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Research Article
Phylogeny of the supertribe Nebriitae (Coleoptera, Carabidae) based on analyses of DNA sequence data
expand article infoDavid H. Kavanaugh, David R. Maddison§, W. Brian Simison, Sean D. Schoville|, Joachim Schmidt, Arnaud Faille#, Wendy Moore¤, James M. Pflug§, Sophie L. Archambeault«, Tinya Hoang, Jei-Ying Chen»
‡ California Academy of Sciences, San Francisco, United States of America
§ Oregon State University, Corvallis, United States of America
| University of Wisconsin, Madison, United States of America
¶ University of Rostock, Rostock, Germany
# Stuttgart State Museum of Natural History, Stuttgart, Germany
¤ University of Arizona, Tucson, United States of America
« University of California, Berkeley, United States of America
» University of California, Santa Cruz, United States of America
Open Access

Abstract

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.

Keywords

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

Introduction

The carabid beetle supertribe Nebriitae is a moderately diverse group comprised of small to medium-sized beetles, varied in form (see Figs 13), 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 2D3F) 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).

Table 1.

Genus-group names in supertribe Nebriitae. All published genus-group names are represented in this list in order of publication and include author, year of publication, type species and current taxonomic status (Ledoux and Roux 2005; Lorenz 2005). The “Type” column indicates if the type species for the name is represented (Y) or not (N) in our taxon sample. The “Div” column lists the number of known species for each valid genus-group name. The “Rep” column indicates how many species-group taxa are represented in our taxon sample, and the “% Rep” column indicates what percentage of the known diversity is represented in our taxon sample. The “Proposed status” column indicates the appropriate status of the names based on the results of our study, with any changes in status noted in the “Result” column.

Genus group name Type species Current status Type Div Rep % Rep Proposed status Result
Leistus Frölich, 1799 Leistus testaceus Frölich = Carabus ferrugineus Linnaeus valid genus and subgenus Y 52 11 21 valid genus and subgenus
Pogonophorus Latreille, 1802 Carabus spinibarbis Fabricius valid subgenus of Leistus N 60 3 5 valid subgenus of Leistus
Nebria Latreille, 1802 Carabus brevicollis Fabricius valid genus and subgenus Y 109 6 6 valid genus and subgenus
Manticora Panzer, 1803 Manticora pallipes Panzer = Leistus spinibarbis Fabricius junior homonym of Manticora Fabricius 1792 N junior homonym of Manticora Fabricius 1792
Notiophilus Duméril, 1805 Cicindela aquatica Linnaeus valid genus N 66 4 6 valid genus
Alpaeus Bonelli, 1810 Carabus hellwigii Panzer subjective junior synonym of Nebria s. str. N subjective junior synonym of Nebria s. str.
Pelophila Dejean, 1821 Carabus borealis Paykull valid genus Y 2 2 100 valid genus
Helobia Stephens, 1828 Carabus brevicollis Fabricius objective junior synonym of Nebria s. str. Y objective junior synonym of Nebria s. str.
Eonebria Semenov & Znojko, 1828 Nebria komarovi Semenov & Znojko valid subgenus of Nebria Y 77 6 7 valid subgenus of Nebria
Opisthius Kirby, 1837 Opisthius richardsoni Kirby valid genus Y 1 1 100 valid genus
Harpazobia Gistel, 1856 Carabus brevicollis Fabricius objective junior synonym of Nebria s. str. Y objective junior synonym of Nebria s. str.
Eurynebria Ganglbauer, 1891 Carabus complanatus Linnaeus valid subgenus of Nebria Y 1 1 100 valid subgenus of Nebria
Leistidius Daniel, 1903 Leistus piceus Frölich subjective junior synonym of Leistus s. str. N subjective junior synonym of Leistus s. str.
Oreonebria Daniel, 1903 Alpaeus castaneus Bonelli valid subgenus of Nebria Y 26 9 35 valid subgenus of Nebria
Oreobius Daniel, 1903 Leistus gracilis Fuss subjective junior synonym of Pogonophorus N subjective junior synonym of Pogonophorus
Nebriola Daniel, 1903 Nebria laticollis Dejean valid subgenus of Nebria Y 19 2 11 valid subgenus of Nebria
Epinebriola Daniel & Daniel, 1904 Nebria oxyptera Daniel & Daniel valid subgenus of Nebria Y 28 6 21 valid subgenus of Nebria
Chaetoleistus Semenov,1904 Leistus relictus Semenov subjective junior synonym of Pogonophorus N subjective junior synonym of Pogonophorus
Acroleistus Reitter, 1905 Leistus denticollis Reitter subjective junior synonym of Leistus s. str. N subjective junior synonym of Leistus s. str.
Euleistulus Reitter, 1905 Leistus ellipticus Reitter = Leistus fulvus Chaudoir subjective junior synonym of Leistus s. str. Y subjective junior synonym of Leistus s. str.
Leistophorus Reitter, 1905 Leistus fulvibarbis Dejean subjective junior synonym of Leistus s. str. Y subjective junior synonym of Leistus s. str.
Spelaeonebria Peyerimhoff, 1911 Spelaeonebria nudicollis Peyerimhoff valid subgenus of Nebria Y 2 1 50 valid subgenus of Nebria
Eurinoleistus Breit, 1914 Leistus depressus Breit subjective junior synonym of Pogonophorus N subjective junior synonym of Pogonophorus
Paropisthius Casey, 1920 Opisthius indicus Chaudoir valid genus Y 5 4 80 valid genus
Patrobonebria Bänninger, 1923 Nebria desgodinsi Oberthür valid subgenus of Nebria Y 11 4 36 subjective junior synonym of Epinebriola New Synonymy
Psilonebria Andrewes, 1923 Nebria superna Andrewes valid subgenus of Nebria Y 4 2 50 valid subgenus of Nebria
Nebrileistus Bänninger, 1925 Leistus ellipticus Wollaston valid subgenus of Leistus N 2 1 50 valid subgenus of Leistus
Archastes Jedlička, 1935 Archastes sterbai Jedlička valid genus N 42 1 2 valid subgenus of Nebria New Status
Boreonebria Jeannel, 1937 Carabus rufescens Ström = Carabus gyllenhali Schönherr valid subgenus of Nebria Y 42 19 45 valid subgenus of Nebria
Eunebria Jeannel, 1937 Carabus psammodes Rossi valid subgenus of Nebria Y 54 15 28 valid subgenus of Nebria
Paranebria Jeannel, 1937 Carabus lividus Linnaeus valid subgenus of Nebria Y 3 2 67 subjective junior synonym of Epinebriola New Synonymy
Neonebria Hatch, 1939 none designated nomen nudum nomen nudum
Nebriorites Jeannel, 1941 Alpaeus gagates Bonelli valid subgenus of Nebria Y 2 1 50 valid subgenus of Nebria
Alpaeonebria Csiki, 1946 Nebria fuscipes Fuss valid subgenus of Nebria N 32 1 3 subjective junior synonym of Nebria s. str. New Synonymy
Nippononebria Uéno, 1955 Nebria pusilla Uéno valid subgenus of Nebria N 7 2 29 valid genus and subgenus New Status
Agonoamara Jedlička, 1962 Agonoamara chujoi Jedlička = Nebria chalceola Bates subjective junior synonym of Nippononebria Y subjective junior synonym of Nippononebria s. str.
Evanoleistus Jedlička, 1965 Leistus nepalensis Jedlička valid subgenus of Leistus N 154 9 5 valid subgenus of Leistus
Neoleistus Erwin, 1970 Leistus ferruginosus Mannerheim valid subgenus of Leistus Y 3 3 100 subjective junior synonym of Leistus s. str. New Synonymy
Germaria Jeanne, 1972 Nebria bremii Germar junior homonym of Germaria Robineau-Desvoidy, 1830 Y junior homonym of Germaria Robineau-Desvoidy, 1830
Catonebria Shilenkov, 1975 Carabus nitidulus Fabricius = Nebria banksii Crotch valid subgenus of Nebria Y 42 39 93 valid subgenus of Nebria
Orientonebria Shilenkov, 1975 Nebria coreica Solsky valid subgenus of Nebria Y 1 1 100 valid subgenus of Nebria
Reductonebria Shilenkov, 1975 Nebria ochotica Sahlberg valid subgenus of Nebria Y 17 15 83 valid subgenus of Nebria
Sardoleistus Perrault, 1980 Leistus sardous Baudi di Selve valid subgenus of Leistus Y 1 1 100 valid subgenus of Leistus
Tetungonebria Shilenkov, 1982 Nebria tetungi Shilenkov subjective junior synonym of Eunebria N incertae sedis
Asionebria Shilenkov, 1982 Nebria roborowskii Semenov valid subgenus of Nebria Y 9 3 33 subjective junior synonym of Psilonebria New Synonymy
Notiokasis Kavanaugh & Nègre, 1983 Notiokasis chaudoiri Kavanaugh & Nčgre valid genus N 1 0 0 valid genus
Archileistobrius Shilenkov & Kryzhanovskij, 1983 Archileistobrius hwangtienyuni Shilenkov & Kryzhanovskij valid genus N 1 0 0 incertae sedis
Germarina Jeanne, 1985 Nebria bremii Germar junior homonym of Germarina Mesnil 1963 Y junior homonym of Germarina Mesnil 1963
Himalayonebria Ledoux, 1985 Nebria nouristanensis Ledoux subjective junior synonym of Epinebriola N incertae sedis
Pseudonebriola Ledoux & Roux, 1989 Nebria saurica Shilenkov valid subgenus of Nebria N 18 7 39 subjective junior synonym of Boreonebria New Synonymy
Latviaphilus Barsevskis, 1994 Elaphrus biguttatus Fabricius subjective junior synonym of Notiophilus N subjective junior synonym of Notiophilus
Makarovius Barsevskis, 1994 Notiophilus rufipes Curtis subjective junior synonym of Notiophilus N subjective junior synonym of Notiophilus
Sphodronebria Sciaky & Pavesi, 1994 Nebria paradoxa Sciaky & Pavesi = Nebria tetungi Shilenkov objective junior synonym of Tetungonebria N objective junior synonym of Tetungonebria
Vancouveria Kavanaugh, 1995 Nebria virescens Horn valid subgenus of Nebria Y 3 3 100 valid subgenus of Nippononebria
Ledouxnebria Deuve, 1998 Ledouxnebria brisaci Deuve valid genus (fossil only) 1 valid genus (fossil only)
Epispadias Ledoux & Roux, 1999 Nebria janschneideri Ledoux & Roux valid subgenus of Nebria N 2 1 50 valid subgenus of Nebria
Falcinebria Ledoux & Roux, 2005 Nebria reflexa Bates valid subgenus of Nebria Y 16 2 13 valid subgenus of Nebria
Nakanebria Ledoux & Roux, 2005 Nebria kurosawai Nakane valid subgenus of Nebria N 6 1 17 valid subgenus of Nebria
Sadonebria Ledoux & Roux, 2005 Nebria sadona Bates valid subgenus of Nebria N 17 4 24 valid subgenus of Nebria
Tyrrhenia Ledoux & Roux, 2005 Carabus rubicundus Quesnel valid subgenus of Nebria Y 18 2 11 valid subgenus of Nebria
Marggia Huber, 2014 Nebria bremii Germar valid subgenus of Nebria Y 2 1 50 subjective junior synonym of Oreonebria New Synonymy
Barbonebriola Huber & Schmidt, 2017 Nebria barbata Andrewes valid subgenus of Nebria N 6 1 17 subjective junior synonym of Epinebriola New Synonymy
Archaeonebria Kavanaugh & Schmidt, 2019 Archaeonebria inexspectata Schmidt & Kavanaugh valid genus (fossil only) 1 valid genus (fossil only)
Parepinebriola subgen. nov. Nebria delicata Huber & Schmidt part of Epinebriola Daniel Y 5 5 ? new subgenus New Subgenus
Insulanebria subgen nov. Nebria snowi Bates part of Reductonebria Shilenkov Y 2 2 100 new subgenus New Subgenus
Erwinebria subgen. nov. Nebria sahlbergii Fisher von Waldheim part of Reductonebria Shilenkov Y 21 20 95 new subgenus New Subgenus
Nivalonebria subgen. nov. Nebria paradisi Darlington part of Nakanebria Ledoux & Roux Y 2 2 100 new subgenus New Subgenus
Neaptenonebria subgen. nov. Nebria ovipennis LeConte part of Catonebria Shilenkov Y 6 6 100 new subgenus New Subgenus
Palaptenonebria subgen. nov. Nebria mellyi Gebler part of Catonebria Shilenkov Y 14 11 79 new subgenus New Subgenus
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 19th and 20th 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.

Materials and methods

Taxon sampling

In total, 264 exemplar specimens were used in this study. These included representatives of 241 different nebriite species-group taxa plus 14 replicates and eight outgroup taxa plus one replicate (Table 2). Locality data for these specimens are provided in Appendix A. Outgroup selection was based on morphological phylogenetic studies by Kavanaugh (1978, 1996, 1998) and the molecular phylogenetic study by Maddison et al. (1999), subject to the availability of material suitable for extraction. Nebriite taxa included in the study represent ca. 25% of the known species-group taxa and 95% (39 out of 41) of the currently accepted genus-group taxa for the supertribe. For the purposes of this study, taxa currently ranked as species and subspecies are all treated as distinct species here and lumped together as “species-group taxa” in most statements about the diversity of groups. From the Nearctic fauna, sampling included four (27%) of 15 Notiophilus species, the single Opisthius species, both Pelophila species, all four Leistus species, all three Nippononebria species, 84 (98%) of the 86 described Nebria species, and all eight genus-group taxa represented. Sampling from the Palearctic fauna was much less comprehensive, but with 35 (97%) of the 36 genus-group taxa represented. The sample included none of the Palearctic Notiophilus species, four (80%) of five of the Paropisthius species, the single Pelophila species, 24 (9%) of 259 Leistus species, two (29%) of seven Nippononebria species, only one (2%) of 42 Archastes species and 125 (ca. 25%) of approximately 500 described Nebria species-group taxa. The only currently accepted nebriite genera not represented in our sample were Notiokasis Kavanaugh and Nègre (1983) and Archileistobrius Shilenkov and Kryzhanovskij (1983). We tried to include the type species for each genus-group name in our sample and were successful for 26 (63%) of the 41 currently accepted generic and subgeneric names and three (23%) of the 13 subgeneric names currently ranked as subjective junior synonyms. Table 1 lists all published nebriite genus-group names, their date of publication, type species, representation (or not) in our taxon sample, known species and subspecies diversity, current taxonomic status, and proposed new taxonomic status based on the results of this study.

Table 2.

Sampling of Nebriitae and outgroup species. The ID column indicates how each voucher specimen was identified. A number indicates that the specimen was identified by the lead author (DK) using the corresponding numbered references as follows: (1) Lindroth (1961), (2) Andrewes (1929), (3) Bousquet and Smetana (1996), (4) Kasahara (1989), (5) Uéno (1955), (6) Perrault (1981), (8) Jeannel (1941), (9) Perrault (1985), (10) Minowa (1932), (11) Farkač (1995), (12) Perrault (1986), (13) Erwin (1970), (14) Dudko (2003), (15) Perrault (1991), (16) Huber and Schmidt (2016), (17) Ledoux and Roux (2005), (18) Shilenkov (1975), (19) Ledoux and Roux (2009), (20) Trautner (1987), (21) Huber and Schmidt (2013), (22) Dudko (2006) and (23) Sasakawa (2020). A “T” indicates that the specimen was compared with the primary type specimen. A two-letter code indicates that the specimen was identified by one of the following: AF = A. Faille; DM = D.R. Maddison; JS = J. Schmidt; and RD = R.Y. Dudko. Three-letter codes indicate the following: CRC = compared with material in Philippe Roux collection and matching no taxon therein; and NID = could not be identified to species. In the remaining columns, GenBank numbers (MW359101 through MW361062) are provided for each specimen from which the listed gene sequence was successfully recovered in this study. Additional information on these voucher specimens is provided in Appendix A. Other GenBank numbers are for previously published sequences from Kavanaugh et al. (2011), Maddison (2008), Maddison et al. (2009), Ober (2002), and Wild and Maddison (2008). Sequences of the five specimens marked with an asterisk in the ID column are paratypes and the sequences are thus “genseq-2”, with the remainder being “genseq-4” (Chakrabarty et al. 2013).

