The phylogeny of the carabid beetle supertribe Nebriitae is inferred from analyses of DNA sequence data from eight gene fragments including one nuclear ribosomal gene (28S), four nuclear-protein coding genes (CAD, topoisomerase 1, PEPCK, and wingless), and three mitochondrial gene fragments (16S + tRNA-Leu + ND1, COI (“barcode” region) and COI (“Pat/Jer” region)). Our taxon sample included 264 exemplars representing 241 species and subspecies (25% of the known nebriite fauna), 39 of 41 currently accepted genera and subgenera (all except Notiokasis and Archileistobrius), and eight outgroup taxa. Separate maximum likelihood (ML) analyses of individual genes, combined ML analyses of nuclear, nuclear protein-coding, and mitochondrial genes, and combined ML and Bayesian analyses of the eight-gene-fragment matrix resulted in a well-resolved phylogeny of the supertribe, with most nodes in the tree strongly supported. Within Nebriitae, 167 internal nodes of the tree (out of the maximum possible 255) are supported by maximum-likelihood bootstrap values of 90% or more. The tribes Notiophilini, Opisthiini, Pelophilini, and Nebriini are well supported as monophyletic but relationships among these are not well resolved. Nippononebria is a distinct genus more closely related to Leistus than Nebria. Archastes, Oreonebria, Spelaeonebria, and Eurynebria, previously treated as distinct genera by some authors, are all nested within a monophyletic genus Nebria. Within Nebria, four major clades are recognized: (1) the Oreonebria Series, including eight subgenera arrayed in two subgeneric complexes (the Eonebria and Oreonebria Complexes); (2) the Nebriola Series, including only subgenus Nebriola; (3) the Nebria Series, including ten subgenera arrayed in two subgeneric complexes, the Boreonebria and Nebria Complexes, with the latter further subdivided into three subgeneric subcomplexes (the Nebria, Epinebriola, and Eunebria Subcomplexes)); and (4) the Catonebria Series, including seven subgenera arrayed in two subgeneric complexes (the Reductonebria and Catonebria Complexes). A strong concordance of biogeography with the inferred phylogeny is noted and some evident vicariance patterns are highlighted. A revised classification, mainly within the Nebriini, is proposed to reflect the inferred phylogeny. Three genus-group taxa (Nippononebria, Vancouveria and Archastes) are given revised status and seven are recognized as new synonymies (Nebriorites Jeannel, 1941 and Marggia Huber, 2014 = Oreonebria Daniel, 1903; Pseudonebriola Ledoux & Roux, 1989 = Boreonebria Jeannel, 1937; Patrobonebria Bänninger, 1923, Paranebria Jeannel, 1937 and Barbonebriola Huber & Schmidt, 2017 = Epinebriola Daniel & Daniel, 1904; and Asionebria Shilenkov, 1982 = Psilonebria Andrewes, 1923). Six new subgenera are proposed and described for newly recognized clades: Parepinebriola Kavanaugh subgen. nov. (type species: Nebria delicata Huber & Schmidt, 2017), Insulanebria Kavanaugh subgen. nov. (type species: Nebria carbonaria Eschscholtz, 1829), Erwinebria Kavanaugh subgen. nov. (type species Nebria sahlbergii Fischer von Waldheim, 1828), Nivalonebria Kavanaugh subgen. nov. (type species: Nebria paradisi Darlington, 1931), Neaptenonebria Kavanaugh subgen. nov. (type species: Nebria ovipennis LeConte, 1878), and Palaptenonebria Kavanaugh subgen. nov. (type species: Nebria mellyi Gebler, 1847). Future efforts to better understand relationships within the supertribe should aim to expand the taxon sampling of DNA sequence data, particularly within subgenera Leistus and Evanoleistus of genus Leistus and the Nebria Complex of genus Nebria.

