ZooKeys 416: 113–155, doi: 10.3897/zookeys.416.7706
An unexpected clade of South American ground beetles (Coleoptera, Carabidae, Bembidion)
David R. Maddison 1
1 Department of Integrative Biology, Oregon State University, Corvallis, OR 97331, USA

Corresponding author: David R. Maddison (david.maddison@science.oregonstate.edu)

Academic editor: T. Assmann

received 12 April 2014 | accepted 23 May 2014 | Published 17 June 2014
(C) 2014 David R. Maddison. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
For reference, use of the paginated PDF or printed version of this article is recommended.

Citation: Maddison DR (2014) An unexpected clade of South American ground beetles (Coleoptera, Carabidae, Bembidion). ZooKeys 416: 113–155. doi: 10.3897/zookeys.416.7706


Phylogenetic relationships of the Antiperyphanes Complex of the genus Bembidion are inferred using DNA sequences from seven genes (two nuclear ribosomal, four nuclear protein coding, and one mitochondrial protein coding). Redefined subgenera within the complex are each well-supported as monophyletic. Most striking was the discovery that a small set of morphologically and ecologically heterogeneous species formed a clade, here called subgenus Nothonepha. This unexpected result was corroborated by the discovery of deep pits in the lateral body wall (in the mesepisternum) of all Nothonepha, a trait unique within Bembidion. These pits are filled with a waxy substance in ethanol-preserved specimens. In one newly discovered species (Bembidion tetrapholeon sp. n., described here), these pits are so deep that their projections into the body cavity from the two sides touch each other internally. These structures in Bembidion (Nothonepha) are compared to very similar mesepisternal pits which have convergently evolved in two other groups of carabid beetles. The function of these thoracic pits is unknown. Most members of subgenus Nothonepha have in addition similar but smaller pits in the abdomen. A revised classification is proposed for the Antiperyphanes Complex.


Carabidae, Bembidiini, Bembidion, phylogeny, systematics, DNA, South America, ground beetles


Ground beetles of the genus Bembidion are distributed throughout the temperate regions of the world (Maddison 2012). The fauna of South America is diverse, with about 140 species described (Jeannel 1962; Toledano 2002; Toledano 2008), mostly occurring in cooler, southern regions of the continent, and northward in the Andes. Although many species resemble northern-hemisphere subgenera scattered throughout the two major clades of Bembidion (the Bembidion Series and the Ocydromus Series), all known species in South America are in fact members of only three groups within the Bembidion Series: the subgenera Notaphus and Nothocys, as well as the Antiperyphanes Complex (Maddison 2012).

The Antiperyphanes Complex is a clade restricted to South and Central America. Eight subgenera are considered to belong to the complex (Maddison 2012; Maddison et al. 2013): Antiperyphanes Jeannel, Antiperyphus Jeannel, Chilioperyphus Jeannel, Plocamoperyphus Jeannel, Nothonepha Jeannel, Pacmophena Jeannel, Notholopha Jeannel, and Ecuadion Moret and Toledano, with two subgenera suspected of belonging (Notoperyphus Bonniard de Saludo (1969), Pseudotrepanes Jeannel (1962)). Members of this complex are moderately diverse in form, and are typically shades of brown, orange, and yellow, although a few species have metallic colors (Figs 13) They are abundant along edges of bodies of water (rivers, creeks, ponds, lakes, snowfields, and ocean; Figs 4A, B) in south temperate regions (especially Argentina, Chile, Peru, and Bolivia), and at higher elevations from Patagonia north into Central America. In the mountains of Ecuador, Peru, and nearby areas, one group (subgenus Ecuadion) has radiated into alpine grasslands (Fig. 4C), cloud forest leaf litter (Fig. 4D), clay cliffs, and other habitats distant from open water.

Figure 1.

Adults of subgenera Antiperyphanes and Chilioperyphus. A Bembidion (Antiperyphanes) rufoplagiatum. Argentina: Neuquén: Arroyo Queñi at Lago Queñi, DRM voucher V100796 B Bembidion (Antiperyphanes) hirtipes, Argentina: Mendoza: Pampa Palauco, DRM voucher V100792 C Bembidion (Antiperyphanes) spinolai, Argentina: Chubut: Rio Azul at Lago Puelo, DRM voucher V100788 D Bembidion (Antiperyphanes) zanettii, Ecuador: Napo: Rio Quijos W of Baeza, DRM voucher V100791 E Bembidion (Antiperyphanes) mandibulare. Chile: Reg. X, Chiloé: Cucao, DRM voucher V100789 F Bembidion (Chilioperyphus) orregoi, Argentina: Chubut: Rio Azul at Lago Puelo, DRM voucher V100674. Scale bar 1 mm.

Figure 2.

Adults of subgenera Antiperyphus and Notholopha. A Bembidion (Antiperyphus) philippii, Argentina: Neuquén: Rio Collón Curá ca 13 km S La Rinconada, DRM voucher V100787 B Bembidion (Notholopha) scitulum, CHILE: Reg. VII: Los Niches E of Curicó, DRM voucher V100790 C Bembidion (Notholopha) sexfoveolatum, CHILE: Reg. IX: 16.3 km E Malalcahuello, Cuesta Las Raices, DRM voucher V100598. Scale bar 1 mm.

Figure 3.

Adults of subgenus Ecuadion. A Bembidion chimborazonum, ECUADOR: Pichincha: Paso de la Virgen, DRM voucher V100793 B Bembidion sanctaemarthae, ECUADOR: Napo: Rio Chalpi Grande, DRM voucher V100798 C Bembidion andersoni, ECUADOR: Pichincha: Reserva Yanacocha, start Andean Snipe Trail, DRM voucher V100655 D Bembidion walterrossi, ECUADOR: Napo: Vinillos, 4.1 km S Cosanga, DRM voucher V100794 E Bembidion cotopaxi, ECUADOR: Pichincha: km 17 on route 28 W of Papallacta DRM voucher V100797 F Bembidion ricei, ECUADOR: Napo: Rio Chalpi Grande, DRM voucher V100622. Scale bar 1 mm.

Figure 4.