ID 28S 16S-ND1 CO1 BC CO1 PJ CAD2 PEPCK Topo wg
Supertribe Nebriitae
Tribe Notiophilini
Genus Notiophilus Duméril
Notiophilus borealis Harris 1 MW359101 MW360814 MW359348 MW359611 MW360101 MW360345 MW359863 MW360574
Notiophilus semistriatus Say 1 MW359102 MW360815 MW359349 MW359612 MW360102 MW360346 MW359864 MW360575
Notiophilus sierranus Casey 1 MW359103 MW360816 MW359350 MW359613 MW360103 MW360347 MW359865 MW360576
Notiophilus sylvaticus Dejean 1 MW359104 MW360817 MW359351 MW359614 MW360104 MW360348 MW359866 MW360577
Tribe Opisthiini
Genus Opisthius Kirby
Opisthius richardsoni Kirby 1 MW359105 MW360818 MW359352 MW359615 MW360105 MW360349 MW359867 MW360578
Genus Paropisthius Casey
Paropisthius davidis (Fairmaire) 2 MW359106 MW360819 MW359353 MW359616 MW360106 MW360350 MW359868 MW360579
Paropisthius indicus chinensis Bousquet & Smetana 3 MW359107 MW360820 MW359354 MW359617 MW360107 MW360351 MW359869 MW360580
Paropisthius indicus indicus (Chaudoir) 3 MW359108 MW360821 MW359355 MW359618 MW360108 MW360352 MW359870 MW360581
Paropisthius masuzoi Kasahara 4 MW359109 MW360822 MW359356 MW359619 MW360109 MW360353 MW359871 MW360582
Tribe Pelophilini
Genus Pelophila Dejean
Pelophila borealis (Paykull) AK 1 MW359110 MW360823 MW359357 MW359620 MW360110 MW360354 MW359872 MW360583
Pelophila borealis (Paykull) RFE 1 MW359111 MW359358 MW359621 MW360111 MW359873 MW360584
Pelophila rudis (LeConte) 1 MW359112 MW360824 MW359359 MW359622 MW360112 MW360355 MW359874 MW360585
Tribe Nebriini
Genus Nippononebria Uéno
Subgenus Nippononebria Uéno
Nippononebria chalceola (Bates) 5 MN982525 MN982525
Nippononebria changbaiensis Kavanaugh & Liang T MW359113 MW360825 MW359360 MW359623 MW360113 MW360356 MW359875 MW360586
Subgenus Vancouveria Kavanaugh
Nippononebria altisierrae Kavanaugh T MW359114 MW360826 MW359361 MW359624 MW360114 MW360357 MW359876 MW360587
Nippononebria campbelli Kavanaugh T MW359115 MW360827 MW359362 MW359625 MW360115 MW360358 MW359877 MW360588
Nippononebria virescens (Horn) T MW359116 MW360828 MW359363 MW359626 MW360116 MW360359 MW359878 MW360589
Genus Leistus Frölich
Subgenus Nebrileistus Bänninger
Leistus nubivagus Wollaston 6 MW359117 MW360829 MW359364 MW359627 MW360117 MW360360 MW359879 MW360590
Subgenus Sardoleistus Perrault
Leistus sardous Baudi di Selve AF MW359118 MW360830 MW359365 MW359628 MW360118 MW360591
Subgenus Pogonophorus Latreille
Leistus parvicollis Chaudoir 8 MW359119 MW360831 MW359366 MW359629 MW360119 MW360361 MW360592
Leistus pyrenaeus Kraatz 8 MW359120 MW360832 MW359367 MW359630 MW360120 MW359880 MW360593
Leistus rufomarginatus (Duftschmid) 8 MW359121 MW360833 MW359368 MW359631 MW360121 MW360362 MW359881 MW360594
Subgenus Evanoleistus Jedlička
Leistus birmanicus Perrault 9 MW359122 MW360834 MW359369 MW359632 MW360122 MW360363 MW359882 MW360595
Leistus gaoligongensis Kavanaugh & Long T MW359123 MW360835 MW359370 MW359633 MW360123 MW360364 MW359883 MW360596
Leistus lihengae Kavanaugh & Long T MW359124 MW360836 MW359371 MW359634 MW360124 MW360365 MW359884 MW360597
Leistus niitakaensis Minowa 10 MW359125 MW360837 MW359372 MW359635 MW360125 MW360366 MW359885 MW360598
Leistus nokoensis Minowa 10 MW359126 MW360838 MW359373 MW359636 MW360126 MW360367 MW359886 MW360599
Leistus smetanai Farkač 11 MW359127 MW360839 MW359374 MW359637 MW360127 MW360368 MW359887 MW360600
Leistus taiwanensis Perrault 12 MW359128 MW360840 MW359375 MW359638 MW360128 MW360369 MW359888 MW360601
Leistus sp NEP NID MW359129 MW360841 MW359376 MW359639 MW360129 MW359889 MW360602
Leistus sp YUN 1 NID MW359130 MW360842 MW359377 MW359640 MW360130 MW360370 MW359890 MW360603
Subgenus Leistus Frölich
Leistus crenatus Fairmaire 8 MW359131 MW360843 MW359378 MW359641 MW360131 MW360371 MW359891 MW360604
Leistus ferrugineus (Linnaeus) 8 MW359132 MW360844 MW359379 MW359642 MW360132 MW360372 MW359892 MW360605
Leistus ferruginosus Mannerheim 13 MW359133 MW360845 MW359380 MW359643 MW360133 MW360373 MW359893 MW360606
Leistus kryzhanovskii frateroides Dudko 14 MW359134 MW360846 MW359381 MW359644 MW360134 MW360374 MW359894 MW360607
Leistus kryzhanovskii kryzhanovskii Dudko 14 MW359135 MW360847 MW359382 MW359645 MW360135 MW360375 MW359895 MW360608
Leistus fulvibarbis Dejean 8 MW359136 MW360848 MW359383 MW359646 MW360136 MW360609
Leistus fulvus Chaudoir 8 MW360849 MW359384 MW359647
Leistus longipennis Casey 13 MW359137 MW360850 MW359385 MW359648 MW360137 MW360376 MW359896 MW360610
Leistus madmeridianus Erwin 13 MW359138 MW360851 MW359386 MW359649 MW360138 MW360377 MW359897 MW360611
Leistus niger Gebler 15 MW359139 MW360852 MW359387 MW359650 MW360139 MW360378 MW359898 MW360612
Leistus nitidus (Duftschmid) 8 MW359140 MW360853 MW359388 MW359651 MW360140 MW360379 MW360613
Leistus sp JIL 2 NID MW359141 MW360854 MW359389 MW359652 MW360141 MW360380 MW359899 MW360614
Leistus sp SCH NID MW359142 MW360855 MW359390 MW359653 MW360142 MW360381 MW359900 MW360615
Leistus sp YUN 2 NID MW359143 MW360856 MW359391 MW359654 MW360143 MW360382 MW359901 MW360616
Genus Nebria Latreille
Oreonebria Series
Eonebria Complex
Subgenus Pseudepinebriola New Subgenus
Nebria delicata Huber & Schmidt1 16 MW359144 MW360857 MW359392 MW359655 MW360144 MW360383 MW359902 MW360617
Nebria retingensis Huber & Schmidt1 16* MW359145 MW360858 MW359393 MW359656 MW360145 MW360384 MW359903 MW360618
Nebria sp YUN 4 NID MW359146 MW360859 MW359394 MW359657 MW360146 MW360385 MW359904 MW360619
Nebria sp YUN 5 NID MW359147 MW360860 MW359395 MW359658 MW360147 MW360386 MW359905 MW360620
Nebria sp XIZ 14 NID MW359148 MW360861 MW359396 MW359659 MW360148 MW360387 MW359906 MW360621
Subgenus Sadonebria Ledoux & Roux
Nebria chinensis Bates 17 MW359149 MW360862 MW359397 MW359660 MW360149 MW360388 MW359907 MW360622
Nebria niitakana Kano 17 MW359150 MW360863 MW359398 MW359661 MW360150 MW360389 MW359908 MW360623
Nebria ohdaiensis Nakane 17 MW359151 MW360864 MW359399 MW359662 MW360151 MW360390 MW359909 MW360624
Nebria saeviens Bates 17 MW359152 MW360865 MW359400 MW359663 MW360152 MW360391 MW359910 MW360625
Subgenus Eonebria Semenov & Znojko
Nebria komarovi Semenov & Znojko 17 MW359153 MW360866 MW359401 MW359664 MW360153 MW359911
Nebria sp GAN CRC MW359154 MW360867 MW359402 MW359665 MW360154 MW359912
Nebria sp YUN 13 CRC MW359155 MW360868 MW359403 MW359666 MW360155 MW360392 MW359913 MW360626
Nebria sp YUN 16 CRC MW359156 MW360869 MW359404 MW359667 MW360156 MW360393 MW359914 MW360627
Nebria sp YUN 17 CRC MW359157 MW360870 MW359405 MW359668 MW360157 MW360394 MW359915 MW360628
Nebria sp YUN 18 CRC MW359158 MW360871 MW359406 MW359669 MW360158 MW360395 MW359916 MW360629
Oreonebria Complex
Subgenus Epispadias Ledoux & Roux
Nebria sp YUN 6 CRC MW359159 MW360872 MW359407 MW359670 MW360159 MW360396 MW359917 MW360630
Subgenus Falcinebria Ledoux & Roux
Nebria formosana Habu 17 MW359160 MW360873 MW359408 MW359671 MW360160 MW360397 MW359918 MW360631
Nebria niohozana Bates 23 MW359161 MW360874 MW359409 MW359672 MW360161 MW360398 MW359919 MW360632
Subgenus Orientonebria Shilenkov
Nebria coreica Solsky 18 MW359162 MW360875 MW359410 MW359673 MW360162 MW360399 MW359920 MW360633
Subgenus Archastes Jedlička
Nebria yuae (Ledoux & Roux) 19 MW359163 MW360876 MW359411 MW359674 MW360163 MW360400 MW359921 MW360634
Subgenus Oreonebria Daniel
Nebria angustata Dejean 17 MW359164 MW360877 MW359412 MW359675 MW360164 MW360401 MW359922 MW360635
Nebria angusticollis Bonelli 17 MW359165 MW360878 MW359413 MW359676 MW360165 MW360402 MW359923 MW360636
Nebria austriaca Ganglbauer 17 MW359166 MW360879 MW359414 MW359677 MW360166 MW359924 MW360637
Nebria boschi Winkler 17 MW359167 MW360880 MW359415 MW359678 MW360167 MW360403 MW359925 MW360638
Nebria bremii Germar2 17 MW359168 MW360881 MW359416 MW359679 MW360168 MW360404 MW359926 MW360639
Nebria castanea Bonelli 17 MW359169 MW360882 MW359417 MW359680 MW360169 MW360405 MW359927 MW360640
Nebria diaphana Daniel & Daniel 17 MW359170 MW360883 MW359418 MW359681 MW360170 MW359928 MW360641
Nebria gagates (Bonelli)3 17 MW359171 MW360884 MW359419 MW359682 MW360171 MW359929 MW360642
Nebria ligurica Daniel 17 MW359172 MW360885 MW359420 MW359683 MW360172 MW359930 MW360643
Nebria lombarda Daniel & Daniel 17 MW359173 MW359421 MW359684 MW360173 MW359931 MW360644
Nebria picea Dejean 17 MW359174 MW360886 MW359422 MW359685 MW360174 MW360406 MW359932 MW360645
Nebriola Series
Subgenus Nebriola Daniel
Nebria fontinalis Daniel & Daniel 17 MW359175 MW360887 MW359423 MW359686 MW360175 MW360407 MW359933 MW360646
Nebria laticollis Dejean 17 MW359176 MW360888 MW359424 MW359687 MW360176 MW360408 MW359934 MW360647
Nebria Series
Boreonebria Complex
Subgenus Nakanebria Ledoux & Roux
Nebria shiretokoana Nakane 17 MW359177 MW360889 MW359425 MW359688 MW360177 MW360409 MW359935 MW360648
Subgenus Boreonebria Jeannel
Nebria baicalica Motschulsky 18 JN847585 JN847610 MW359426 JN847535 JN847560 MW360410 JN847510 JN847635
Nebria bellorum Kavanaugh T JN847603 JN847628 MW359427 JN847553 JN847578 MW360411 JN847528 JN847653
Nebria castanipes Kirby T MW359178 MW360890 MW359428 MW359689 MW360178 MW360412 MW359936 MW360649
Nebria changaica Horvatovich 17 MW359179 MW360891 MW359429 MW359690 MW360179 MW359937 MW360650
Nebria crassicornis Van Dyke 1 JN847581 JN847606 MW359430 JN847531 JN847556 JN847506 JN847631
Nebria dabanensis Shilenkov4 17 MW359180 MW360892 MW359431 MW359691 MW360180 MW359938 MW360651
Nebria frigida Sahlberg 1 MW359181 MW360893 MW359432 MW359692 MW360181 MW360413 MW359939 MW360652
Nebria gouleti Kavanaugh T JN847587 JN847612 MW359433 JN847537 JN847562 MW360414 JN847512 JN847637
Nebria gyllenhali (Schönherr) T JN847582 JN847607 MW359434 JN847532 JN847557 JN847507 JN847632
Nebria hudsonica LeConte T JN847589 JN847614 MW359435 JN847539 JN847564 MW360415 JN847514 JN847639
Nebria intermedia Van Dyke OR 1 MW359182 MW360894 MW359436 MW359693 MW360182 MW359940 MW360653
Nebria intermedia Van Dyke UT 1 MW359183 MW360895 MW359437 MW359694 MW360183 MW359941 MW360654
Nebria intermedia Van Dyke WY 1 MW359184 MW360896 MW359438 MW359695 MW360184 MW359942 MW360655
Nebria kaszabi Shilenkov4 17 MW359185 MW360897 MW359439 MW359696 MW360185 MW359943 MW360656
Nebria lacustris Casey T JN847590 JN847615 MW359440 JN847540 JN847565 MW360416 JN847515 JN847640
Nebria lassenensis Kavanaugh T MW359186 MW360898 MW359441 MW359697 MW360186 MW360417 MW359944 MW360657
Nebria lindrothi Kavanaugh T MW359187 MW360899 MW359442 MW359698 MW360187 MW360418 MW359945 MW360658
Nebria murzini Ledoux & Roux4 17 MW359188 MW360900 MW359443 MW359699 MW360188 MW359946 MW360659
Nebria nivalis (Paykull) AK 1 JN847584 JN847609 MW359444 JN847534 JN847559 MW360419 JN847509 JN847634
Nebria nivalis (Paykull) NU 1 JN847583 JN847608 MW359445 JN847533 JN847558 MW360420 JN847508 JN847633
Nebria nivalis gr sp NID MW359189 MW360901 MW359446 MW359700 MW360189 MW360421 MW359947 MW360660
Nebria rubrofemorata Shilenkov 18 MW359190 MW360902 MW359447 MW359701 MW360190 MW360422 MW359948 MW360661
Nebria sajanica Bänninger4 17 MW359191 MW360903 MW359448 MW359702 MW360191 MW359949 MW360662
Nebria sajanica gr sp 14 NID MW359192 MW360904 MW359449 MW359703 MW360663
Nebria sajanica gr sp 24 NID MW359193 MW360905 MW359450 MW359704 MW360192 MW360423 MW359950 MW360664
Nebria subdilatata Motschulsky 18 JN847586 JN847611 MW359451 JN847536 JN847561 MW360424 JN847511 JN847636
Nebria tekesensis Ledoux & Roux4 17 MW359194 MW360906 MW359452 MW359705 MW360193 MW359951 MW360665
Nebria sp MG 1 NID MW359195 MW360907 MW359453 MW359706 MW360194 MW360425 MW359952 MW360666
Nebria sp MG 2 NID MW359196 MW360908 MW359454 MW359707 MW360195 MW360426 MW359953 MW360667
Nebria Complex
Nebria Subcomplex
Subgenus Tyrrhenia Ledoux & Roux
Nebria fulviventris Bassi 17 MW359197 MW360909 MW359455 MW359708 MW360196 MW360427 MW359954 MW360668
Nebria rubicunda maroccana Antoine 17 MW359198 MW360910 MW359456 MW359709 MW360197 MW359955 MW360669
Subgenus Nebria Latreille
Nebria brevicollis (Fabricius) OR 20 JN847580 MW360911 MW359457 JN847530 JN847555 MW360428 JN847505 JN847630
Nebria brevicollis (Fabricius) SP 20 MW359199 MW360912 MW359458 MW359710 MW360198 MW360429 MW359956 MW360670
Nebria lafresnayi Audinet-Serville 8 MW359200 MW360913 MW359459 MW359711 MW360199 MW359957 MW360671
Nebria olivieri Dejean 8 MW359201 MW360914 MW359460 MW359712 MW360200 MW360430 MW359958 MW360672
Nebria rubripes Audinet-Serville 8 MW359202 MW360915 MW359461 MW359713 MW360201 MW360431 MW359959 MW360673
Nebria tibialis Bonelli 17 MW359203 MW360916 MW359462 MW359714 MW360202 MW360432 MW359960 MW360674
Nebria turcica Chaudoir 17 MW359204 MW360917 MW359463 MW359715 MW360203 MW360675
Subgenus Alpaeonebria Csiki
Nebria germari Heer AU 17 MW359205 MW360918 MW359464 MW359716 MW360204 MW360433 MW359961 MW360676
Nebria germari Heer GE 17 MW359206 MW360919 MW359465 MW359717 MW360205 MW360434 MW359962 MW360677
Subgenus Spelaeonebria Peyerimhoff
Nebria nudicollis Peyerimhoff AF MW359207 MW360920 MW359466 MW359718 MW360206 MW359963 MW360678
Epinebriola Subcomplex
Subgenus Epinebriola Daniel
Nebria businskyorum Ledoux & Roux 16 MW359208 MW360921 MW359467 MW359719 MW360207 MW360435 MW359964 MW360679
Nebria capillosa Ledoux & Roux6 JS MW359209 MW360922 MW359468 MW359720 MW360208 MW360436 MW359965
Nebria cf. desgodinsi Oberthür6 JS MW359210 MW360923 MW359469 MW359721 MW360209 MW359966
Nebria kagmara Huber & Schmidt5 16* MW359211 MW360924 MW359470 MW359722 MW360210 MW360437 MW360680
Nebria cf. laevistriata Ledoux & Roux NID MW359212 MW360925 MW359471 MW359723 MW360211 MW360438 MW359967 MW360681
Nebria livida (Linnaeus)7 17 MW359213 MW360926 MW359472 MW359724 MW360212 MW360439 MW359968 MW360682
Nebria macrogona Bates7 17 MW359214 MW359473 MW359725 MW360213 MW360440 MW359969 MW360683
Nebria martensi Huber & Schmidt 16 MW359215 MW360927 MW359474 MW359726 MW360214 MW360684
Nebria numburica Huber & Schmidt 16* MW359216 MW360928 MW359475 MW359727 MW360215 MW360441 MW359970 MW360685
Nebria oxyptera Daniel & Daniel 16 MW359217 MW360929 MW359476 MW359728
Nebria pertinax Huber & Schmidt6 JS MW359218 MW360930 MW359477 MW359729 MW360216 MW360442 MW359971 MW360686
Nebria pseudorestias Huber & Schmidt 16 MW359219 MW360931 MW359478 MW359730 MW360217 MW360443 MW359972 MW360687
Nebria sp YUN 16 NID MW359220 MW360932 MW359479 MW359731 MW360218 MW360444 MW359973 MW360688
Subgenus Psilonebria Andrewes
Nebria composita macra Ledoux & Roux9 17 MW359221 MW360933 MW359480 MW359732 MW360219 MW360445 MW359974 MW360689
Nebria mentoincisa Huber & Schmidt8 21* MW359222 MW360934 MW359481 MW359733 MW360220 MW360446 MW359975 MW360690
Nebria nana Ledoux & Roux9 17 MW359223 MW360935 MW359482 MW359734 MW360221 MW360447 MW359976 MW360691
Nebria pharina walteriana Ledoux & Roux8 17 MW359224 MW360936 MW359483 MW359735 MW360222 MW360448 MW359977 MW360692
Nebria przewalskii Semenov9 17 MW359225 MW360937 MW359484 MW359736 MW360223 MW360449 MW359978
Nebria roborowskii Semenov8 17 MW359226 MW360938 MW359485 MW359737 MW360224 MW360450 MW359979 MW360693
Nebria cf. superna Andrewes JS MW359227 MW360939 MW359486 MW359738 MW360225 MW360451 MW359980 MW360694
Nebria sp XIZ 20 NID MW359228 MW360940 MW359487 MW359739 MW360226 MW360452 MW359981 MW360695
Subgenus Eurynebria Ganglbauer
Nebria complanata (Linnaeus) 8 MW359229 MW360941 MW359488 MW359740 MW360227 MW359982 MW360696
Subgenus Eunebria Jeannel
Nebria jockischii Sturm 17 MW359230 MW360942 MW359489 MW359741 MW360228 MW360453 MW359983 MW360697
Nebria lewisi Bates 17 MW359231 MW360943 MW359490 MW359742 MW360229 MW360454 MW359984 MW360698
Nebria limbigera Solsky 17 MW359232 MW360944 MW359491 MW359743 MW360230 MW360455 MW359985
Nebria morvani Ledoux & Roux JS MW359233 MW360945 MW359492 MW359744 MW360231 MW360456 MW359986 MW360699
Nebria perlonga Heyden 17 MW359234 MW360946 MW359493 MW359745 MW360232 MW360457 MW359987 MW360700
Nebria picicornis (Fabricius) 17 MW359235 MW360947 MW359494 MW359746 MW360233 MW360458 MW359988
Nebria psammodes (Rossi) 17 MW359236 MW360948 MW359495 MW359747 MW360234 MW360459 MW359989
Nebria uenoiana Habu 17 MW359237 MW360949 MW359496 MW359748 MW360235 MW360460 MW359990 MW360701
Nebria yunnana Bänninger 17 MW359238 MW360950 MW359497 MW359749 MW360236 MW360461 MW359991 MW360702
Nebria sp YUN 9 CRC MW359239 MW360951 MW359498 MW359750 MW360237 MW360462 MW359992 MW360703
Nebriasp YUN 10-dark CRC MW359240 MW360952 MW359499 MW359751 MW360238 MW360463 MW359993 MW360704
Nebria sp YUN 10-pale CRC MW359241 MW360953 MW359500 MW359752 MW360239 MW360464 MW359994 MW360705
Nebria sp YUN 11 CRC MW359242 MW360954 MW359501 MW359753 MW360240 MW360465 MW359995 MW360706
Catonebria Series
Reductonebria Complex
Subgenus Insulanebria New Subgenus
Nebria carbonaria Eschscholtz10 18 MW359243 MW360955 MW359502 MW359754 MW360241 MW360466 MW359996 MW360707
Nebria snowi Bates10 18 MW359244 MW360956 MW359503 MW359755 MW360242 MW360467 MW359997 MW360708
Subgenus Reductonebria Shilenkov
Nebria altaica Gebler 18 MW359245 MW360957 MW359504 MW359756 MW360243 MW360468 MW359998 MW360709
Nebria appalachia Darlington T MW359246 MW360958 MW359505 MW359757 MW360244 MW360469 MW359999 MW360710
Nebria chuskae Kavanaugh T MW359247 MW360959 MW359506 MW359758 MW360245 MW360470 MW360000 MW360711
Nebria darlingtoni Kavanaugh T MW359248 MW360960 MW359507 MW359759 MW360246 MW360471 MW360001 MW360712
Nebria desolata Kavanaugh T MW359249 MW360961 MW359508 MW359760 MW360247 MW360472 MW360002 MW360713
Nebria diversa LeConte T MW359250 MW360962 MW359509 MW359761 MW360248 MW360473 MW360003 MW360714
Nebria eschscholtzii Ménétriés 1 MW359251 MW360963 MW359510 MW359762 MW360249 MW360474 MW360004 MW360715
Nebria georgei Kavanaugh T* MW359252 MW360964 MW359511 MW359763 MW360250 MW360475 MW360005 MW360716
Nebria japonica Bates 17 MW359253 MW360965 MW359512 MW359764 MW360251 MW360476 MW360006 MW360717
Nebria mannerheimii Fischer von Waldheim 1 MW359254 MW360966 MW359513 MW359765 MW360252 MW360477 MW360007 MW360718
Nebria navajo Kavanaugh T MW359255 MW360967 MW359514 MW359766 MW360253 MW360478 MW360008 MW360719
Nebria obliqua LeConte CO T MW359256 MW360968 MW359515 MW359767 MW360254 MW360479 MW360009 MW360720
Nebria obliqua LeConte UT T MW359257 MW360969 MW359516 MW359768 MW360255 MW360480 MW360010 MW360721
Nebria ochotica Sahlberg 18 MW359258 MW360970 MW359517 MW359769 MW360256 MW360481 MW360011 MW360722
Nebria pallipes Say 1 MW359259 MW360971 MW359518 MW359770 MW360257 MW360482 MW360012 MW360723
Nebria suturalis LeConte AB T MW359260 MW360972 MW359519 MW359771 MW360258 MW360483 MW360013 MW360724
Nebria suturalis LeConte CO T MW359261 MW360973 MW359520 MW359772 MW360259 MW360484 MW360014 MW360725
Nebria suturalis LeConte NH T MW359262 MW360974 MW359521 MW359773 MW360260 MW360485 MW360015 MW360726
Subgenus Erwinebria New Subgenus
Nebria acuta Lindroth10 T MW359263 MW360975 MW359522 MW359774 MW360261 MW360486 MW360016 MW360727
Nebria arkansana Casey10 T MW359264 MW360976 MW359523 MW359775 MW360262 MW360487 MW360017 MW360728
Nebria charlottae Lindroth10 T MW359265 MW360977 MW359524 MW359776 MW360263 MW360488 MW360018 MW360729
Nebria danmanni Kavanaugh10 T MW359266 MW360978 MW359525 MW359777 MW360264 MW360489 MW360019 MW360730
Nebria edwardsi Kavanaugh10 T MW359267 MW360979 MW359526 MW359778 MW360265 MW360490 MW360020 MW360731
Nebria fragilis Casey10 T MW359268 MW360980 MW359527 MW359779 MW360266 MW360491 MW360021 MW360732
Nebria gregaria Fischer von Waldeim10 1 MW359269 MW360981 MW359528 MW359780 MW360267 MW360492 MW360022 MW360733
Nebria haida Kavanaugh10 T MW359270 MW360982 MW359529 MW359781 MW360268 MW360493 MW360023 MW360734
Nebria jeffreyi Kavanaugh10 T MW359271 MW360983 MW359530 MW359782 MW360269 MW360494 MW360024 MW360735
Nebria lituyae Kavanaugh10 T MW359272 MW360984 MW359531 MW359783 MW360270 MW360495 MW360025 MW360736
Nebria louiseae Kavanaugh10 T MW359273 MW360985 MW359532 MW359784 MW360271 MW360496 MW360026 MW360737
Nebria lyelli Van Dyke10 T MW359274 MW360986 MW359533 MW359785 MW360272 MW360497 MW360027 MW360738
Nebria modoc Kavanaugh10 T MW359275 MW360987 MW359534 MW359786 MW360273 MW360498 MW360028 MW360739
Nebria oowah Kavanaugh10 T MW359276 MW360988 MW359535 MW359787 MW360274 MW360499 MW360029 MW360740
Nebria quileute Kavanaugh10 T MW359277 MW360989 MW359536 MW359788 MW360275 MW360500 MW360030 MW360741
Nebria sahlbergii Fischer von Waldheim10 T MW359278 MW360990 MW359537 MW359789 MW360276 MW360501 MW360031 MW360742
Nebria sonorae Kavanaugh10 T MW359279 MW360991 MW359538 MW359790 MW360277 MW360502 MW360032 MW360743
Nebria triad Kavanaugh10 T MW359280 MW360992 MW359539 MW359791 MW360278 MW360503 MW360033 MW360744
Nebria wallowae Kavanaugh10 T MW359281 MW360993 MW359540 MW359792 MW360279 MW360504 MW360034 MW360745
Nebria zioni Van Dyke10 T MW359282 MW360994 MW359541 MW359793 MW360280 MW360505 MW360035 MW360746
Catonebria Complex
Subgenus Nivalonebria New Subgenus
Nebria paradisi Darlington OR11 T MW359283 MW360995 MW359542 MW359794 MW360281 MW360506 MW360036 MW360747
Nebria paradisi Darlington WA11 T MW359284 MW360996 MW359543 MW359795 MW360282 MW360507 MW360037 MW360748
Nebria turmaduodecima Kavanaugh11 T MW359285 MW360997 MW359544 MW359796 MW360283 MW360508 MW360038 MW360749
Subgenus Neaptenonebria New Subgenus
Nebria balli Kavanaugh12 T MW359286 MW360998 MW359545 MW359797 MW360284 MW360509 MW360039 MW360750
Nebria carri Kavanaugh12 T MW359287 MW360999 MW359546 MW359798 MW360285 MW360510 MW360040 MW360751
Nebria kincaidi Schwarz12 T MW359288 MW361000 MW359547 MW359799 MW360286 MW360511 MW360041 MW360752
Nebria ovipennis LeConte12 T MW359289 MW361001 MW359548 MW359800 MW360287 MW360512 MW360042 MW360753
Nebria sierrae Kavanaugh12 T MW359290 MW361002 MW359549 MW359801 MW360288 MW360513 MW360043 MW360754
Nebria spatulata Van Dyke12 T MW359291 MW361003 MW359550 MW359802 MW360289 MW360514 MW360044 MW360755
Subgenus Palaptenonebria New Subgenus
Nebria arinae Dudko & Shilenkov12 RD MW359292 MW361004 MW359551 MW359803 MW360290 MW360515 MW360045 MW360756
Nebria baenningeri baenningeri Dudko & Shilenkov12 RD MW359293 MW361005 MW359552 MW359804 MW360291 MW360516 MW360046 MW360757
Nebria baenningeri korgonica Dudko & Shilenkov12 RD MW359294 MW361006 MW359553 MW359805 MW360292 MW360517 MW360047 MW360758
Nebria lyubechanskii Dudko12 RD MW359295 MW361007 MW359554 MW359806 MW360293 MW360518 MW360048 MW360759
Nebria mellyi mellyi Gebler12 RD MW359296 MW361008 MW359555 MW359807 MW360294 MW360519 MW360049 MW360760
Nebria mellyi teletskiana Dudko & Shilenkov12 RD MW359298 MW361010 MW359557 MW359809 MW360296 MW360521 MW360051 MW360762
Nebria roddi Dudko & Shilenkov12 RD MW359297 MW361009 MW359556 MW359808 MW360295 MW360520 MW360050 MW360761
Nebria sajana dubatolovi Dudko & Shilenkov12 RD MW359299 MW361011 MW359558 MW359810 MW360297 MW360522 MW360052 MW360763
Nebria sajana sajana Dudko & Shilenkov12 RD MW359300 MW361012 MW359559 MW359811 MW360298 MW360523 MW360053 MW360764
Nebria sajana sarlyk Dudko & Shilenkov12 RD MW359301 MW361013 MW359560 MW359812 MW360299 MW360524 MW360054 MW360765
Nebria sajana sitnikovi Dudko & Shilenkov12 RD MW359302 MW361014 MW359561 MW359813 MW360300 MW360525 MW360055 MW360766
Subgenus Catonebria Shilenkov
Nebria aenea Gebler RD MW359303 MW361015 MW359562 MW359814 MW360301 MW360526 MW360056 MW360767
Nebria albimontis Kavanaugh T MW359304 MW361016 MW359563 MW359815 MW360302 MW360527 MW360057 MW360768
Nebria banksii Crotch 22 MW359305 MW361017 MW359564 MW359816 MW360303 MW360528 MW360058 MW360769
Nebria baumanni Kavanaugh T MW359306 MW361018 MW359565 MW359817 MW360304 MW360529 MW360059 MW360770
Nebria beverlianna Kavanaugh T MW359307 MW361019 MW359566 MW359818 MW360305 MW360530 MW360060 MW360771
Nebria calva Kavanaugh T MW359308 MW361020 MW359567 MW359819 MW360306 MW360531 MW360061 MW360772
Nebria cascadensis Kavanaugh T MW359309 MW361021 MW359568 MW359820 MW360307 MW360532 MW360062 MW360773
Nebria catenata Casey T MW359310 MW360812 MW359569 MW359821 MW360308 MW360533 MW360063 MW360774
Nebria catenulata Fischer von Waldheim 22 MW359311 MW361022 MW359570 MW359822 MW360309 MW360534 MW360064 MW360775
Nebria coloradensis Van Dyke T MW359312 MW361023 MW359571 MW359823 MW360310 MW360535 MW360065 MW360776
Nebria fragariae Kavanaugh T MW359313 MW361024 MW359572 MW359824 MW360311 MW360536 MW360066 MW360777
Nebria fulgida Gebler 22 MW359314 MW361025 MW359573 MW359825 MW360312 MW360537 MW360067 MW360778
Nebria gebleri Dejean T MW359315 MW361026 MW359574 MW359826 MW360313 MW360538 MW360068 MW360779
Nebria giulianii Kavanaugh T MW359316 MW361027 MW359575 MW359827 MW360314 MW360539 MW360069 MW360780
Nebria holzunensis Dudko & Shilenkov 22 MW359317 MW361028 MW359576 MW359828 MW360315 MW360540 MW360070 MW360781
Nebria ingens Horn T MW359318 MW361029 MW359577 MW359829 MW360316 MW360541 MW360071 MW360782
Nebria labontei Kavanaugh T MW359319 MW361030 MW359578 MW359830 MW360317 MW360542 MW360072 MW360783
Nebria lamarckensis Kavanaugh T MW359320 MW361031 MW359579 MW359831 MW360318 MW360543 MW360073 MW360784
Nebria meanyi Van Dyke CA T MW359321 MW361032 MW359580 MW359832 MW360319 MW360544 MW360074 MW360785
Nebria meanyi Van Dyke WA T MW359322 MW361033 MW359581 MW359833 MW360320 MW360545 MW360075 MW360786
Nebria metallica Fischer von Waldheim 1 MW359323 MW361034 MW359582 MW359834 MW360321 MW360546 MW360076 MW360787
Nebria pasquineli Kavanaugh T MW359324 MW361035 MW359583 MW359835 MW360322 MW360547 MW360077 MW360788
Nebria pektusanica Horvatovich 22 MW359325 MW361036 MW359584 MW359836 MW360323 MW360548 MW360078 MW360789
Nebria piperi Van Dyke T MW359326 MW361037 MW359585 MW359837 MW360324 MW360549 MW360079 MW360790
Nebria piute Erwin & Ball T MW359327 MW360813 MW359586 MW359838 MW360325 MW360550 MW360080 MW360791
Nebria praedicta Kavanaugh & Schoville T MW359328 MW361038 MW359587 MW359839 MW360326 MW360551 MW360081 MW360792
Nebria purpurata LeConte CO T MW359329 MW361039 MW359588 MW359840 MW360327 MW360552 MW360082 MW360793
Nebria purpurata LeConte NM T MW359330 MW361040 MW359589 MW359841 MW360328 MW360553 MW360083 MW360794
Nebria rathvoni LeConte T MW359331 MW361041 MW359590 MW359842 MW360329 MW360554 MW360084 MW360795
Nebria riversi Van Dyke T MW359332 MW361042 MW359591 MW359843 MW360330 MW360555 MW360085 MW360796
Nebria schwarzi Van Dyke T MW359333 MW361043 MW359592 MW359844 MW360331 MW360556 MW360086 MW360797
Nebria sevieri Kavanaugh T MW359334 MW361044 MW359593 MW359845 MW360332 MW360557 MW360087 MW360798
Nebria sierrablancae Kavanaugh T MW359335 MW361045 MW359594 MW359846 MW360333 MW360558 MW360088 MW360799
Nebria siskiyouensis Kavanaugh T MW359336 MW361046 MW359595 MW359847 MW360334 MW360559 MW360089 MW360800
Nebria splendida Fischer von Waldheim 22 MW359337 MW361047 MW359596 MW359848 MW360335 MW360560 MW360090 MW360801
Nebria steensensis Kavanaugh T MW359338 MW361048 MW359597 MW359849 MW360336 MW360561 MW360091 MW360802
Nebria sylvatica Kavanaugh T MW359339 MW361049 MW359598 MW359850 MW360337 MW360562 MW360092 MW360803
Nebria trifaria LeConte T MW359340 MW361050 MW359599 MW359851 MW360338 MW360563 MW360093 MW360804
Nebria utahensis Kavanaugh T MW359341 MW361051 MW359600 MW359852 MW360339 MW360564 MW360094 MW360805
Nebria vandykei Bänninger 1 MW359342 MW361052 MW359601 MW359853 MW360340 MW360565 MW360095 MW360806
Nebria wyeast Kavanaugh T MW359343 MW361053 MW359602 MW359854 MW360341 MW360566 MW360096 MW360807
Outgroup taxa
Trachypachidae
Trachypachus inermis Motschulsky 1 MW359344 MW361054 MW359603 MW359855 MW360342 MW360808
Trachypachus slevini Van Dyke T MW359345 MW361055 MW359604 MW359856 DHK0513a MW360809
Carabidae
Carabini
Calosoma scrutator (Fabricius) 1 EU677684 MW361056 MW359605 MW359857 EU677530 MW360567 EU677634 EU677661
Cychrini
Scaphinotus petersi Roeschke DR EU658920 MW361058 MW359607 MW359859 EU677531 MW360569 EU677635 EU658922
Metriini
Metrius contractus Eschscholtz 1 MW359346 MW361059 MW359608 MW359860 MW360343 MW360570 MW360097 MW360810
Loricerini
Loricera pilicornis (Fabricius) 1 MW359347 MW361060 MW359609 MW359861 MW360344 MW360571 MW360098 MW360811
Bembidiini
Asaphidion yukonense Wickham DM EU677686 EU677540 EU677638 EU677666
Bembidion antiquum Dejean DM EF648850 MW361061 EF649127.1 EF649127.1 EF649402 MW360572 MW360099 EF649489
Pterostichini
Pterostichus melanarius (Illiger) DM AF398707 MW361062 MW359610 MW359862 EU677533 MW360573 MW360100 AF398623

