Research Article |
Corresponding author: Matteo Montagna ( matteo.montagna@unina.it ) Academic editor: Jorge Santiago-Blay
© 2016 Giulia Magoga, Davide Sassi, Mauro Daccordi, Carlo Leonardi, Mostafa Mirzaei, Renato Regalin, Giuseppe Lozzia, Matteo Montagna.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Magoga G, Sassi D, Daccordi M, Leonardi C, Mirzaei M, Regalin R, Lozzia G, Montagna M (2016) Barcoding Chrysomelidae: a resource for taxonomy and biodiversity conservation in the Mediterranean Region. In: Jolivet P, Santiago-Blay J, Schmitt M (Eds) Research on Chrysomelidae 6. ZooKeys 597: 27–38. https://doi.org/10.3897/zookeys.597.7241
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The Mediterranean Region is one of the world’s biodiversity hot-spots, which is also characterized by high level of endemism. Approximately 2100 species of leaf beetle (Coleoptera; Chrysomelidae) are known from this area, a number that increases year after year and represents 5/6% of the known species. These features, associated with the urgent need to develop a DNA-based species identification approach for a broad spectrum of leaf beetle species, prompted us to develop a database of nucleotide sequences, with a solid taxonomic background, for all the Chrysomelidae Latreille, 1802 sensu latu inhabiting the Mediterranean region. The Mediterranean Chrysomelidae Barcoding project, which has started in 2009, involves more than fifty entomologists and molecular biologists from different European countries. Numerous collecting campaigns have been organized during the first seven years of the project, which led to the collection of more than 5000 leaf beetle specimens. In addition, during these collecting campaigns two new allochthonous species for Europe, namely Ophraella communa LeSage, 1986 and Colasposoma dauricum Mannerheim, 1849, were intercepted and some species new to science were discovered (e.g., Pachybrachis sassii Montagna, 2011 and Pachybrachis holerorum
Leaf beetles, molecular taxonomy, DNA barcoding, Cytochrome c oxidase subunit 1, C-bar project
In the last decades we have witnessed what has been defined as the “taxonomy impediment” (
In this project are involved taxonomists, specialized in different leaf beetle clades, in order to guarantee the accurate specimen identification. In our view, the adoption of this strategy is a way to bring together traditional (intended as based on morphology) and molecular taxonomy in order to tentatively overcome the “taxonomy impediment” (
The purpose of this paper is to report the preliminary results achieved during the first seven years of the project in order to show the potential of a cooperation between molecular biologists and traditional taxonomists. In particular, we report: i) the method adopted and issues arisen in the development of the sequence dataset; ii) the list of subfamilies, genera and the number of species for which cox1 gene sequences were obtained; iii) the metadata associated with the processed organisms; iv) mean values of intraspecific and interspecific nucleotide divergence v) the new species described and the important faunistic findings.
More than 50 entomologists, from different European Countries, have joined the C-Bar project and have actively participated in samples collection. During the first seven years of the project (from 2009 to 2015) numerous collecting campaigns were organized from March to September of each year. The specimens were collected using different methods: from the vegetation by sweep net or by beating sheet, and directly by hand in specific habitats (e.g. under stones or digging the host plant roots). All the collected specimens were placed in 5 ml vials filled with absolute ethanol in order to preserve the genomic DNA. Within an hour of specimen collection, the mixture in the vials was replaced with fresh absolute ethanol in order to obtain better sample dehydration and preservation for long-term storage. Each vial was preserved at -20°C and was labeled by a unique identifier plus other metadata related to the sampling locality (i.e. Country, Province, Region, exact site, latitude, longitude and elevation), the date of collection, the collector/s and other ecological information related to the specimens.
