Research Article |
Corresponding author: Michael J. Raupach ( raupach@snsb.de ) Academic editor: Harald Letsch
© 2020 Michael J. Raupach, Karsten Hannig, Jérome Morinière, 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 (2020) A DNA barcode library for ground beetles of Germany: the genus Agonum Bonelli, 1810 (Insecta, Coleoptera, Carabidae). Deutsche Entomologische Zeitschrift 67(2): 197-207. https://doi.org/10.3897/dez.67.56163
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The ground beetle genus Agonum Bonelli, 1810 is a large genus of the tribe Platynini with many species that show high amounts of intraspecific variations, making a correct identification challenging. As part of the German Barcode of Life initiative, this publication provides a comprehensive DNA barcode library for species of Agonum that are reported for Germany. In total, DNA barcodes from 258 beetles and 23 species were analysed using the Barcode of Life Data System (BOLD) workbench, including sequences from former studies and 68 newly-generated sequences. The neighbour-joining analyses, based on K2P distances, revealed distinct clustering for all studied species, with unique Barcode Index Numbers (BINs) for 15 species (65%). BIN sharing but distinct clustering was found for three species pairs: Agonum micans/Agonum scitulum, Agonum impressum/Agonum sexpunctatum and Agonum duftschmidi/Agonum emarginatum. The given dataset and its analysis represent another important step in generating a comprehensive DNA barcode library for the ground beetles of Germany and Central Europe in terms of modern biodiversity research.
BOLD, Central Europe, cytochrome c oxidase subunit I, German Barcode of Life, mitochondrial DNA, molecular specimen identification, sibling species
Species identification represents a pivotal component for biodiversity studies and conservation planning, but represents a challenge for many taxa when using morphological traits only (e.g. the correct identification of juveniles or larval stages). As a consequence of tremendous technological advances in molecular biology during the last 20 years, molecular data have become increasingly popular in species identification. In this context, DNA barcoding represents the central component in the modern diagnostic toolbox of molecular biodiversity assessment studies and taxonomic research (e.g.
Nevertheless, it should be noted that various effects can seriously limit the use of DNA barcoding or mitochondrial markers in general, in terms of molecular species identification (e.g.
Not surprisingly, the build-up of comprehensive DNA barcode libraries represents a pivotal task (e.g.
Within the Carabidae, the genus Agonum Bonelli, 1810 is a species-rich taxon of shiny black or metallic ground beetles that imitate large representatives of Bembidion Latreille, 1802 (
Representative images of analysed Agonum species. A. Agonum (Olisares) duftschmidi Schmidt, 1994; B. Agonum (Olisares) ericeti (Panzer, 1809); C. Agonum (Olisares) hypocrita (Apfelbeck, 1904); D. Agonum (Olisares) sexpunctatum (Linné, 1758); E. Agonum (Agonum) marginatum (Linné, 1758); F. Agonum (Agonum) muelleri (Herbst, 1784); G. Agonum (Europhilus) fuliginosum (Panzer, 1809); H. Agonum (Europhilus) piceum (Linné, 1758) and I. Agonum (Europhilus) thoreyi Dejean, 1828. Scale bars: 1 mm. All images were obtained from http://www.eurocarabidae.de.
In this study, we present, as part of the on-going GBoL project, another step in generating a comprehensive DNA barcode library for the molecular identification of Central European ground beetle species, here focusing on the genus Agonum. The analysed sequence library included 23 species of Agonum and Oxypselaphus obscurus (Paykull, 1790) as outgroup. We generated 68 new barcodes including some sequences of old pinned museum specimens and analysed a total number of 258 DNA barcodes in detail.
Most of the sampled ground beetles (n = 186, 78%) were collected between 1999 and 2015 using various sampling methods (i.e. hand collecting, pitfall traps). All beetles were stored in ethanol (96%). The analysed specimens were identified using the identification key provided in
Most of the studied beetles were collected in Germany (n = 191, 73.9%), but for comparison, a number of specimens were included from Austria (n = 10, 3.6%), Czech Republic (n = 1, 0.4%), Estonia (n = 1, 0.4%), Finland (n = 49, 18.9%), France (n = 2, 0.8%), Italy (n = 1, 0.4%), Montenegro (n = 2, 0.8%) and Sweden (n = 2, 0.8%). The number of analysed specimens per species ranged from one (A. gracilipes (Duftschmid, 1812), A. impressum (Panzer, 1796)) to a maximum of 31 for A. fuliginosum (Panzer, 1809).
