OR885986 Xenacriconemella paraiberica OR885987 Xenocriconemella paraiberica 0.97; OR885993 Xenocriconemella paraiberica OR885992 Xenocriconemella paraiberica 0.85 FOR885988 Xenocriconemella paraiberica OR885989 Xenocriconemella paraiberica OR885990 Xenocriconemella paraiberica OR885991 Xenocriconemella paraiberica OR886005 Xenocnconemella paraiberica OR886000 Xenocriconemeila paraiberica OR885998 Xenocriconemella paraiberica OR885997 Xenocriconemella paraiberica OR885999 Xenocriconemella paraiberica 1 of OR886002 Xenocriconemelia paraiberica = OR886001 Xenocriconemelia paraiberica ‘OR886003 Xenocriconemella paraiberica OR886004 Xenocriconemelia paraiberica OR885994 Xenocriconemella paraiberica PP210897 Xenocriconemella costaricense 1.00_}-PP210898 Xenocriconemella costaricense PP210899 Xenocriconemella costaricense PP210900 Xenocriconemella costaricense 0,93. OR885966 Xenocriconemella iberica TT OR885968 Xenocriconemella iberica OR885967 Xenocriconemella iberica 'OR885965 Xenocriconemella iberica OR885971 Xenocriconemella iberica ¥ OR885970 Xenocriconemella iberica O.8t LOR885964 Xenocriconemella iberica 0.97 OR885977 Xenocriconemella iberica OR885975 Xenocriconemella iberica ‘OR885976 Xenocriconemella iberica OR885957 Xenocriconemella iberica ‘OR885956 Xenocriconemella iberica OR885960 Xenocriconemella iberica 'OR885961 Xenocriconemella iberica OR885962 Xenocriconemella iberi OR885963 Xenocriconemella iberi OR885953 Xenocriconemella iberica ‘OR885959 Xenocriconemella iberica o.g9f7 OR885954 XXenocriconemelia iberica —r OR885958 Xenocriconemelia iberica OR885955 Xenocriconemelia iberica 00 OR885973 Xenocriconemella iberica OR885980 Xenocriconemella iberica ‘OR885933 Xenocriconemella iberica 'OR885937 Xenocriconemella iberica 4.00 OR885934 Xenocriconemella iberica ‘OR885936 Xenocriconemella iberica ‘OR885935 Xenocriconemella iberica 'OR885938 Xenocriconemelia iberica OR885939 Xenocriconemella iberica OR885940 Xenocriconemella iberica OR885941 Xenocriconemelia iberica 'OR885969 Xenocriconemella iberica ‘OR885972 Xenocriconemella iberica OR885974 Xenocriconemelila iberica ‘OR885979 Xenocriconemelia iberica 'OR885978 Xenocriconemella iberica 'OR885981 Xenocriconemella iberica OR885943 Xenocriconemella iberica OR885946 Xenocriconemelia iberica OR885942 Xenocriconemella iberica OR885944 Xenocriconemelia iberica ‘OR885982 Xenocriconemelia iberica OR885945 Xenocriconemella iberica OR885947 Xenocriconemella iberica 1.00 ‘OR885948 Xenocriconemella iberica - ‘OR885949 Xenocriconemella iberica OR885950 Xenocriconemella iberica OR885951 Xenocriconemella iberica OR885952 Xenocriconemelia iberica OR886029 Xenocriconemelia pradense OR886028 Xenocriconemelia pradense OR886027 Xenocriconemelia pradense OR886026 Xenocriconemelia pradense OR886025 Xenocriconemelia pradense ‘OR886024 Xenocriconemella pradense ‘OR886023 Xenocriconemella pradense OR886022 Xenocriconemella pradense 0.79/-OR886021 Xenocriconemella pradense OR886020 Xenocriconemella pradense 4,00 MZ820008 Discocriconemelia limitanea --IMZ820007 Discocriconemella limitanea 1.00 MW?797005 Paratylenchus indalus MW797016 Paratylenchus hamatus - — 2262220 Paratylenchus baldaccii 0.02 Figure 5. Phylogenetic relationships of Xenocriconemella andreae sp. nov. with other Xenocriconemella spp. Bayes- ian 50% majority-rule consensus trees as inferred from cytochrome c oxidase subunit I (COI) mtDNA gene sequence alignments under the TPM3uf + I+ G model (—InL = 2464.7614; AIC = 5639.5228; freqA = 0.3727; freqC = 0.0511; freqG = 0.0810; freql = 0.4953; R(a) = 1.7003; R(b) = 9.5660; R(c) = 1.0000; R(d) = 1.7003; R(e) = 9.5660; R(f) = 1.0000; Pinva = 0.3460; and Shape = 0.4040). Posterior probabilities greater than 0.70 are given for appropriate classes. The newly obtained sequences in this study are shown in bold, and colored boxes indicate the clade association of the new species. Scale bar = expected changes per site. zse.pensoft.net Zoosyst. Evol. 100 (3) 2024, 1175-1190 but the phylogenetic relationship with them was not well resolved (Fig. 5). Sequences from X. macrodora from the USA appeared together in a well-supported (PP = 1.00) subclade. Taxonomy Phylum: Nematoda Rudolphi, 1808 Class: Chromadorea Inglis, 1983 Order: Rhabditida Chitwood, 1933 Suborder: Tylenchina Chitwood, 1950 Superfamily: Criconematoidea Khan & Ahmad, 1975 Family: Criconematidae Taylor, 1936 Genus: Xenocriconemella De Grisse & Loof, 1965 Xenocriconemella andreae sp. nov. https://zoobank.org/1 A7BEE7F-3C2A-4E9F-803 1-F5AD3C1300E2 Description. Females. Body ventrally arcuate to straight, slightly narrowing anteriorly and posteriorly. Body annuli smooth and retrorse 2.6 (2.5—3.0) um wide, without anas- tomosis (Fig. 6). Lip region with two annul, not offset, not separated from body contour; first lip annulus par- I ee is ban 7 \ 1185 tially covering the second lip annulus (Fig. 6); second lip annulus retrorse and slightly wider than first annulus 9.1 (8.0-10.0) um wide. Stylet thin, long, and flexible (Figs 6, 7, Table 3), occupying 31 (27.2—-35.0)% of the body length, with short basal portion 7.2 (7.0—8.0) um long and knobs slightly rounded 5.1 (5.0-6.0) um wide. Pharynx typical criconematoid, with a cylindroid procorpus wid- ening to a large muscular oval median bulb containing well-developed valves (8.0—9.5 um long), istmus slender, and amalgamated with basal bulb. Excretory pore located from two to three annuli posterior to level of stylet knobs, at 102 (87.0-107.0) um from anterior end. Nerve ring located at the level of istmus, 116 (103-124) um from the anterior end. Vagina ventrally curved (14.0-17.0 um long). Female genital tract monodelphic, prodelphic, out- stretched, and occupying 43 (34.4—-52.4)% of the body length; spermatheca almost hemispherical (11.0-14.0 x 12.5—18.0) um, sperm absent. Anus located at 7.7 (6-9) annuli from the terminus. Tail short, conoid, and bluntly rounded terminus. Males. Not found. Juveniles. Body similar to females, including tail shape, but shorter. Edge of body annuli without append- ages, marked with delicate irregular punctations. Figure 6. Xenocriconemella andreae sp. nov. (drawings). A. Entire female; B. Female anterior region; C, D. Detail of female posterior region showing vulva and anus. zse.pensoft.net Cantalapiedra-Navarrete, C. et al.: Xenocriconemella andreae sp. nov. Figure 7. Light micrographs of Xenocriconemella andreae sp. nov. A, E. Entire female; C. Entire female showing body annuli without anastomosis; B, G—J. Female anterior body region showing stylet (arrowed); D, F, K—-N. Vulval region showing vulva and anus (arrowed). Abbreviations: a = anus; st = stylet; V = vulva. Scale bars: 50 um (A, C, E); 20 um (B, D, F-N). Diagnosis and relationships. Xenocriconemella an- dreae sp. nov. 1s characterized by the following measure- ments and ratios: a short-sized female body 307 (274-353) um, a long and flexible