Continuing the nematode surveys for deciphering the molecular and morphological diversity of Xenocriconemella 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 (Table 1). Samples were taken from the rhizospheric soil of the selected plants and mixed to establish a single sample from each sample point. The soil samples were taken from a depth of 5 to 40 cm. Afterward, nematode specimens were isolated from a 500 cm³ 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 following abbreviations (i.e., measurements and ratios) are used in the morphological descriptions, data analyses, and figures: 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 a percentage of stylet length; R = total number of body annuli; Roes = number of annuli in the pharyngeal region; Rex = number of annuli between the anterior end of the body and the 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 v. 22.5.2 (San Francisco, CA, USA).

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 molecular markers for each Xenocriconemella isolate were obtained 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 specified 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 described 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 Archidona-Yuste et al. 2024 for details). The sequence chromatograms 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 numbers specified on the phylogenetic trees and in Table 1.

We used two independent approaches to species delineation 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 (Legendre and Legendre 2012). We established the species delimitation amongst the new Xenocriconemella populations found in the Iberian Peninsula and other species recently described within this genus, and we further evaluated the relationships between these new isolates and those previously designated within Xenocriconemella. PCA was constructed upon the following morphological 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.l. populations previously characterized and recorded from numerous countries (Archidona-Yuste et al. 2024), as well as 28 Iberian populations previously studied under an integrative taxonomical approach, including 13 belonging to X. iberica, 12 belonging to X. paraiberica, 3 belonging to X. pradense, and 1 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 collinearity 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’ (Lê 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 compute 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 calculated 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 probability of species identification is high (Masters et al. 2011). The P ID (liberal) value (Ross et al. 2008) means the likelihood 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 species clade). Taxa with a P ID (liberal) ≥ 0.93 were considered to be satisfactorily demarcated (Hamilton et al. 2014). Rosenberg’s P_AB means the likelihood that the monophyly of a group of sequences is the result of random branching; significant values were <0.05 (Rosenberg 2007).

Methods and software programs for aligning, sequence edition, 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; Etongwe et al. 2020; Nguyen et al. 2022; Archidona-Yuste et al. 2024). The best-fit models for each marker were: the transitional model with invariable sites and a gamma-shaped distribution (TIM3 + I + G) for the D2-D3 expansion domains of 28S rRNA; the general time-reversible model with invariable sites and a gamma-shaped distribution (GTR + I + G) for ITS and the partial 18S rRNA gene; and the 3-parameter model with invariable sites and a gamma-shaped distribution (TPM3uf + I + G) for the COI gene.

Our PCA results showed a wide intraspecific variation amongst the specimens of Xenocriconemella spp., especially 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 morphological variation displayed by X. macrodora (Archidona-Yuste et al. 2024). PCA clearly distinguished between almost all the individuals of X. andreae sp. nov., X. pradense, and X. costaricense, as well as those identified within X. iberica and X. paraiberica. However, this spatial separation 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, stylet 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 nematode. Except in specific cases, the delimitation of species boundaries provided by the PCA occurs through a linear combination of multiple diagnostic characters (Archidona-Yuste et al. 2016, 2024). Thus, we detected that species separations were mainly based on a combination of the following 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 Dim1 (i.e., X. paraiberica and X. andreae sp. nov.). Likewise, specimens 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 Dim2 (i.e., X. pradense). Ultimately, we could conclude that Roes, Rst, Rex, and Rv were the most useful morphometrics for separating species within this cryptic complex in the genus Xenocriconemella. However, most of the specimens of X. iberica and 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 (i.e., X. iberica, X. paraiberica, and X. pradense) encompass a model complex of cryptic species (i.e., the X. macrodora species complex). However, PCA allowed us to separate the new taxa X. andreae sp. nov. and the already described species (X. costaricense) from this species cryptic complex.

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 morphometric 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.

Species delineation using ribosomal and mitochondrial markers proved that X. andreae sp. nov., X. iberica, X. paraiberica, X. pradense, and X. costaricense were undoubtedly distinguished among them, as were X. macrodora 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 Iberian 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 domains of 28S rRNA and ITS genes undoubtedly displayed intra- and inter-species molecular variation (Table 2), signifying 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 probability 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 adequately 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).