All but nine of the specimens used in this study were collected directly into 95–100% ethanol, which was subsequently replaced with fresh ethanol at least once. Six specimens were collected directly into silica gel and allowed to fully desiccate. All specimens were stored as soon as possible in a freezer at -20 °C awaiting DNA extraction. The remaining three specimens were pinned museum specimens killed by unknown means. Depositories for all voucher specimens are listed in Appendix A.

Identification

Nearctic Nebria and Nippononebria specimens were identified by DHK, based on his revision (Kavanaugh 1978) of that fauna with subsequent additions (Kavanaugh 1979a, 1981, 1984, 2008, 2015; Kavanaugh and Schoville 2009). Most Himalayan nebriite specimens were identified by JS and European and North African specimens by AF. Identifications of all Palearctic specimens were confirmed as far as possible by DHK using the worldwide revisions of Nebria and Archastes by Ledoux and Roux (2005, 2009), taxonomic keys and descriptions in papers listed in Table 2, or comparisons with holotypes or reliably determined museum specimens. Philippe Roux generously provided DHK access to his personal collection, which greatly facilitated identifications of specimens from China. Those specimens in our taxon sample that are without formal scientific names could not be identified by any of these means and are likely undescribed. The method used to identify each specimen is listed in Table 2.

DNA extraction and sequencing

Specimens preserved for DNA

DNA was extracted from specimens preserved in ethanol or desiccated in silica gel using Qiagen DNeasy (Qiagen) Blood & Tissue kits and the manufacturer’s recommended protocol. The first 25 extractions (in 2004) were made using thoracic muscle tissue; but all subsequent extractions were made from one or more legs, depending on the size of the specimen. The eight gene fragments used in this study and their abbreviations are: 28S: ca. 955 bases of the nuclear ribosomal DNA subunit 28S (D1–D3 regions); 16S-ND1: ca. 812 bases of the mitochondrial gene fragment including 16S ribosomal RNA gene (partial sequence), tRNA-leucine gene (complete sequence), and NADH dehydrogenase subunit 1 (ND1) (partial sequence); COI BC: 658 bases of the “barcode” region of mitochondrial protein-coding cytochrome c oxidase subunit I; COI PJ: 829 bases of the ”Pat/Jer” region of mitochondrial protein-coding cytochrome c oxidase subunit I (no overlap with COI BC); CAD2: 804 bases of the nuclear protein-coding carbamoyl-phosphate synthetase domain of the “rudimentary” gene, corresponding to region 2 of Moulton and Wiegmann (2004); PEPCK: ca. 550 bases of the nuclear protein-coding phosphoenolpyruvate carboxykinase gene; Topo: ca. 550 bases of the nuclear protein-coding triosephosphate isomerase I gene; and wg: 471 bases of the nuclear protein-coding wingless gene. For sake of simplicity of the text, these eight fragments will be referred to as “genes”, even though each corresponds to a portion of a gene or a portion of multiple genes.

Gene fragments were amplified successfully using polymerase chain reaction (PCR) on several different thermal cyclers, including an Eppendorf Mastercycler 5333, an MJ Research PTC-100 Thermal Cycler, or a BioRad MyCycler Thermal Cycler, and using different Taq polymerases, including Eppendorf Hotmaster Taq, Invitrogen Taq, or TaKaRa Ex Taq, each with the basic protocols recommended by the manufacturer. Cycling conditions and primers used in this study are presented in Appendix B. PCR amplification of PEPCK was unsuccessful for several taxa, in spite of as many as 10 attempts for some samples. PCR products were then cleaned, quantified, and sequenced at either the California Academy of Sciences’ Center for Comparative Genomics, using an Applied Biosystems 3130xl DNA Analyzer, or the University of Arizona’s Genomic and Technology Core Facility using either a 3730 or 3730 XL Applied Biosystems DNA Analyzer.

Multiple chromatograms for each gene fragment were assembled using SEQUENCHER (several versions, concluding with v 5.2.2) (Gene Codes Corporation) or PHRED (Green and Ewing 2002) and PHRAP (Green 1999) and the CHROMASEQ package in MESQUITE v3.2 (Maddison and Maddison 2016, 2017). Initial base calls were made using these programs. Early in the project, these base calls were checked using MACCLADE v4.08 (Maddison and Maddison 2005), and final modifications were made using CHROMASEQ and manual inspection. Multiple base reads at a single position were coded using the IUPAC ambiguity codes.