Habitats of the Antiperyphanes Complex. A River shore, Argentina: Neuquén: Rio Collón Curá, about 13 km S La Rinconada, 625m. On the sandy bank in the foreground Bembidion philippii is abundant, as is Bembidion (Nothonepha) sp. nr. lonae. Also occurring on the sand banks are Bembidium orregoi and Bembidion (Nothonepha) eburneonigrum. On the upper sand banks across the river are Bembidium mandibulare, and in the gravel are Bembidium spinolai B Edges of snowfields at Chile: Reg. IX: Volcán Lonquimay, 1910m. Habitat of Bembidion (Notholopha) sexfoveolatum C Open high-elevation grassland at Ecuador: Pichincha: Paso de la Virgen, 4060m, habitat of three species of subgenus Ecuadion: Bembidion chimborazonum, Bembidion guamani, and Bembidion humboldti D Leaf litter in cloud forest, Ecuador: Pichincha: Reserva Yanacocha, 0.1152°S, 78.5837°W, 3540m, habitat of Bembidion andersoni, Bembidion georgeballi, and Bembidion onorei.

Although monophyly of the Antiperyphanes Complex is well supported (Maddison 2012), details about its phylogenetic structure are poorly known. Only 20 species of the 95 or so known species have been included in phylogenetic studies, with some key taxa missing. For example, only two species of what is considered the heterogeneous subgenus Antiperyphus have been sampled, and its type species (Bembidium philippii Germain) has not previously been examined. Similarly, only two of the more than 50 species of Ecuadion were included in previous studies.

The current more in-depth investigation into phylogeny of the Antiperyphanes Complex was inspired by discovery, on the gravel shores of Rio Puntra on Isla Grande de Chiloé, Chile, of a large, distinctive, undescribed species of Bembidion (Figs 5A and 5B). This unusual species appeared to fall outside any named subgenus, and is given the name Bembidion tetrapholeon in this paper. In order to infer its relationships, additional members of the Antiperyphanes Complex were gathered and sequenced. Preliminary results from the sequences of one gene indicated the existence of a clade so surprising that I initially considered it fallacious, a result of errors in sample labeling, but when additional samples and genes provided stronger support, that explanation was no longer tenable. This apparent clade, including the new species, consisted of taxa that are much more diverse in form (Fig. 5) and habitat (Fig. 6) than other small clades of similar molecular diversity. This paper reports the results of sequencing of seven genes which together provide very strong support for this clade. The discovery of the clade led to the search for morphological synapomorphies of its members, and a striking, derived character was found in thoracic structure. Although the focus of the paper is on this unexpected clade, the relationships of other members of the Antiperyphanes Complex are explored, and a new classification is proposed for the group.

Figure 5.

Adults of subgenus Nothonepha. A Bembidion tetrapholeon (black form), Argentina: Neuquén: Arroyo Queñi at Lago Queñi, DRM voucher V100781 B Bembidion tetrapholeon (orange form), Argentina: Chubut: Rio Azul at Lago Puelo, DRM voucher V100780 C Bembidion germainianum, Argentina: Neuquén: Rio Salado at route 40, DRM voucher V100782 D Bembidion lonae, Argentina: Mendoza: Salinas del Diamante, DRM voucher V100786 E Bembidion eburneonigrum, Argentina: Neuquén: Rio Neuquén at Chos Malal, DRM voucher V100785 F Bembidion tucumanum, Argentina: Mendoza: Salinas del Diamante, DRM voucher V100783 G Bembidion engelhardti engelhardti, Argentina: Neuquén: Rio Salado at route 40, DRM voucher V100784. Scale bar 1 mm.

Figure 6.

Habitats of the subgenus Nothonepha. A Habitat of Bembidion (Nothonepha) eburneonigrum (on sand patches) and Bembidion (Nothonepha) sp. nr. lonae (on sand patches and in gravel). This habitat (Chile: Reg. IX: Rio Allipén at route 119, 132m) is also home to Bembidion spinolai Solier and Bembidion rufoplagiatum B Habitat of Bembidion (Nothonepha) tucumanum, Bembidion (Nothonepha) lonae, and Bembidion (Nothonepha) engelhardti (Argentina: Mendoza: Salinas del Diamante, 1280m). The beetles are common under rocks and around vegetation on the salt-encrusted clay and sand banks of this saline pond; in the same habitat Bembidion (Notaphus) cillenoides Jensen-Haarup and two other Notaphus are common C On the sand shores of this desert river Bembidion (Nothonepha) germainianum, Bembidion (Nothonepha) engelhardti, and Bembidion (Nothonepha) lonae are common (Argentina: Neuquén: Rio Salado at route 40, 725m) D Type locality of Bembidion (Nothonepha) tetrapholeon (Argentina: Neuquén: Arroyo Queñi at Lago Queñi, 830m). The beetles are found under rocks along the river shore; Bembidion rufoplagiatum is also common in this habitat.


Specimens examined and depositories. Specimens examined are from or will be deposited in the collections listed below. Each collection’s listing begins with the coden used in the text.

BMNH The Natural History Museum, London, UK

CMNH Carnegie Museum of Natural History, Pittsburgh, Pennsylvania, USA

CTVR Luca Toledano collection, Verona, Italy

EMEC Essig Museum Entomology Collection, University of California, Berkeley, USA

IADIZA Instituto Argentino de Investigaciones de las Zonas Aridas, Mendoza, Argentina

MACN Museo Argentino de Ciencias Naturales “Bernardino Rivadavia”, Buenos Aires, Argentina.

MNHN Muséum National d’Histoire Naturelle, Paris, France

MNNC Museo Nacional de Historia Natural, Santiago, Chile

NHMW Naturhistorisches Museum, Wien, Austria

OSAC Oregon State Arthropod Collection, Oregon State University, Corvallis, USA

USNM National Museum of Natural History, Washington, USA

ZMUC Natural History Museum of Denmark, University of Copenhagen, Copenhagen, Denmark

Collecting methods. Specimens were collected by hand or using an aspirator; specimens were found during the day in their habitat after splashing the soil with water, or with the aid of a headlamp at night, when the beetles are more actively moving on the surface.

Most specimens were killed and preserved in Acer sawdust to which ethyl acetate was added. Specimens collected specifically for DNA sequencing were killed and stored in 95% or 100% ethanol, with best results obtained when the abdomen was slightly separated from the rest of the body to allow better penetration, or when the reproductive system was dissected out through the rear of the abdomen within a few minutes of the beetle’s death in ethanol. Ethanol was decanted and refilled at least two times within the first few weeks after death. Storage was then at 4° or -20°C.

