Paratrichodorus allius

Decraemer, Wilfrida, Archidona-Yuste, Antonio, Clavero-Camacho, Ilenia, Vovlas, Alessio, Cantalapiedra-Navarrete, Carolina, Alba, Ruiz-Cuenca, Castillo, Pablo, Juan & Palomares-Rius, 2024, Unravelling cryptic diversity in the Paratrichodorus allius- group species complex to resolve eight new species of the genus and new insights on the molecular phylogeny % Nematoda: Trichodoridae), Zoological Journal of the Linnean Society 202 (3), pp. 1-44 : 3-5

Species delimitation within Paratrichodorus allius species-complex

Two different approaches of species delimitation were used to determine species boundaries within this P. allius species-complex, including morphometric and molecular data. Only morphologically closely related species were included in the delimitation analyses.

Species delineation using morphometry was conducted with principal component analysis (PCA) to estimate the degree of association among some selected and morphological closely related populations within the P. allius species-complex (Legendre and Legendre 2012). PCA was performed on three populations belonging to two new species ( Paratrichodorus uliaensis sp. nov. and Paratrichodorus benalupensis sp. nov.) using, separately, male and female specimens. Other taxa belonging to the P. allius species-complex were not included in this analysis due to the unavailability of suitable and complete morphometric data (i.e. P. allius ) and morphological non-similarity (i.e. Paratrichodorus paraallius sp. nov.). To do this, PCA was based on the main morphometric features used in the tabular identification keys and based on importance in species diagnosis in literature for males and females ( Loof 1975, Decraemer 1995, Decraemer and Baujard 1998) that included: (i) in both sexes: L (body length) and onchiostyle length; (ii) in females: V [(distance from anterior end to vulva/body length) × 100], the vagina shape (i.e. length vagina as % of width at midbody), vaginal sclerotized pieces length and distance between sclerotized pieces, the distance from anterior end to secretory-excretory pore (EP), and the ratio a (body length/maximum body width); and (iii) in males: the distance from anterior end to ventromedian cervical papilla (CP), and the spicule length. Prior to the statistical analysis, key diagnostic characters were tested for collinearity ( Zuur et al. 2010). We used the collinearity test based on the values of the variance inflation factor (VIF) method that iteratively excludes numeric covariates showing VIF values>10, as suggested by Montgomery et al. (2012). In PCA, we used an orthogonal varimax rotation to estimate the factor loadings where only components with sum of squares (SS) loadings>1 were extracted (Legendre and Legendre 2012). All data analyses were done with the R v.4.2.2 ( R Core Team 2022; https://www.R-project.org).

Species delineation was also based on molecular data achieved by using species delimitation plugin ( Masters et al. 2011) from the program GENEIOUS PRIME v. 2022.1.1. (Geneious, Auckland, New Zealand), and was applied to compute intra- and interspecies variation by means of the Probability (P) Intra Distance (ID) liberal and the Rosenberg’s P AB value (The probability that species A represented by a sequences, in a clade of a + b sequences, will be reciprocally monophyletic with the remaining b sequences under the null model of random coalescence). Here, this approach was carried out on nematode populations considering two sperate species-pair comparisons based on morphologically similar species groups ( P. uliaensis sp. nov. vs. P. benalupensis sp. nov., and P. paraallius sp. nov. vs. P. allius ; please see species description below). The intra-/ interspecies molecular variation was 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 (the average pairwise tree distance among members of a putative species/the average pairwise tree distance between the members of one putative species, and the members of the closest second putative 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) represents the probability that a correct species identification would be made using best sequence alignment (BLAST), closest genetic distance, or placement on a tree (falling within or being sister to a monophyletic species clade). Species with P ID (Liberal) ≥0.93 were considered to be adequately delimited ( Hamilton et al. 2014). The Rosenberg’s P AB represents the probability that the monophyly of a group of sequences is the result of random branching ( Rosenberg 2007).

Phylogenetic analysis

D2-D3 expansion segments of 28S rRNA, ITS rRNA, and partial 18S rRNA gene sequences from the P. allius species-complex, as well as different accessions belonging to the Trichodoridae family that are available in GenBank, were used for phylogenetic reconstruction. Outgroup taxa for each dataset were chosen according to previously published data (Kumari and Subbotin 2012, Decraemer et al. 2019, 2021a, Subbotin et al. 2020) that included all the molecular variation in the analysed sequences ( van Megen et al. 2009). Multiple sequence alignments of the different genes were completed using the FFT-NS-2 algorithm of MAFFT v.7.450 ( Katoh et al. 2019). BioEdit program v.7.2.5 ( Hall 1999) was used for sequence alignments’ visualization 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 some computation time as suggested by Tan et al. (2015), since 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, the base frequency, the proportion of invariable sites, and the gamma distribution shape parameters and substitution rates in the AIC were then used in MrBayes for the phylogenetic analyses. The transversion model with invariable sites and a gamma-shaped distribution (TVM+I+G) for the D2-D3 expansion segments of 28S rRNA, the one-parameter model with invariable sites and gamma distribution model (TPM2uf+I+G) for the partial ITS rRNA gene, and the symmetrical model with invariable sites and gamma distribution (SYM+I+G) for the partial 18S rRNA gene, were run with four chains for 4 × 106 generations. The sampling for Markov chains was carried out 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. On each appropriate clade, posterior probabilities (PP) were given. FigTree software v.1.4.3 ( Rambaut 2016) was used for visualizing trees from all analyses.

R E SU LTS

Morphological characterization of the Paratrichodorus allius species-complex

Within the P. allius species group, the main morphological diagnostic features to differentiate species in males are, apart from morphometrics, the number and position of CP and the number (total and in relation to retracted spicules) and position of precloacal supplements (SP) and, to a lesser extent, the appearance of sperm in the testis; in females, the vaginal shape and length, the size, shape and position of the vaginal sclerotized pieces in optical section, and sperm location are of main diagnostic importance. Within the Paratrichodorus allius species-complex, seven new Spanish species were found and compared with a new species from Italy. For all species, except one species with unknown males, males were present either as common as females or rare. We start with the description of the species with males with one CP followed by those without.