Pinned specimens

Three of the specimens from which DNA was extracted were pinned specimens whose killing and preservation history was unknown: Nebria coreica DRM4721, Nebria komarovi DRM5171, and Nebria oxyptera DRM5172. We prepared specimens following Kanda et al. (2015) and extracted DNA from whole or part of the body using a QIAamp DNA Micro kit (Qiagen) using the optional carrier RNA. Extraction was conducted in a laminar flow hood, sterilized with UV light before each use, in a clean room designed to minimize contamination from non-target DNA.

The extracted DNA was quantified using a Qubit Fluorometer (Life Technologies) with a Quant-iT dsDNA HS Assay Kit. The fragment length distribution was measured using a 2100 Bioanalyzer (Agilent Technologies) using the High Sensitivity DNA Analysis Kit and 1 μl of sample. The DNA of Nebria oxyptera was treated with NEBNext FFPE DNA Repair Mix (New England BioLabs) to repair damaged bases prior to library preparation. Libraries were prepared using NEBNext DNA Ultra II (New England BioLabs) kits and dual-indexed using NEBNext Multiplex Oligos for Illumina. Sequencing (150 base paired-end) was performed at the Oregon State University Center for Genome Research and Biocomputing on an Illumina HiSeq 3000. The Nebria libraries were sequenced on two separate lanes with libraries of other carabids (including bembidiines, chlaeniines, oodines, licinines, and scaritines). Nebria coreica DRM4721 was the only Nebriitae on its lane, while Nebria komarovi DRM5171 and Nebria oxyptera DRM5172 were sequenced on the same lane. In the latter case, the two Nebria libraries were given unique multiplex indices to minimize the possibility of adapter cross-talk.

Demultiplexing was performed using bcl2fastq v2 Conversion Software (Illumina). Paired-end reads were imported into CLC GENOMICS WORKBENCH version 9.5.3 (QIAGEN Aarhus A/S), using default options except for the minimum and maximum paired-read distances, which we determined by analyzing a dilution of the enriched library on a Bioanalyzer 2100 (Agilent Technologies). Failed reads were removed during import. We used the “Trim Sequences” tool in CLC (with default parameters) to remove read ends with low quality, ambiguous base calls, and adapter contamination.

De novo assemblies were generated using CLC GENOMICS WORKBENCH from paired, trimmed reads using an automatic word and bubble size, with the minimum contig length set to 200. The assemblies were converted to local, BLASTable databases using NCBI’s makeblastdb tool, and these databases were queried using the local BLAST tool in MESQUITE. The query sequences were those from Nebria (Boreonebria) gouleti DHK0027, chosen because it is expected to be equally distant from all expected placements for the pinned specimens that were sequenced. For each BLAST conducted, up to ten top matches from each database with eValues less than 1 E -80 were imported into the matrix in MESQUITE for analysis.

Two reference-based assemblies were conducted for each of the three pinned specimens. The first reference-based assembly for each used sequences of Nebria (Catonebria) piperi DHK0965 for the reference; the second used sequences from Nippononebria virescens DHK0348. Reference based assembly was performed using the “Map Reads to Reference” tool in CLC GENOMICS WORKBENCH. Default settings were used except for the “Length Fraction” and Similarity Fraction” parameters, which were both increased to 0.8 to decrease the possibility of spurious read mappings.

Some contigs produced by de novo and reference-based assemblies were discarded. For the de novo assembly, several samples yielded multiple contigs; in these cases, one of the contigs was much larger than the remainder (e.g., two contigs were returned when 16S-ND1 was BLASTed to the Nebria komarovi de novo assembly, one of 4677 bases, the other of 528 bases). In such cases, the shorter contig always appeared to be a contaminant, with one exception. The apparent contaminants were identical to a non-nebriine that was included in the multiplex pool on the same Illumina lane [e.g., Clivina bipustulata (Fabricius), Chlaenius tomentosus (Say)], suggesting that cross-talk occurred (Wright and Vetsigian 2016; Wang et al. 2017), even though the libraries were dual-indexed. The contaminant sequences were discarded. The one exception was a smaller fragment for Nebria coreica 16S-ND1, which partly overlapped with the longer contig; in this case, the union of the contigs was created after alignment, and that union was the sequence analyzed. For the reference-based assemblies, some small contigs less than 250 bases were produced in addition to the longer ones; similar examination of the small ones also suggested that they represented contamination, or that they were for a conserved region of the gene and contained no signal for placement; these were also discarded. For each gene for each of the three pinned specimens, there are thus up to three sequences (one de novo sequence, and two reference-based sequences).

In order to judge the consistency of the two or three sequences (de novo assembly, reference-based to Nippononebria, and reference-based to Catonebria) from each pinned specimen, preliminary analyses were conducted on each of the seven genes and on the concatenated data. After model choice with JMODELTEST version 2.1.10 (Guindon and Gascuel 2003; Darriba et al. 2012) and PARTITIONFINDER 2.1.1 (Lanfear et al. 2012) (resulting in the same models as chosen for the finalized matrix, as described under Results), a search for ML trees was conducted with RAXML version 8.1.4 (Stamatakis 2014) using five search replicates. For all eight matrices, if there was more than one sequence for each gene for a specimen, then these formed a clade on the gene tree, with short branches, indicating that the data were at least consistent. The final gene sequence or a pinned specimen was formed from the union of the two or three sequences from different assembly techniques, with sites thereby having multiple states treated as ambiguities and assigned IUPAC codes.

Alignment and data exclusion

The gene fragments containing ribosomal DNA were aligned differently from the protein-coding genes. An alignment of 28S was conducted in MAFFT version 7.130b (Katoh and Standley 2013), using the L-INS-i search option and otherwise default parameter values. Boundaries of 16S, tRNA leu and ND1 were determined by aligning our sequences to the annotated complete mitochondrial genome of Trachypachus inermis (GenBank Accession NC_011329; Sheffield et al. 2008). However, the boundary between 16S and tRNA Leu was not completely clear; there were between zero and seven bases whose assignment was unclear in some taxa; these sites were excluded from further analyses. Once the boundaries were determined, the 16S region was aligned using MAFFT from within MESQUITE; the tRNA was separately aligned, again using MAFFT from within MESQUITE.

The methods used to align the protein-coding genes varied from gene to gene. COI BC, COI PJ, and Topo showed no evident insertion or deletion events in the history of the taxa studied, and so they could easily be aligned by inspection. Variation in wg suggested several insertion or deletion events present in the history of that taxa. This gene was aligned by first translating the sequence to amino acids in MESQUITE, aligning amino acids in MAFFT, and then forcing that amino acid alignment onto the original nucleotide alignment using MESQUITE. CAD2 and PEPCK contained introns, but few other insertions and deletions, so they were most easily aligned by inspection within MESQUITE’s data editors. CAD2 contained one intron present in Trachypachus, as well as one three-base insertion in Metrius; there were no insertions or deletions evident within nebriites. PEPCK contained one variable-length intron present in most nebriites as well as Pterostichus and Scaphinotus. In addition, there were two three-base deletions in two lineages of Nebria.

Site exclusion also varied from gene to gene. For the protein-coding genes, introns were excluded as well as starting region of PEPCK and Topo, as that region was sequenced in only a few taxa. For 28S and 16S-ND1, sites were excluded from consideration using the modified GBLOCKS (Talavera and Castresana 2007) analysis present in MESQUITE with the following options: minimum fraction of identical residues for a conserved position = 0.2, minimum fraction of identical residues for a highly-conserved position = 0.4, counting fraction within only those taxa that have non-gaps at that position, maximum length of contiguous non-conserved blocks = 4, minimum length of a block = 4, and allowed fraction of gaps within a position = 0.5. In addition, seven bases at the start and three bases at the end of 28S were excluded to make the starting and ending points more even across sequences.

Phylogenetic inference

Models of nucleotide evolution for single genes (except for the 16S-ND1 fragment) were chosen with the aid of JMODELTEST. For concatenated analyses and for the 16S-ND1 fragment, PARTITIONFINDER was used to determine the optimal partitioning, using the greedy algorithm and Bayesian Information Criterion (BIC). The beginning partition for the PARTITIONFINDER analysis had each codon position in each protein-coding gene in a separate part, and the components of the 16S-ND1 fragment in separate parts; thus, for the full concatenated matrix, the initial partition had 25 parts (three parts for each of COI BC, COI PJ, CAD2, ND1, PEPCK, Topo, and wg, as well as one part for each of 28S, 16S, tRNA-Leu, and the non-coding section of the 16S-ND1 fragment). For the final, concatenated matrix the “--raxml” option was used in PARTITIONFINDER, as otherwise program failure occurred during optimization.

Maximum likelihood estimates of phylogenetic relationships were reconstructed using RAXML version 8.2.12 (Stamatakis 2014) under the General Time Reversible (GTR) model of substitution rates, the gamma model of rate heterogeneity, an estimated proportion of invariable sites (GTRGAMMAI), and 100 independent heuristic searches. Non-parametric bootstrap support values were generated from 1,000 replicates employing the standard or “slow” bootstrap algorithm. Bootstrap (MLB) values were mapped onto the highest scoring likelihood tree generated from the 100 heuristic searches using MESQUITE’s Clade Frequencies in Trees feature. Separate RAXML analyses were performed on each of the eight genes. We also ran RAXML analyses on concatenated matrices of all eight genes (8G ML), as well as of nuclear genes (Nuc G), nuclear protein-coding genes (NPC G), and mitochondrial gene fragments (Mito G).

Bayesian analyses were conducted on the eight-fragment concatenated matrix (8G B) with MRBAYES v 3.2.7 (Ronquist and Huelsenbeck 2003; Ronquist et al. 2012), using two runs with four chains each, with 100,000,000 generations run, sampling every 1,000 generations, and with a burn-in period of 25%. The average standard deviation of split frequencies was 0.00092 at the end of the run, with the effective sample size for all parameters being at least 550, as determined by Tracer v 1.7.1 (Rambaut et al. 2018). The final sample consisted of a total of 150,000 trees. Bayesian Posterior Probability (BPP) values are reported as percentages.

Support values

Bootstrap support for and against various clades was calculated using MESQUITE’s “Clade Frequencies in Trees” feature and the bootstrap trees produced by RAXML. Alternative trees with specific contradictory nodes were also included among the bootstrap support trees, and support values for or against those specific nodes were calculated by the same method. Bayesian posterior probability (BPP) values for and against clades were calculated in similar fashion, but using the trees produced by the Bayesian sampling.

Unique molecular synapotypies

To find unique molecular synapotypies for clades, MESQUITE’s “With State Distinguishing Selected Taxa” feature was used. In brief, the “Select Taxa in Clade” tool was used in the tree window, and then the “With State Distinguishing Selected Taxa” feature selected all characters with a unique state in the selected taxa. The unique state was then determined by visually examining those characters in the data matrix. Unique amino acids characterizing clades were found using the same procedure after converting each protein-coding gene to the implied amino acids using MESQUITE’s “Translate DNA to Protein” feature. To find unique base insertions and deletions in 28S sequences, the aligned matrix, including well-aligned internal portions of sequences excluded from ML and Bayesian analyses, was examined visually. Unique amino acid insertions and deletions in protein-coding gene sequences were identified by visually examining the matrix for each fragment converted to a protein matrix.

Data resources

New sequences generated in this study have been deposited in GenBank with accession numbers MW359101 through MW361062. Two files have been deposited in the Dryad Digital Repository, at https://doi.org/10.5061/dryad.6wwpzgmxn: (1) a NEXUS file containing aligned sequence data as well as inferred maximum likelihood trees, and (2) a NEXUS file containing data about PEPCK intron lengths and the ancestral state reconstruction shown in Suppl. material 2: Fig. S13.

Results

Sequencing

We were able to acquire sequence data for 97% (2041) of the 2112 possible sequences for the eight gene fragments for our 264 exemplar specimens (see Table 2). These included 1956 newly generated sequences and 85 sequences from GenBank (including 65 from Kavanaugh et al. 2011, three from Maddison 2008, two from Maddison et al. 2009, two from Ober 2002, two from Weng et al. 2020 and 11 from Wild and Maddison 2008). The numbers and percentages of sequences obtained are listed by gene fragment in Table 3.

Table 3.

Numbers and percentages of sequences used in analyses by gene.

28S 16S-ND1 COI PJ COI BC CAD PEPCK TOPO wg totals
specimens sampled 264 264 264 264 264 264 264 264 2112
sequences obtained 262 260 264 264 259 228 251 253 2041
% success 99 98 100 100 98 86 95 96 97

The 16S-ND1 sequences for Nebria piute DHK1098 and N. catenata DHK1067 each contain a stop codon in the ND1 gene, providing evidence that those particular sequences might be nuclear copies of the mitochondrial genes (“numts”, Thalmann et al. 2004) rather than the true mitochondrial copies, although we have no additional evidence of this (e.g., in the form of double peaks in the chromatograms). As these sequences appear in the phylogenies exactly where expected (see below), we have included these sequences in the analyses.

The only gene fragment for which we obtained less than 95% of the possible number was PEPCK; we speculate that failures in this gene may be the result of the variable-length intron within the gene, which may be long enough in some specimens to prevent amplification under the protocols we used. Of the 228 taxa from which we obtained sequences of PEPCK, 208 shared an intron, although the length of the intron varied between 27 and 830 bases. The intron was lacking from all outgroups except for Pterostichus and Scaphinotus; within nebriites it was absent from Notiophilus, Nebria (Parepinebriola), and the Orientonebria-Archastes-Oreonebria clade, but otherwise present. The only large group in which PEPCK was sequenced in all species was the Catonebria Series, a group in which the intron is relatively short.

Models of evolution and partitions

GTR+I+G was chosen by the BIC for all single-gene analyses. This includes the 16S-ND1 fragment, for which PARTITIONFINDER found that the optimal partition had all sites in one part.

For the concatenated matrices, the best model scheme chosen by PARTITIONFINDER for all parts was GTR+I+G. The optimal partition scheme varied among concatenated matrices. For the eight-gene concatenated matrices (with or without the merging of the pinned specimens’ sequences), third positions of the mitochondrial genes were in one part, and all of the remaining sites in a second part. For the concatenated nuclear protein-coding gene matrix and concatenated nuclear gene matrix, all sites were in one part. For the concatenated mtDNA matrix, all third positions were in one part, with all remaining sites in a second part.

Inferred phylogeny

From both ML and Bayesian analyses, we infer a well-resolved tree (Fig. 4 and Suppl. material 2: Fig. S1) with strong support in multigene analyses for most of the clades (Fig. 5, Chart 1). A fully resolved tree for the 256 nebriite taxon samples in our matrix would have had 255 internal nodes, including the basal node for nebriites. Our ML bootstrap tree for the concatenated eight-gene matrix (8G ML) (Fig. 5) had 229 internal nodes, of which 167 (65% of the possible 255 internal nodes) had MLB support of 90% or more, 194 (76%) had support of 75% or more, and 211 (83%) had support of 60% or more. The majority rule consensus tree from our Bayesian analysis (8G B) of the same matrix (Suppl. material 2: Fig. S1) had 253 internal nodes and thus was 99% resolved. Of these nodes, 228 (89%) had BPP values of 90 or more, 236 (93%) had values of 75 or more, and 244 (96%) had values of 60 or more. The 8G ML and 8G B trees were very similar in structure, with all but 18 of the 229 clades (92%) in the 8G ML tree shared with the 8G B tree. Those not shared are identified in the ML tree (Fig. 4) by an open circle on basally pale branches. Thirteen of the 18 differing clades were found within the Catonebria Series, the most densely sampled part of the tree. Instances of ambiguity are addressed where appropriate in the Discussion.

Chart 1. 

Support for or against various clades. All columns provide maximum likelihood bootstrap values for or against a particular clade, except for column “8G B,” which shows the Bayesian posterior probability estimates for the eight-gene matrix. “8G ML” shows the bootstrap values for the eight-gene concatenated matrix, “Nuc G” for the concatenated nuclear genes, “NPC G” for the concatenated nuclear protein-coding genes, and “Mito G” for the concatenated mitochondrial genes. The remaining eight columns provide values for the single gene analyses. All values are expressed as percentages, with positive numbers indicating support for a clade and negative numbers indicating support for a contradictory clade having the highest support. Specific contradictory clades from alternative trees are highlighted in medium grey. Cells with bootstrap values ≥ 90 are shown in black, with values between 75 and 89 in dark grey, and values from 50 to 74 in light grey. Cells in white indicate clades present in the ML tree, but with bootstrap values < 50. Cells in red have bootstrap values for a contradictory clade ≥ 50. Cells in pink have bootstrap values for or against a clade < 50, and the clade is not present in the ML tree. A “-“ in a cell indicates that taxon sampling for that gene was not sufficient to assess monophyly of that clade. “#g” shows the number of single-gene analyses (maximum of eight) that support a clade with bootstrap values of 50 or more.

Chart 1. 

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Figure 4. 

Maximum likelihood tree for concatenated matrix of all genes. Scale bar: 0.1 units, as estimated by RAXML.

Figure 4. 

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Figure 4. 

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Figure 4. 

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Figure 5. 

Majority rule consensus tree of trees from bootstrap replicates. The first number under a branch is the percentage of bootstrap replicates with that clade, the second number is the estimate of the Bayesian posterior probability of that clade expressed as a percentage.

Figure 5. 

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Figure 5. 

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Figure 5. 

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ML trees and majority rule consensus trees of bootstrap trees resulting from analyses of concatenated matrices for Nuc G, NPC G, and Mito G and for each of the single-gene matrices are presented in Suppl. material 2: Figs S2–S12. Support for and against particular clades from each of the concatenated and single-gene analyses is summarized in Chart 1. Phylogenetic results of interest are discussed in detail in the Discussion.

Unique bases found to support particular clades are listed in Suppl. material 1: Table S1, unique amino acids in Suppl. material 1: Table S2, and unique insertions and deletions in Suppl. material 1: Table S3.

The intron length of PEPCK varies across the phylogeny, with some clades having small or no introns, and with a few clades with longer introns (Suppl. material 2: Fig. S13).

Discussion

Monophyly of Nebriitae

Morphology has provided only weak support for the monophyly of a supertribe Nebriitae because most features that have been used to characterize it are both plesiomorphic within carabids and not unique to this group. Kavanaugh and Nègre (1983) suggested that the only known synapomorphy for the entire group is the asetose parameres of the male genitalia. Arndt (1993) suggested three additional larval morphological synapomorphies. In fact, the most parsimonious tree for a matrix of 153 morphological characters suggested that the Nebriitae represent a grade rather than a clade (Kavanaugh 1998). Other “basal grade” carabid groups, including cicindines (Erwin 1991; Lorenz 2005), gehringiines, omophronines, elaphrines (Liebherr and Will 1998) and a few other lineages, have been suggested as members of the Nebriitae, so both the monophyly and the inclusiveness of the supertribe have remained uncertain.

Based on results from analyses of our molecular data, we find very strong evidence for a monophyletic Nebriitae (Fig. 4; Chart 1, line 1), of course with Notiokasis unsampled for the present. Both our ML (Fig. 5) and Bayesian analyses (Suppl. material 2: Fig. S1) of the eight-gene matrix show support (MLB and BPP) values of 100 for this clade. ML analyses of concatenated nuclear gene (Suppl. material 2: Fig. S2) and nuclear protein-coding gene (Suppl. material 2: Fig. S3) matrices, as well as of single-gene matrices for 28S, CAD2 and wg (Suppl. material 2: Figs S5, S9 and S12, respectively) show MLB values ≥ 90. This clade also appears in the tree for concatenated mitochondrial genes (Suppl. material 2: Fig. S4), albeit with weak support. Monophyly of the supertribe is also supported by three unique amino acids (Suppl. material 1: Table S2). Evidence contradictory to this clade is provided by ML analyses of single-gene matrices for PEPCK (Suppl. material 2: Fig. S10), Topo (Suppl. material 2: Fig. S11), COI BC (Suppl. material 2: Fig. S7) and COI PJ (Suppl. material 2: Fig. S8), each of which shows one or more of notiophilines, opisthiines, or pelophilines more closely related to one or more of the outgroup taxa than to the other nebriite taxa but with only weak support and no consistent pattern of relationship. Because we were unable to include Notiokasis in our sample, our conclusions about nebriite monophyly cannot yet include the Notiokasiini. A more thorough test of the monophyly of nebriites would not only include Notiokasis, but also a more thorough sampling of outgroups.