Taxon sampling for DNA studies. DNA was newly sequenced from 25 species of the Antiperyphanes Complex of Bembidion; to these sequences were added sequences of 20 more species of this complex acquired during previous studies (Table 1). Forty additional species of Bembidion were also included in the analyses (27 species of the Bembidion Series exclusive of the Antiperyphanes Complex, plus 13 species of Bembidion outside the Bembidion Series). The 27 additional Bembidion Series species are evident in Fig. 7; details about these species and specimens sequenced are available in Maddison (2012). The 13 species outside of the Bembidion Series included are Bembidion variegatum Say, Bembidion planum (Haldeman), Bembidion stephensi Crotch, Bembidion tetracolum Say, Bembidion hastii Sahlberg, Bembidion punctulatum Drapiez, Bembidion properans (Stephens), Bembidion concolor (Kirby), Bembidion chalceum (Dejean), Bembidion lunulatum (Geoffroy), Bembidion wickhami Hayward, Bembidion genei illigeri Netolitzky, and Bembidion cf. exquisitium Andrewes.

Figure 7.

Tree of highest likelihood found for the combined, seven-gene matrix with partitioning scheme chosen by PartitionFinder. Only members of the Bembidion Series shown; more distant outgroups not depicted. Scale bar: 0.01, branch lengths as reconstructed by RAxML.

Table 1.

Sampling of Bembidion species of the Antiperyphanes Complex. The ID column indicates how the specimens were identified: if there is a number in square brackets, then the specimen was identified by me using one of the following references: 1: Jeannel (1962); 2: Toledano (2008); 3: Vigna Taglianti and Toledano (2008); 4: Moret and Toledano (2002); 5: Erwin (1982). If there is a single letter, then the specimen is the holotype of the species (“H”), or I compared the specimen to the primary type or syntypes (“T”) of the species, in consultation with the relevant literature; the species marked “LT” was identified by Luca Toledano. Thus, all of these are “genseq-4” sequences (Chakrabarty et al. 2013), except for DNA2653 (genseq-1) and DNA2236 (genseq-3). In the third column, four-digit numbers in entries are D.R. Maddison voucher numbers for sequenced specimens; if a “*” appears in that column, the data from these specimens were obtained from GenBank from previous studies (Maddison 2012; Maddison and Ober 2011; Maddison and Toledano 2012; Maddison et al. 2013), and more data about the specimens is present in those papers. Locality details for newly sequenced specimens (those without a “*”) are given in the Appendix 1. For those three species with a “*” in the “#” column but without a number, details of specimens sequenced is available in Maddison (2012). For each gene, GenBank accession numbers are listed.

ID # CAD wg ArgK Topo 28S 18S COI
Subgenus Antiperyphanes Jeannel
Bembidion caoduroi Toledano [2] 1987* JN170771 JN171375 JN170525 JN171188 JN170305 JN170158 JN171006
Bembidion sp. nr. caoduroi Toledano 2677 KJ653108 KJ653211 KJ653079 KJ653177 KJ653045 KJ653141
Bembidion chilense Solier [1] 1466* JN170779 JN171382 JN170533 JN171196 JN170313 JN171014
Bembidion sp. nr. chilense Solier * JN170920 GU556037 JN170677 JN171297 GU556088 JN170236 JN171117
Bembidium spinolai Solier T * JN170925 JN171523 JN170682 JN171302 JN170448 JN171122
Bembidion neodelamarei Toledano [1] 2342 KJ653102 KJ653205 KJ653073 KJ653171 KJ653040 KJ653136
Bembidion zanettii Toledano [2] 2679* KC140267 KC140241 KC140273 KC140251 KC140281 KC140285 KC140261
Bembidion hirtipes (Jeannel) T 2335* JN170822 JN171424 JN170576 JN171227 JN170354 JN171045
Bembidion rufoplagiatum Germain T * JN170902 JN171501 JN170659 JN171282 JN170426 JN170227 JN171102
Bembidium mandibulare Solier [1] 2203* EU677545 EU677669 JN170603 EU677643 EU677689 JN170200 JN171065
Subgenus Chilioperyphus Jeannel
Bembidium orregoi Germain T 2333* KC140265 KC140238 KC140271 KC140246 KC140278 KC140283 KC140256
Bembidion mendocinum Jensen-Haarup T 2337* KC140266 KC140239 KC140272 KC140247 KC140279 KC140284 KC140257
Bembidion sp. n.“Cal” 2700* KC140262 KC140234 KC140268 KC140243 KC140274 KC140282 KC140252
Subgenus Ecuadion Moret & Toledano
Bembidion agonoides Vigna Taglianti & Toledano [3] 2675 KJ653090 KJ653193 KJ653061 KJ653159 KJ653028 KJ653020 KJ653124
Bembidion andersoni Toledano [2] 2651 KJ653091 KJ653194 KJ653062 KJ653160 KJ653029 KJ653125
Bembidion chimborazonum Moret & Toledano [4] 2659 KJ653093 KJ653196 KJ653064 KJ653162 KJ653031 KJ653127
Bembidion cotopaxi Moret & Toledano [4] 2658 KJ653094 KJ653197 KJ653065 KJ653163 KJ653032 KJ653128
Bembidion georgeballi Toledano [2] 2661 KJ653097 KJ653200 KJ653068 KJ653166 KJ653035 KJ653022 KJ653131
Bembidion guamani Moret & Toledano [4] 2660 KJ653099 KJ653202 KJ653070 KJ653168 KJ653037 KJ653133
Bembidion humboldti Moret & Toledano [4] 2673 KJ653100 KJ653203 KJ653071 KJ653169 KJ653038 KJ653134
Bembidion jimburae Moret & Toledano [4] 2674 KJ653101 KJ653204 KJ653072 KJ653170 KJ653039 KJ653135
Bembidion onorei Moret & Toledano [4] 2678 KJ653103 KJ653206 KJ653074 KJ653172 KJ653041 KJ653137
Bembidion paulinae paulinae Moret & Toledano [4] 2783 KJ653104 KJ653207 KJ653075 KJ653173 KJ653042 KJ653138
Bembidion rawlinsi Moret & Toledano [4] 1462* JN170893 JN171492 JN170650 JN171275 JN170418 JN171096
Bembidion ricei Maddison & Toledano H 2653 KJ653106 KJ653209 KJ653077 KJ653175 JX971116 JX971117
Bembidion rogersi Bates [5] 2414* JN170897 JN171496 JN170654 JN171279 JN170422 JN170225 JN171100
Bembidion sanctaemarthae Darlington [4] 2652 KJ653107 KJ653210 KJ653078 KJ653176 KJ653044 KJ653140
Bembidion sp. “Mendoza” 2701 KJ653088 KJ653191 KJ653059 KJ653157 KJ653026 KJ653019 KJ653122
Bembidion sp. “Papallacta” 2657 KJ653092 KJ653195 KJ653063 KJ653161 KJ653030 KJ653126
Bembidion walterrossii Toledano [2] 2650 KJ653121 KJ653220 KJ653087 KJ653190 KJ653058 KJ653154
Subgenus Antiperyphus Jeannel
Bembidium philippii Germain T 2327 KJ653105 KJ653208 KJ653076 KJ653174 KJ653043 KJ653023 KJ653139
Subgenus Notholopha Jeannel
Bembidion rugosellum (Jeannel) LT 1348* JN170903 JN171502 JN170660 JN171283 JN170427 JN170228 JN171103
Bembidion sexfoveatum Germain T 2208* JN170916 JN171515 JN170673 JN171293 JN170439 JN170233 JN171113
Bembidion melanopodum Solier T 2307* JN170853 JN171453 JN170609 JN171249 JN170383 JN170202 JN171069
Bembidium scitulum Erichson [1] 1347* JN170911 JN171510 JN170668 JN171288 JN170435 JN171109
Bembidion stricticolle Germain T 2240 KJ653109 KJ653212 KJ653080 KJ653178 KJ653046 KJ653142
Bembidion sp. “Lago Roca” 2046* JN170747 JN171352 JN170500 KC140249 JN170281 KC140259
Bembidion sp. “Nahuelbuta” 2239 KJ653089 KJ653192 KJ653060 KJ653158 KJ653027 KJ653123
Subgenus Nothonepha Jeannel
Bembidion eburneonigrum Germain T 2204 KJ653095 KJ653198 KJ653066 KJ653164 KJ653033 KJ653021 KJ653129
Bembidion engelhardti Jensen-Haarup T 2334 KJ653096 KJ653199 KJ653067 KJ653165 KJ653034 KJ653130
Bembidion germainianum Toledano T 2336 KJ653098 KJ653201 KJ653069 KJ653167 KJ653036 KJ653132
Bembidion lonae Jensen-Haarup T 1321* JN170844 JN171444 JN170599 JN171242 JN170374 JN170196 JN171061
Bembidion sp. nr. lonae Jensen-Haarup 1457* JN170921 JN171519 JN170678 JN171298 JN170444 JN171118
Bembidion tetrapholeon sp. n. T 2236 KJ653111 KJ653214 KJ653081 KJ653180 KJ653048 KJ653024 KJ653144
Bembidion tucumanum (Jeannel) T 1430 KJ653120 KJ653219 KJ653086 KJ653189 KJ653057 KJ653025 KJ653153

