Research Article |
Corresponding author: Michael Raupach ( raupach@snsb.de ) Academic editor: Borislav Guéorguiev
© 2016 Michael Raupach, Karsten Hannig, Jerome Moriniere, Lars Hendrich.
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:
Raupach MJ, Hannig K, Morinière J, Hendrich L (2016) A DNA barcode library for ground beetles (Insecta, Coleoptera, Carabidae) of Germany: The genus Bembidion Latreille, 1802 and allied taxa. ZooKeys 592: 121-141. https://doi.org/10.3897/zookeys.592.8316
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As molecular identification method, DNA barcoding based on partial cytochrome c oxidase subunit 1 (COI) sequences has been proven to be a useful tool for species determination in many insect taxa including ground beetles. In this study we tested the effectiveness of DNA barcodes to discriminate species of the ground beetle genus Bembidion and some closely related taxa of Germany. DNA barcodes were obtained from 819 individuals and 78 species, including sequences from previous studies as well as more than 300 new generated DNA barcodes. We found a 1:1 correspondence between BIN and traditionally recognized species for 69 species (89%). Low interspecific distances with maximum pairwise K2P values below 2.2% were found for three species pairs, including two species pairs with haplotype sharing (Bembidion atrocaeruleum/Bembidion varicolor and Bembidion guttula/Bembidion mannerheimii). In contrast to this, deep intraspecific sequence divergences with distinct lineages were revealed for two species (Bembidion geniculatum/Ocys harpaloides). Our study emphasizes the use of DNA barcodes for the identification of the analyzed ground beetles species and represents an important step in building-up a comprehensive barcode library for the Carabidae in Germany and Central Europe as well.
Asaphidion , Central Europe, cytochrome c oxidase subunit I, German Barcode of Life, mitochondrial DNA, molecular specimen identification, Ocys , Sinechostictus
The Carabidae (ground beetles) is a large cosmopolitan family of the Coleoptera, with an estimated number of 40,000 species world-wide, about 2,700 in Europe and 567 in Germany (
Within the Carabidae, the genus Bembidion Latreille, 1802 is the largest in this family, with more than 1,200 described species mostly in the temperate regions of the world (
Representative images of analyzed beetle species. A Asaphidion caraboides (Schrank, 1781) B Asaphidion flavipes (Linnaeus, 1761) C Ocys harpaloides (Audinet-Serville, 1821) D Ocys quinquestriatus (Gyllenhal, 1810) E Sinechostictus elongatus (Dejean, 1831) F Sinechostictus ruficornis (Sturm, 1825) GBembidion (Bembidion) quadrimaculatum (Linnaeus, 1761) HBembidion (Bembidionetolitzkya) fasciolatum (Duftschmid, 1812) IBembidion (Emphanes) azurescens Dalla Torre, 1877 JBembidion (Notaphus) semipunctatum (Donovan, 1806) KBembidion (Ocydromus) testaceum (Duftschmid, 1812) LBembidion (Bracteon) litorale (Olivier, 1790) MBembidion (Philochthus) biguttatum (Fabricius, 1779) NBembidion (Talanes) aspericolle (Germar, 1829) OBembidion (Trepanedoris) doris (Panzer, 1796), and PBembidion (Trepanes) octomaculatum (Goeze, 1777). Scale bars = 1 mm. All images were obtained from www.eurocarabidae.de.
During the last years the analysis of DNA sequence data, in particular the use of an approx. 650 base pair (bp) fragment of the mitochondrial cytochrome
However, the application of COI (and other mitochondrial markers in general) for species identification is not without problems. Recent speciation and hybridization events (e.g.
In this study we present as part of the German Barcode of Life project a comprehensive DNA barcode library of a variety of Central European species of the genus Bembidion and associated taxa. Our new barcode library includes 65 species of the genus Bembidion as well as five species of the closely related genera Asaphidion Des Gozis, 1886, two species of the genus Ocys Stephens, 1828 and six species of the genus Sinechostictus Motschulsky, 1864. In total, our library comprised 819 sequences of 78 species.