Monophyly of the nebriite tribes and relationships among them

Monophyly of the tribes Notiophilini, Pelophilini, and Opisthiini are well supported morphologically (Ball and Bousquet 2001), and our results strongly support their being clades. Analyses of all concatenated and single-gene matrices produced MLB and BPP values of 100 for both the Notiophilini and Pelophilini (Chart 1, lines 2 and 5, respectively). Monophyly of each of these two tribes is supported also by unique amino acids (19 and two, respectively; Suppl. material 1: Table S2). The notiophiline clade is also supported by one unique base insertion and one unique deletion in 28S and by one amino acid insertion and three amino acid deletions in wg (Suppl. material 1: Tables S3). The pelophiline clade is also supported by 18 unique bases (Suppl. material 1: Tables S1). Our ML and Bayesian analyses of the eight-gene matrix and ML analyses of the concatenated nuclear gene and nuclear protein-coding gene matrices produced support values of 100 for Opisthiini (Chart 1, line 6). Opisthiine monophyly was also strongly supported in single-gene analyses for 28S, CAD2 and Topo, and moderately supported in the concatenated mitochondrial gene analysis (Chart 1, line 6), as well as by one unique amino acid and seven unique bases (Suppl. material 1: Tables S1, S2). Paropisthius is also strongly supported as monophyletic in all analyses (MLB and BPP values > 95) except for wg (Chart 1, line 7), where it does not appear in that resulting tree (Suppl. material 2: Fig. S12). It is also supported by five unique amino acids and 17 unique bases (Suppl. material 1: Tables S1, S2) and by a unique three-base insertion in 28S (Suppl. material 1: Table S3).

In spite of the fact that the diverse Nebriini is not well characterized morphologically, being recognized mainly on the basis of symplesiomorphic features, monophyly of the tribe is strongly supported by our molecular data. Both ML and Bayesian analyses of the eight-gene matrix and ML analyses of the concatenated nuclear gene and nuclear protein-coding gene matrices produced support values of 100 for the tribe (Chart 1, line 11). Nebriini is also strongly supported in analyses for CAD2 and Topo, moderately supported in the wg analysis and weakly supported in the 28S and PEPCK analyses (Chart 1, line 11), as well as by one unique amino acid (Suppl. material 1: Table S2). The nebriine clade appears in six of the eight single-gene trees (Suppl. material 2: Figs S5–S12).

Phylogenetic relationships among the nebriite tribes remain somewhat less clearly defined than their monophyly. Our results show moderate to strong support for a clade including Opisthiini, Pelophilini and Nebriini as sister to Notiophilini (Fig. 5; Chart 1, line 3). Strongest support (≥ 94) for this clade comes from the eight-gene Bayesian analysis (BPP = 95) and the single-gene 28S analysis (MLB = 95) with moderate additional support from the concatenated nuclear gene analysis (MLB = 82) and from one unique amino acid (Suppl. material 1: Tables S1) and one unique amino acid insertion in the wg gene fragment (Suppl. material 1: Table S3) not seen in notiophilines or any of the outgroups. We found no support for clades including Nebriini + Notiophilini (Chart 1, line 8) or Nebriini + Opisthiini (Chart 1, line 9). Our results about tribal relationships within nebriites should be considered with some caution, as they are an old enough group with distant enough relatives that determining the placement of the root within Nebriitae may not be easy. For example, it is possible that notiophilines are reconstructed as the sister to other nebriites as they are on a long branch, which might then be the place of attachment to the relatively minimally sampled and thus long-branched outgroups through long-branch attraction (Felsenstein 1978).

Our results do not strongly speak to the treatment of Pelophila as belonging to a separate monogeneric tribe. Pelophila had been included among the Nebriini (see Thomson 1859; Ganglbauer 1891a; Reitter 1908; Ball 1960; Lindroth 1961) until Kavanaugh (1996) proposed separate tribal status for it based a parsimony analysis of morphological data that found a clade including Notiophilini + Notiokasiini + Opisthiini more closely related to other nebriines than was Pelophila. In a second parsimony analysis using a slightly different morphological dataset, Kavanaugh (1998) found Pelophila more closely related to other nebriines than was a clade including Notiokasiini + Notiophilini + Opisthiini; but in this reconstruction, the Nebriitae represented a grade rather than a clade. Ambiguity in the relationships of Pelophila to the other nebriites is reflected in our molecular results as well. A clade including Pelophila and the opisthiines (Chart 1, line 4) is supported weakly (MLB = 51-52) in the ML eight-gene concatenated and 28S bootstrap analyses and it appears in only half of the single-gene ML trees (28S, COI BC, PEPCK, and wg) (Suppl. material 2: Figs S5, S7, S10, S12). It is supported also by one amino acid unique for the entire taxon sample and three others unique among nebriites and by a single unique base (Suppl. material 1: Tables S1, S2). Conversely, a clade including Pelophila with the other nebriine genera (Suppl. material 2: Fig. S1) has strong support from the Bayesian eight-gene concatenated analysis (BPP = 97) and weak support from the ML concatenated nuclear protein-coding gene bootstrap analysis (MLB = 64) and the Topo bootstrap analysis (MLB = 61) (Chart 1, line 10); but this clade appears only in the single-gene ML tree for Topo (Suppl. material 2: Fig. S12). This difference in results from the ML and Bayesian eight-gene concatenated analyses is the single most important disagreement between them, so at least for the present, we continue to treat Pelophilini as a distinct nebriite tribe but part of an unresolved polytomy with notiophilines, opisthiines, and nebriines (see overview tree of nebriite phylogeny, Fig. 6).

Figure 6. 

Summary tree of nebriite phylogeny illustrating the revised classification; clade representation in Europe (including North Africa and the Middle East), Asia, and North America is indicated in the three-box bar.

Relationships among the Nebriini

As noted in the Introduction, there have been differences of opinion among taxonomists concerning the number of distinct genera that should be recognized among the Nebriini as defined here (i.e., excluding Pelophila). Nevertheless, Leistus has been universally accepted as a distinct genus, based on its wonderful suite of mouthpart co-adaptations for small prey capture. Nebria also has been universally regarded as a distinct genus, but its inclusiveness has differed among authors. In the following sections, we discuss how our results provide new evidence to support phylogenetic relationships among Leistus subgenera and species and better determine which groups are part of a monophyletic genus Nebria and which ones are not.

Phylogeny of Leistus

Perrault (1980) introduced and later modified (Perrault 1991) a classification of Leistus that is currently in wide use, recognizing six subgenera. His conclusions were based mainly on detailed studies of adult mouthparts and male genitalia. Particularly striking are the differing degrees of development and fusion of digitiform processes on the maxillary stipes and submentum on which stout, spiniform setae insert, as well as differences in the form and development of the trident-like anterior extension of the ligular sclerite seen in members of this genus. Our results based on analyses of molecular data provide support for his classification and the phylogenetic relationships he proposed within the genus.

The monophyly of genus Leistus is strongly supported in all of our concatenated and single-gene analyses (see Fig. 5A; Chart 1, line 17) as well as by one unique amino acid, one unique base and three unique base deletions in 28S (Suppl. material 1: Tables S1–S3).

We had only one of the two known species of subgenus Nebrileistus Bänninger, 1925 in our sample, so we cannot comment further on its monophyly. In our analyses, Nebrileistus is shown as sister to a clade including the other Leistus subgenera (Figs 4A, 5A; Chart 1, line 18) that is very strongly supported as monophyletic in the 8G B analysis (BPP = 100) (Suppl. material 2: Fig. S1), slightly less strongly supported by the 8G ML (Fig. 5A), Nuc G (Suppl. material 2: Fig. S2) and NPC G (Suppl. material 2: Fig. S3) and moderately supported by analyses for 28S (Suppl. material 2: Fig. S5), CAD2 (Suppl. material 2: Fig. S9) and wg (Suppl. material 2: Fig. S12). That clade is also supported by one unique amino acid (Suppl. material 1: Table S2). This relationship is not supported by any of the mitochondrial genes in single-gene or concatenated bootstrap analyses, but still is shown in the ML trees from both the Mito G and 16S-ND1 analyses (Suppl. material 2: Figs S4, S6–S8). A sister group relationship of Nebrileistus to a clade including all other Leistus is consistent with the absence of several relatively apomorphic morphological features present in their members.

Subgenus Sardoleistus Perrault, 1980 is monobasic, as it includes only Leistus sardous Baudi di Selve, 1883. Results from our analyses suggest that this subgenus is sister to a clade including Pogonophorus Latreille, 1802, Evanoleistus Jedlička, 1965 and Leistus s. str. (Fig. 4A), but support for the latter clade is mixed (Chart 1, line 19). It is strongly supported in the 8G B analysis (BPP = 92) (Suppl. material 2: Fig. S1) but only weakly supported in the 8G ML (Fig. 5A), Nuc G (Suppl. material 2: Fig. S2) and NPC G (Suppl. material 2: Fig. S3) analyses. The clade does not appear in ML trees from the Mito G analysis (Suppl. material 2: Fig. S4) or from any of the single-gene analysis except for 28S (Suppl. material 2: Fig. S5), where it has low MLB support. Other possible relationships suggested in individual analyses include (1) a sister-group relationship to all other Leistus subgenera (Suppl. material 2: Figs S7–S9), or (2) sister to a clade including only Evanoleistus and Leistus s. str. (Suppl. material 2: Fig. S4), or (3) as part of an unresolved trichotomy with Pogonophorus and Evanoleistus + Leistus s. str. (Suppl. material 2: Figs S6, S12).

The subgenus Pogonophorus is strongly supported as monophyletic in all concatenated gene analyses and all single-gene analyses except for 28S (Chart 1, line 20). In the tree from the 28S analysis (Suppl. material 2: Fig. S5), Pogonophorus is paraphyletic in an unresolved trichotomy with a clade including subgenera Evanoleistus + Leistus s. str. Because our sample included only ca. 5% of the known diversity currently included in this subgenus (there are 40 species and more than 20 subspecies beyond the three species we sampled) as well as only a limited geographical sample (European species only) of the range of this taxon, monophyly for the subgenus should be confirmed with additional sampling.

A clade including Evanoleistus and Leistus s. str. is strongly supported as monophyletic in all concatenated gene analyses and strongly to moderately supported in all single-gene ML analyses except for PEPCK (Chart 1, line 21). It is also supported by one base unique among nebriites (Suppl. material 1: Table S1) and by two unique base deletions in 28S (Suppl. material 1: Table S3). In the ML tree from the PEPCK single-gene analysis (Suppl. material 2: Fig. S10), groups of species from both subgenera are shown in an unresolved polytomy that also includes Leistus nubivagus Wollaston, 1864 (i.e., subgenus Nebrileistus), a pattern of relationships not seen in any of the other single-gene or concatenated gene trees. We therefore suggest that Evanoleistus and Leistus s. str. together form a clade. As noted below, each of them is strongly supported as monophyletic in most of our concatenated gene analyses. However, the phylogenetic distinction between them is not yet completely clear.

Evanoleistus is the most diverse subgenus of Leistus with more than 150 species described. Our sample included only nine species, two of which are undescribed, so our sampling was small (ca. 5%) relative to that diversity and our results must be judged in that light. Nonetheless, the species in our sample form a monophyletic group (Figs 4A and 5A) that is strongly supported in all the concatenated gene analyses except the Mito G analysis (Chart 1, line 22) and also in the 28S and Topo analyses (Suppl. material 2: Figs S5 and S11, respectively). This clade also appears in ML trees from CAD2, PEPCK, and wg analyses (Suppl. material 2: Figs S9, S10, and S12, respectively) but with only moderate support. Evidence against a monophyletic Evanoleistus clade for the species in our sample involves only the mitochondrial genes and mainly the relationships of Leistus sp NEP to the other species of Evanoleistus and to Leistus s. str. In the ML tree from the Mito G analysis (Suppl. material 2: Fig. S4), L. sp NEP is part of a clade with the species of Leistus s. str. rather than with Evanoleistus species, but that clade has only moderate support (MLB = 61). Stronger support (MLB = 84) for this same clade is provided by the COI PJ analysis (Suppl. material 2: Fig. S8). In the tree from the 16S-ND1 analysis (Suppl. material 2: Fig. S6), L. sp NEP is shown as sister to a clade including the remaining Evanoleistus species plus all the Leistus s. str. species, but with only weak support (MLB = 40); and in the tree from the COI BC analysis (Suppl. material 2: Fig. S7), it is part of a weakly supported clade with the other Evanoleistus species that also includes Leistus nitidus (Duftschmidt,1812) from among our Leistus s. str. species. A clade that includes all the Evanoleistus species excluding L. sp NEP (Chart 1, line 23) is strongly supported in all concatenated gene analyses, including the Mito G analysis, in all three mitochondrial and in the wg single-gene analysis, but it is only moderately supported in CAD2 and PEPCK analysis and contradicted in the 28S and Topo analyses. The above results suggest that Leistus sp NEP is unique among the species in our sample, representing either a sister taxon to all our other Evanoleistus or an outlier related more closely to one or more species of Leistus s. str. As the only Himalayan species in our sample out of the 18 species described in Evanoleistus from that region, it may represent a more diverse lineage with relationships intermediate between Evanoleistus and Leistus s. str. We therefore include L. sp NEP in an unresolved trichotomy in our summary tree (Fig. 6) to reflect this ambiguity. Molecular data from additional members of the Himalayan fauna may help clarify relationships.

Leistus s. str. currently includes ca. 50 species and our sample represents a little more than 20% of them. Our sample of this subgenus is strongly supported as monophyletic in all concatenated analyses except for the Mito G analysis (Chart 1, line 24), in which it is only weakly supported (MLB = 42). This clade is shown also in ML trees for all but three of the single-gene analyses (COI BC, COI PJ and PEPCK; Suppl. material 2: Figs S6–S8, respectively) but with lower support values. Contradictory clades involve the inclusion of L. sp NEP from Evanoleistus in Leistus s. str. (MLB = 84; COI PJ tree, Suppl. material 2: Fig. S8), the inclusion of L. nitidus instead in Evanoleistus (COI BC tree, Suppl. material 2: Fig. S7), and, in the PEPCK tree (Suppl. material 2: Fig. S10), Leistus crenatus Fairmaire, 1855 and L. ferrugineus, the type species of Leistus, in a clade with L. nitidus that is more closely related to the Evanoleistus species than to the remaining “Leistus s. str.” species.

Erwin (1970) described subgenus Neoleistus for the three endemic Leistus species in western North America. Perrault (1980) originally treated Neoleistus as a junior synonym of Leistus s. str.; but later (Perrault 1991) assigned species of his niger group of Leistus s. str. to Neoleistus and resurrected it as a valid subgenus. Lorenz (2005) followed Perrault’s reclassification, but Farkač (2016) retained the Palearctic species (niger group) in Leistus s. str. Our results confirm a monophyletic North American clade (Figs 4A, 5A) that is strongly supported in all concatenated and single-gene analyses (Chart 1, line 25). However, this clade is deeply nested within subgenus Leistus s. str., so recognizing it as a separate subgenus would render Leistus s. str. paraphyletic. We therefore consider Neoleistus as a junior synonym of Leistus s. str.

Status of Nippononebria

Uéno (1955) described Nippononebria as a subgenus of Nebria. Ledoux and Roux (2005) treated Nippononebria and Vancouveria as separate subgenera in their “Nippononebrides” group of subgenera within the “Vetanebri”, one of their two primary divisions of Nebria. In contrast, Kavanaugh (1995, 1996), had proposed that Nippononebria should be considered as a genus distinct from Nebria and with Vancouveria (Fig. 2D) as its subgenus and, further, that this clade was sister to Leistus rather than to Nebria. This conclusion was based on phylogenetic analyses of morphological data, as was that of Ledoux and Roux. However, the latter do not appear to have included morphological data for Leistus, Archastes, or any genera from other nebriite tribes in their formal analysis of Nebria, and this limitation would have prevented them from recognizing a sister group relationship between Nippononebria and any other group except Nebria.

Results from our analyses of molecular data provide strong support for a clade including only Nippononebria and Leistus (Chart 1, line 12). Our 8G ML, 8G B, Nuc G, and NPC G concatenated analyses and CAD2 single-gene analyses show strong support for this clade, PEPCK shows moderate support, and Mito G and 16S-ND1 show weak support. Conversely, a clade including only Nippononebria and Nebria (Chart 1, line 13) is unsupported, except weakly so by the 28S single gene analysis (MLB = 41). Monophyly of Nippononebria (including both Nippononebria s. str. and Vancouveria) (Chart 1, line 14) is also strongly supported in all concatenated analyses except Mito G and in 28S, CAD2, PEPCK and wg analyses. Two unique amino acids also support this clade (Suppl. material 1: Table S2). Subgenera Nippononebria s. str. (Chart 1, line 15) and Vancouveria (Chart 1, line 16) are both strongly supported as monophyletic by all available results, as well as by three and five unique amino acids, respectively (Suppl. material 1: Table S2). The fossil Archaeonebria inexspectata, recently described from Baltic amber and dated at 50–35 Mya (Schmidt et al. 2019), appears to be a stem Nippononebria and, as such, supports our molecular results in attesting to a deep split between this lineage and Leistus. We conclude that Nippononebria should be classified as a nebriine genus distinct from both Nebria and Leistus and more closely related to the latter (Figs 4A, 6). An alternative change would be to expand the scope of Nebria to include Nippononebria, Archaeonebria and Leistus, which we view as excessive consolidation obscuring rather than clarifying relationships.

Status of Archastes

Unlike Nippononebria, Archastes (Fig. 2C) was described as a distinct nebriine genus (Jedlička 1935) and has always been treated as such (Ledoux and Roux 2009). All of the 37 described species share a similar general body form and habitus, all are flightless and together they occupy a relatively small area, mainly in central Sichuan Province, China. We had only one species represented in our sample, but for the purpose of exploring the phylogenetic relationships of Archastes to other nebriines and to Nebria in particular, this sample may be sufficiently representative, given the morphological and geographical cohesion of the group.

All our concatenated analyses, as well as 28S and Topo single-gene analyses, very strongly support a clade including Archastes within Nebria (Chart 1, line 26). Conversely, a Nebria clade excluding Archastes is strongly contra-indicated. This is shown whether Archastes is excluded alone (Chart 1, line 29) or along with the other Nebria subgenera to which our results show close its relationship (see further discussion below and Chart 1, lines 27 and 28). Consequently, we conclude that Archastes does not warrant status as a separate genus, but should be classified within genus Nebria.

Status of Oreonebria

Daniel (1903) described Oreonebria (Fig. 2E) as a subgenus of Nebria. Jeannel (1937, 1941) recognized this taxon as a genus separate from Nebria, based mainly on differences between the two groups in male genitalic features. Huber (2004, 2017) and Lorenz (2005) followed Jeannel in giving Oreonebria generic status, but Ledoux and Roux (2005) included it as a subgenus in their treatment of Nebria.

Just as for Archastes, our analyses strongly support a monophyletic Nebria including Oreonebria (Chart 1, line 26), and a Nebria clade excluding Oreonebria is strongly contra-indicated. This is shown whether Oreonebria is excluded alone (Chart 1, line 30) or along with the other Nebria subgenera found from our analyses to be closely related (see further discussion below and Chart 1, lines 27). We conclude that Oreonebria does not warrant status as a separate genus and should be classified within genus Nebria.

