Species of the ground beetle genus Pterostichus Bonelli, 1810 are some of the most common carabids in Europe. This publication provides a first comprehensive DNA barcode library for this genus and allied taxa including Abax Bonelli, 1810, Molops Bonelli, 1810, Poecilus Bonelli, 1810, and Stomis Clairville, 1806 for Germany and Central Europe in general. DNA barcodes were analyzed from 609 individuals that represent 51 species, including sequences from previous studies as well as more than 198 newly generated sequences. The results showed a 1:1 correspondence between BIN and traditionally recognized species for 44 species (86%), whereas two (4%) species were characterized by two BINs. Three BINs were found for one species (2%), while one BIN for two species was revealed for two species pairs (8%). Low interspecific distances with maximum pairwise K2P values below 2.2% were found for four species pairs. Haplotype sharing was found for two closely related species pairs: Pterostichus adstrictus Eschscholtz, 1823/Pterostichus oblongopunctatus (Fabricius, 1787) and Pterostichus nigrita Paykull, 1790/Pterostichus rhaeticus Heer, 1837. In contrast to this, high intraspecific sequence divergences with values above 2.2% were shown for three species (Molops piceus (Panzer, 1793), Pterostichus panzeri (Panzer, 1805), Pterostichus strenuus (Panzer, 1793)). Summarizing the results, the present DNA barcode library does not only allow the identification of most of the analyzed species, but also provides valuable information for alpha-taxonomy as well as for ecological and evolutionary research. This library represents another step in building a comprehensive DNA barcode library of ground beetles as part of modern biodiversity research.

Abax, Central Europe, cytochrome c oxidase subunit I, German Barcode of Life, mitochondrial DNA, molecular specimen identification, Molops, Poecilus, Stomis

As part of the global International Barcode of Life initiative (IBoL; https://ibol.org), the German Barcode of Life initiative (GBoL; www.bolgermany.de) aims at capturing the genetic diversity of animals, fungi and plants of Germany using DNA barcodes in terms of modern biodiversity research (Hebert et al. 2003a, b). Despite the fact that various effects may limit the efficiency of a successful species identification, for example recent or ongoing hybridization events (e.g., Rougerie et al. 2012; Mutanen et al. 2016; Havemann et al. 2018), mitochondrial DNA-like sequences in the nucleus (numts) (e.g., Rogers and Griffiths-Jones 2012; Jordal and Kambestand 2014), or effects of Wolbachia infections (e.g., Smith et al. 2012; Klopfstein et al. 2016; Kolasa et al. 2018; Kajtoch et al. 2019), DNA barcoding has become the method of choice in terms of modern molecular species identification, including the identification of single specimens as well as metabarcoding of bulk samples (e.g., Casiraghi et al. 2010; Brandon-Mong et al. 2015). In recent years, various barcode libraries for numerous animal groups of Germany were established, including both marine and freshwater fish (Knebelsberger et al. 2014; Knebelsberger et al. 2015), amphibians and reptiles (Hawlitschek et al. 2016), echinoderms (Laakmann et al. 2017), molluscs (Gebhardt and Knebelsberger 2015; Barco et al. 2016), crustaceans (Raupach et al. 2015), spiders (Astrin et al. 2016), myriapods (Spelda et al. 2011), and numerous insect taxa, e.g., Coleoptera (Hendrich et al. 2015), Ephemeroptera, Plecoptera, Trichoptera (Morinière et al. 2017), Heteroptera (Raupach et al. 2014; Havemann et al. 2018), Hymenoptera (Schmidt et al. 2015; Schmidt et al. 2017; Schmid-Egger et al. 2019), Lepidoptera (Hausmann et al. 2011), Neuroptera (Morinière et al. 2014), and Orthoptera (Hawlitschek et al. 2017). Previous studies also laid the groundwork of a comprehensive DNA barcode library for the ground beetles (Coleoptera: Carabidae) of Germany (Raupach et al. 2010; Raupach et al. 2011; Hendrich et al. 2015; Raupach et al. 2016; Raupach et al. 2018; Raupach et al. 2019).