Nine additional specimens of Bembidion tetrapholeon sp. n., were sequenced (Table 2) to examine variation. The 10 specimens sequenced in total included specimens from two localities in Chile and three localities in Argentina (Tables 1, 2), and included the typical uniformly black specimens, and others that have a large orange spot on their elytra.

Table 2.

Additional sampling of Bembidion tetrapholeon to examine DNA sequence variation. The DNA voucher number is listed in the “#” column. Color of elytra: BL: nearly black; OR: black with large orange spot. * indicates the holotype. Further details about the localities are provided under the description of Bembidion tetrapholeon and in the Appendix 1. All of these are “genseq-2” sequences (Chakrabarty et al. 2013), except for DNA2356 (genseq-1) and DNA1752 (genseq-3).

# Color CAD wg ArgK Topo 28S COI
1752 BL KJ653110 KJ653213 KJ653179 KJ653047 KJ653143
2356* BL KJ653112 KJ653215 KJ653082 KJ653181 KJ653049 KJ653145
2357 OR KJ653113 KJ653216 KJ653083 KJ653182 KJ653050 KJ653146
2562 BL KJ653115 KJ653217 KJ653084 KJ653184 KJ653052 KJ653148
2566 BL KJ653119 KJ653218 KJ653085 KJ653188 KJ653056 KJ653152
2555 OR KJ653114 KJ653183 KJ653051 KJ653147
2564 OR KJ653117 KJ653186 KJ653054 KJ653150
2565 BL KJ653118 KJ653187 KJ653055 KJ653151
2563 BL KJ653116 KJ653185 KJ653053 KJ653149

Vouchers are housed in the David Maddison voucher collection at Oregon State University, with the exception of voucher number DNA2356, the holotype of Bembidion tetrapholeon, which is deposited in IADIZA.

DNA sequencing. The genes studied, and abbreviations used in this paper, are: 28S or 28S rDNA: 28S ribosomal DNA; 18S or 18S rDNA: 18S ribosomal DNA; ArgK: arginine kinase; CAD: carbamoyl phosphate synthetase domain of the rudimentary gene; COI: cytochrome oxidase I; Topo: topoisomerase I; wg: wingless.

Fragments for these genes were amplified using the Polymerase Chain Reaction on an Eppendorf Mastercycler Thermal Cycler ProS, using TaKaRa Ex Taq and the basic protocols recommended by the manufacturer. Primers and details of the cycling reactions used are given in Maddison (2012). The amplified products were then cleaned, quantified, and sequenced at the University of Arizona’s Genomic and Technology Core Facility using a 3730 XL Applied Biosystems automatic sequencer.

Assembly of multiple chromatograms for each gene fragment and initial base calls were made with Phred (Green and Ewing 2002) and Phrap (Green 1999) as orchestrated by Mesquite’s Chromaseq package (Maddison and Maddison 2011a; Maddison and Maddison 2011b) with subsequent modifications by Chromaseq and manual inspection. Multiple peaks at a single position in multiple reads were coded using IUPAC ambiguity codes.

Sequences of COI for two species showed evidence of nuclear copies of this mitochondrial gene (“numts”) (Thalmann et al. 2004). For Bembidion georgeballi (voucher 2661) and Bembidion walterrossii (voucher 2650), sequences obtained using the LC1490-HC2198 primer pair yielded different sequences from the B1490-Bcoi2R primer pair. The former had numerous double-peaks, suggesting that the sequences included numts (Maddison 2012); the LC1490-HC2198 sequence for Bembidion walterrossii also had one stop codon near its 5’ end. The reads from the B1490-Bcoi2R primers are much cleaner and show no double-peaks. Although all of these sequences have been submitted to GenBank, only the sequences from the B1490-Bcoi2R primers have been included in the analyses.

Alignment and data exclusion. The appropriate alignment was obvious for all protein-coding genes. There were no insertion or deletions (indels) evident in the sampled CAD, ArgK, Topo, or COI sequences. In wingless, there were two small, well-separated indels, restricted to only three taxa: three inserted nucleotides in Bembidion (Zemetallina) parviceps Bates, and six inserted nucleotides in a different region in the two species of subgenus Omotaphus Netolitzky sampled. These inserted nucleotides were excluded from analyses.