stylet = 94.6 (88.0-99.0) um long, zse.pensoft.net V = 92 (90.2—-92.5), a = 10.2 (8.4-12.2), b = 2.3 (2.1- 2.6), c = 26.3 (21.9-32.5), c’? = 0.7 (0.6-0.8), R = 113 (105-119), RV = 10.7 (9-12), Ran = 7.7 (6-9), VL/VB = 1.0 (0.8-1.1). Morphologically and morphometrically, Zoosyst. Evol. 100 (3) 2024, 1175-1190 1187 Table 3. Morphometrics of Xenocriconemella andreae sp. nov. from the rhizosphere of mastic tree, cork oak, and chestnut from Linho, Sintra region, Portugal; Aroche, Huelva province, Spain; and Trabadelo, Le6n province, Spain '. Character! Portugal Holotype Paratype Females n 1 20 L 302 307.2 + 21.0 (274-353) R 114 112.5 + 4.1 (105-119) Rst 35 36.0 + 2.4 (31-40) Roes 47 47.7 + 2.6 (42-52) Rex 38 38.5 + 2.6 (33-43) RV 10 10.7 + 0.8 (9-12) Rvan 3 3.0 + 0.0 (3-3) Ran 7 7.7 + 0.7 (6-9) O 0.9 8.2 + 0.4 (7.4-8.9) A 8.9 10.2 + 1.1 (8.4-12.2) B 2.2 2.3 + 0.1 (2.1-2.6) C 22.4 26.3 + 3.4 (21.9-32.5) Cc’ 0.6 0.7 + 0.05 (0.6-0.8) V 91.1 91.5 + 0.7 (90.2-92.5) VL/VB 0.9 1.0 + 0.1 (0.8-1.1) Stylet 95.0 94.6 + 2.9 (88.0-99.0) Pharynx 135 132.5 + 5.1 (122-140) Max. body width 34 30.5 + 3.3 (24.0-37.0) Anal body width 21 17.6 + 1.9 (14.5-21.0) Vulva to anus distance 14 12.7 + 1.8 (10.0-16.0) Tail 13.5 11.9 + 1.5 (10.0-14.0) ‘All measurements are in um and in the form: mean + s.d. (range). X. andreae sp. nov. resembles members of the X. macro- dora-species complex (including X. macrodora, X. iberi- ca, X. paraiberica, X. pradense, and X. costaricense), from which it can be separated by several morphometric traits and ratios. From _X. macrodora, it is almost undis- tinguishable but mainly differs by a slightly higher c ratio 26.3 (21.9-32.5) vs. 19.6 (12.8—25.3). From_X. iberica, it is also almost undistinguishable, but differs by a slightly shorter tail length 11.9 (10.0-14.0) um vs. 16.4 (11.0— 24.5) um and a slightly higher c ratio 26.3 (21.9-32.5) vs. 18.3 (12.1—27.3). From X. paraiberica, it 1s also al- most undistinguishable, but mainly differs by a slightly longer stylet length 94.6 (88.0-99.0) um vs. 89.6 (80.0- 100.0) um, a higher number of body annuli (R) 112.5 (105-119) vs. 104 (95-116), and a slightly higher c ratio 26.3 (21.9-32.5) vs. 20.2 (13.0-28.6). From_X. pradense, it mainly differs by a slightly lower VL/VB ratio 1.0 (0.8— 1.1) vs. 1.4 (1.1-1.5), a lower number of body annuli from vulva to posterior end (RV) 10.7 (9-12) vs. 16 (14— 18), a slightly shorter tail length 11.9 (10.0-14.0) um vs. 20.2 (15.5—25.0) um, a higher c ratio 26.3 (21.9-32.5) vs. 16.6 (13.7—21.3), and a lower c’ ratio 0.7 (0.60.8) vs. 0.9 (0.8—1.2). Finally, X. andreae sp. nov. clearly differs from X. costaricense by a shorter body length 307.2 (274-353) um vs. 349 (276-404) um, a shorter stylet length 94.6 (88.0-99.0) um vs. 125 (113.0-133.0) um, a slightly higher number of body annuli (R) 112.5 (105-119) vs. 124 (117-130), a slightly higher c ratio 26.3 (21.9-32.5) vs. 22.8 (16.0—28.8), and a slightly lower VL/VB ratio 1.0 (0.8—1.1) vs. 1.1 (0.9-1.3). Etymology. The species epithet refers to the name of the daughter of the last author, Miss. Andrea Archidona Ro- sales, who helped to take the sample of the type population. Spain Aroche, Huelva province Trabadelo, Leon province 3 4 331.3 + 24.7 (303-348) 341.3 + 12.9 (323-353) 110.7 + 2.9 (109-114) 114.3 + 2.9 (111-118) 34.7 + 0.6 (34-35) 34.5 + 1.3 (33-36) 45.3 + 1.2 (44-46) 46.0 + 1.4 (45-48) 37.0 + 1.0 (36-38) 36.8 + 1.0 (36-38) 12.3 + 0.6 (12-13) 11.3 + 1.0 (10-12) 3.0 + 0.0 (3-3) 3.0 + 0.0 (3-3) 9.3 + 0.6 (9-10) 8.3 + 1.0 (7-9) 7.6 + 0.5 (7.4-8.2) 7.5 + 0.5 (7.1-8.2) 11.6 + 0.6 (11.2-12.3) 11.5 + 0.6 (10.7-11.9) 2.5 + 0.1 (2.4-2.5) 2.6 + 0.1 (2.5-2.7) 18.7 + 0.8 (17.8-19.3) 20.2 + 1.5 (18.7-22.3) 0.8 + 0.03 (0.8-0.9) 0.8 + 0.04 (0.7-0.9) 90.8 + 0.8 (90.2-91.7) 90.9 + 0.7 (90.4-92.0) 1.2 + 0.06 (1.2-1.3) 1.1 + 0.05 (1.1-1.2) 96.0 + 1.7 (95.0-98.0) 96.3 + 1.5 (95.0-98.0) 133.3 + 5.7 (127-138) 133.3 + 6.1 (127-139) 28.7 + 2.1 (27.0-31.0) 29.8 + 2.2 (28.0-33.0) 20.8 + 1.0 (20.0-22.0) 21.1 + 1.7 (19.5-23.0) 13.2 + 1.0 (12.0-14.0) 13.3 + 2.0 (11.5-16.0) 17.7 + 0.6 (17.0-18.0) 17.0 + 1.8 (14.5-18.5) Type host and locality. The new species was recov- ered from the rhizosphere of a mastic tree (Pistacia len- tiscus L.) at Linho, Sintra region, Portugal (coordinates 38°46'07.78"N, 9°23'41.96"W). Additional specimens were detected from the rhizosphere of cork oak (Quercus suber L.) and chestnut (Castanea sativa Mill.) at Aro- che, Huelva province, Spain (coordinates 37°54'13.06"N, 6°37'02.95"W), and Trabadelo, Leon province, Spain (coordinates 42°38'38.3"N, 6°52'14.0"W), respectively. Type material. Holotype female and 16 female para- types deposited at the nematode collection of the institute for sustainable agriculture (IAS) of the Spanish National Research Council (CSIC; collection nos. XEN-AND-01/ XEN-AND-16), Cordoba, Spain; and two females at the USDA Nematode Collection (T-8065p). Discussion Late studies based on integrative taxonomy on profuse X. macrodora-species complex populations from the Ibe- rian Peninsula and a population from Costa Rica clearly demonstrate that the cosmopolitan species X. macrodo- ra need to be considered a species complex including at least five species, viz. X. iberica, X. macrodora, X. para- iberica, X. pradense, and X. costaricense, and probably comprising additional new cryptic species all over the world (Archidona-Yuste et al. 2024; Peraza-Padilla et al. 2024). Because of their basic morphology and a wide morphometric range of populations all over the world (Archidona-Yuste et al. 2024), accurate species identifi- cation within the genus Xenocriconemella has only been possible after applying integrative taxonomical studies, zse.pensoft.net 1188 allowing to decipher the presence of cryptic species (Ar- chidona-Yuste et al. 2024; Peraza-Padilla et al. 2024). The main goal here was to describe and identify morpho- logically and molecularly the three new populations of Xenocriconemella found in natural environments on the Iberian Peninsula, as well as clarify phylogenetic rela- tionships within this genus. Our results corroborate that the three new Xenocriconemella populations studied here are morphologically and morphometrically related to the X. macrodora-species complex, except for some minor morphometric features. Nevertheless, all the molecular markers certainly delineated the three new Iberian Penin- sula populations from all other species within this genus, confirming that they comprise a new valid species within the genus Xenocriconemella. These data provide a clear indication that the global biodiversity of this genus 1s much greater than previously suspected, as has been sug- gested recently by Archidona- Yuste et al. (2024) and Per- aza-Padilla et al. (2024). Certainly, although more studies are required to confirm this assumption on a global scale, the present results indicate that additional new taxa can be detected within the widely reported populations of X. macrodora s.