Specimen manipulation and dissection (when necessary) were completed with the auxiliary use of a stereomicroscope. Images of the specimen habitus were acquired by a reflex camera (Canon EOS 450D, macro objective 60 mm or 100 mm with a set of macro extension tubes) or with Axiocam 506 mounted on Zeiss Axio Zoom V16. The specimens were morphologically identified by Italian taxonomists expert in different leaf beetle clades (most of them are listed among the authors of the present article). The nomenclature adopted in the C-bar project follows the work of
DNA extraction was performed in two different ways since it took place in different laboratories (Biodiversity Institute of Ontario, University of Guelph and Department of Agricultural and Environmental Sciences, Università degli Studi di Milano): for 950 samples the DNA was extracted from one hind leg while for the 56 remaining samples the DNA was extracted from the whole specimen, after the removal of the abdomen. The latter procedure ensures to keep specimen morphology intact. In both cases, DNA was purified using the Qiagen DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany). Here we describe the adopted non-destructive procedure: the specimen was taken off from absolute ethanol and dried in single 1.5 ml vials for 45 minutes at 30°C; after the removal of the abdomen with the use of sterile pins and tweezers the specimen was placed in 180 µL of ATL lysis buffer (Qiagen) with 200 ng/mL proteinase K (Sigma Aldrich, St. Louis, MO, USA) at 56°C for 12 hours. The following steps of the DNA extraction were performed according to the manufacturer’s instructions of Qiagen DNeasy Blood and Tissue Kit. After DNA extraction, the specimens were dry mounted on pins together with genitalia and kept for future reference. A quote of the extracted DNA was preserved in the C-bar DNA library at -80°C for long term storage and a rate was preserved at -20° in order to perform the following amplifications. A fragment of 658 bp at the 5’-end of the mitochondrial cytochrome c oxidase subunit 1 gene (cox1) was amplified with primers LCO1490 5’-GGT CAA CAA ATC ATA AAG ATA TTG G / HCO2198 5’-TAA ACT TCA GGG TGA CCA AAA AAT CA (
The obtained cox1 gene sequences were aligned at codon level using MUSCLE (
Until now, C-Bar collecting campaigns have investigated some areas of Bulgaria, France, Greece, Italy, Morocco, Romania, Spain, Switzerland, Turkey and Tunisia (Figure
Among the collected samples, the DNA was extracted from 1006 specimens and PCRs targeting a fragment of the cox1 gene performed. PCRs with the selected primer pairs lead to successful amplification in 93.8% of the cases (62 specimens failed the amplification). Among the specimens for which the amplification failed, 43 specimens belong to the subfamily Cryptocephalinae Gyllenhaal, 1813: 18 species of Cryptocephalus (40 specimens); interestingly cox1 sequences have never been obtained for Cryptocephalus therondi Franz, 1949, Cryptocephalus cantabricus Franz, 1958 and Cryptocephalus etruscus Sassi, 1995. We can hypothesize the presence of mutations in the annealing region of the used primers. Sequences obtained from Clytra laeviuscula Ratzeburg, 1837, Clytra quadripunctata Linnaeus, 1758, Cryptocephalus cristula Dufour, 1843, Cryptocephalus octoguttatus Linnaeus, 1767, Lachnaia tristigma Lacordaire, 1848 and Oomorphus concolor Sturm, 1807 did not possess an open reading frame and were thus considered as nuclear pseudogenes. Twenty-seven sequences were discarded because of contamination from exogenous DNA. A total of 910 cox1 sequences (267 species corresponding to ~13% of those inhabiting the Mediterranean region) were obtained, the size of the sequences was > 400 bp in ~99% of the cases.
We observed that only two species, namely Cryptocephalus violaceus Laicharting, 1781 and Cryptocephalus duplicatus Suffrian, 1845, sharing the same haplotype can not be discriminated through DNA barcoding. In this and in similar cases a barcoding failure can be confirmed only ensuring the correct identification of the samples by expert taxonomists. Therefore 99.3% of the species (265) for which we obtained cox1 sequences possessed unique haplotypes, allowing their molecular identification. The mean intraspecific nucleotide distance value is of 2%, while the mean interspecific and intrageneric distances result of, respectively, 25.2% and of 19.8%. The obtained intraspecific value are higher than that inferred in a previous study on Coleoptera (
Among the nine subfamilies for which cox1 sequences were obtained (Table
List of the barcoded subfamilies and genera with the number of species and specimens belonging to each taxon.
Subfamily | Genus |
Ns |
|
N |
---|---|---|---|---|
Zeugophorinae Böving & Craighead, 1931 | Zeugophora Kunze, 1818 | 1 | 1 | 1 |
Orsodacninae Thomson, 1859 | Orsodacne Latreille, 1802 | 3 | 7 | 2.3 |
Donacinae Kirby, 1837 | Donacia Fabricius, 1775 | 2 | 6 | 3 |
Criocerinae Latreille, 1804 | Crioceris Muller, 1764 | 3 | 18 | 3 |