The ribosomal genes showed a slightly more complex history of insertions and deletions. Both genes were first subjected to multiple sequence alignment in MAFFT version 7.130b (Katoh and Standley 2013), using the L-INS-i search option and otherwise default parameter values. Visual inspection suggested no needed improvements, and no ambiguously aligned regions that required exclusion.

Molecular phylogenetic analysis. Models of nucleotide evolution were chosen with the aid of jModelTest version 2.1.1 (Darriba et al. 2012; Guindon and Gascuel 2003) (for each gene) and PartitionFinder version 1.1.1 (Lanfear et al. 2012) (for parts of the partition chosen by PartitionFinder). Among the models supported by RAxML, the model chosen for all genes by the Bayesian Information Criterion was GTR+I+Γ.

Likelihood analyses of nucleotide data were conducted using RAxML version 7.2.6 (Stamatakis 2006). Analyses were conducted on each gene individually, as well as a matrix of seven genes concatenated together. Two different partitioning schemes were examined: (1) with seven parts, one for each gene; (2) as chosen using the Bayesian Information Criterion (BIC) using PartitionFinder (Lanfear et al. 2012). The partition chosen by BIC contained three parts: one part with third positions of COI; a second part with third positions of the nuclear protein-coding genes; a third part with all remaining sites. For bootstrap analyses 2000 replicates were conducted; maximum likelihood bootstrap (MLB) values are reported as percentages. In addition to these bootstrap analyses, searches for maximum likelihood trees were conducted using 1000 search replicates.

Most-parsimonious trees (MPTs) were sought using PAUP* (Swofford 2002). To search for most parsimonious trees, 2000 replicates were conducted, each beginning with a starting tree formed with the random addition sequence option, with each replicate saving no more than 25 trees. For parsimony bootstrap analyses in PAUP*, 1000 bootstrap replicates were examined, each of which used a heuristic search with four replicates, each beginning with a starting tree formed by the random addition sequence option, with TBR branch rearrangement, with each replicate saving no more than 25 trees; the estimated bootstrap values are reported as parsimony bootstrap percentages (PB).

Morphological methods. General methods of specimen preparation for morphological work, and terms used, are given in Maddison (1993; 2008). After dissection from the body, genitalia were prepared by treatment in 10% KOH at 65 °C for 10 minutes followed by multi-hour baths of distilled water, 5% glacial acetic acid, distilled water, and then ethanol. Male genitalia, when studied, have been mounted in Euparal between two small coverslips attached to archival-quality heavyweight watercolor paper. Measurements for body length are from apex of the labrum to apex of the longer elytron.

An examination of external and internal features of the exoskeleton was conducted to search for possible apomorphies of one of the discovered clades, focusing on externally-visible pits in the mesothorax and anterior region of the abdomen. Internal skeletal elements were studied on specimens whose soft tissue was dissolved by placing the opened body in a 10% KOH solution at 65 °C for 10 minutes.

Studies of muscles and other internal soft tissue were conducted on specimens killed and preserved in 100% ethanol. This is not the ideal preservation medium, as it leaves muscles brittle and more difficult to trace. However, since the unexpected discovery of structures requiring internal examination, better-fixed specimens have not been available.

Photographs of body parts were taken with a Leica Z6 and JVC KY-F75U camera. For pronotal, elytral, and genitalic images, a stack of photographs at different focal planes was taken using Microvision’s Cartograph software; these TIFF images were then merged using the PMax procedure in Zerene Systems’s Zerene Stacker; the final images thus potentially have some artifacts caused by the merging algorithm.

Data resources

Sequences have been deposited in GenBank with accession numbers KJ653019 through KJ653220. GenBank numbers for the two apparent numts sequences are KJ653155 for DNA2661 and KJ653156 for DNA2650. Aligned data for each specimen as well as files containing inferred trees for each gene and concatenated matrices are available in Suppl. material 1 and 2, and have been deposited in the Dryad Digital Repository, http://doi.org/10.5061/dryad.47r16.

Molecular results

The inferred phylogeny is presented in Figs 7 and 8, with support values for notable clades given in Table 3. Maximum likelihood trees and maximum likelihood bootstrap trees for each gene and the concatenated matrices are illustrated in Suppl. material 3 and 4. They are also contained in the NEXUS file S1 in the Suppl. material 1.

Figure 8.

Maximum likelihood bootstrap tree showing only those clades appearing in 90% of the bootstrap replicates; taxa outside of the Antiperyphanes Complex not shown. Numbers below branches indicate maximum likelihood bootstrap percentage / parsimony bootstrap percentage. Circled letters on branches correspond to groups documented in Table 3.

Table 3.

Support for and against various clades. The letter at the left corresponds to the circled letters of clades in Fig. 8 (although clade i, consisting of clades j and k, is absent in Fig. 8). ML: Maximum likelihood analysis; P: parsimony analysis. Numbers indicate the bootstrap support expressed as a percentage. Cells shaded in gray to black indicate that the clade is present with bootstrap support greater than 50 or present in the optimal (maximum likelihood or most parsimonious) trees but with bootstrap value below 50. Cells in white indicate that the clade has a bootstrap value less than 50 and is not present in the optimal tree; if the bootstrap value is listed in parentheses, it means that a contradictory clade was present in the optimal trees. For no analysis was there bootstrap support greater than 50 against any of these clades. Abbreviations: “exc.” = “excluding”.

Clade Combined CAD wg ArgK Topo 28S 18S COI
a Antiperyphanes Complex 98 94 88 86 27 19 (4) 1 (1) 0 (0) 0 (2) 1 (0) (0)
b Nothonepha 100 100 97 92 69 70 (1) 0 (2) 1 50 34 79 59 (0) (0)
c Nothonepha exc. Bembidion tetrapholeon 95 (48) 81 71 55 62 42 43 (1) 1 (9) 7 46 40 (0) (0)
d Antiperyphanes Complex exc. Nothonepha 100 99 80 79 (15) 20 38 22 (3) 9 81 75 54 23 (0) 0
e Antiperyphus + Notholopha 100 98 98 98 78 70 8 2 (20) 20 (1) 3 (8) 46 (0) (0)
f Notholopha 100 100 63 80 83 81 16 43 43 53 (2) 3 72 68 49 63
g Antiperyphanes + Chilioperyphus + Ecuadion 100 100 97 97 22 32 (1) 0 (29) 45 65 53 65 34 (0) 0
h Antiperyphanes + Chilioperyphus 100 100 99 99 48 57 (7) 3 61 60 65 71 87 70 (0) 0
i Antiperyphanes 68 71 54 59 (15) 16 (6) 7 33 58 68 73 75 62 (0) 0
j Bembidion caoduroi group 100 100 100 100 95 96 98 98 96 96 93 92 66 57 62 57
k Antiperyphanes exc. Bembidion caoduroi group 100 100 99 100 96 97 32 32 61 77 94 91 (38) 34 (29) 12
l Ecuadion 100 100 54 46 47 53 44 33 66 59 99 100 55 43 (2) 0
m Bembidion chimborazonum group 100 100 100 100 53 56 95 95 97 93 68 68 93 90 17 11
n Ecuadion exc. Bembidion chimborazonum group 100 100 46 33 (38) 5 (23) 21 46 39 99 99 89 83 (1) 0

