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Complete DNA Barcode Reference Library for a Country's Butterfly Fauna Reveals High Performance for Temperate Europe

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Complete DNA Barcode Reference Library for a Country's Butterfly Fauna Reveals High Performance for Temperate Europe

Vlad Dinca et al. Proc Biol Sci.

Abstract

DNA barcoding aims to accelerate species identification and discovery, but performance tests have shown marked differences in identification success. As a consequence, there remains a great need for comprehensive studies which objectively test the method in groups with a solid taxonomic framework. This study focuses on the 180 species of butterflies in Romania, accounting for about one third of the European butterfly fauna. This country includes five eco-regions, the highest of any in the European Union, and is a good representative for temperate areas. Morphology and DNA barcodes of more than 1300 specimens were carefully studied and compared. Our results indicate that 90 per cent of the species form barcode clusters allowing their reliable identification. The remaining cases involve nine closely related species pairs, some whose taxonomic status is controversial or that hybridize regularly. Interestingly, DNA barcoding was found to be the most effective identification tool, outperforming external morphology, and being slightly better than male genitalia. Romania is now the first country to have a comprehensive DNA barcode reference database for butterflies. Similar barcoding efforts based on comprehensive sampling of specific geographical regions can act as functional modules that will foster the early application of DNA barcoding while a global system is under development.

Figures

Figure 1.
Figure 1.
Statistical results of DNA barcoding performance. (a) Performance based on genetic distances. (b) Performance based on taxon clustering.
Figure 2.
Figure 2.
DNA barcoding can reliably distinguish the sibling species Leptidea sinapis and L. reali, which otherwise can be identified only based on morphometry of their genitalia. (a) Bivariate scatter plot using the phallus length (PL) and saccus length (SL) as discriminative characters. Both variables were normalized by the vinculum width (VW). The upper image indicates how measurements of the male genitalia were performed. (b) Neighbour-joining (NJ) tree of COI barcodes of Romanian Leptidea sinapis and L. reali with bootstrap values more than 50% indicated.
Figure 3.
Figure 3.
Carcharodus flocciferus is paraphyletic with respect to C. orientalis. The two species are morphologically very similar and accurate identifications were possible based on the combined results of geometric and linear morphometry of the male genitalia. (a) Scatter plot of relative warp 1 and relative warp 2 explaining most of the variance reveals the presence of two discrete clusters of cuiller tip shape. The upper left image illustrates the landmarks (open circles) and semi-landmarks (black dots) used to describe the shape of the cuiller. (b) NJ tree of COI barcodes of Romanian Carcharodus orientalis and C. flocciferus with bootstrap values more than 50% indicated.
Figure 4.
Figure 4.
Examples of taxa with deep intraspecific divergence. (a) Sampling sites of the two COI lineages found in Pyrgus armoricanus. In one locality specimens of both lineages were present (scale bar, 100 km). (b) NJ tree of COI barcodes of Romanian P. armoricanus with bootstrap values more than 50% indicated. (c) Sampling sites of the three COI lineages found in Thymelicus sylvestris. In three localities specimens of two lineages were sympatric (scale bar, 100 km). (d) NJ tree of COI barcodes of Romanian T. sylvestris with bootstrap values more than 50% indicated.
Figure 5.
Figure 5.
The endangered butterfly Maculinea nausithous, whose larvae are obligate parasites of Myrmica ants, displays geographically and biologically correlated barcode divergence, highlighting the need for deeper taxonomic studies. (a) Distribution of M. nausithous in Romania and its relationship to the ant hosts (scale bar, 100 km). Upper right image illustrates a specimen resting on S. officinalis, the species larval host plant. (b) NJ tree of COI barcodes of Romanian M. nausithous with bootstrap values more than 50% indicated.

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