All analyzed ground beetles were collected between 1997 and 2015 using various sampling methods (i.e. hand collecting, pitfall traps). All specimens were stored in ethanol (96%). The analyzed beetles were identified by one of the authors (KH) using the keys in
Most specimens were collected in Germany (n = 617, 75%), but for comparison some specimens were also included from Austria (n = 107, 13%), Belgium (n = 3, 0.04%), Czech Republic (n = 1, 0.01%), Italy (n = 41, 0.5%), France (n = 34, 0.4%), Slovenia (n = 15, 0.2%) and Sweden (n = 1, 0.01%). The number of analyzed specimens per species ranged from one (8 species, 10.3%) to a maximum of 38 in the case of Bembidion tetracolum Say, 1823.
Laboratory operations were carried out either at the Canadian Center for DNA Barcoding (CCDB), University of Guelph, following standardized high-throughput protocols for COI amplification and sequencing (
Polymerase chain reaction (PCR) has been used for amplifying the COI barcode fragment using the primer pair LCO1480 and HCO2198 (
Intra- and interspecific distances of the analyzed ground beetle species were based on the Kimura 2-parameter (K2P;
A neighbor joining cluster analysis (NJ;
Overall, 819 DNA barcode sequences of 78 carabid beetle species were analyzed. A full list of the analyzed species is presented in the supporting information (Suppl. material
Fragment lengths of the analyzed DNA barcode fragments ranged from 352 to 657 bp. As it is typically known for arthropods, our sequence data also revealed a high AT-content for the DNA barcode region: the mean sequence compositions were A=16.6%, C=15.9%, G=29.8% and T=37.7%. Intraspecific K2P distances within a genus ranged from zero to 9.62% (Ocys harpaloides (Audinet-Serville, 1821)) whereas interspecific distances within the analyzed genera had values between zero and 14.72%. In this context, the lowest interspecific distances of distinct barcode clusters were revealed for Bembidion ascendens K. Daniel, 1902 and Bembidion fasciolatum (Duftschmid, 1812) with values ranging from 0.49% to 0.82%. Both species had the same BIN (ACJ7842).
In total, unique BINs were revealed for 69 species (89%), two BINs for 3 species (4%), and one BIN for two species for 6 species (7%). Interspecific distances of zero were found for two species pairs (5.1%): Bembidion atrocaeruleum Stephens, 1828 vs. Bembidion varicolor Fabricius, 1803 and Bembidion guttula (Fabricius, 1792) vs. Bembidion mannerheimii C.R. Sahlberg, 1827. In contrast to this, maximum intraspecific pairwise distances >2.2% were found for three species (3.8%): Bembidion decorum (Panzer, 1799) (2.56%), Bembidion geniculatum Heer, 1837 (4.49%), and Ocys harpaloides (Audient-Serville, 1821) (9.62%). The NJ analyses based on K2P distances revealed non-overlapping clusters with bootstrap support values >95% for 63 species (81%) with more than one analyzed specimen (Fig.
Neighbor joining topology of the analyzed ground beetle species based on Kimura 2-parameter distances. Triangles indicate the relative number of individual’s sampled (height) and sequence divergence (width). Green triangles indicate species with intraspecific maximum pairwise distances >2.2%, blue triangles species pairs with interspecific distances <2.2%. Numbers next to nodes represent non-parametric bootstrap values >90% (1,000 replicates). Asterisks indicate species not recorded in Germany. All images were obtained from www.eurocarabidae.de.
Our statistical maximum parsimony analysis showed multiple sharing of haplotypes for two species pairs: Bembidion atrocaeruleum (n = 32) and Bembidion varicolor (n = 22) (Fig.
Maximum statistical parsimony network of Bembidion species sharing COI haplotypes: A Bembidion atrocaeruleum Stephens, 1828 and Bembidion varicolor Fabricius, 1803 B Bembidion guttula (Fabricius, 1792) and Bembidion mannerheimii C.R. Sahlberg, 1827. Used settings included a user specified maximum of connection steps at 50, gaps were treated as fifth state. Each line represents a single mutational change whereas small black dots indicate missing haplotypes. The numbers of analyzed specimens (n) are listed, whereas the diameter of the circles is proportional to the number of haplotypes sampled (see given open half circles with numbers). Scale bars = 1 mm. Beetle images were obtained from www.eurocarabidae.de.