The monophyly of each subgenus (indicated by color in Figs 7 and 8) is well supported by analyses of the concatenated matrix (MLB=100 and PB=100 for all but one subgenus) and at least four genes (Table 3), except for subgenus Antiperyphanes. Antiperyphanes is monophyletic in the maximum likelihood trees of four genes, but bootstrap support is low (Table 3).

The basal split in the Antiperyphanes Complex appears to be between clade b and clade d (Fig. 8). Clade b is strongly supported in seven-gene analyses (MLB=100, PB=100), and there is bootstrap support in individual analyses of four genes (Table 3). As a subgenus of Bembidion, this clade would take the name Nothonepha Jeannel, as it contains Bembidion lonae, the type species of Nothonepha. Clade d is also strongly supported in the seven-gene analyses (MLB=100, PB=99), and there is moderate to weak bootstrap support from four genes (Table 3).

Within the Antiperyphanes Complex, strongly supported relationships between subgenera include a clade containing Antiperyphanes and Chilioperyphus, and a sister-group relationship between that clade and the subgenus Ecuadion (Fig. 8, Table 3). Bembidion (Antiperyphus) philippii appears as the sister group to subgenus Notholopha.

As a whole, the Antiperyphanes Complex is supported as monophyletic (clade a in Figs 7 and 8, Table 3), although not as strongly as in an earlier study with more limited sampling of the group (Maddison 2012). Individual gene support for the clade is only provided by CAD and to a lesser extent wingless (Table 3), but the concatenated analyses have MLB=88 and PB=86. Lack of monophyly of the complex in some analyses (e.g., individual gene analyses of 28S and 18S) is a result of Nothonepha falling in the Bembidion Series separate from the rest of the complex.

Different analytical methods yielded similar results for the concatenated, seven-gene matrices. The two partition schemes examined (by gene and as chosen by PartitionFinder) resulted in maximum likelihood trees that differ only in the placement of Bembidion georgeballi within subgenus Ecuadion. In maximum likelihood bootstrap analyses, clades with MLB>90 were the same in both partition schemes. Parsimony analyses showed similar results to maximum likelihood (Table 3).

Within Bembidion tetrapholeon sp. n., the 10 specimens sequenced from five localities showed little variation in DNA, and the variation observed was not correlated with presence of an orange spot. There was no variation observed in CAD (n=10), ArgK (n=5), Topo (n=10), and 28S (n=10), over a total of more than 3090 bases. In the wingless gene (n=6) variation was observed at three third-position sites, all of which represent synonymous differences, and for each of which some other specimens were heterozygous for the variants. COI showed the most variation with variability at eight sites, seven of which represented synonymous differences and one a non-synonymous difference. At seven of these sites, nine of the ten specimens had the same nucleotide, with the tenth specimen being unique; the specimen that was unique varied from site to site. The most distinct specimen was DNA2236, from Chiloé, with three unique nucleotides in the more than 650 bases of COI sequenced.

Morphological results

With the unexpected discovery that Bembidion tetrapholeon sp. n., belongs in a clade with an assortment of morphologically and ecologically diverse Bembidion, the search for synapomorphies for this clade became compelling.

Mesothoracic pits. The most striking derived feature observed was presence in all Nothonepha species of a pit in each lateral wall of the mesothorax. This mesepisternal pit (Fig. 9A) appears empty in many specimens killed in ethyl acetate, but in most specimens preserved in ethanol, a waxy substance is visible within it (Fig. 9A). When extracted and placed in glycerin on a microscope slide, this substance appears slightly yellowish-gray and contains no obvious substructure or particles (including no evident bacteria or fungal spores) when examined at 400× with transmitted, brightfield light (n=2, from Bembidion tetrapholeon). In contrast, all other members of Bembidion examined to date lack such a pit (e.g., Fig. 9B).

Figure 9.

Left lateral region of the prothorax and mesothorax; top of each photograph is anterior. msst: mesosternum; msepst: mesepisternum; msep: mesepimeron (note that the boundaries between these sclerites are not evident externally in A. A Bembidion (Nothonepha) tetrapholeon, DRM voucher V100810 B Bembidion (Antiperyphanes) zanettii, DRM voucher V100811. Scale bar 0.1 mm.

In Bembidion tetrapholeon these paired structures, one on either side, internally manifest as large intrusions which touch in the center of the body cavity (Figs 10A, C, E). Typical Bembidion have no such structures internally (Figs 10B, D, F). There is variation within Nothonepha in the size of the intrusions, with Bembidion engelhardti having relatively small intrusions (and thus relatively shallow pits) (Fig. 11).

Figure 10.

Mesothorax, dorsal surface and soft tissue removed. msst: mesosternum; msepst: mesepisternum; msep: mesepimeron. Scale bar 0.1 mm. (A, C and E) Bembidion (Nothonepha) tetrapholeon, DRM voucher V100766 B, D, and F Bembidion (Antiperyphanes) zanettii, DRM voucher V100767 A, B oblique ventral view; view from lower left side, slightly in front of mesothorax. C, D oblique dorsal view; view from upper right side, slightly behind the mesothorax. E, F anterior view.

Figure 11.

Mesothorax, dorsal surface and soft tissue removed, of Bembidion (Nothonepha) engelhardti rayoda Toledano. Oblique dorsal view; view from upper right side, slightly behind the mesothorax. Scale bar 0.1 mm.

Examination of musculature in Bembidion tetrapholeon (n=4) and Bembidion tucumanum (n=1) revealed no muscles attached to the internal intrusions, although the course of some muscles appeared to be bent by the necessity to wrap around the structures. There were no evident large glands associated with the intrusions, although there were small patches of tissue on their internal surfaces.