The Carabidae are a cosmopolitan family with an estimated number of probably more than 40,000 species world-wide (Lindroth 1986). The margined pronotum, large head, prominent mandibles, and striate elytra help to characterize this family (Arnett and Thomas 2000). These features, however, vary considerably throughout this taxon. Ground beetles can be found in all habitats except deserts and polar regions. Most adults of this family present a somber appearance, that is, a uniformly dark color, although some species are bi- or tricolored dorsally, and can have striking patterns (e.g., Callistus Bonelli, 1810, Omophron Latreille, 1802 or Panagaeus, Latreille, 1804). Adult ground beetles range in size from 2 up to 70 mm (genus Hyperion Castelnau, 1834). Most carabids are predators of invertebrates and consume many pest species, and are therefore typically considered as beneficial organisms (e.g., Lövei 1996). Within the Carabidae, the genus Pterostichus Bonelli, 1810 is a very large and diverse taxon with a Holarctic distribution, also reaching the Oriental and Neotropical regions (Hůrka 1996). More than 1,000 species are known world-wide to date, with more than 200 species are recorded for Europe (Hůrka 1996; Luff 2007) and 36 documented in Germany, including some of the commonest carabids of Germany, e.g., Pterostichus niger (Schaller, 1783), Pterostichus nigrita (Paykull, 1790), or Pterostichus strenuus (Panzer, 1796) (Trautner et al. 2014). The genus is in the present concept, however, undoubtedly not monophyletic and has been subdivided into numerous subgenera or sometimes even genera in the past (e.g., Lindroth 1986; Hůrka 1996; Luff 2007). Unfortunately, a thorough and comprehensive revision is still missing. In order to accommodate this situation, the subgeneric arrangement used in this study follows the traditional arrangement (see Marggi 2006). In general, adults of the genus Pterostichus have a body length between 5 to 25 mm, with most species above average. They have normally a somewhat uniform appearance with a strongly sclerotized and stout pronotum, thick antennae, and rather long legs with pronounced tibiae (Lindroth 1986). The overwhelming majority of species are carnivorous, night-active black-colored insects; those with a metallic coloration are often diurnal (Lindroth 1986) (Fig. 1). Due to the fact that various species of this genus represent important and highly abundant elements of the carabid fauna of many habitats world-wide (e.g., Igondová and Majzlan 2015; Hong et al. 2017; Baranová et al. 2018), they have been intensively studied in the past, for example their general ecology (e.g., Rushton et al. 1990; Fournier and Loreau 2001; Allema et al. 2012; Brigić et al. 2014), feeding strategies (e.g., Symondson et al. 2000; Langan et al. 2001; Dinis et al. 2016), and zoogeography/phylogeography (e.g., Lagisz et al. 2010; Schmidt et al. 2012; Sasakawa et al. 2017).

Figure 1. 

Representative images of analyzed beetle species A Abax parallelepipedus (Piller & Mitterbacher, 1783) B Molops piceus (Panzer, 1793) C Poecilus versicolor (Sturm, 1824) D Pterostichus (Eosteropus) aethiops (Panzer, 1796) E Pterostichus (Omaseus) melanarius (Illiger, 1798) F Pterostichus (Oreophilus) multipunctatus (Dejean, 1828) G Pterostichus (Bothriopterus) quadrifoveolatus Letzner, 1852 H Pterostichus (Argutor) vernalis Panzer, 1796 and I Stomis pumicatus (Panzer, 1795). Scale bars: 1 mm. All images were obtained from www.eurocarabidae.de.

In this study we present as part of the GBoL project a further step in generating a comprehensive DNA barcode library for the molecular identification of Central European ground beetle species, focusing on the genus Pterostichus and allied taxa. This barcode library included 36 species of Pterostichus as well as additional species of other genera belonging to the Pterostichini, including five species of the genus Abax Bonelli, 1810, three species of the genus Molops Bonelli, 1810, six species of the genus Poecilus Bonelli, 1810 and one species of the genus Stomis Clairville, 1806. In sum, 198 new barcodes were generated and a total number of 609 DNA barcodes examined in detail, including DNA barcodes of pinned museum specimens up to 39 years old.