Status of Eurynebria

Eurynebria was described as a new genus by Ganglbauer (1891b) with Nebria complanata (Carabus complanatus Linnaeus, 1758) (Fig. 2F) as the type species. Jeannel (1937, 1941), followed by Lorenz (2005) and Huber (2017), treated this taxon as a distinct genus. Although N. complanata exhibits several autapomorphic features, such as more setae on the labrum and penultimate labial palpomere than in any other Nebria species, Ledoux and Roux (2005) included it as a subgenus in their treatment of Nebria.

Once again, our analyses strongly support genus Nebria including Eurynebria as monophyletic (Chart 1, line 26) and a Nebria clade excluding Eurynebria as strongly contra-indicated (see further discussion below and Chart 1, line 31). We conclude that this taxon does not warrant generic status and should be classified within genus Nebria.

Phylogeny of Nebria

As noted above, the evidence for the monophyly of Nebria, including Archastes, Oreonebria, and Eurynebria while excluding Nippononebria and Vancouveria, is strong. All concatenated analyses and the 28S analysis yielded MLB support values ≥ 97 (Chart 1, line 26). The Topo analysis also provides solid support (MLB value = 89) for this clade, and it appears in ML trees from five of the remaining six single-gene analyses (Suppl. material 2: Figs S6–S12) but with only weak support. Results from the COI BC analysis, which generated the only tree not showing this clade, differed only in having Bembidion antiquum from the outgroup included within the Nebria clade, a relationship not suggested by any of the other results. This Nebria clade is also supported by two unique bases (Suppl. material 1: Table S1).

Ledoux and Roux (2005: 75, “Graphique 2”) provided a tree illustrating proposed phylogenetic relations and classification of subgenera of Nebria based on their analyses of morphological data. Their analyses included a custom-designed, distance-based clustering method, as well as unspecified analyses using PHYLIP (Felsenstein 1989). Their clustering method was also designed to determine boundaries of taxa, including boundaries of subgenera, based upon a distance-based threshold. Their classification method found 12 distinct groups of subgenera, four of which included a single subgenus, the remainder including from two to four subgenera. They arranged these subgeneric groups into two larger assemblages, the “Vetanebri” and “Notanebri”. The main distinguishing features of these assemblages were as follows. Most Vetanebri male genitalia have a well-developed sagittal aileron at the base of the median lobe and females have a long, spiraled or convoluted spermathecal duct, or one that is abruptly expanded basally near its insertion on the bursa copulatrix. In contrast, most Notanebri males lack or have only a vestigial sagittal aileron and females typically have short and simple (not spiraled or convoluted) spermathecal ducts. Ledoux and Roux considered the absence of a sagittal aileron and presence of a long, spiraled or convoluted spermathecal duct as apotypic states. These distinguishing features explain why they included Nippononebria and Vancouveria in their Vetanebri. Indeed, those two clades have males with a well-developed sagittal aileron and females with very long, slender and convoluted spermathecal ducts. However, the fact that these features are shared also with Leistus males and females, respectively, suggests that both features may be symplesiomorphies within the Nebriini. This would mean that Ledoux and Roux’s Notanebri is supported as monophyletic by two synapomorphies while their Vetanebri is based mainly on two likely symplesiomorphies.

Results of our analyses of molecular data also support several large clades within Nebria, most including multiple subgenera. Following the system of informal hierarchic ranks between genus and subgenus used by Maddison (2012) for Bembidion Latreille, 1802, we call these clades “complexes”, with some of them subdivided into “subcomplexes” of subgenera. Major clades that include one or more complexes we call “series”. Our reconstruction of phylogenetic relationships within Nebria shows the genus divided into four series (Fig. 4A).

The first of these is the Oreonebria Series (Fig. 4B), which corresponds in many ways to Ledoux and Roux’s Vetanebri. However, it differs from the latter in key details. It does not include Nippononebria or Vancouveria but does include Archastes and a group of species that previously have been ascribed to Epinebriola Daniel & Daniel, 1904. This clade is strongly supported in both 8G analyses, the Nuc G analysis, and the 28S analysis (Chart 1, line 32). There is also moderate support from the Topo analysis and weak support from the NPC G and COI PJ analyses. One unique base (Suppl. material 1: Table S1) and one amino acid (Suppl. material 1: Table S2) unique to this clade among Nebria also support this clade. We find no consistent alternative pattern of relationships for members of this clade seen among the ML trees in which the clade does not appear (Suppl. material 2: Figs S4, S6, S7, S9, S10, S12). However, ML trees from the Mito G, CAD2, PEPCK, and wg analyses (Suppl. material 2: Figs S4, S9, S10, S12) show the Oreonebria Series as a grade rather than a clade, but each with different relationships shown among the taxa included in the grade.

The Oreonebria Series is an endemic Eurasian lineage that includes more than 180 described species-group taxa. This clade occupies a disjunct geographical distribution comprising the Alps mountain system of Europe in the west and central Asia to far eastern Asia, including Japan and Taiwan, in the east. It includes two groups of subgenera, the Eonebria and Oreonebria Complexes (Fig. 4B). Homoplasy is so widespread in key characters among Nebria in general and this clade in particular that we cannot cite any synapotypic features shared by all clade members. The main morphological characters that Ledoux and Roux (2005) cited as characteristic of this clade are either inconsistent or not unique to it. For example, males of this clade were characterized as having a sagittal aileron at the base of the median lobe of the genitalia, but males of some subgenera or species groups in this series do not have an evident sagittal aileron. Similarly, females of this clade were said to have long and helical spermathecal ducts or ducts with the proximal portion thickened and abruptly narrowed distally. This is correct as far as we have observed, but long and convoluted ducts or proximally thickened spermathecal ducts are found also in females of many Notanebri taxa, so this feature does not distinguish this clade from the latter.

The remaining three series of Nebria are strongly supported as a monophyletic group in both 8G ML and 8G B analyses (Fig. 5A), as well as in the Nuc G, NPC G, and 28S analyses (Chart 1, line 44). The Mito G, COI PJ, CAD2 PEPCK and wg analyses also provide independent but weak support. This clade is also supported by three bases unique in the entire dataset, one additional base unique among Nebriitae and two unique base insertions in 28S (Suppl. material 1: Tables S1, S3). Strong support for a sister group relationship between this clade and the Oreonebria Series (Fig. 4A) is shown in trees from the 8G ML, 8G B, Nuc G, and NPC G concatenated analyses and the 28S analysis. This is a large clade, including more than 460 described species-group taxa, with a Holarctic distribution. As for the Oreonebria Series, we cannot cite any morphological feature unique to all members of this clade.

The Nebriola Series, which includes only subgenus Nebriola Daniel, 1903, is a small group comprised of only 19 described species-group taxa. Members of all described species are small and flightless due to atrophied hindwings. Their combined geographical distribution is confined to mountain systems of western Europe, from the Pyrenees Mountains of northern Spain and southern France northeast through the Alps to the Jura Mountains of eastern France and Switzerland and the Black Forest of southern Germany. Ledoux and Roux (2005) included Nebriola in their “Boreonebrides” among their Notanebri and cited the main morphological features characterizing members of this subgenus. Most distinctive among these are (1) males with protarsomeres 1 to 4 dilated; (2) male mesotarsomeres (and to a less extent metatarsomeres) 2 to 4 dilated, ca. as wide as long; and (3) females with the spermathecal duct long and convoluted and thickened proximally. Our results show the monophyly of subgenus Nebriola strongly supported (MLB values ≥ 98) in all concatenated and single-gene analysis except that for PEPCK (in which MLB support is a modest 58) (Chart 1, line 45). The clade is also supported by three unique amino acids, six unique bases and three unique insertions in 28S (Suppl. material 1: Tables S1–S3).

The Nebria Series is the most diverse and widespread group in the genus. More than 320 species-group taxa have been described, and the geographical range of the group encompasses most of the Holarctic Region and the northern edge of the Oriental Region in Asia. The group includes two main groups of subgenera, the Boreonebria and Nebria Complexes, and the latter includes three subgeneric subgroups, the Nebria, Epinebriola and Eunebria Subcomplexes (Fig. 4C). This series is equivalent to Ledoux and Roux’s (2005) Notanebri with the exclusion of subgenera Nebriola, Reductonebria, Catonebria and the Nearctic species included in their Nakanebria Ledoux & Roux, 2005. Monophyly of this group, thus comprised, is well supported by the 8G ML, 8G B, and Nuc G concatenated-gene analyses, as well as by the 28S and PEPCK analyses (but the latter only weakly) (Chart 1, line 49). It is also supported by two unique bases (Suppl. material 1: Table S1). Modest support for a contradictory clade including Nebriola within the Nebria Series and excluding Boreonebria and the Palearctic Nakanebria species in our sample is provided by the NPC G and Topo analyses (Suppl. material 2: Figs S3 and S11, respectively). Results from the mitochondrial gene analyses, whether in single- or concatenated- gene analyses, show no consistent pattern of relationship among the included taxa. We conclude that the evidence for the monophyly of this group outweighs the evidence against it. However, we recognize that our sample includes only ca. 20% of described species-group taxa, so additional taxon sampling for molecular data is needed for this clade.

The Catonebria Series is a moderately diverse group with 104 described species, representing a majority of Nearctic Nebria species. It has a Holarctic distribution with a geographical range extending from central Asia east to eastern North America. It includes two subgeneric groups, the Reductonebria and Catonebria Complexes (Fig. 4D). Unlike any of the other complexes in genus Nebria, these two are more diverse in the Nearctic than in the Palearctic Region. This group corresponds to Ledoux and Roux’s (2005) “Serinebrides”, except that we exclude the Palearctic species of Nakanebria from it. The clade is supported by all our concatenated- and single-gene analyses (Chart 1, line 94) as well as by seven unique bases (Suppl. material 1: Table S1) and a two-base deletion in 28S unique among Nebria (Suppl. material 1: Table S3). With 95% of the species-group taxa described for this clade included in our sample, we have high confidence in these results.

Although our results provide evidence that the Nebriola, Nebria, and Catonebria Series together form a clade and that each individually is monophyletic, relationships among them are not yet as clear. Trees from both the 8G ML (Fig. 4A) and 8G B (Fig. 5A) show a clade including the Nebria and Catonebria Series as sister to Nebriola, with moderate and strong support, respectively (Chart 1, line 46). The Nuc G, CAD2, and PEPCK analyses also support this clade, but there is no strong support from any single-gene analysis, and the NPC G, 28S, and Topo results provide MLB support ≥ 50 against this clade. Results from the 28S analysis show support (MLB value = 86) for a contradictory clade including Nebriola and the Nebria Series (Suppl. material 2: Fig. S5), but this clade has no other support (Chart 1, line 47). Although results from the NPC G concatenated analysis show support against the Nebria + Catonebria clade (MLB value = -57) and the ML tree from that analysis (Suppl. material 2: Fig. S3) shows a clade including Nebriola and the Catonebria Series, no single-gene or concatenated analysis shows much support for it (Chart 1, line 48). Consequently, our evidence suggests that a clade including the Nebria and Catonebria Series is likely sister to the Nebriola clade, but additional molecular sampling of species from both Nebriola and the Nebria Series would further test this hypothesis.

Oreonebria Series: Eonebria Complex

The group of subgenera that we refer to as the Eonebria Complex (Figs 4B, 5B) is similar to Ledoux and Roux’s (2005) “Eonebrides” in that it includes the subgenera Eonebria Semenov & Znojko, 1928 and Sadonebria Ledoux & Roux, 2005. However, it also includes a group of species previously classified in subgenus Epinebriola. We recognize this last group of species as a distinct subgenus, Parepinebriola, which is described below (see the Taxonomy section). Together, these three subgenera represent an endemic Palearctic lineage that includes over 90 species with a combined geographical range extended from the southern margin of the Tibetan Plateau to Japan. Support for this clade is provided by all the single-gene analyses except for PEPCK and wg, and by all the concatenated analyses (Chart 1, line 33).

The most diverse subgenus in this clade is Eonebria (Fig. 3A) with 77 described species-group taxa. Most of these species occupy the eastern part of the Tibet-Qinghai Plateau east to western Hubei Province, and from eastern Qinghai in the north to northern Yunnan Province in the south. However, a group of three species, including the type species, Nebria komarovi Semenov & Znojko, 1928, occurs in the Russian Far East and North Korea and no species are known from the intervening area. This represents a significant disjunction in the range of the subgenus. A monophyletic Eonebria, with N. komarovi most basal among species included in our sample, is well supported by analyses of the mitochondrial genes both individually and concatenated, and independently by PEPCK and wg results (Chart 1, line 37). Strong support is also provided by the concatenated 8G ML analysis. However, the trees from the 8G B and 28S analyses (Suppl. material 2: Figs S1, S5) show N. komarovi as more distantly related to the other Eonebria species in our sample than are the species of Nakanebria. Additional molecular sampling of species from each of the vicariant regions might either further support a monophyletic Eonebria or reveal a more significant phylogenetic split between the subfaunas.

Diversity within subgenus Sadonebria has increased from the three species recognized by Ledoux and Roux (2005) to 15, based on morphometric analyses of pronota, elytra and male genitalia and detailed examination of the internal sac of the median lobe of males by Sasakawa (2006, 2008, 2009, 2010, 2016; Sasakawa and Toki 2011). All but one of these species occur in Japan. The lone exception is Nebria niitakana Kano, 1930 from Taiwan. Only one of the Japanese species, Nebria chinensis Bates, 1872, also occurs on the Asian mainland as far west as eastern Sichuan and Shaanxi Provinces. Based on the four species in our sample, the monophyly of Sadonebria is well supported by all our analyses (all MLB or BPP values ≥ 93) (Chart 1, line 36).

One of the most unexpected results of our study was the discovery of the group of five species represented in our sample, including Nebria delicata Huber & Schmidt, 2017 and Nebria retingensis Huber & Schmidt, 2017 and three undescribed species (Figs 4B, 5B), which we assign to a new subgenus, Parepinebriola (for description see Taxonomy section). The geographical range of the known species of this group occupies a very limited area extending from the Central Trans-Himalaya in southern Tibet eastward to the Gaoligong Shan of northwestern Yunnan. This group is well supported as monophyletic in all our single-gene and concatenated analyses (Chart 1, line 34). All but one of these species had been identified as members of subgenus Epinebriola [the lead author had initially identified the fifth sample as an Eonebria species]. We had DNA from only eight (28%) of the 29 described species of Epinebriola available for our study, including the two that our results suggest are not part of an Epinebriola clade. Consequently, we do not yet know if other species currently assigned to Epinebriola actually belong to the Parepinebriola clade. Only three species among those remaining in Epinebriola occur near or within the known geographical range of Parepinebriola species. Two of them, Nebria businskyorum Ledoux & Roux, 1997 and Nebria laevistriata Ledoux & Roux, 1998 represented in our taxon sample are clearly part of the Epinebriola clade and form a distinct subclade within that taxon (see further discussion below under that subgenus). The third, Nebria zayula Andrewes, 1936 occurs near the eastern end of the range of Parepinebriola and remains a candidate for this taxon. As might be expected from the past taxonomic intermixing, morphological characters that distinguish members of Parepinebriola species from those of Epinebriola species are few and mainly involve internal features (see the Taxonomy section for discussion of these characters).

The monophyly of a group including Sadonebria and Eonebria as sister to Parepinebriola (Fig. 5B) is well supported by all the concatenated analyses and by the 28S, 16S-ND1 and Topo single-gene analyses (Chart 1, line 35) (Suppl. material 2: Figs S5, S6, S11).

Oreonebria Series: Oreonebria Complex

The Oreonebria Complex of subgenera (Figs 4B, 5B) is similar to Ledoux and Roux’s Vetanebri but with the addition of Archastes and the exclusion of their Nippononebrides and Eonebrides. We recognize five subgenera within the group, which include 90 described species-group taxa. The geographical range of the group is widely disjunct, with subgenus Oreonebria restricted to the Alps mountain system of western Europe and the remaining subgenera found in eastcentral to far eastern Asia. This group is well supported as monophyletic by the 8G ML, 8G B, and Nuc G concatenated analyses and the 28S analysis (Chart 1, line 38) and, to a lesser degree, by the COI BC, COI PJ, and PEPCK analyses (Suppl. material 2: Figs S7, S8, S10).

Within the complex, two main groups of subgenera are apparent. The first is a clade including Falcinebria Ledoux & Roux, 2005 and Epispadias Ledoux & Roux, 2005. It is well supported (MBL and BPP values ≥ 96) in all our analyses except for wg (Chart 1, line 39), as well as by three unique amino acids (Suppl. material 1: Table S2) and a unique base insertion in 28S (Suppl. material 1: Table S3). Falcinebria currently includes 15 species, with seven of these only recently distinguished by Sasakawa (2020) using the same character systems as noted above for Sadonebria. It is a group of morphologically very similar species. The geographical range of this subgenus includes Japan (11 species), Taiwan (two species), and the Asian mainland (one species known from Guangxi and one from Sichuan Provinces in China). The two species represented in our sample (one from Taiwan and one from Japan) form a monophyletic group based on evidence from every one of our analyses (Chart 1, line 40). Epispadias was previously known from a single species, Nebria janschneideri Ledoux & Roux, 1999, based on two specimens from the Jinfo Shan in southeast Sichuan Province (now part of Chongqing Municipality). The species in our sample is undescribed but clearly related to N. janschneideri because males of both species share a genitalic median lobe that is completely membranous dorsally except at the basal bulb, a feature unique among nebriites. They also share exceptionally low-elevation habitats for Nebria in the regions where they have been found.

The other major group of subgenera in this complex includes Orientonebria Shilenkov, 1975, Archastes and Oreonebria (Fig. 4B). Orientonebria includes a single species, Nebria coreica Solsky, 1875 (Fig. 3B), which is restricted to central Japan and the adjacent Asian mainland in southern Primorsky Krai in Russia, Jilin Province in China and North and South Korea. The diversity and overall distributions of Archastes and Oreonebria already have been discussed (see above). This clade is well supported in all our analyses except the 16S-ND1 analysis (Fig. 5B) (Chart 1, line 41), as well as by one base unique for the entire taxon sample and one unique among nebriites (Suppl. material 1: Tables S1). In addition, a clade including Orientonebria and Archastes is well supported as sister to Oreonebria by all our analyses (Chart 1, line 42).

Ledoux and Roux (2005) followed Jeannel in treating Nebria gagates Bonelli, 1810 as a distinct, monobasic subgenus, Nebriorites Jeannel, 1941. They also treated Nebria bremii Germar, 1831 as a separate subgenus, Germarina Jeanne, 1985 (= Marggia Huber, 2014). A second species, Nebria bluemlisalpicola Szallies & Huber, 2014, has been described in this subgenus. Results from every one of our concatenated and single-gene analyses support a monophyletic Oreonebria including both N. gagates and N. bremii (Chart 1, line 43, Figs 4B, 5B). In none of the trees from these analyses is either of these species shown anywhere but embedded within Oreonebria (Suppl. material 2: Figs S1 to S10). Within Oreonebria, our results support Ledoux and Roux’s division of the subgenus into the austriaca and castanea species groups, although we did not have all of the species available for our analyses. In addition, ML trees from all our analyses except the 28S and 16S-ND1 analyses show N. gagates as a member of the austriaca group clade and N. bremii as part of the castanea group clade (Fig. 4B).

Nebria Series: Boreonebria Complex

The Boreonebria Complex is comprised of two subgenera, Boreonebria and Nakanebria. This group includes 66 species-group taxa with a combined geographical range that covers most of the Holarctic Region. It differs from Ledoux and Roux’s (2005) “Boreonebrides” in that Nebriola is excluded from the group and the Palearctic species of Nakanebria are included in it. All females of this group have the spermathecal duct inserted on the ventral face of the spermathecal chamber of the bursa copulatrix, a feature not shared with females of any other subgenus of Nebria. This clade is well supported in all our concatenated analyses and in all but one (Topo) single-gene analyses (Chart 1, line 50; Figs 4C, 5C). These two subgenera also share one unique amino acid and six unique bases (Suppl. material 1: Tables S1, S2).