\. in those regions of the Iberian Peninsula where the species complex has been reported (Archido- na-Yuste et al. 2024). Ribosomal and mitochondrial markers (D2-D3 expan- sion domains of the 28S and ITS rRNA and the mtDNA gene COI) are again demonstrated to be important tools for the accurate identification of Xenocriconemella spp. and other Criconematidae (Subbotin et al. 2005; Etongwe et al. 2020; Powers et al. 2021; Nguyen et al. 2022; Archi- dona- Yuste et al. 2024). In our studies on the molecular diversity of Xenocriconemella spp. in the Iberian Penin- sula, ribosomal markers looked like the best molecular tools for identifying Xenocriconemella species since they showed the lowest intraspecific variability. Phylogenetic analyses based on D2-D3, ITS, 18S, and COI genes using BI mostly clearly demonstrated the monophyly of the ge- nus Xenocriconemella, were consistent with those given by previous phylogenetic analyses (Subbotin et al. 2005; Etongwe et al. 2020; Nguyen et al. 2022; Archidona- Yuste et al. 2024; Peraza-Padilla et al. 2024), and confirmed the validity of Xenocriconemella within Criconematidae. Our data also suggest that the 18S rRNA accession of X. mac- rodora from Portugal (MT229843, differing by 12 bp from X. andreae sp. nov.) was most likely misidentified as hypothesized by Archidona- Yuste et al. (2024). Unfor- tunately, no additional molecular data were available in GenBank from this population, and further studies will be needed to clarify this identification, linking morpho- logical and molecular data through integrative taxonomy. Similarly, the high diversity (up to 25%, 101—115 bp and 40-45 indels) among ITS sequences of Xenocriconemel- la spp. from the Iberian Peninsula and Costa Rica with the sequence of X. macrodora from the USA (JQ708139) suggests a misidentification that should be confirmed with additional studies since no other molecular markers of this population are available (Cordero et al. 2012). zse.pensoft.net Cantalapiedra-Navarrete, C. et al.: Xenocriconemella andreae sp. nov. Conclusions This study expands our understanding of the biodiversity of the genus Xenocriconemella in the Iberian Peninsula. It also confirms the effectiveness of using an integrative approach that combines morphometric and morphological character- istics with the genotyping of rRNA and mtDNA markers for accurate species identification among Xenocriconemel- la species. Additionally, the study highlights the need for ongoing nematode surveys in natural habitats to uncover the uncharted biodiversity of this genus globally. Acknowledgements This work was supported by the Consejeria de Univer- sidad, Investigacion e Innovacion-Junta de Andalu- cia, Qualifica Project (QUAL21 023 IAS). A. Archi- dona-Yuste is funded by the Ramon y Cajal program (RY C2021-031108-I), and I. Criado-Navarro is funded by the Juan de la Cierva programs (JDC2022-048855-I), funded by MCIN/AEI/10.13039/501100011033 and UE “Next Generation EU/PRTR.” The authors thank Gra- cia Liébanas and Maria Rodriguez Santamaria for their help with sampling. In addition, the authors thank Jorge Martin Barbarroja (IAS-CSIC), Guillermo Leon-Ropero (I[ASCSIC), and Inmaculada Casero Godoy for their ex- cellent technical assistance. References Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. 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