DNA, evolutionary tree, ground beetles, molecular phylogenetics, nomenclature, systematics, taxonomy

The carabid beetle supertribe Nebriitae is a moderately diverse group comprised of small to medium-sized beetles, varied in form (see Figs 1–3), but all sharing a suite of morphological features most of which are plesiomorphic among carabids. Most recognizable of these features include mandibles with a scrobal seta present, procoxal cavities open and confluent, mesocoxal cavities disjunct and confluent, metacoxal cavities conjunct and confluent, and protibiae with a simple, sulcate antennal cleaner (Grades “A” and “B” of Hlavac 1971) and both tibial spurs inserted apically or nearly so. At present, the supertribe includes nearly 1,100 described species and subspecies assigned to five tribes. These include the Notiophilini, Notiokasiini, Opisthiini, Pelophilini, and Nebriini.

Among these, Notiokasiini is the least diverse, represented by a single known species (Fig. 1A) in the genus Notiokasis Kavanaugh & Nègre, 1983, and most geographically divergent, known only from two localities in the Neotropical Region, one in Brazil and one in Uruguay. Virtually nothing is known about the habitat or life history of members of this group, and, as far as we are aware, no specimens have been collected in at least 60 years.

Figure 1. 

Habitus images of Nebriitae A Notiokasis chaudoiri Kavanaugh & Nègre B Pelophila borealis (Paykull) C Opisthius richardsoni Kirby D Paropisthius indicus chinensis Bousquet & Smetana E Notiophilus palustris Duftschmid F Archileistobrius hwangtienyuni Shilenkov & Kryzhanovskij. Scale bars: 1.0 mm. Photograph credits: A, C David Maddison; B, E Kiril Makarov; D, F Alexander Anischenko.

Pelophilini includes only two described species, both in genus Pelophila Dejean, 1821 (Fig. 1B), one with a Holarctic distribution, the other apparently strictly Nearctic. Both species live in boreal and subarctic areas, typically in moist to wet habitats along the margins of streams, lakes and marshes.

Opisthiini is comprised of six described species-group taxa, with one species in the Nearctic genus Opisthius Kirby, 1837 (Fig. 1C) and four species and one additional subspecies in the Palearctic genus Paropisthius Casey, 1920 (Fig. 1D). Adults and larvae of Opisthius are found on the bare or sparsely vegetated and exposed banks of medium-sized to large streams with soft clay or sandy soil and at least some rocky cover for hiding. Paropisthius adults are found under stones and other cover in open disturbed habitats, such as road-cuts, trail margins, and landslides at middle to high elevations. Adults of both genera are active mainly at night but occasionally can be found active during daylight hours.

Notiophilini includes ca. 60 described species and six additional subspecies, all in genus Notiophilus Duméril, 1806 (Fig. 1E). Members of all species share a body form that is unique among Carabidae and easily recognizable. Two species are Holarctic in distribution, 15 species (two of which have been introduced from Europe) occur in the Nearctic Region, two species are found in the mountainous northern part of the Neotropical Region in Middle America, and the remaining species occur in the Palearctic Region. Members of this group occur mainly in open exposed habitats or those with thin forest cover, often on gravel substrate. They prefer drier conditions than almost all other nebriites and are active in daytime, even in direct sunlight, as well as at night.

Nebriini is the most diverse tribe of the supertribe with ca. 1,000 described species and subspecies, and additional new species are discovered, mainly in Asia, almost every year. The tribe is Holarctic in distribution, with more than 90% of the described species-group taxa and most of the genus-group taxa found in the Palearctic Region. The number of extant genera currently recognized as distinct ranges from four to seven, depending on the authors. At present, the widely accepted genera include Leistus Frölich, 1799, Nebria Latreille, 1802, Archastes Jedlička, 1935, and Archileistobrius Shilenkov & Kryzhanovskij, 1983. Lorenz (2005) and Huber (2017) considered Eurynebria Ganglbauer, 1891b, Oreonebria Daniel, 1903, and Nippononebria Uéno, 1955 as distinct genera rather than as subgenera of Nebria as ranked by Ledoux and Roux (2005). Lorenz treated Archileistobrius as a junior synonym of Archastes, but Huber (2017) followed Ledoux and Roux (2009) in maintaining these as separate genera. Habu (1958), Kavanaugh (1995, 1996) and Bousquet (2012) have also treated Nippononebria as a distinct genus.

