Comprehensive characterisation of Culicoides clastrieri and C. festivipennis (Diptera: Ceratopogonidae) according to morphological and morphometric characters using a multivariate approach and DNA barcode

Leila Hadj-Henni,Denis Augot,Zoubir Djerada,Christine Millot

doi:10.1038/s41598-020-78053-3

Abstract

Biting midges are widespread around the world and play an essential role in the epidemiology of over 100 veterinary and medical diseases. For taxonomists, it is difficult to correctly identify species because of affinities among cryptic species and species complexes. In this study, species boundaries were examined for C. clastrieri and C. festivipennis and compared with six other Culicoides species. The classifiers are partial least squares discriminant analysis (PLS-DA) and sparse partial least squares discriminant analysis (sPLS-DA).The performance of the proposed method was evaluated using four models: (i) geometric morphometrics applied to wings; (ii) morphological wing characters, (iii) "Full wing" (landmarks and morphological characters) and (iv) "Full model" (morphological characters—wing, head, abdomen, legs—and wing landmarks). Double cross-validation procedures were used to validate the predictive ability of PLS-DA and sPLS-DA models. The AUC (area under the ROC curve) and the balanced error rate showed that the sPLS-DA model performs better than the PLS-DA model. Our final sPLS-DA analysis on the full wing and full model, with nine and seven components respectively, managed to perfectly classify our specimens. The C. clastrieri and C. festivipennis sequences, containing both COI and 28S genes, revealed our markers’ weak discrimination power, with an intraspecific and interspecific divergence of 0.4% and 0.1% respectively. Moreover, C. clastrieri and C. festivipennis are grouped in the same clade. The morphology and wing patterns of C. clastrieri and C. festivipennis can be used to clearly distinguish them. Our study confirms C. clastrieri and C. festivipennis as two distinct species. Our results show that caution should be applied when relying solely on DNA barcodes for species identification or discovery.

Highlights

Culicoides is a large and diverse genus which includes approximately 1,340 extant species[7]
Our molecular analysis (Fig. 1) with both markers generated seven supported clusters, six of which were in agreement with the morphological determination (i.e. C. alazanicus, C. brunnicans, C. circumscriptus, C. furcillatus, C. nubeculous and C. pictipennis)
C. pictipennis is the sister species of C. brunnicans while C. circumscriptus is positioned between the two clades

Summary

Introduction

Culicoides is a large and diverse genus which includes approximately 1,340 extant species[7]. There have been attempts to clarify Culicoides systematics by using approaches other than traditional morphological diagnostic characters, such as via (i) traditional morphometrics; (ii) geometric morphometrics (GM); (iii) nuclear, mitochondrial and ribosomal DNA analyses and (iv) matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF)[7]. Recent taxonomic revisions based on these alternative characters (morphology characters, molecular and/or morphometric tools) have led to the description of new species within the g enus[18,19,20,21,22,23]. Large-scale integrative taxonomic efforts incorporating morphological, ecological, and independent multi-locus sequence data from species sampled across their known ranges provide the best means to test species boundaries and refine essential species distribution d ata[31]. We focus on different kinds of characters, from (i) morphology; (ii) GM; (iii) mitochondrial DNA; and (iv) ribosomal DNA

Methods

Results

Conclusion