Two other groups of carabids reported to have mesepisternal pits were also examined, members of subgenus Tachylopha of the genus Elaphropus (Bruneau de Miré 1966; Erwin 1970) and the genus Oodinus (Spence 1982). I have specimens of these preserved in 95% ethanol, and both have large pits in the mesepisternum in the same place as Bembidion (Nothonepha). In Elaphropus (Tachylopha) leleupi Basilewsky from South Africa the pits are filled with a waxy substance similar to that seen in Nothonepha (Fig. 12A). Internally these pits appear as two large intrusions that join in mid-thorax to form a tunnel (Fig. 12B); in the two specimens I have dissected there is no evidence of a septum at the point of joining, and the waxy substance fills the tunnel. I have seen ethanol-preserved specimens of Elaphropus (Tachylopha) basilewskyi Bruneau de Miré from Gabon (identified with Bruneau de Miré (1966)) and Elaphropus (Tachylopha) spenceri (Sloane) from Australia (identified with Baehr (1988)) that have similar pits also filled with a waxy substance. The apparently related subgenus Sphaerotachys also has mesepisternal pits, although they are much smaller than those seen in Tachylopha; a specimen I have examined from Hans Merensky Nature Reserve, Republic of South Africa, has pits similar to those shown in Fig. 11. The single ethanol-preserved Oodinus alutaceus (Bates) (identified with Bousquet (1996)) that I have examined, from south Texas, also has mesepisternal pits, but internally the intrusions do not touch, and are more similar in structure to those of Bembidion (Nothonepha) than Elaphropus (Tachylopha). In some specimens of Oodinus, the pits are also filled with a waxy substance (Spence 1982).

Figure 12.

Mesothoracic structures of Elaphropus (Tachylopha) leleupi Basilewsky. A Left lateral region of the prothorax and mesothorax B Anterior view of mesothorax, dorsal surface and soft tissue removed. Scale bars 0.1 mm.

Abdominal pits. In additional to mesepisternal pits, Bembidion tetrapholeon has a prominent pit on each side of the abdomen, ventrally, between abdominal segments II and III (Fig. 13A). In ethanol-preserved specimens, this pit is filled with a waxy substance similar to that in the mesepisternal pits. Almost all other species of subgenus Nothonepha have similar pits (e.g., Bembidion (Nothonepha) sp. nr. lonae, Fig. 13C); they are lacking only in Bembidion (Nothonepha) lonae (Fig. 13D), the sister to Bembidion sp. nr. lonae.

Figure 13.

Ventral surface of anterior end of abdomen and posterior region of metathorax. The second and third abdominal segments are marked by II and III. A Bembidion tetrapholeon, DRM voucher V100773. B Bembidium mandibulare, DRM voucher V100772 C Bembidion sp. nr. lonae, DRM voucher V100771 D Bembidion lonae, DRM voucher V100770. Scale bar 0.1 mm.

Internally these abdominal pits are evident as knob-shaped intrusions (Fig. 14A, C). Consistent with the lack of externally visible pits, Bembidion lonae lacks these intrusions, and has instead only a ridge between the abdominal segments (Fig. 14D), as is typical in Bembidion.

Figure 14.

Inner surface of the anterior portion of the abdomen, soft tissue removed. The abdomen is slightly tilted to the left. sp: sclerotized patch on membrane that bounds the front of the abdomen on the ventral side. All species shown are members of the Bembidion Series; A–F are members of the Antiperyphanes Complex; A, C and D are members of the subgenus Nothonepha A Bembidion tetrapholeon, DRM voucher V100766 B Bembidium mandibulare, DRM voucher V100805 C Bembidion sp. nr. lonae, DRM voucher V100803 D Bembidion lonae, DRM voucher V100802 E Bembidium philippii, DRM voucher V100804 F Bembidion chimborazonum, DRM voucher V100809 G Bembidion charile Bates, DRM voucher DNA1171 H Bembidion quadrimaculatum oppositum Say, DRM voucher V100807. Scale bar 0.1 mm.

Outside of Nothonepha I have seen no species of Bembidion with as prominent abdominal pits, and most species lack them entirely. Bembidion (Antiperyphanes) mandibulare, for example, lacks abdominal pits and has only a slight linear depression in that region (Fig. 13B), and internally a simple ridge is evident (Fig. 14B). There is much variation between members of the Antiperyphanes Complex in this feature, however, with some species having an evident pit/internal intrusion (e.g., Bembidion (Antiperyphus) philippii, Fig. 14E, and Bembidion (Antiperyphanes) zanettii), and others (e.g., Bembidion (Ecuadion) chimborazonum, Fig. 14F) having a low, wide hump internally. Outside of the Antiperyphanes Complex all species examined either have a sinuate (Fig. 14G) or straight (Fig. 14H) ridge internally in this region.

Whatever internal structure is present between abdominal segments II and III, whether a ridge, or low mound, or knob-like intrusion, in the species examined this structure serves (at least in part) as an apodeme. In Bembidion tetrapholeon (n=5), for example, a muscle bundle is attached to the apex of the internal intrusion, and extends forward to the small sclerotized patch (sp in Fig. 14C) in the membrane that serves as the front boundary above the ventral margin of the abdomen. Another muscle bundle extends from this sclerotized patch forward and laterally to the lateral wall of the body, where it attaches to an external rod-like sclerite that is connected to the posterior lateral corner of the metanotum; this rod-like sclerite extends posterior laterally from that point to near the metepimeron. I have examined Bembidion (Ecuadion) chimborazonum (n=1) and it has a similar muscle attached to the low mound in the same region; Bembidion (Bracteon) foveum Motschulsky (n=1), Bembidion (Ocydromus Complex) nebraskense (n=2), and the bembidiine Lionepha casta (n=2) all have a similar muscle connecting the intersegmental ridge at the equivalent region to the small sclerotized patch.


Monophyly of Nothonepha. DNA data strongly support Nothonepha as a clade. Four genes (CAD, wg, 28S, 18S) independently have bootstrap support for the clade (Table 3), and the concatenated seven-gene analysis has MLB=100 and PB=100. Combined with the striking synapomorphy of mesothoracic pits, evidence for this clade becomes convincing. As Darwin (1859) noted, “We may err in this respect in regard to single points of structure, but when several characters, let them be ever so trifling, occur together throughout a large group of beings having different habits, we may feel almost sure, on the theory of descent, that these characters have been inherited from a common ancestor.”