Overall, 609 DNA barcode sequences of 51 ground beetle species of the Pterostichini were analyzed. A full list of the analyzed species is presented in the supporting information (Suppl. material 1). For the genus Pterostichus we analyzed 31 species which represent 86% of all recorded species (n = 36) of this genus for Germany. Beside this, the given sampling covered four species of the genus Abax (recorded species for Germany: n = 4, therefore 100%), two species of the genus Molops (n = 2, 100%), six species of the genus Poecilus (n = 6, 100%), and the only known species for Germany of the genus Stomis. Seven additional analyzed species are actually not recorded from Germany but included for comparison: Abax beckenhauptii (Duftschmid, 1812) (n = 3), Molops striolatus (Fabricius, 1801) (n = 2), Pterostichus adstrictus Eschscholtz, 1823 (n = 5), Pterostichus illigeri (Panzer, 1803) (n = 3), Pterostichus muehlfeldii (Duftschmid, 1812) (n = 3), Pterostichus schmidtii (Chaudoir, 1838) (n = 3), and Pterostichus ziegleri (Duftschmid, 1812) (n = 8).

In total, fragment lengths of the analyzed DNA barcode fragments ranged from 420 to 658 base pairs. As it is typically known for arthropods, the DNA barcode region was characterized by a high AT-content: the mean sequence compositions were A = 29.7%, C = 15.1%, G = 16.2%, and T = 39%. Intraspecific K2P distances ranged from 0 to a maximum of 3.15% (Molops piceus), whereas interspecific distances within the analyzed species had values between 0 and 11.19%. Lowest interspecific distances were found for Pterostichus adstrictus Eschscholtz, 1823 and Pterostichus oblongopunctatus (Fabricius, 1787) (0%; BIN: ABY4764) as well as Pterostichus nigrita Paykull, 1790 and Pterostichus rhaeticus Heer, 1837 (0%; BIN: AAM9738). In total, unique BINs were revealed for 44 species (86%), two BINs for two species (4%), three BINs for one species (2%) and one BIN for two species for two species pairs (8%). Due to the fact that the numbers of unspecified nucleotides (“Ns”) exceeds more than 1% of their total length, a distinct cluster of two sequences for Pterostichus panzeri (Panzer, 1803) received no BIN. The NJ analyses based on K2P distances revealed non-overlapping clusters with bootstrap support values > 95% for 40 species (78%) with more than one analyzed specimen (Fig. 2). A more detailed topology of all analyzed specimens is presented in the supporting information (Suppl. material 2).

Figure 2. 

Neighbor-joining (NJ) topology of the analyzed ground beetle species based on Kimura 2-parameter distances. Triangles show the relative number of individual’s sampled (height) and sequence divergence (width). Red 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). Images are provided for species recorded in Germany whereas asterisks indicate species not recorded in Germany. All beetle images were obtained from www.eurocarabidae.de except of Poecilus sericeus (photographer: Katja Neven, Lars Hendrich).

Our statistical maximum parsimony analysis showed multiple sharing of haplotypes for Pterostichus nigrita (n = 29)/Pterostichus rhaeticus (n = 11) and Pterostichus adstrictus (n = 5)/Pterostichus oblongopunctatus (n = 26) (Fig. 3). For Pterostichus nigrita and Pterostichus rhaeticus a number of 13 different haplotypes was found (Fig. 3A) One dominant haplotype (h1) was shared by 22 specimens of Pterostichus nigrita and three specimens of Pterostichus rhaeticus (Fig. 3A). Most other haplotypes, however, were revealed only for one specimen (singletons; Pterostichus nigrita: h5-h13, Pterostichus rhaeticus: h4) and located at the tips of the network, separated from haplotype h1 or other core haplotypes (h2, h3, h10) by up to nine additional mutational steps. In case of Pterostichus adstrictus and Pterostichus oblongopunctatus, the analysis identified six haplotypes (Fig. 3B). The dominant haplotype h1 was shared by four specimens of Pterostichus adstrictus and 21 specimens of Pterostichus oblongopunctatus, representing the 81% of the analyzed specimens. All others were connected to this haplotype by a maximum of four mutational steps in a star-like pattern, generating a compact network.

Figure 3. 

Maximum statistical parsimony networks of the sibling species pairs A Pterostichus nigrita Paykull, 1790 (green) and Pterostichus rhaeticus Heer, 1837 (blue) and B Pterostichus adstrictus Eschscholtz, 1823 (yellow) and Pterostichus oblongopunctatus (Fabricius, 1787) (red). 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 analyzed 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 www.eurocarabidae.de except Pterostichus adstrictus (photographer: Ditta Balke).