Our study clearly confirms the usefulness of DNA barcodes for the identification of species of the genera Asaphidion, Bembidion, Ocys, and Sinechostictus of Central Europe, in particular Germany. Unique BINs were found for 69 species (89%) of the analyzed 78 beetle species, coinciding with high rates of successful species identification of previous barcoding studies of ground beetles (
Haplotype sharing of COI sequences was found for two species pairs. In the case of Bembidion guttula and Bembidion mannerheimii identical COI sequences are not surprising (Fig.
A somewhat similar situation was revealed for Bembidion atrocaeruleum and Bembidion varicolor. Nevertheless, the statistical maximum parsimony network revealed a more complex structure (Fig.
Maximum intraspecific pairwise distances >2.2% were observed for three species. Whereas Bembidion decorum showed no conspicuous substructure for the analyzed COI sequences (see Suppl. material
Subtrees of the neighbor joining topology based on Kimura 2-parameter distances of all analyzed specimens of A Ocys harpaloides (Audinet-Serville, 1821) and nearest neighbor, and B Bembidion geniculatum Heer, 1837 and nearest neighbor. Branches with specimen ID-number from BOLD, species names and sample localities. Numbers next to internal nodes are non-parametric bootstrap values (in %).
Based on the given data we are unable to clarify the reasons of the observed distinct lineages which can be caused by various effects, including phylogeographic events (e.g.
Carabid beetles are one of the best-known taxa in entomology that have been studied intensively by numerous generations of coleopterists, clarifying their taxonomy and phylogeny, biogeography, habitat associations and ecological requirements, life history and adaptations, especially in Central Europe (see review in
We would like to thank Christina Blume and Claudia Etzbauer (both ZFMK, Bonn) as well as Jana Deppermann (DZMB, Wilhelmshaven) and Laura Ney (RUB, Bochum) for their laboratory assistance. We also thank Joachim Schmidt for his help in identifying various analyzed beetle specimens. Furthermore we are very grateful to Ortwin Bleich for giving permission to use his excellent photos of ground beetles from www.eurocarabidae.de. David Maddison and Yuichi Oba provided helpful comments on the manuscript. This publication was partially financed by German Federal Ministry for Education and Research (FKZ01LI1101A, FKZ01LI1101B, FKZ03F0664A), the Land Niedersachsen and the German Science Foundation (INST427/1-1), as well as by grants from the Bavarian State Government (BFB) and the German Federal Ministry of Education and Research (GBOL2: 01LI1101B). We are grateful to the team of Paul Hebert in Guelph (Ontario, Canada) for their great support and help and in particularly to Sujeevan Ratnasingham for developing the BOLD database infrastructure and the BIN management tools. Sequencing work was partly supported by funding from the Government of Canada to Genome Canada through the Ontario Genomics Institute, whereas the Ontario Ministry of Research and Innovation and NSERC supported development of the BOLD informatics platform.
Barcode analysis using the BOLD workbench
Data type: Data table
Explanation note: Molecular distances based on the Kimura 2-parameter model of the analyzed specimens of the analyzed species of the genera Asaphidion, Bembidion, Ocys and Sinechostictus. Divergence values were calculated for all studied sequences, using the Nearest Neighbour Summary implemented in the Barcode Gap Analysis tool provided by the Barcode of Life Data System (BOLD). Align sequencing option: BOLD aligner (amino acid based HMM), ambiguous base/gap handling: pairwise deletion. ISD = intraspecific distance. BINs are based on the barcode analysis from 01-02-2016. Asterisks indicate species not recorded from Germany. Species with intraspecific maximum pairwise distances >2.2% and species pairs with interspecific distances <2.2% are marked in bold. Country codes: A = Austria, B = Belgium, CZ = Czech Republic, D = Germany, F = France, I = Italy, S = Sweden and SLO = Slovenia.
Neighbor joining topology
Data type: Neighbor joining topology
Explanation note: Neighbor joining topology of all analyzed carabid beetles based on Kimura 2-parameter distances. Specimens are classified using ID numbers from BOLD and species name. Numbers next to nodes represent non-parametric bootstrap values (1,000 replicates, in %).