Integrated Taxonomy and DNA Barcoding of Alpine Midges (Diptera: Chironomidae)

RESEARCH ARTICLE Integrated Taxonomy and DNA Barcoding of Alpine Midges (Diptera: Chironomidae) Matteo Montagna1*, Valeria Mereghetti1, Valeria Lencioni2, Bruno Rossaro3 1 Dipartimento di Scienze Agrarie e Ambientali—Università degli Studi di Milano, Via Celoria 2, I-20133, Milano, Italy, 2 MUSE—Museo delle Scienze, Corso del Lavoro e della Scienza 3, I-38122, Trento, Italy, 3 Dipartimento di Scienze per gli Alimenti, la Nutrizione e l’Ambiente—Università degli Studi di Milano, Via Celoria 2, I-20133, Milano, Italy * Abstract OPEN ACCESS Citation: Montagna M, Mereghetti V, Lencioni V, Rossaro B (2016) Integrated Taxonomy and DNA Barcoding of Alpine Midges (Diptera: Chironomidae). PLoS ONE 11(3): e0149673. doi:10.1371/journal. pone.0149673 Editor: Xiao-Yue Hong, Nanjing Agricultural University, CHINA Received: October 19, 2015 Accepted: February 2, 2016 Published: March 3, 2016 Copyright: © 2016 Montagna et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: Sequence data are deposited at ENA archive (accession numbers: LN897576- LN897687). Morphological voucher specimens (MR-1 to MR-117) are deposited in Bruno Rossaro collection at Dipartimento di Scienze degli Alimenti—Università degli Studi di Milano. DNA vouchers of each specimen are stored in Insect Ecology and Evolution Lab at Dipartimento di Scienze Agrarie e Ambientali—Università degli Studi di Milano. Rapid and efficient DNA-based tools are recommended for the evaluation of the insect biodiversity of high-altitude streams. In the present study, focused principally on larvae of the genus Diamesa Meigen 1835 (Diptera: Chironomidae), the congruence between morphological/molecular delimitation of species as well as performances in taxonomic assignments were evaluated. A fragment of the mitochondrial cox1 gene was obtained from 112 larvae, pupae and adults (Diamesinae, Orthocladiinae and Tanypodinae) that were collected in different mountain regions of the Alps and Apennines. On the basis of morphological characters 102 specimens were attributed to 16 species, and the remaining ten specimens were identified to the genus level. Molecular species delimitation was performed using: i) distance-based Automatic Barcode Gap Discovery (ABGD), with no a priori assumptions on species identification; and ii) coalescent tree-based approaches as the Generalized Mixed Yule Coalescent model, its Bayesian implementation and Bayesian Poisson Tree Processes. The ABGD analysis, estimating an optimal intra/interspecific nucleotide distance threshold of 0.7%-1.4%, identified 23 putative species; the tree-based approaches, identified between 25–26 entities, provided nearly identical results. All species belonging to zernyi, steinboecki, latitarsis, bertrami, dampfi and incallida groups, as well as outgroup species, are recovered as separate entities, perfectly matching the identified morphospecies. In contrast, within the cinerella group, cases of discrepancy arose: i) the two morphologically separate species D. cinerella and D. tonsa are neither monophyletic nor diagnosable exhibiting low values of between-taxa nucleotide mean divergence (0.94%); ii) few cases of larvae morphological misidentification were observed. Head capsule color is confirmed to be a valid character able to discriminate larvae of D. zernyi, D. tonsa and D. cinerella, but it is here better defined as a color gradient between the setae submenti and genal setae. DNA barcodes performances were high: average accuracy was ~89% and precision of ~99%. On the basis of the present data, we can thus conclude that molecular identification represents a promising tool that could be effectively adopted in evaluating biodiversity of high-altitude streams. Funding: This research was funded by the Systematic Research Fund jointly administered by the Linnean Society of London and Systematic PLOS ONE | DOI:10.1371/journal.pone.0149673 March 3, 2016 1 / 20 Integrated Taxonomy and DNA Barcoding of Alpine Midges Association round 2014 (http://www.systass.org/ awards/srf13-14results.shtml; B. Rossaro and M. Montagna, Università degli Studi di Milano – Italy). Competing Interests: The authors have declared that no competing interests exist. Introduction Recent climatic warming has had a strong impact on habitats and species occurring at high elevation [1]. Amongst other effects, extensive environmental change occurs during glacial retreat, which affects hydrological and thermal regimes of glacier-fed streams [2]. Stream biodiversity is expected to dramatically change in relation to retreating glaciers, favoring an upstream shift of lowland species associated with local extinction of kryal species [3–7]. Unfortunately, a monitoring method and dedicated biotic indices, able to evaluate the biotic components of high-altitude habitats, have not yet been developed. There are several reasons for this, including and most importantly difficulties in identifying larvae belonging to the genus Diamesa Meigen 1835 (Diptera; Chironomidae), which dominate species richness and abundance in glacial streams and cold spring habitats [7–9]. Identification of midges based on morphology can be accurately achieved for adult males [10–11] or to a lesser extent for pupal exuviae [10], as clear discriminating features are visible for these stages. In addition, at present, despite updated keys identifying Diamesa larvae being available [12], the implementation of innovative tools, able to accurately identify larvae of the genus Diamesa by integrating different sources of information (e.g., molecular and morphological diagnostic characters), are required. Such approaches will promote the exploitation of ecological information provided by the presence/ absence of these species in the habitats under study [3]. The West Palaearctic species belonging to the genus Diamesa have been separated into nine different groups [13] according to adult male and pupal morphology. A combination of qualitative and quantitative characters observable in larvae (head capsule color, mouth-parts and posterior body appendages) only partially confirm this separation. This is because D. aberrata Lundbeck, 1898 and D. incallida (Walker 1856), included by Serra-Tosio in the aberrata group, have very different larvae, suggesting the separation into two distinct groups, while D. bertrami, Edwards, 1935, included by Serra-Tosio in the zernyi–insignipes–cinerella groups, has a larva very similar to the larvae of the latitarsis group [12]. Within each group, determination to species level is generally hampered by the lack of diagnostic characters or by the degradation of valid taxonomic characters, such as mental and mandibular teeth, in field-collected samples [12]. Moreover, quantitative characters should be used with cau (...truncated)