Ledoux and Roux (2005) included Nakanebria with Reductonebria and Catonebria in their “Serinebrides”, but whereas the Nearctic species in their Nakanebria appear to be closely related to the latter two subgenera (see below), the Palearctic species do not. Because the type species of this subgenus, Nebria kurosawai Nakane, 1960, is part of the Palearctic fauna, the subgeneric name remains with the Palearctic species as part of the Boreonebria Complex. The group includes six species-group taxa, four found only on the island of Hokkaido in northern Japan, one in the southern Kuril Islands, and one on the Asian mainland in northern North Korea. While our sample includes only one of these taxa (Nebria shiretokoana Nakane, 1960), we have examined specimens of all the included species except Nebria kumgangi Shilenkov, 1983 and can confirm close relationships based on morphology in general and the ventral insertion of the spermathecal duct on the bursa in particular.

Subgenus Boreonebria includes the 60 remaining species of this complex. Their combined geographical range extends across the Holarctic region, from Greenland and Iceland eastward across Eurasia, Beringia and across North America from Alaska to Ellesmere Island and the Island of Newfoundland. In Eurasia, they range from above the Arctic Circle to southern Europe (but not northern Africa), the Tian Shan, Altai, and Qilian mountain systems of central Asia, northern China, North Korea, and northern Japan. In North America, they range south into the northern Sierra Nevada in California, the southern Rocky Mountains in northern New Mexico, and into the southern Appalachian Mountains of Tennessee and North Carolina. Ledoux and Roux (2005) treated Pseudonebriola Ledoux & Roux, 1989 as a distinct subgenus closely related to Boreonebria. Our results strongly support a clade including Pseudonebriola nested within Boreonebria (Chart 1, line 51; Figs 4C, 5C) but not sister to it. This relationship is seen in ML trees from all concatenated gene analyses and all but one (Topo) single-gene analyses Suppl. material 2: Figs S2 to S12). At the same time, a clade including all Boreonebria species but excluding Pseudonebriola species is strongly contra-indicated (Chart 1, line 52), as is a clade including all Pseudonebriola species and excluding all Boreonebria species (Chart 1, line 53). This evidence suggests that either Pseudonebriola should be considered as a junior synonym of Boreonebria or the latter should be split into two or more subgenera. Even within Boreonebria, Pseudonebriola is shown either as a grade or as polyphyletic rather than a clade in all ML trees except the NPC G tree (Suppl. material 2: Fig. S3). Consequently, we conclude that these names should be synonymized.

Within Boreonebria, several well-supported clades can be recognized (Fig. 5C). First and most basal is the hudsonica species group (sensu Ledoux and Roux 2005). This group of four species is endemic to North America, with two species each in the western and eastern parts of the continent. This clade is supported by all concatenated and single-gene analyses except for wg (Chart 1, line 54). A clade including the remaining Boreonebria species as sister to the hudsonica group is also supported by the concatenated analyses and all single-gene analyses except for 28S and Topo (Chart 1, line 55). Within this latter clade, the nivalis group, which includes some but not all species from several of the species groups recognized by Ledoux and Roux (2005), is seen as sister to the remainder of the subgenus. Members of all of these species share an antennal scape that is more or less elongate and sinuate. Their combined east/west geographical range is that of the subgenus, but they occupy areas north of 50°N latitude except in Mongolia, the Asian Far East and the northernmost Appalachian Mountains and Island of Newfoundland in eastern North America. This group includes at least eleven species, one of which (Nebria nivalis (Paykull), 1798) is Holarctic, one (Nebria gaspesiana Kavanaugh, 1979) occurs only in northeastern North America, and the remainder are Eurasian. Two of the species in our taxon sample are from Mongolia and are not yet described. This clade is well supported by all our concatenated and single-gene analyses except 28S (Chart 1, line 56).

Our results suggest that sister to the nivalis group is a clade that includes all the species currently assigned to subgenus Pseudonebriola and the remaining Boreonebria species, which we call the gyllenhali group Fig. 4C. This clade is well supported by all the concatenated analyses except the Mito G analysis, as well as by the CAD2 and wg analyses (Chart 1, line 57). Modest additional support is shown by the COI BC and COI PJ results. Species currently assigned to Pseudonebriola appear to represent either a grade or a polyphyletic assemblage, which, as a group, occupy mountain ranges in the region in central Asia extending from the western end of the Tian Shan just south of Issyk Lake northward and eastward to the southern end of Lake Baikal in southern Buryatia, Russia (Huber and Schnitter 2020). One species, Nebria mingyii Ledoux & Roux, 2014, is known from the Qilian Shan of Qinghai Province, China, more than 1000 km south and east of the range of any other species in the group. Fifteen species have been included in the group and two additional undescribed species (both in our taxon sample) appear to be closely related to them. Ledoux and Roux (2005) recognized five species groups within Pseudonebriola. In our sample, which included five (33%) of the described species and the two undescribed species, two moderately well supported clades are represented. These do not appear to be sister taxa. The first includes two species, Nebria tekesensis Ledoux & Roux, 2005 and Nebria murzini Ledoux & Roux, 2000, which Ledoux and Roux had placed in two different species groups. This clade is supported by all the concatenated analyses and by three nuclear and two mitochondrial single-gene analyses (Chart 1, line 58). It appears to be sister to a clade including the other group of Pseudonebriola species and the gyllenhali group of Boreonebria. This latter clade is supported by the 8G ML, 8G B, and Mito G concatenated analyses, as well as by the COI BC, COI PJ, and, to a lesser extent, the CAD2 analyses (Chart 1, line 59). The second Pseudonebriola group, which we are calling the sajanica group, is probably equivalent to Ledoux and Roux’s species group of the same, but we were not able to include Nebria stanislavi Dudko & Matalin, 2002 in our taxon sample. This group also is supported as monophyletic by all concatenated analyses and by COI BC and COI PJ (mitochondrial) and CAD2 and wg (nuclear protein-coding) single-gene analyses (Chart 1, line 60). These two groups of species appear to occupy opposite ends of the range of the group as a whole, with the first group in the southwestern portion of the range and the sajanica group occupying the northeastern portion. It should be interesting to see how the species not included in our sample assort between these two clades or if additional clades are found with wider molecular sampling.

Our gyllenhali group of Boreonebria is only partially equivalent to that of Ledoux and Roux’s (2005) species group of the same name in that we include Nebria frigida Sahlberg, 1844 and exclude several nivalis group species that Ledoux and Roux included. This group is supported as monophyletic by the 8G ML, 8G B, Nuc G, and NPC G analyses and only modestly by one single-gene analysis (CAD2) (Chart 1, line 61). Nonetheless, it is the only pattern of relationship among these taxa that is supported with any consistency. Relationships within this group are only partially resolved with our findings. Kavanaugh (1978, 1979) initially considered Nebria castanipes Kirby, 1837, Nebria lassenensis Kavanaugh, 1979 and Nebria lindrothi Kavanaugh, 1979 as subspecies of a Holarctic Nebria gyllenhali (Schönherr, 1806). Results from our 8G ML, 8G B and Mito G concatenated analyses and COI BC and COI PJ analyses support a Nearctic clade, including those three species and Nebria crassicornis Van Dyke, 1925 and Nebria intermedia Van Dyke, 1949, but not including N. gyllenhali (Chart 1, line 63). Conversely, a clade including N. gyllenhali with N. castanipes, N. lassenensis, and N. lindrothi but excluding N. crassicornis and N. intermedia (Chart 1, line 62) is contra-indicated. This suggests that N. gyllenhali is an endemic Palearctic species, not most closely related to any of the Nearctic taxa.

We have not had the opportunity to include in our study several of the species of Boreonebria that Ledoux and Roux included in this group from southern and eastern Europe, China (including Tibet), Kazakhstan, or Afghanistan. However, those authors provided illustrations of the female bursa copulatrix and spermathecal duct and reservoir for several of these, all but one of which appear to have the spermathecal duct inserted ventrally or apicoventrally on the bursa. The lone exception is Nebria klapperichi Bänninger, 1956, which appears to have the duct inserted mid-dorsally on the bursa (Ledoux and Roux 2005, pg. 116, fig. 53). This feature, coupled with the fact that the area where this species occurs (in Afghanistan) is more than 1000 km southeast of the range of the nearest confirmed members of this group, suggests that N. klapperichi is not a member of Boreonebria or the Boreonebria Complex.

Nebria Series: Nebria Complex

The group of taxa that we recognize as the Nebria Complex (Fig. 4C) includes eleven (> 40%) of the subgenera recognized by Ledoux and Roux (2005) and 286 species-group taxa. It is the most diverse subgeneric complex within Nebria and restricted to the Palearctic Region, except for a few accidental introductions of Nebria brevicollis (Fabricius, 1792) into North America. This clade is supported in all our concatenated analyses and in the 28S, CAD2, PEPCK and Topo analyses (Chart 1, line 64). As noted above, our results show that this group is comprised of three distinct groups of subgenera that we recognize as the Nebria, Epinebriola and Eunebria Subcomplexes, respectively. The large and diverse Nebria Subcomplex is strongly supported as monophyletic by results from all concatenated and single-gene analyses except for COI PJ (Chart 1, line 65). Sister to the Nebria Subcomplex is a group including the Epinebriola and Eunebria Subcomplexes. Monophyly of this group is well supported by the 8G ML, 8G B, and Nuc G concatenated analyses and the 28S and Topo analyses and also (but weakly) by the Mito G analysis (Chart 1, line 71) and there is no other consistent pattern of relationship supported by our results.

Nebria Subcomplex

This group of subgenera is equivalent to the “Nebrides” of Ledoux and Roux (2005) except that we also include here subgenus Tyrrhenia Ledoux & Roux, 2005, which they included in their “Eunebrides”. Currently, nearly 160 species-group taxa are arrayed among four subgenera: Nebria s. str., Alpaeonebria Csiki, 1946, Spelaeonebria Peyerimhoff, 1911, and Tyrrhenia. This group is mainly a European lineage, with highest diversity in southern Europe, northern Turkey, and the Caucasian mountain region. The geographical area of the group covers all of Europe, extreme North Africa, the Mediterranean fringe of the Middle East and Asia Minor to the southern end of the Caspian Sea, and no species (except for Nebria brevicollis in the north), occurs east of northcentral Iran. This group is the least well represented in our taxon sample with only ten (ca. 6%) of the described taxa included, but all of the subgenera are represented.

Tyrrhenia includes 18 species-group taxa with a combined geographical range extending from Portugal and Morocco across southern Europe and northern Africa to southeastern Turkey. Although we had only two species of this subgenus represented in our sample, they form a well- supported clade in all our analyses (Chart 1, line 66). Two base insertions and two deletions in 28S are unique to the clade (Suppl. material 1: Table S3). They also appear in all but one (COI PJ) of our ML trees as sister to a clade including other members of this subcomplex (Figs 4C, 5C; Suppl. material 2: Figs S1–S12). This latter clade, the Nebrides of Ledoux and Roux, is also well supported by our results, including those from all the concatenated and single-gene analyses except for PEPCK (for which we obtained insufficient data) (Chart 1, line 67). One unique base (Suppl. material 1: Table S1) and one unique three-base insertion in 28S (Suppl. material 1: Table S3) also support this clade.

Spelaeonebria includes a single species, Nebria nudicollis Peyerimhoff, 1911, restricted to the Djurdjura Mountains of northern Algeria, where these large and elegant beetles (3C) live in limestone caves at high elevation. Alpaeonebria, as currently comprised, includes 33 species-group taxa arrayed in nine species groups (Ledoux and Roux 2005). This subgenus occupies two separate regions. The first is an area extending from the Carpathian Mountains in the northeast west through the Alps of Austria, Switzerland, and northern Italy (but not into France or Germany) and south through the Balkan countries to northern Albania, Macedonia, and western Bulgaria. The second region includes the mountains of northern Africa in Algeria and Morocco and Tenerife and Grand Canary in the Canary Islands. The subgenus is absent from the Iberian, Italian, and southern Balkan peninsulas and the islands of Mediterranean. Our taxon sample was insufficient to test whether or not Alpaeonebria is a clade.

Ledoux and Roux (2005) treated Alpaeus Bonelli, 1810 as a synonym of subgenus Nebria because they found the morphological features traditionally used to distinguish between them unreliable (see also Ledoux and Roux 1990). As presently constituted, the nominate subgenus includes more than 130 species-group taxa with a combined geographical range extended from northern Africa through all of Europe except the Arctic, eastward to the Urals in the north and to the Middle East and the southern edge of the Caspian Sea in Asia Minor in the south. Ledoux and Roux recognized fifteen different species groups within this subgenus.

The number of taxa represented in our sample for this group is low in relation to its high diversity. While this prevents us from drawing firm conclusions about relationships within it, our results allow us to clarify a few issues. First, our representatives of both Alpaeonebria (Nebria germarii Heer, 1837) and Spelaeonebria (N. nudicollis) are nested within a Nebria clade in ML trees from all concatenated and single-gene analyses except for Topo (Suppl. material 2: Fig. S11), in which N. germarii is shown as sister to a group including the Nebria s. str. species and N. nudicollis. The relationship suggested by the Topo result is not supported by evidence from most other genes (Chart 1, line 69). A clade including Nebria s. str. species but excluding either N. nudicollis alone (Chart 1, line 68) or along with N. germari (Chart 1, line 70) is strongly contra-indicated by our results. This suggests that treating Spelaeonebria and Alpaeonebria as separate subgenera renders Nebria s. str. as a paraphyletic or even a polyphyletic assemblage. In most of the trees, Nebria turcica Chaudoir, 1843 is shown as sister to the other species in our sample (Fig. 4C; Suppl. material 2: Figs S1–S6, S8), but in the ML trees from the COI BC and CAD2 analyses (Suppl. material 2: Figs S7, S9), it is shown as sister to N. germarii (the Alpaeonebria representative in our sample). Nebria nudicollis is shown either as sister to Nebria tibialis Bonelli, 1810) and nested within a European clade of Nebria species (Fig. 4C) or as sister to that clade (Suppl. material 2: Figs S4, S10, S12). Peyerimhoff (1911) suggested a close relationship between Spelaeonebria and Nebria exul Peyerimhoff, 1910, which was included by Ledoux and Roux (2005) in Alpaeonebria. Both Nebria turcica and N. tibialis were previously included in Alpaeus (Farkač and Janata 2003). Clearly, there is more complexity to this clade than we can resolve with our limited sample. Our choice at this point is to either synonymize both Spelaeonebria and Alpaeonebria with Nebria s. str. and create an even larger single taxon or accept the current status, which includes a para- or polyphyletic Nebria s. str. We choose the latter option pending much broader taxon sampling.

Epinebriola Subcomplex

This group is comprised of 46 described species-group taxa currently arrayed among four subgenera: Epinebriola, Barbonebriola Huber & Schmidt, 2017, Patrobonebria Bänninger, 1923 and Paranebria Jeannel, 1937. Ledoux and Roux (2005) included Epinebriola (including the species of Barbonebriola known to them) and Patrobonebria with Psilonebria Andrewes, 1923 in their “Patrobonebrides”, whereas Paranebria was part of their “Eunebrides” with Tyrrhenia, Eunebria Jeannel, 1937 and Asionebria Shilenkov, 1982. The monophyly of this group is well supported by all our concatenated and single-gene analyses (Chart 1, line 72). Paranebria includes only two species. Nebria livida (Linnaeus, 1758) ranges across Eurasia at latitudes from slightly south of the Arctic Circle to mid-latitudes, from the United Kingdom east to Japan but with large gaps in the record from western and central Asia. The second species, Nebria macrogona Bates, 1873, occurs only in Japan. Members of both of these species have full-sized hindwings and are likely capable of flight. Patrobonebria includes ten described and at least one undescribed species. This group is restricted to the western and southern margins of High Asia, with a combined geographical range extended from northwestern Afghanistan to ranges of the Hengduan Shan in western Yunnan Province, China. All except two of these species (Nebria assidua Huber & Schmidt, 2009 and Nebria pertinax Huber & Schmidt, 2009) have members with full-sized, functional hindwings. Huber and Schmidt (2017) distinguished members of Barbonebriola from those of Epinebriola mainly by the presence of laterally bulging, seta-bearing tubercules on the maxillary stipes (not seen in Epinebriola members). They recognized six species in this group, with a combined geographical range extended from extreme northern Pakistan narrowly along the Himalayan Mountain system to western Nepal. All of these species have vestigial hindwings. The remaining 28 species of this subcomplex are currently included in subgenus Epinebriola and all have vestigial hindwings. Males of the species that we have examined share a median lobe of their genitalia with well-developed basolateral lobes on the basal bulb and an apical orifice slightly to markedly deflected right. Females share a short spermathecal duct and medium-length spermathecal reservoir. The combined geographical range of this group is similar to that of Patrobonebria except that, in the eastern part of its range, on the eastern margin of the Tibet-Qinghai Plateau, it is also extended north to the Kunlun Mountains of extreme western Qinghai Province, China.

Our analyses of relationships within this clade have produced some unexpected findings. As already noted above, two described species (N. delicata and N. retingensis) previously included in Epinebriola have been shown instead to be part of the Eonebria Complex. A clade including them with members of the Epinebriola Subcomplex is contra-indicated by all of our results (Chart 1, line 73). Patrobonebria is well supported as a clade in all of our analyses (Chart 1, line 79) and Paranebria is supported as monophyletic (Chart 1, line 78) in all trees except the 28S ML tree (Suppl. material 2: Fig. S5). However, both of these groups are nested within Epinebriola as currently comprised (Fig. 4C) in all trees except the 28S tree, which shows a clade including Patrobonebria and N. macrogona (but not N. livida) as sister to Epinebriola including Nebria kagmara Huber & Schmidt, 2017, our only representative of Barbonebriola. Nebria kagmara also is nested within Epinebriola in ML trees from all analyses. This evidence suggests that Paranebria, Patrobonebria, and Barbonebriola should be considered junior synonyms of Epinebriola, but we wanted to look at this group in greater detail to determine if it would be possible to conserve any of these names.

We only had access to a very old pinned specimen of Nebria oxyptera Daniel & Daniel, 1904, the type species of Epinebriola, for DNA extraction. This may explain why we were unable to obtain sequence data from it for any of the nuclear protein-coding genes. Consequently, this important taxon is missing from several of our analyses and resulting ML trees. Nonetheless, in five of the eight ML trees in which it appears, it is shown as sister to the Paranebria species, forming a clade with them that is sister to Patrobonebria (Fig. 4C; Suppl. material 2: Figs S1, S2, S4, S8). The monophyly of N. oxyptera + Paranebria is supported in all the concatenated analyses in which data for N. oxyptera are included and in the single-gene analysis for COI PJ (Chart 1, line 77). In the tree for 16S-ND1 (Suppl. material 2: Fig. S6), N. oxyptera is seen as sister to a clade including Paranebria with Patrobonebria. In either case, the type species of Epinebriola is shown to be more closely related to Paranebria and Patrobonebria than to the other Epinebriola species in our sample. Consequently, we see no way to recognize either Paranebria or Patrobonebria as a separate subgenus without rendering Epinebriola paraphyletic, so the three should be synonymized. What is perhaps most unexpected here is the fact that taxa with full-sized hindwings are, in most trees, nested well within larger clades of taxa with reduced hindwings. This requires that the loss of functional wings has occurred repeatedly in this group and probably independently.