Archileistobrius (Fig. 1F) includes a single described species, A. hwangtienyuni Shilenkov & Kryzhanovskij, 1983, known only from a single mountain, Emei Shan, in Sichuan Province, China.

Genus Leistus (Fig. 2A, B) currently includes ca. 250 species and 25 additional subspecies arranged among six subgenera. The genus is Holarctic in distribution but only four species occur in the Nearctic Region and one of these has been introduced from Europe, so the genus is predominantly Palearctic. All species share a distinctive suite of modifications to the mouthparts, the ventral face of the head, and the setae and their insertion points associated with these structures that together form a basket thought to function in small prey (e.g., springtails) capture (see Bauer 1985). Different Leistus species occupy a broad elevational range, occurring in moist woodlands at various elevations and also above treeline at the edges of alpine streams and persistent snowfields.

Archastes (Fig. 2C) includes 37 described species and an additional five subspecies. Its known geographical range is confined to southcentral China, including only Sichuan, Gansu, Shaanxi, and Ningxia provinces, mainly in mountain ranges along the dissected southern edge of the Tibetan Plateau. Species occur in both forested areas and alpine steppes and talus slopes.

Figure 2. 

Habitus images of Nebriini A Leistus (Nebrileistus) nubivagus Wollaston B L. (Leistus) ferruginosus Mannerheim C Archastes solitarius (Ledoux & Roux) D Nippononebria (Vancouveria) virescens (Horn) E Nebria (Oreonebria) castanea Bonelli F N. (Eurynebria) complanata (Linnaeus). Scale bars: 1.0 mm. Photograph credits: A, D–F David Maddison; B, C Alexander Anischenko.

Nebria (Figs 2D–3F) is by far the most diverse genus within the tribe Nebriini with nearly 500 species and more than 100 additional subspecies described. Of these, 82 species occur in the Nearctic Region, including one introduced from Europe, and the remainder can be found in the Palearctic and northern Indomalayan regions. Members of the genus occupy cool to cold habitats from sea level to more than 5500 m in elevation, including sea beaches, the open or shaded shores of streams and lakes, the edges of persistent snowfields and glaciers, alpine talus slopes and fellfields, and even alpine caves. This diversity is arrayed among 27 currently accepted subgenera (see Table 1). As noted above, three of these have been considered separate genera by some authors. If treated as a separate genus, Nippononebria (Fig. 2D) includes nine described species and one additional subspecies in two subgenera: (1) the nominate subgenus, comprised of six species and one subspecies and known only from Japan and Jilin Province on the Chinese mainland; and (2) subgenus Vancouveria Kavanaugh, 1995, comprised of three described species and restricted to western North America. Members of this group range from moist lowland forests to the margins of alpine streams, seeps, and snowfields at and above treeline. Oreonebria (Fig. 2E) includes 14 described species and 13 additional subspecies, all restricted to the Alps mountain system of Europe and all with reduced hindwings. Eurynebria (Fig. 2F) includes a single species, E. complanata (Linnaeus), which, at least historically, inhabited sandy sea beach habitat along the Atlantic and Mediterranean coasts of southern Europe (from the southern British Isles to Turkey), and northern Africa (at least from Morocco to Tunisia).

Figure 3. 

Habitus images of Nebria A N. (Eonebria) djakonovi Semenov & Znojko B N. (Orientonebria) coreica Solsky C N. (Spelaeonebria) nudicollis Peyerimhoff D N. (Psilonebria) superna Andrewes E N. (Reductonebria) ochotica Sahlberg F N. (Catonebria) banksii Crotch. Scale bars: 1.0 mm. Photograph credits: A, B, F Kiril Makarov; C, D David Maddison; E Alexander Anischenko.