Xenocriconemella andreae sp. nov. was molecularly characterized by the sequences of three ribosomal genes, D2-D3 expansion domains of 28S rRNA, ITS rRNA, and partial 18S rRNA, and the mitochondrial gene COI. The amplification of these regions yielded single fragments 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 sequences 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 species. Intraspecific sequence variations in ribosomal and mitochondrial markers were low in D2-D3 expansion domains 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), 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 expansion domains of 28S rRNA of X. andreae sp. nov. (PP833567–PP833576) were 95.0–94.6% similar (differing by 33–41 bp, 2–4 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, 1 indel) to X. pradense from Spain (OR880203–OR880217), and 91.4–91.2% similar (differing by 47–48 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. costaricense 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. pradense (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 indels) 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 (differing 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 (differing by 32–35 bp, 5 indels) to X. pradense (OR878360–OR878361), and 97.8% similar (differing by 37 bp, 5 indels) 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 X. iberica from Spain and Portugal (OR885936–OR885976), 90.3–92.0% similar (differing by 28–36 bp, 0–1 indel) to X. paraiberica 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, 1 indel) to X. pradense from Spain (OR886020–OR886029).

Phylogenetic analysis among Xenocriconemella species, 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 included 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 marker, all six species belonging to the genus Xenocriconemella 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 X. 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).

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 (−lnL = 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; Pinva = 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 relationships 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–OR878349), and X. costaricense (PP209397–PP209398), and the second one (PP = 1.00), comprising sequences from X. pradense and X. iberica. Xenocriconemella macrodora 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).

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 (−lnL = 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.

In 18S rRNA phylogeny (Fig. 4), all the sequences 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 subclades, each one separating a different Xenocriconemella 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).

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 (−lnL = 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). 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.

Lastly, using COI gene sequences, the phylogenetic position 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), 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.

Figure 5. 

Phylogenetic relationships of Xenocriconemella andreae sp. nov. with other Xenocriconemella spp. Bayesian 50% majority-rule consensus trees as inferred from cytochrome c oxidase subunit I (COI) mtDNA gene sequence alignments under the TPM3uf + I + G model (−lnL = 2464.7614; AIC = 5639.5228; freqA = 0.3727; freqC = 0.0511; freqG = 0.0810; freqT = 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.

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/1A7BEE7F-3C2A-4E9F-8031-F5AD3C1300E2

Description

Females. Body ventrally arcuate to straight, slightly narrowing anteriorly and posteriorly. Body annuli smooth and retrorse 2.6 (2.5–3.0) µm wide, without anastomosis (Fig. 6). Lip region with two annuli, not offset, not separated from body contour; first lip annulus partially covering the second lip annulus (Fig. 6); second lip annulus retrorse and slightly wider than first annulus 9.1 (8.0–10.0) µm 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) µm long and knobs slightly rounded 5.1 (5.0–6.0) µm wide. Pharynx typical criconematoid, with a cylindroid procorpus widening to a large muscular oval median bulb containing well-developed valves (8.0–9.5 µm 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) µm from anterior end. Nerve ring located at the level of istmus, 116 (103–124) µm from the anterior end. Vagina ventrally curved (14.0–17.0 µm long). Female genital tract monodelphic, prodelphic, outstretched, and occupying 43 (34.4–52.4)% of the body length; spermatheca almost hemispherical (11.0–14.0 × 12.5–18.0) µm, sperm absent. Anus located at 7.7 (6–9) annuli from the terminus. Tail short, conoid, and bluntly rounded terminus.

Table 3.

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Morphometrics of Xenocriconemella andreae sp. nov. from the rhizosphere of mastic tree, cork oak, and chestnut from Linhó, Sintra region, Portugal; Aroche, Huelva province, Spain; and Trabadelo, León province, Spain ¹.

