Where Taxonomy Based on Subtle Morphological Differences Is Perfectly Mirrored by Huge Genetic Distances: DNA Barcoding in Protura (Hexapoda)

<div><p>Background</p><p>Protura is a group of tiny, primarily wingless hexapods living in soil habitats. Presently about 800 valid species are known. Diagnostic characters are very inconspicuous and difficult to recognize. Therefore taxonomic work constitutes an extraordinary challenge which requires special skills and experience. Aim of the present pilot project was to examine if DNA barcoding can be a useful additional approach for delimiting and determining proturan species.</p><p>Methodology and Principal Findings</p><p>The study was performed on 103 proturan specimens, collected primarily in Austria, with additional samples from China and Japan. The animals were examined with two markers, the DNA barcoding region of the mitochondrial COI gene and a fragment of the nuclear 28S rDNA (Divergent Domain 2 and 3). Due to the minuteness of Protura a modified non-destructive DNA-extraction method was used which enables subsequent species determination. Both markers separated the examined proturans into highly congruent well supported clusters. Species determination was performed without knowledge of the results of the molecular analyses. The investigated specimens comprise a total of 16 species belonging to 8 genera. Remarkably, morphological determination in all species exactly mirrors molecular clusters. The investigation revealed unusually huge genetic COI distances among the investigated proturans, both maximal intraspecific distances (0–21.3%), as well as maximal congeneric interspecifical distances (up to 44.7%).</p><p>Conclusions</p><p>The study clearly demonstrates that the tricky morphological taxonomy in Protura has a solid biological background and that accurate species delimitation is possible using both markers, COI and 28S rDNA. The fact that both molecular and morphological analyses can be performed on the same individual will be of great importance for the description of new species and offers a valuable new tool for biological and ecological studies, in which proturans have generally remained undetermined at species level.</p></div

Comparison of COI and 28S rDNA in species discrimination of the genera <i>Acerentomon</i> (<i>Aco</i>), <i>Acerentulus</i> (<i>Acu</i>) and <i>Acerella</i> (<i>Ace</i>).

<p>Mirrored NJ tree based on K2P distances of COI (left) and 28S (right). Bootstrap support (maximal support marked with full circles) derived from 5000 replicates. Color code for genera: <i>Acerentomon</i> = violet, <i>Ionescuellum</i> = green, <i>Acerentulus</i> = orange, <i>Acerella</i> = red, <i>Eosentomon</i> = blue; Austrian sample sites are coded with different icons: Leopoldsberg = square, Eichkogel = triangle, and Twimberger Graben = circle.</p

List of morphologically determined species of Protura investigated in this study.

<p>Genus abbreviation: <i>Ace</i> = <i>Acerella</i>, <i>Aco</i> = <i>Acerentomon</i>, <i>Acu</i> = <i>Acerentulus, Eos</i> = <i>Eosentomon</i>, <i>Ion</i> = <i>Ionescuellum</i>; Locality abbreviation: LB = Leopoldsberg, TG = Twimberger Graben, EK = Eichkogel.</p

K2P distances in COI among species of Protura collected in Austria.

<p>Species described by their maximal intraspecific (Max intra), and the range of the best intraspecific matches (Best intra), as well as the maximal congeneric interspecific distances (Max congen), the name of the best matching species (Best match), along with the range of their best interspecific distances (Best inter). In species, covered by a single representative, missing distances are marked by a short line. Species abbreviations as used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090653#pone-0090653-t003" target="_blank">Tab. 3</a>.</p

List of primer pairs and starting positions within the selected markers used in the present study.

<p>Reference sequence <i>Sinentomon erythranum</i> (NCBI accession number: NC015982), * indicates internal primers additionally used in some specimens.</p

Comparison of COI and 28S rDNA in species discrimination of the genera <i>Ionescuellum</i> (<i>Ion</i>) and <i>Eosentomon</i> (<i>Eos</i>).

<p>NJ tree based on K2P distances of COI (left) and the mirrored 28S rDNA results (right). Bootstrap support (maximal support marked with full circles) derived from 5000 replicates is maximal for all species and polpulations. Color code for genera: <i>Acerentomon</i> = violet, <i>Ionescuellum</i> = green, <i>Acerentulus</i> = orange, <i>Acerella</i> = red, <i>Eosentomon</i> = blue; Austrian sample sites are coded with different icons: Leopoldsberg = square, Eichkogel = triangle, and Twimberger Graben = circle.</p

NJ tree based on K2P distances from 91 COI sequences of Protura.

<p>Newly sequenced specimens labeled with lab code number (HP), abbreviation for genus, and species name. Color code for genera: <i>Acerentomon</i> = violet, <i>Ionescuellum = </i>green, <i>Acerentulus</i> = orange, <i>Acerella</i> = red, <i>Eosentomon</i> = blue; Austrian sample sites are coded with different icons: Leopoldsberg = square, Eichkogel = triangle, and Twimberger Graben = circle. Bootstrap support (given below nodes) derived from 5000 replicates. Maximally supported clusters and subclusters are indicated by black dots. Genus abbreviations: <i>Aco</i> = <i>Acerentomon</i>, <i>Ion</i> = <i>Ionescuellum</i>, <i>Acu</i> = <i>Acerentulus</i>, <i>Ace</i> = <i>Acerella</i>, and <i>Eos</i> = <i>Eosentomon</i>.</p