Two additional nebriine genera have been described from fossil remains. Ledouxnebria Deuve (1998) was described from diatomite deposits in the Massif Central of France and dated to the Upper Miocene (10 to 5 million years ago). Archaeonebria Kavanaugh and Schmidt (in Schmidt et al. 2019) was described from Baltic amber and dated to the Eocene (50–35 Mya). The latter appears to be a stem lineage of the Nippononebria clade (Schmidt et al. 2019), but the affinities of Ledouxnebria are unclear.

The taxonomic history of nebriite carabids began with Linnaeus, who, in the Tenth Edition of his Systema Naturae (Linnaeus 1758), described one species in his genus Cicindela (C. aquaticus) and two in his genus Carabus (C. ferruginosus and C. lividus). Later, Linnaeus (1767) added a fourth species destined to be included among nebriites: Carabus complanatus. Three of these four would later be designated as the type species of different nebriite genera. All four of them occurred in Europe or North Africa, where new species continue to be discovered (e.g., Farkač et al. 2010; Szallies and Huber 2014). Description of endemic Nearctic nebriite species began with Elaphrus aeneus Herbst (1806) (now included in Notiophilus), Nebria pallipes Say (1823), Leistus ferrugineus Dejean (1831) (= L. ferruginosus Mannerheim, 1843, not L. ferrugineus (Linnaeus, 1758)), Opisthius richardsoni Kirby (1837), and Nebria rudis LeConte (1863) (now included in Pelophila). The Nearctic fauna is now reasonably well-known, but, again, with occasional additions (e.g., Kavanaugh and Schoville 2009, Kavanaugh 2015). Knowledge of the nebriite fauna of Asia was slower and generally later to develop, and the fauna of that vast and largely inaccessible region is still poorly known. Early descriptions for each genus from Asia included Nebria carbonaria Eschscholtz, 1829, Leistus niger Gebler, 1847, Opisthius indicus Chaudoir, 1863 (now included in Paropisthius), Archastes sterbai Jedlička (1935), and Archileistobrius hwangtienyuni Shilenkov & Kryzhanovskij, 1983. As shown graphically by Ledoux and Roux (2005:35), the accumulation curve for described species and subspecies of Nebria has still not begun to level off. The same is true for Leistus and Archastes species, with the main increases for each due to the discovery of new taxa from previously unsampled parts of Asia. Just a few examples of recent major contributions to the knowledge of this fauna include Deuve (2009, 2010, 2011), Dudko (2006), Dudko and Shilenkov (2001), Huber and Schmidt (2007, 2017, 2018) and Ledoux and Roux (2005, 2009). The last nebriite fauna to be described was that of the Neotropical Region, which currently includes only three species: two Notiophilus species (N. specularis Bates, 1881 and N. chihuahuae Casey, 1920) and Notiokasis chaudoiri Kavanaugh & Nègre, 1983.

As the number of described species increased rapidly in the 19^th and 20^th centuries and it became clear that groups of more or less similar species could be recognized, the description of nebriite genera and subgenera began, first through extracting species from genus Carabus of Linnaeus and then adding new genus-group taxa to accommodate species unknown to Linnaeus as needed. The first of these described was genus Leistus Frölich, 1799, followed by Nebria Latreille, 1802 and Notiophilus Duméril, 1805, and each of these included Linnaean species. Subsequently, Pelophila Dejean, 1821, Opisthius Kirby, 1837, Eurynebria Ganglbauer, 1891b, Paropisthius Casey, 1920, Archastes Jedlička, 1935, Nippononebria Uéno, 1955, Notiokasis Kavanaugh & Nègre, 1983, and Archileistobrius Shilenkov & Kryzhanovskij, 1983 were described. For three of these genera, one or more additional subgenera have been proposed. Barševskis (1994) proposed two new subgenera for Notiophilus, but these have not been widely accepted and are currently considered as junior synonyms of Notiophilus. A total of 13 additional subgenera has been proposed for Leistus; but in an excellent series of papers published from 1979 through 1994, Perrault treated the genus worldwide and recognized only six subgenera, including Leistus s. str. (Perrault 1980, 1991). His subgeneric classification is the currently accepted standard (Lorenz 2005). In total, 37 additional genus-group names have been proposed for Nebria in the broad sense. Of these, Ledoux and Roux (2005) recognized 27 as valid subgenera, the rest as junior synonyms or junior homonyms and one as a nomen nudum. As noted above, three of these have been considered as distinct genera by some authors, but only two of them (Nebria and Eurynebria) were originally described as distinct genera. See Table 1 for a list of all published nebriite genus-group names and their current status.