Nebria kagmara is not the most genetically distinct taxon within our Epinebriola sample. That distinction belongs to a clade including Nebria businskyorum Ledoux & Roux, 1997 and Nebria laevistriata Ledoux & Roux, 1998, two of the three species Ledoux and Roux (2005) included in their pindarica species group. This species pair is strongly supported as monophyletic in all our analyses (Chart 1, line 74) and as sister to all other members of the subcomplex in our taxon sample in all but the 28S and COI BC analyses (Fig. 4C) (Chart 1, line 75). A unique two-base insertion in 28S (Suppl. material 1: Table S3) also supports this clade. Another small clade recovered with strong support from all of our analyses (Chart 1, line 76) includes three species (Nebria pseudorestias Huber & Schmidt, 2017, Nebria martensi Huber & Schmidt, 2012 and Nebria numburica Huber & Schmidt, 2017), the martensi species group. Members of this clade share a unique three-amino acid insertion in the wg gene fragment (Suppl. material 1: Table S3). Different analyses suggest different relationships of this clade to the others in this lineage (Suppl. material 2: Figs S1–S12), but in most results, it occupies a middle level in the diversification of the subcomplex (Fig. 4C). Based on morphological data, it seems likely that most if not all of the other species of Epinebriola that were not included in our analyses are more closely related to this clade than to any of the others in the subcomplex. Although the six species included in Barbonebriola certainly share at least one outstanding morphological feature, maintaining them as more than a distinctive species group within Epinebriola would render the latter paraphyletic unless it is further subdivided, which we discourage based on data available at this time. Consequently, these two subgeneric names should also be synonymized (see proposed revised classification of Nebriitae below, Table 4).

Table 4.

A revised classification of the supertribe Nebriitae.

Supertribe Nebriitae
Tribe Notiokasiini
[Genus Notiokasis Kavanaugh & Nègre, 1983]
Tribe Notiophilini
Genus Notiophilus Duméril, 1805
Tribe Opisthiini
Genus Opisthius Kirby, 1837
Genus Paropisthius Casey, 1920
Tribe Pelophilini
Genus Pelophila Dejean, 1821
Tribe Nebriini
[Genus Archaeonebria Kavanaugh & Schmidt, 2019 Fossil]
Genus Nippononebria Uéno, 1955 Revised Status
Subgenus Nippononebria Uéno, 1955
Subgenus Vancouveria Kavanaugh, 1995 Revised Status
Genus Leistus Frölich, 1799
Subgenus Nebrileistus Bänninger, 1925
Subgenus Sardoleistus Perrault, 1980
Subgenus Pogonophorus Latreille, 1802
[Manticora Panzer, 1803]
[Oreobius Daniel, 1903]
[Chaetoleistus Semenov, 1904]
Eurinoleistus Breit, 1914
Subgenus Evanoleistus Jedlička, 1965
Subgenus Leistus Frölich, 1799
[Leistidius Daniel, 1903]
[Acroleistus Reitter, 1905]
Euleistus Reitter, 1905
Leistophorus Reitter, 1905
Neoleistus Erwin, 1970
Genus Nebria Latreille, 1802
Oreonebria Series
Eonebria Complex
Subgenus Parepinebriola New Subgenus
Subgenus Sadonebria Ledoux & Roux, 2005
Subgenus Eonebria Semenov & Znojko, 1828
Oreonebria Complex
Subgenus Epispadius Ledoux & Roux, 1999
Subgenus Falcinebria Ledoux & Roux, 2005
Subgenus Orientonebria Shilenkov, 1975
Subgenus Archastes Jedlička, 1935 New Status
Subgenus Oreonebria Daniel, 1903
Nebriorites Jeannel, 1941 New Synonymy
Germaria Jeanne, 1972
Germarina Jeanne, 1985
Marggia Huber, 2014 New Synonymy
Nebriola Series
Subgenus Nebriola Daniel, 1903
Nebria Series
Boreonebria Complex
Subgenus Nakanebria Ledoux & Roux, 2005
Subgenus Boreonebria Jeannel, 1937
Pseudonebriola Ledoux & Roux, 1989 New Synonymy
Nebria Complex
Nebria Subcomplex
Subgenus Tyrrhenia Ledoux & Roux, 2005
Subgenus Nebria Latreille, 1802*
Alpaeus Bonelli, 1810
Helobia Stephens, 1828
Harpazobia Gistel, 1856
Subgenus Spelaeonebria Peyerimhoff, 1911
Subgenus Alpaeonebria Csiki, 1946
Epinebriola Subcomplex
Subgenus Epinebriola Daniel & Daniel, 1904
Patrobonebria Bänninger, 1923 New Synonymy
Paranebria Jeannel, 1937 New Synonymy
[Himalayonebria Ledoux, 1985]
Barbonebriola Huber & Schmidt, 2016 New Synonymy
Eunebria Subcomplex
Subgenus Psilonebria Andrewes, 1923
Asionebria Shilenkov, 1982 New Synonymy
Subgenus Eurynebria Ganglbauer, 1891
Subgenus Eunebria Jeannel, 1937
[Tetungonebria Shilenkov, 1982]
[Sphodronebria Sciaky & Pavesi, 1994]
Catonebria Series
Reductonebria Complex
Subgenus Insulanebria New Subgenus
Subgenus Reductonebria Shilenkov, 1975
Subgenus Erwinebria New Subgenus
Catonebria Complex
Subgenus Nivalonebria New Subgenus
Subgenus Neaptenonebria New Subgenus
Subgenus Palaptenonebria New Subgenus
Subgenus Catonebria Shilenkov, 1975
Incerta sedis
[Genus Archileistobrius Shilenkov & Kryzhanovskij, 1983]
[Genus Ledouxnebria Deuve, 1998 Fossil]

Eunebria Subcomplex

This subcomplex includes 68 described species-group taxa presently arrayed in four subgenera: Asionebria, Eunebria, Psilonebria and Eurynebria. Ledoux and Roux (2005) included the first two along with Tyrrhenia and Paranebria in their Eunebrides and Psilonebria with Patrobonebria and Epinebriola in their Patrobonebrides. Eurynebria was assigned to its own monobasic group, the “Halonebrides”. This group ranges widely but discontinuously across the Palearctic Region in mid- and southern latitudes, from extreme northern Africa and the United Kingdom east to Japan and Taiwan, and along the southern margin of High Asia. Monophyly of the group (Fig. 4C) is supported by results from all our concatenated analyses as well as from 28S and CAD2 analyses (Chart 1, line 80). This clade also appears in ML trees from the PEPCK and wg analyses (Suppl. material 2: Figs S10, S12).

Relationships among the taxa included in our analyses proved to be somewhat unexpected. The group is clearly divided into two well-supported clades (Fig. 4C). The first includes all the species of Asionebria and Psilonebria in our sample, but also three species from Ledoux and Roux’s (2005) przewalskii group of Eunebria. A clade retaining the przewalskii group with the other species of Eunebria in our sample is strongly contra-indicated by results from all analyses (Chart 1, line 81). This clade probably includes all 20 of the described species-group taxa in these three groups, including those species and subspecies in the przewalskii species group not included in our sample; however, confirming this will require additional DNA sampling. In addition, several undescribed taxa are also included in this group. The combined geographical range of this group includes the southern and eastern parts of the Tibetan Plateau north of the Himalayan Mountains, extending from the central parts of the Tibetan Himalaya and Trans-Himalaya in southern Tibet to the Qilian Mountain system north and northeast of Qinghai Lake in eastern Qinghai and western Gansu Provinces, China. Psilonebria as presently comprised occupies only the southwesternmost part of this range, whereas the other two groups range from the central Tibetan Himalaya and Trans-Himalaya to western Gansu. This means that the three groups overlap in only the central part of the Trans-Himalaya in southern Tibet. A group including all three subgroups is strongly supported as monophyletic in all concatenated and single-gene analyses (Chart 1, line 82) and by eight unique bases and two unique amino acids (Suppl. material 1: Tables S1, S2). The two species of Psilonebria represented in our sample (Nebria cf. superna Andrewes, 1923 (Fig. 3D) and Nebria sp XIZ 20 [undescribed]) are supported as a monophyletic group (Fig. 4C) in all of our analyses (Chart 1, line 83), whereas a monophyletic Asionebria is contra-indicated in all analyses (Chart 1, line 84). Instead, both Psilonebria and the przewalskii group species are nested within Asionebria in trees from all concatenated analyses except the Mito G analysis (Suppl. material 2: Fig. S4)) and all single-gene analyses except COI BC, COI PJ and CAD2 (Suppl. material 2: Figs S7–S9). Our results suggest that this group represents a single lineage that should be recognized as a single subgenus. Members of this group share the following morphological features: head distinctly wider than average for the genus relative to other body proportions; antennomeres 3 and 4 laterally compressed basally (except in Nebria przewalskii Semenov, 1889); metepisterna impunctate; and median lobe of male genitalia without a sagittal aileron but with a sclerotized basal collar. The name Psilonebria has priority over Asionebria, so that should be the name of this subgenus.

The second clade in the Eunebria Subcomplex includes Eurynebria (N. complanata) and the remaining species of Eunebria in our sample (Fig. 4C). Nebria complanata (Fig. 2F) once occupied sandy sea beach habitats on the southern Irish Sea and Atlantic coasts of Ireland and the United Kingdom and along Atlantic coastal Europe and northern Africa from Belgium and France to central Morocco. It has been recorded also from the sandy Mediterranean shores of France, Italy, Turkey, Algeria, and Tunisia [the record from Iran (Azadbakhsh and Nozari 2015, Huber 2017) requires confirmation]. Unfortunately, many of the former populations of this attractive species have been extirpated (A. Casale, pers. comm.). The subgenus Eunebria (excluding the przewalskii group) is comprised of almost 50 described species-group taxa, collectively ranging from northern Africa and western Europe to Japan and Taiwan, but with endemic clusters of species in largely disjunct areas between these extremes. Three species occur in Europe, with a fourth species (likely related to them, Ledoux and Roux 2005) in Morocco. Five species occur in the area including the Caucasus, Elbruz, and Zagros mountains from Georgia to central Iran. Twenty-five species occur in central Asia, arrayed in a broad semicircular pattern around the Taklimakan Desert and edge of the Tibetan Plateau, from northern Mongolia and Tuva in Russia, southwest to eastern Uzbekistan and Afghanistan, then southeast along the Himalayan Mountains to central Nepal. Another group of five described and at least three undescribed species occupies an area on the southeastern edge of High Asia in northeasternmost India, Yunnan and central to southern Sichuan. Four species occur in far eastern Asia (including two in Japan, one in Taiwan, and one on the Chinese mainland in Fujian Province). Finally, a single, distinctive species, Nebria tetungi Shilenkov, 1982, occupies an area from northern Sichuan to easternmost Qinghai Province in China. This last species has been described twice as the type species of a new subgenus (i.e., Tetungonebria Shilenkov, 1982 and Sphodronebria Sciaky & Pavesi, 1994), but Ledoux and Roux (2005) included it in Eunebria. We were not able to include this species in our molecular sample and its relationships to other Nebria species remain unclear. This group is supported as monophyletic by all our analyses (Chart 1, line 85) and by three shared unique bases, one unique amino acid and one unique base insertion in 28S (Suppl. material 1: Tables S1–S3).

We had only ten (22%) of the described taxa included in our sample of this subcomplex, so our results represent only a preliminary assessment of relationships within the group. With respect to the relationship between Eurynebria and Eunebria, several possibilities remain viable. There is some support for a clade including all Eunebria as sister to Eurynebria (Chart 1, line 86) from the 8G ML (Fig. 5C), 8G B and Mito G (Suppl. material 2: Figs S1, S4) concatenated analyses and also from the COI PJ and Topo analyses (Suppl. material 2: Figs S6, S11). Although none of the evidence is compelling, the Nuc G and NPC G concatenated analyses and 28S, COI BC, CAD2, and wg analyses all support Eurynebria as nested within Eunebria and related to one or another of the clades included within the latter (Chart 1, lines 87–89). Each of these clades occupies one or two of the focal areas for Eunebria described above. One clade includes the European and central Asian species in our sample. This clade is supported by all our analyses except the 28S analysis (Chart 1, line 90) and by two shared unique bases (Suppl. material 1: Table S1). Results from the CAD2 analyses show Eurynebria as sister to this clade (Suppl. material 2: Fig. S9). Within this clade the European and central Asian species each form sister clades (Fig. 4C) supported by all our concatenated analyses and by three or more single-gene analysis (Chart 1, lines 91 and 92). Results from the 28S analysis suggest that Eurynebria is most closely related to the European clade, a result that makes sense geographically. Another clade within Eunebria is one including the species in our sample from southeastern Asia (centered in Yunnan Province) and far eastern Asia. This east Asian clade is well supported by all our analyses (Chart 1, line 93). Results from the Nuc G and NPC G concatenated analyses and the wg single-gene analysis suggest that Eurynebria is sister to this clade (Suppl. material 2: Figs S2, S3, S12).

Given the conflicting evidence from our results, we cannot establish unambiguously whether Eurynebria is sister to Eunebria or nested within it. This is an important distinction for taxonomy because Eurynebria, which has included only N. complanata since its introduction, has priority over Eunebria if the two groups are united. We choose to leave these subgenera as separate pending additional evidence supporting the alternative.

Catonebria Series: Reductonebria Complex

This complex corresponds to subgenus Reductonebria as treated by Ledoux and Roux (2005). The group includes 40 described species-group taxa with a combined geographical range that extends from the Altai Mountain system of central Asia eastward to eastern North America, but with gaps inside this range. The group is well supported by all of our concatenated and single-gene analyses (Chart 1, line 95) as well as by four synapotypic amino acids and two unique bases (Suppl. material 1: Tables S1, S2). Within this complex, three distinct lineages are apparent (Fig. 4D). The first includes just two species, Nebria carbonaria Eschscholtz, 1829 and Nebria snowi Bates, 1883. This group corresponds to Ledoux and Roux’s “carbonaria group”. Nebria carbonaria occurs on the Kamchatka Peninsula and the northern Kuril Islands (Paramushir and Onekotan Islands) and N. snowi is endemic to the Kuril Islands. This clade is so strongly supported in all our analyses (Chart 1, line 96), as well as by two unique bases (Suppl. material 1: Tables S1, S2), that we recognize it as a distinct subgenus, Insulanebria. Morphological features that unambiguously distinguish members of this group from those of other members of the Reductonebria Complex are few and mainly involve internal features (see the Taxonomy section for discussion of these characters).

The second lineage in the complex is subgenus Reductonebria (Fig. 4D). This group is comprised of 17 species, some of which occupy very large geographical ranges. The combined range of the group is that of the entire complex. This clade is also very strongly supported in all of our analyses (Chart 1, line 99) and by one synapotypic amino acid (Suppl. material 1: Table S2) and one unique base insertion in 28S (Suppl. material 1: Table S3). Within this clade, an initial divergence among the 15 taxa in our sample resulted in vicariant Palearctic and Nearctic sister clades. The Palearctic clade, equivalent to Ledoux and Roux’s “ochotica group”, includes at least three species, one geographically widespread, the others more restricted in distribution. Nebria altaica Gebler, 1847 is restricted to the region from the southern end of Lake Baikal west through the Eastern and Western Sajan Mountains to the western end of Altai Mountains. Nebria ochotica Sahlberg, 1844 ranges from Yakutia in central Siberia east to the Russian Far East, Sakhalin Islands, Kamchatka and the Kuril Islands, Hokkaido Island in Japan, and south along the mainland coastal region to North Korea. Nebria japonica Bates, 1883 appears to be restricted to the mountains of Honshu Island, Japan. The only species of this subgenus not represented in our taxon sample were Nebria angustula Motschulsky, 1866, which is endemic to the Kamchatka Peninsula, and Nebria nicolasi Ledoux & Roux, 2006, which is known only from the Qionglai Mountains of central Sichuan Province, China. Members of these two species each have unique features that put their close relationship to other species in this group in question, but we have no evidence at present to place them anywhere but in the Palearctic group. Results from all our concatenated analyses as well as the COI BC, COI PJ, CAD2, and Topo analyses support the three species of this group on our sample as a clade (Chart 1, line 100).

The Nearctic group of Reductonebria is comprised of 12 species (Fig. 4D), including all those assigned to the “pallipes”, “obliqua”, and “mannerheimi” groups by Ledoux and Roux (2005) or the “appalachia”, “pallipes”, “mannerheimi”, and “obtusa” groups of Lindroth (1961). Nine of these occur only in the west, all living at relatively low elevations for Nebria species, with a combined geographical range extended from the Kenai Peninsula in southcentral Alaska south to southern California, northern Arizona and New Mexico, and east as far as the Black Hills of western South Dakota and northwestern Nebraska. Nebria mannerheimii Fischer von Waldheim, 1828, Nebria eschscholtzii Ménétriés, 1844, and Nebria obliqua LeConte, 1866 occupy large geographical ranges while the six other western species are geographically more restricted. Two species occur only in the east: Nebria pallipes Say, 1823 ranges broadly from the Canadian Maritime Provinces south along the Appalachian Mountains to northern Georgia and South Carolina and west to Michigan, Illinois, and Kentucky. Nebria appalachia Darlington, 1932 is restricted to the highest elevations of the southern Appalachian Mountains in North Carolina and Tennessee. The last species, Nebria suturalis LeConte, 1850, has a glacio-relictual distribution including the tops of the highest peaks in the Green Mountains of Vermont and White Mountains of New Hampshire, streams along the Labrador coast and coasts of Ungava and Hudson Bays in Québec, the north shore of Lake Superior in Ontario, the margins of glaciers in the Rocky Mountains of central Alberta, and high mountain summits in the southern Rocky Mountains of central Colorado. The monophyly of this group is supported by results from all of our concatenated analyses and from the 16S-ND1, COI PJ, and Topo analyses (Chart 1, line 101; Fig. 5D; Suppl. material 2: Figs S1–S4, S8, S11). Within this group, relationships generally are not well resolved by our molecular data. The species pair, N. mannerheimi and Nebria darlingtoni Kavanaugh, 1979, is supported as monophyletic in all analyses and as sister to the other species in the group in the 8G ML, 8G B, Nuc G, and NPC G concatenated analyses and CAD2, PEPCK, Topo, and wg analyses. Two other species pairs, Nebria diversa LeConte, 1863 with N. eschscholtzii and N. pallipes with N. appalachia, are also supported as monophyletic in all or most analyses, respectively (Fig. 5D; Suppl. material 2: Figs S1–S12). We find no other consistent pattern of relationships within this clade.

The third lineage in the Reductonebria Complex (Fig. 4D) is comprised of 21 species and equivalent to the “sahlbergi” and “gregaria” groups of Ledoux and Roux (2005) or the “gregaria group” of Lindroth (1961). This is an endemic western Nearctic lineage with a combined geographical range extending from the outer Aleutian Islands of Alaska south to the central Sierra Nevada of California, northern Arizona and New Mexico, and east to streams draining the eastern slopes of the Rocky Mountains from Alberta to New Mexico. Monophyly of this group is supported by all of our analyses except for the Topo analysis (Chart 1, line 102), and we recognize this group as a distinct subgenus, Erwinebria. The morphological features that unambiguously distinguish members of this group from those of other members of the Reductonebria Complex mainly involve internal features (see the Taxonomy section for discussion of these characters). As for subgenus Reductonebria in North America, branch lengths in this group are very short, suggesting recent divergences, and resolution of relationships among the included species is generally poor. However, some patterns are still evident. Nebria lyelli Van Dyke, 1925 from high elevations in the central Sierra Nevada of California is shown as sister to a clade including all other species in trees from all concatenated analyses and the COI BC, COI PJ, and wg analyses (Fig. 4D, Suppl. material 2: Figs S1–S4, S7, S8, S12). Nebria danmanni Kavanaugh, 1981 from high elevations on the Olympic Peninsula in Washington is shown as sister to a clade including the remaining group taxa in these same analyses except for COI BC. In general, several subgroups recognized by Kavanaugh (1978) are shown as monophyletic only in some but not all analyses or with changes in their species composition. For example, he included Nebria edwardsi