All laboratory operations were carried out, following standardised protocols for COI amplification and sequencing (
Detailed information about used primers, PCR amplification and sequencing protocols can be found in a previous publication (see
Detailed voucher information, taxonomic classifications, photos, DNA barcode sequences, primer pairs used and trace files (including their quality) were uploaded to the public dataset “DS-BAAGO” (Dataset ID: dx.doi.org/10.5883/DS-BAAGO) on the Barcode of Life Data Systems (BOLD; www.boldsystems.org) (
The complete dataset was analysed by using various approaches. First, the comprehensive analysis tools of the BOLD workbench were employed to calculate the nucleotide composition of the sequences and distributions of Kimura-2-parameter distances (K2P;
Second, maximum parsimony networks were constructed with TCS 1.21, based on default settings (
Finally, all sequences were aligned using MUSCLE (
Overall, 252 DNA barcode sequences of 24 Agonum species were analysed, representing 96% of all documented species (n = 25) of this genus for Germany, except Agonum nigrum Dejean, 1828. A full list of the analysed species is presented in the supporting information (Suppl. material
In total, unique BINs were revealed for 15 species (65%) and one BIN for two species for three species pairs (13%), namely Agonum micans (Nicolai, 1822)/Agonum scitulum Dejean, 1828), Agonum impressum (Panzer, 1796)/Agonum sexpunctatum (Linne, 1758) and Agonum duftschmidi Schmidt, 1994/Agonum emarginatum (Gyllenhal, 1827) (Fig.
The NJ analyses, based on K2P distances, revealed non-overlapping clusters for all analysed species (Fig.
Maximum statistical parsimony networks of the species pairs. A. Agonum micans (Nicolai, 1822) (blue) and Agonum scitulum Dejean, 1828 (red); B. Agonum impressum (Panzer, 1796) (yellow) and Agonum sexpunctatum (Linne, 1758) (green) and C. Agonum duftschmidi Schmidt, 1994 (violet) and Agonum emarginatum (Gyllenhal, 1827) (light brown). Used parameters included default settings for connection steps, gaps were treated as fifth state. Each line represents a single mutational change, whereas small black dots indicate missing haplotypes. The numbers of analysed specimens (n) are listed, whereas the diameter of the circles is proportional to the number of haplotypes sampled (see given open circles with numbers). Scale bars: 1 mm. Beetle images were obtained from http://www.eurocarabidae.de.
Neighbour-joining (NJ) topology of the analysed ground beetle species, based on Kimura 2-parameter distances. Triangles show the relative number of individual’s sampled (height) and sequence divergence (width). Numbers next to nodes represent non-parametric bootstrap values > 75% (1,000 replicates). Beetle images were obtained from http://www.eurocarabidae.de except for Agonum monachum (Duftschmid, 1812) (Photo taken by Lars Hendrich).
The results of this study highlight the efficiency of DNA barcodes for the determination of most German species of the genus Agonum, but also indicate a close relationship of a number of species with shared BINs, for example, Agonum scitulum (Dejean, 1828) and Agonum micans (Nicolai, 1822) (Suppl. material
In contrast to the previous species pair, a close relationship between Agonum impressum (Panzer, 1796) and Agonum sexpunctatum (Linne, 1758), as well as Agonum duftschmidi Schmidt, 1994 and Agonum emarginatum (Gyllenhal, 1827), has been already suggested in the past (
In comparison with other Central European ground beetles, for instance species of the genera Bembidion Latreille, 1802 (
At this point it should be kept in mind that not all beetles were collected in Germany. For instance, all specimens of Agonum antennarium (Duftschmid, 1812) were sampled in Montenegro (see Suppl. material
As central part of modern biodiversity research, DNA barcoding will become more and more prominent in this research field. Therefore, comprehensive DNA barcode libraries of important bio-indicators will become the backbone of any applied analysis, for example, metabarcoding as well as eDNA studies. The present study demonstrates the successful identification of most Agonum species documented for Germany by using DNA barcodes. The dataset also shows a close relationship between various species and their putative recent origin. Only the analysis of additional species and specimens, however, will reveal if the observed patterns of distinct clusters persist.
We would like to thank Christina Blume and Claudia Etzbauer (both
Barcode analysis using the BOLD workbench
Data type: Data table
Explanation note: Molecular distances are based on the Kimura 2-parameter model of the analysed specimens of the analysed species of the genus Agonum and Oxypselaphus obscurus (Paykull, 1790). 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 16-04-2020. Species pairs with interspecific distances < 2.2% are marked in bold. As a consequence of that the number of unspecified nucleotides (“Ns”) exceeds more than 1% of the total length of the sequence, barcodes of Agonum antennarium (Duftschmid, 1812) and A. ericeti (Panzer, 1809) received no BIN. Country codes: D = Germany, A = Austria, B = Belgium, BG = Bulgaria, CZ = Czech Republic, EST = Estonia, FIN = Finland, F = France, I = Italy, RO = Romania, SK = Slovakia, SLO = Slovenia, and SW = Switzerland.
Neighbour-joining topology
Data type: Neighbour-joining topology
Explanation note: Neighbour-joining topology of all analysed 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 %). Three beetles of a previous publication that were identified as Agonum ericeti (Panzer, 1809) (see