The suprageneric classification of nebriites has been relatively stable during the last century and a half compared with that of many other carabid groups, although the ranking and inclusiveness of various taxa has changed somewhat through time. The post-Linnaean era of carabid classification, which Ball (1979) has called the “Latreillean Period”, began with Latreille (1802), when he introduced the family name, Carabidae. He grouped the two nebriite genera known to him (Pogonophorus and Nebria, both newly described in that work) in his “Celerigrades” (equivalent to a subfamily rank) and then in his “Barbus” (equivalent to a tribal rank). Also included in “Barbus” were Loricera Fabricius and Omophron (also newly described in the same work). Laporte de Castelnau (1834) was the first to use the names Nébriites and Nebriidae. His concept of the group was broader than at present, including Notiophilus, Pelophila, Leistus and Nebria, the nebriite genera known at that time, but also Metrius Eschscholtz, Omophron Latreille, Elaphrus Fabricius, Blethisa Bonelli, Notiobia Perty, and even Pteroloma Dejean (now included in the polyphagan family Agyrtidae). Motschulsky (1850) considered Notiophilus as a separate tribe and Dupuis (1912) did the same for Opisthius, whereas Horn (1881) included both of these genera in his “Nebriini”. For Thomson (1859), Ganglbauer (1891a), Reitter (1908), Ball (1960) and Lindroth (1961), among others, Nebriini included only Pelophila, Leistus, and Nebria (although Ganglbauer also treated Eurynebria as a distinct genus within Nebriini). Kavanaugh and Nègre (1983) introduced the tribe Notiokasiini; and Kavanaugh (1996) proposed that Pelophila should be recognized as a tribe separate from the Nebriini. The group as whole and as presently comprised has been ranked as a single tribe, the Nebriini (Horn 1881), a supertribe, the Nebriitae (Kryzhanovskij 1976; Erwin 1985), a subfamily, the Nebriinae (Laporte de Castelnau 1834; Ball and Bousquet 2001; Lorenz 2005; Bousquet 2012; Löbl and Löbl 2017), and as a family, the Nebriidae (Jeannel 1937; Ledoux and Roux 2005). In urging use of the supertribe as a rank between tribe and subfamily, Kryzhanovskij (1978) argued against the excessive proliferation of subfamilies and for a more fine-grained hierarchic classification for groups of closely related tribes, especially within the main subfamilies, Carabinae and Harpalinae. We follow Kryzhanovskij’s suggestion for ranking in this presentation.