Character1	Portugal		Spain
	Holotype	Paratype Females	Aroche, Huelva province	Trabadelo, León province
n	1	20	3	4
L	302	307.2 ± 21.0 (274–353)	331.3 ± 24.7 (303–348)	341.3 ± 12.9 (323–353)
R	114	112.5 ± 4.1 (105–119)	110.7 ± 2.9 (109–114)	114.3 ± 2.9 (111–118)
Rst	35	36.0 ± 2.4 (31–40)	34.7 ± 0.6 (34–35)	34.5 ± 1.3 (33–36)
Roes	47	47.7 ± 2.6 (42–52)	45.3 ± 1.2 (44–46)	46.0 ± 1.4 (45–48)
Rex	38	38.5 ± 2.6 (33–43)	37.0 ± 1.0 (36–38)	36.8 ± 1.0 (36–38)
RV	10	10.7 ± 0.8 (9–12)	12.3 ± 0.6 (12–13)	11.3 ± 1.0 (10–12)
Rvan	3	3.0 ± 0.0 (3–3)	3.0 ± 0.0 (3–3)	3.0 ± 0.0 (3–3)
Ran	7	7.7 ± 0.7 (6–9)	9.3 ± 0.6 (9–10)	8.3 ± 1.0 (7–9)
O	0.9	8.2 ± 0.4 (7.4–8.9)	7.6 ± 0.5 (7.4–8.2)	7.5 ± 0.5 (7.1–8.2)
A	8.9	10.2 ± 1.1 (8.4–12.2)	11.6 ± 0.6 (11.2–12.3)	11.5 ± 0.6 (10.7–11.9)
B	2.2	2.3 ± 0.1 (2.1–2.6)	2.5 ± 0.1 (2.4–2.5)	2.6 ± 0.1 (2.5–2.7)
C	22.4	26.3 ± 3.4 (21.9–32.5)	18.7 ± 0.8 (17.8–19.3)	20.2 ± 1.5 (18.7–22.3)
c’	0.6	0.7 ± 0.05 (0.6–0.8)	0.8 ± 0.03 (0.8–0.9)	0.8 ± 0.04 (0.7–0.9)
V	91.1	91.5 ± 0.7 (90.2–92.5)	90.8 ± 0.8 (90.2–91.7)	90.9 ± 0.7 (90.4–92.0)
VL/VB	0.9	1.0 ± 0.1 (0.8–1.1)	1.2 ± 0.06 (1.2–1.3)	1.1 ± 0.05 (1.1–1.2)
Stylet	95.0	94.6 ± 2.9 (88.0–99.0)	96.0 ± 1.7 (95.0–98.0)	96.3 ± 1.5 (95.0–98.0)
Pharynx	135	132.5 ± 5.1 (122–140)	133.3 ± 5.7 (127–138)	133.3 ± 6.1 (127–139)
Max. body width	34	30.5 ± 3.3 (24.0–37.0)	28.7 ± 2.1 (27.0–31.0)	29.8 ± 2.2 (28.0–33.0)
Anal body width	21	17.6 ± 1.9 (14.5–21.0)	20.8 ± 1.0 (20.0–22.0)	21.1 ± 1.7 (19.5–23.0)
Vulva to anus distance	14	12.7 ± 1.8 (10.0–16.0)	13.2 ± 1.0 (12.0–14.0)	13.3 ± 2.0 (11.5–16.0)
Tail	13.5	11.9 ± 1.5 (10.0–14.0)	17.7 ± 0.6 (17.0–18.0)	17.0 ± 1.8 (14.5–18.5)

¹All measurements are in µm and in the form: mean ± s.d. (range).

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.

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 µm (A, C, E); 20 µm (B, D, F–N).

Males. Not found.

Juveniles. Body similar to females, including tail shape, but shorter. Edge of body annuli without appendages, marked with delicate irregular punctations.

Diagnosis and relationships

Xenocriconemella andreae sp. nov. is characterized by the following measurements and ratios: a short-sized female body 307 (274–353) µm, a long and flexible stylet = 94.6 (88.0–99.0) µm long, 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, X. andreae sp. nov. resembles members of the X. macrodora-species complex (including X. macrodora, X. iberica, 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 undistinguishable 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) µm vs. 16.4 (11.0–24.5) µm and a slightly higher c ratio 26.3 (21.9–32.5) vs. 18.3 (12.1–27.3). From X. paraiberica, it is also almost undistinguishable, but mainly differs by a slightly longer stylet length 94.6 (88.0–99.0) µm vs. 89.6 (80.0–100.0) µm, 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) µm vs. 20.2 (15.5–25.0) µm, a higher c ratio 26.3 (21.9–32.5) vs. 16.6 (13.7–21.3), and a lower c’ ratio 0.7 (0.6–0.8) 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) µm vs. 349 (276–404) µm, a shorter stylet length 94.6 (88.0–99.0) µm vs. 125 (113.0–133.0) µm, 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 Rosales, who helped to take the sample of the type population.

Type host and locality

The new species was recovered from the rhizosphere of a mastic tree (Pistacia lentiscus L.) at Linhó, 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 Aroche, Huelva province, Spain (coordinates 37°54'13.06"N, 6°37'02.95"W), and Trabadelo, León province, Spain (coordinates 42°38'38.3"N, 6°52'14.0"W), respectively.

Type material

Holotype female and 16 female paratypes 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), Córdoba, Spain; and two females at the USDA Nematode Collection (T-8065p).