Cryptic diversity and species delimitation in the Xiphinema americanum-group complex (Nematoda: Longidoridae) as inferred from morphometrics and molecular markers

The Xiphinema americanum-group constitutes a complex of about 55 species of polyphagous plant-ectoparasitic nematodes with a worldwide distribution. This group of plant-parasitic nematodes is one of the most difficult dagger nematode species complexes for diagnosis because the morphology is very conservative and morphometric characters often overlap. We conducted nematode surveys in cultivated and wild olives in southern Spain from 2012 to 2014, from which we identified 16 nematode populations of the X. americanum-group, five of which were tentatively identified as belonging to three new species and are described herein as Xiphinema plesiopachtaicum sp. nov.,Xiphinema vallense sp. nov., and Xiphinema astaregiense sp. nov., and 11 populations belonging to nine known species: Xiphinema brevisicum, Xiphinema duriense, Xiphinema incertum, Xiphinema luci, Xiphinema madeirense, Xiphinema opisthohysterum, Xiphinema pachtaicum, Xiphinema parapachydermum, and Xiphinema rivesi. A phenetic study based on multivariate factor analyses was developed to compare some of these related species by using morphometric features. In the factor analysis the first four factors accounted for 73.1% of the total variance of the selected characters, identifying body length, body length/maximum body width (a), body length/pharyngeal length (b), body length/tail length (c), and tail length/body width at anus (c′) ratios, distance from anterior end to vulva as percentage of body length (V), stylet length, oral aperture-guiding ring distance, and lip region width as key morphometric characters to differentiate a restricted set of species within the X.pachtaicum-subgroup that includes X.plesiopachtaicum sp. nov. and X.vallense sp. nov. Multivariate analysis of variance using these specific characters allowed to differentiate species in the X.pachtaicum complex or groups of them using morphometric characters (body length, a, b, c, c′, V, stylet length, lip region width, oral aperture-guiding ring distance, female tail length, and hyaline region length). Phylogenetic analyses based on nuclear ribosomal DNA genes [D2-D3 expansion segments of large ribosomal subunit 28S, and internal transcribed spacer 1 (ITS1)] and the protein-coding mitochondrial gene, cytochrome c oxidase subunit 1 (coxI) were congruent, showing two main clades separating most of the species of X.americanum-subgroup ‘sensu stricto’ from the X.pachtaicum-subgroup. Agreement between phylogenetic trees and some morphological characters (viz. total stylet length, vulva position, and a ratio) were tested by reconstruction of their histories on rRNA-based trees using parsimony and Bayesian approaches. Thus, integrative taxonomy, based on a combination of multivariate morphological and molecular analyses constitutes a new insight into the identification of X.americanum-group species.
This research is part of the PhD project of the first author and was supported by grant AGL2012-37521 from Ministerio de Economía y Competitividad of Spain, grant 219262 ArimNET_ERANET FP7 2012-2015 Project PESTOLIVE ‘Contribution of olive history for the management of soilborne parasites in the Mediterranean basin’ from Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), grants P12-AGR 1486 and AGR-136 from Consejería de Economía, Innvovación y Ciencia of the Junta de Andalucía, and Union Europea, Fondo Europeo de Desarrollo regional, ‘Una manera de hacer Europa’. A. J. Archidona-Yuste is a recipient of research contract BES-2013-063495 from Ministerio de Ciencia e Innovación of Spain. The authors thank J. Martín Barbarroja and G. León Ropero from IAS-CSIC for the excellent technical assistance.