Few studies have explored phylogenetic relationships among the nebriite taxa through formal analyses and most of them have used morphological data. Kavanaugh (1978) used purely “manual” methods (i.e., without the aid of computer algorithms) proposed by Hennig (1966) to analyze phylogenetic relationships among the Nearctic Nebria and closely related Palearctic species. He mainly used the “outgroup comparison method” (Hennig 1966; see also Watrous and Wheeler 1981) and parsimony on a character-by-character basis to establish the polarity of transformation series of character states for each of 318 morphological and life history characters. Outgroup sampling included representatives of most adephagan families recognized at that time, most carabid tribes, all nebriite genera and most subgenera, including endemic Palearctic Nebria subgenera (see appendix C in Kavanaugh 1978 for a list of outgroup taxa and the text for methods of tree construction and optimization). The resulting phylogenetic hypothesis suggested that the Nearctic Nebria fauna included representatives of only four monophyletic groups, corresponding to the subgenera Nippononebria, Boreonebria Jeannel, 1937, Reductonebria Shilenkov, 1975 and Catonebria Shilenkov, 1995, and that Nippononebria was sister to a group including all the other subgenera of Nebria. Later, Kavanaugh (1996) reported on the results of a parsimony analysis of morphological data for all nebriite genera (except Archileistobrius) and selected Nebria subgenera with a small set of outgroup taxa. The monophyly of supertribe Nebriitae was well supported in this analysis, but that of tribe Nebriini including Pelophila was not, so a new tribe, Pelophilini, was proposed. A clade including Nippononebria and Leistus but not Nebria was also supported, which served as the basis for subsequent assertion of Nippononebria as a genus distinct from Nebria (Kavanaugh 1995). However, results from a second parsimony analysis (Kavanaugh 1998), with an expanded set of outgroup taxa but a reduced set of morphological characters, found the Nebriitae to be paraphyletic and Pelophila to be more closely related to the nebriine genera than a clade including Notiokasiini, Notiophilini, and Opisthiini. These conflicting results from different analyses and datasets were interpreted as resulting more from homoplasy in many of the morphological characters examined than to differences in the composition of the outgroup or suites of characters included. Ledoux and Roux (2005) provided a reclassification of Nebria worldwide based primarily on a custom-built, distance-based clustering method, which used thresholds for defining boundaries between taxa; they created useful groupings by adjusting the values of these thresholds. They recognized clusters and isolated outliers as subgenera, a few of which were monospecific and several of which they described as new, and their classification represents a major advance in our understanding of the most diverse genus of nebriites. They too found homoplasy in morphological characters traditionally used to characterize subgenera of Nebria as a major impediment to phylogenetic reconstruction.

Until now, the use of molecular data for phylogenetic studies of nebriites has been very limited. Clarke et al. (2001) examined phylogenetic relationships within the gregaria infragroup of Nebria in western North America using a rapid amplified polymorphic DNA (RAPD) analysis of molecular data from five mitochondrial DNA regions. Kavanaugh et al. (2011) used morphological, morphometric, and molecular sequence data from four nuclear and two mitochondrial genes to investigate whether Nebria lacustris Casey (1913) represented one or more distinct species. Schoville et al. (2012), Weng et al. (2016) and Weng et al. (2020) have published phylogeographic analyses of several species of Nebria from high elevations in the Sierra Nevada of California and Taiwan and the western North American species of Nippononebria, respectively. Raupach et al. (2019) presented a first analysis of phylogenetic relationships among 15 Notiophilus species and their putative colonization of the continents based on sequence data from the COI barcode region of mitochondrial DNA. The phylogenetic analyses that we present here are the first for the Nebriitae as a whole based on DNA sequence data.

Working with insights gained from phylogenetic studies based on morphological data gathered during the past 50 years and guided by Perrault’s (1980, 1991) classification of Leistus and Ledoux and Roux’s (2005) phylogenetic classification of Nebria for sampling the two most diverse elements of the nebriite fauna worldwide, the lead author (DHK) began collecting material suitably preserved for DNA extraction in mid-1990’s. Co-authors Faille, Maddison, Schmidt, and Schoville collected and subsequently provided important additional samples. We stopped collecting and accepting additional materials for these analyses at the end February 2017. In this work, we provide: (1) the results of our analyses and their implications for the current nebriite phylogenetic hypothesis and classification; (2) the evidence for or against the monophyly of selected nebriite taxa at all levels in the classification; (3) proposed changes to nebriite classification based on our findings; and (4) suggestions for some next steps that would expand and improve on these research results.