Lilioceris Reitter, 1912 | 1 | |||
Lema Fabricius, 1798 | 1 | |||
Oulema Gozis, 1886 | 1 | |||
Cassidinae Gyllenhal, 1813 | Cassida Linnaeus, 1758 | 14 | 61 | 3.4 |
Hypocassida Weise, 1893 | 2 | |||
Hispa Linnaeus, 1767 | 1 | |||
Dicladispa Gestro, 1897 | 1 | |||
Chrysomelinae Latreille, 1802 | Chrysolina Motschulsky, 1860 | 13 | 117 | 3.4 |
Chrysomela Linnaeus, 1758 | 3 | |||
Entomoscelis Chevrolat, 1836 | 1 | |||
Gastrophysa Chevrolat, 1836 | 1 | |||
Gonioctena Motschulsky, 1860 | 3 | |||
Oreina Chevrolat, 1836 | 6 | |||
Plagiosterna Motschulsky, 1860 | 1 | |||
Phratora Chevrolat, 1836 | 1 | |||
Plagiodera Chevrolat, 1836 | 1 | |||
Prasocuris Latreille, 1802 | 1 | |||
Timarcha Latreille, 1829 | 3 | |||
Galerucinae Latreille, 1802 | Agelastica Chevrolat, 1836 | 1 | 274 | 3.1 |
Arima Chapuis, 1875 | 1 | |||
Calomicrus Stephens, 1831 | 3 | |||
Exosoma Jacoby, 1903 | 2 | |||
Diabrotica Chevrolat, 1836 | 1 | |||
Galeruca Geoffroy, 1762 | 5 | |||
Galerucella Crotch, 1873 | 3 | |||
Lochmaea Weise, 1883 | 2 | |||
Luperus Geoffroy, 1762 | 6 | |||
Nymphius Weise, 1900 | 2 | |||
Sermylassa Reitter, 1913 | 1 | |||
Altica Muller, 1764 | 4 | |||
Aphthona Chevrolat, 1842 | 6 | |||
Argopus Fischer von Waldheim, 1824 | 1 | |||
Arrhenocoela Foudras, 1860 | 1 | |||
Chaetocnema Stephens, 1831 | 2 | |||
Crepidodera Chevrolat, 1836 | 5 | |||
Derocrepis Weise, 1886 | 2 | |||
Dibolia Latreille, 1829 | 2 | |||
Epitrix Foudras, 1860 | 1 | |||
Hermaeophaga Foudras, 1860 | 1 | |||
Hippuriphila Foudras, 1860 | 1 | |||
Longitarsus Berthold, 1827 | 9 | |||
Lythraria Bedel, 1897 | 1 | |||
Neocrepidodera Heikertinger, 1911 | 6 | |||
Phyllotreta Chevrolat, 1836 | 4 | |||
Podagrica Chevrolat, 1836 | 1 | |||
Psylliodes Berthold, 1827 | 12 | |||
Sphaeroderma Stephens, 1831 | 2 | |||
Cryptocephalinae Gyllenhal, 1813 | Cryptocephalus Geoffroy, 1762 | 73 | 426 | 3.8 |
Pachybarchis Chevrolat, 1836 | 8 | |||
Stylosomus Suffrian, 1848 | 2 | |||
Clytra Laicharting, 1781 | 4 | |||
Coptocephala Chevrolat, 1836 | 3 | |||
Labidostomis Chevrolat, 1836 | 10 | |||
Lachnaia Chevrolat, 1836 | 3 | |||
Macrolenes Chevrolat, 1836 | 1 | |||
Smaragdina Chevrolat, 1836 | 7 | |||
Tituboea Lacordaire, 1848 | 1 | |||
Eumolpinae Hope, 1840 | Chrysochus Chevrolat, 1836 | 1 | 5 | 1.7 |
Colaspidea Laporte de Castelnau, 1833 | 1 | |||
Macrocoma Chapuis, 1874 | 1 |
The metadata related to the specimens (i.e., specimen identification, collection identifier, collecting date, state, province, exact site of collection, latitude, longitude, elevation and collector/s) from which cox1 gene sequences were obtained, are available in a web site dedicated to the project (http://www.c-bar.org). Regarding the specimens collected within Italian administrative boundaries the metadata associated with the specimens are also available in the Biodiversity Database and GIS platform of the Italian National Network of Biodiversity. These faunistic data are useful because increase the awareness of species presence and distribution in the sampled area.
In this paper, we report that C-Bar project, besides having produced useful data for molecular taxonomy (cox1 sequences were obtained for about 13% of the species inhabiting the investigated area), has obtained important results also from the viewpoint of the classical taxonomy leading to the morphological description of same new species of Chrysomelidae. A further important achievement has been the interception of allochthonous species. These results have been obtained only thanks to the cooperation amongst the taxonomists specialized in different leaf beetle clades, which have ensured the correct identification of samples, the people involved in the extensive collecting campaigns and the molecular biologists.
The promising preliminary results that have been obtained encourage us to continue with this project since they strongly confirm the urgent need to increase the efforts in faunistic studies to uncover the real biodiversity of leaf beetles inhabiting the Mediterranean region. For these reasons, we are confident that the aim of C-bar project of developing a repository of cox1 sequences for the majority of the species of Chrysomelidae s. l. inhabiting the Mediterranean region may be achieved in the near future.
In conclusion, as demonstrated by the relevant results obtained during the first years of the project, we believe that DNA barcoding projects, when developed with the participation of taxonomists and molecular biologists, represent an opportunity to bring together two different worlds and may be considered the driving force able to revive interest in what can be regarded as the milestone of biological studies that is a-taxonomy, helping to fill the “taxonomy impediment”.
The Authors would like to thank Dr. Stefano Zoia for the work performed and precious suggestions. In addition, special thanks go to all those involved in the initiative and have helped in sample collection across the Mediterranean region (http://www.c-bar.org/about/people-actively-involved/).