As a result of preservative, passionate and intensive work in the past centuries, carabid beetles have become one of the most prominent model groups of insects for biodiversity studies (Kotze et al. 2011). Generations of carabidologists clarified most aspects of their taxonomy and phylogeny, geographic distribution, habitat associations and ecological requirements, life history strategies and adaptations, in particular for those species found in Central Europe (Kotze et al. 2011). Due to the habitat specificity of various species, ground beetles are routinely used as biological indicators to assess land use changes among different ecosystems (Lövei and Sunderland 1996; Rainio and Niemelä 2003; Pearce and Venier 2004; Koivula 2011; Kotze et al. 2011).

The present study highlights the use of DNA barcodes for the identification of species of the five genera of Pterostichini found in Germany. Unique BINs were revealed for 44 species (86%) of the analyzed 51 taxa. This result coincides with high rates of successful species identification of previous barcoding studies in terms of carabid beetles (Raupach et al. 2010; Raupach et al. 2011; Pentinsaari et al. 2014; Hendrich et al. 2015; Raupach et al. 2018). Nevertheless, the data revealed some species pairs with low interspecific distances (< 2.2%) and shared haplotypes but also three species with intraspecific distances > 2.2%.

Species with high intraspecific variability

Intraspecific pairwise distances with values > 2.2% were found for three species. Whereas Pterostichus strenuus (Panzer, 1796) showed no conspicuous substructure for the analyzed COI sequences (see Suppl. material 2), three clearly distinct monophyletic cluster/lineages were revealed within Pterostichus panzeri (Panzer, 1803) (Fig. 4, Table 1) and Molops piceus (Panzer, 1793) (Fig. 5, Table 1), respectively, and will be discussed more in detail.

Table 1.

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Intraspecific Kimura 2-distances for all distinct clusters of Pterostichus panzeri (Panzer, 1805).

	Cluster A (Germany) BIN: ACC4332	Cluster B (Romania) BIN: n. a.	Cluster C (Austria) BIN: ACD0986
Cluster A (Germany) BIN: ACC4332	0
Cluster B (Romania) BIN: n. a.	0.014	0
Cluster C (Austria) BIN: ACD0986	0.019 – 0.023	0.018 – 0.02	0 – 0.005

Figure 4. 

Subtree of the Neighbor-joining topology based on Kimura 2-parameter distances of all analyzed specimens of Pterostichus panzeri (Panzer, 1805) 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 %). Cluster (A-C) with BINs (if available) based on the barcode analysis from 11-05-2020. Beetle image was obtained from www.eurocarabidae.de.

Figure 5. 

Subtree of the Neighbor-joining topology based on Kimura 2-parameter distances of all analyzed specimens of Molops piceus (Panzer, 1793) 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 %). Cluster (A-C) with BINs based on the barcode analysis from 11-05-2020. Beetle image was obtained from www.eurocarabidae.de.

The build-up of comprehensive DNA barcode libraries represents a pivotal task for modern molecular biodiversity research and species surveys (e.g., Brandon-Mong et al. 2015, Curry et al. 2018). This is especially true for the hyperdiverse and numerous species of insects. Within the beetles, carabids are highly valuable bioindicators that are used routinely to characterize disturbances in various habitats such as forests, meadows, river banks, or fens for a long time. Our DNA barcode library clearly encourages the application of DNA barcodes as effective method for the molecular identification of species of Pterostichus and allied taxa even if a few species pairs cannot be resolved. The given data, however, also revealed distinct lineages that correlate with high distances within a few species, indicating significant phylogeographic patterns and/or even the possible existence of overlooked cryptic species.

We would like to thank Christina Blume, Claudia Etzbauer (both ZFMK, Bonn) and Jana Deppermann (DZMB, Wilhelmshaven) for their laboratory assistance. Furthermore, we are grateful to Frank Köhler (Bonn), Karl-Hinrich Kielhorn (Berlin), and Wolfgang Lorenz (Tutzing) for providing various specimens, and to Ortwin Bleich for giving permission to use his excellent photographs of ground beetles taken from www.eurocarabidae.de. We also thank David Maddison and one anonymous reviewer for their helpful comments. 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 (Barcoding Fauna Bavarica) and the German Federal Ministry of Education and Research (GBOL1, GBOL2, GBOL3: 01LI1901B). 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.