The unexpectedness of Nothonepha. The relationship between species here grouped into subgenus Nothonepha was so unexpected when first discovered from sequences of 28S that it was dismissed, and considered to be the result of DNA contamination or mislabeled extractions. This small clade, of only ten known species, includes some of the largest Bembidion in South America (Bembidion germainianum, up to 6.4 mm in length, Fig. 5C), and some of the smallest (Bembidion lonae, down to 2.4 mm in length, Fig. 5D). They range in habitat from cobbles shores of small, cold, clear rivers (Fig. 6D) to mixed shores of large rivers (Fig. 6A), and sand shores of desert rivers (Fig. 6C) to warm, exposed salt flats (Fig. 6B). In the field, beetles in this group give the appearance of rather different groups of carabids. In my first field encounter with live Bembidion lonae, I mistook them for tachyines of the genus Elaphropus Motschulsky; Bembidion germainianum is reminiscent of the Nearctic Bembidion perspicuum Casey, a member of the Ocydromus Series of Bembidion; Bembidion eburneonigrum looks very much like a small member of the subgenus Notaphus as it scurries on the sandy shores of rivers.

There are many examples of clades throughout the tree of life that contain species of diverse forms living in diverse habitats. Why then is Nothonepha unexpected? The current classification, to the extent that it might be a predictor of relationships, would suggest that these taxa are not related. Bembidion lonae is the only described species of those sampled that was placed in Nothonepha; the very similar but undescribed Bembidion sp. nr. lonae would have been placed there as well. The other described species (Bembidion germainianum, Bembidion engelhardti, Bembidion tucumanum, Bembidion eburneonigrum, and Bembidion tucumanum) had all been classified in subgenus Antiperyphus, along with Bembidium philippii. When I first discovered Bembidion tetrapholeon, I thought it represented a separate lineage requiring a new subgeneric name, as it is very different in form from any other South American Bembidion. This apparently added a third element to the diverse group. However, the current classification, constructed with limited data and without phylogenetic analyses, may not be the best predictor of phylogenetic relationships.

Nonetheless, Nothonepha is a morphologically heterogeneous group (Fig. 6). I have been studying Bembidion systematics for over three decades, and whatever predictive map my brain has developed from all the data accumulated over the years contained no hint that the species shown in Fig. 6 formed a clade.

However, it may not be the diversity of form within Nothonepha that is unusual, but rather the diversity given the lack of intermediate forms and small size of the group. Other clades in the South American fauna are also diverse in form and size (e.g., Ecuadion, Fig. 3), but they have many more species, some of which are intermediate between the more distinct forms. The lack of intermediate forms in Nothonepha may be a result of low speciation rates with high rates of morphological evolution, or it might be a result of extinction of intermediate forms; it is unlikely to be a lack of sampling, as enough collecting has been done in South America to suggest that there are not a large number of undescribed species of Nothonepha. A full investigation of patterns of morphological and molecular rates, times of divergence, and speciation and extinction rates relative to other clades is beyond the scope of this paper, but would be a worthwhile topic for future studies.

Function of mesepisternal pits. Exoskeletal invaginations are widespread in beetles (Grebennikov and Leschen 2010). These cavities occur on many different body parts, including in the lateral regions of the mesothorax (e.g., in the ptiliid tribe Discheramocephalini (Grebennikov 2008; 2009)); their function probably varies from group to group. Many have been thought to be mycangia for storing fungal spores, but this is well documented in only two of the many independent origins of such cavities (Grebennikov and Leschen 2010). In a few groups of polyphagan beetles (e.g., the Staphylinoidea subfamily Scydmaeninae, the Cucujoid families Cyclaxyridae and Nitidulidae, the Tenebrionoidea family Zopheridae) there are species with pits containing a waxy substance (Grebennikov and Leschen 2010). Wax has been proposed to act as a defensive shield (Lawrence and Hlavac 1979), or as a medium for retaining fungal spores (Grebennikov and Leschen 2010).

The functions of mesepisternal pits in the two carabid groups in which they were previously described is not known, but functions have been hypothesized. Erwin (1970) proposes that pits of Elaphropus (Tachylopha) are insertion points for ant mandibles, allowing ants to carry the adults around. Bousquet (1996) calls the structures in Oodinus “apodemal pits”, which implies a function as internal attachment points for muscles.

The similarities between the wax-filled mesepisternal pits observed in the bembidiine Bembidion (Nothonepha), the tachyines Elaphropus (Tachylopha) and Elaphropus (Sphaerotachys), and the oodine Oodinus are striking (e.g., Figs 9A and 12A in this paper, and Fig. 145 in Bousquet (1996)), enough so that a common function might be hypothesized. Although the shape and nature of the deepest pits and largest intrusions differs between the groups (e.g., there are no Nothonepha known with the merged intrusions of some Tachylopha), the less extreme forms are indistinguishable externally and internally. These features are surely convergent, as the clades are not closely related. Bembidion and Elaphropus are both members of the subfamily Trechinae, but they are each deeply nested within independent clades (Maddison and Ober 2011); Oodinus is a member of the tribe Oodini (Bousquet 1996; Spence 1982), which is nested within several clades in the superfamily Harpalinae, itself a well-supported clade (Maddison et al. 1999; Ober and Heider 2010). It may be that these pits serve different functions in these clades, but consistency of structure suggests they may be serving the beetles in similar ways in the three different groups.

Correlates in way of life might provide some hints about function. Members of the three carabid groups are all presumably generalist predators, as is typical in Carabidae (Thiele 1977). They are also all terrestrial, but associated with shorelines. Bembidion (Nothonepha) species occur at edges of bodies of water in southern South America. Elaphropus (Tachylopha) occurs in similar habitats. I have seen Elaphropus (Tachylopha) spenceri (Sloane) (identified using Baehr (1988)) from multiple localities in Queensland, Australia, labeled as being found at “water’s edge” along creek shores. Bruneau de Miré (1966) reports numerous species along rivers, and states that they can be abundant in swamps and moist forest humus. Oodinus occurs at the edges of marshes and swamps (Bousquet 1996; Spence 1982). However, many other carabid groups lacking these pits are also found in these habitats, including many other Bembidion, Elaphropus, and Oodini.

It appears unlikely that mesepisternal pits are used as ant handles in Bembidion (Nothonepha) and Oodinus, and probably not in Elaphropus (Tachylopha). Erwin’s (1970) hypothesis was based in part on the unusual elytral structure of Tachylopha, which is narrow above the mesepisternum and which possesses a notch into which the base of ant mandibles could fit. Although most Nothonepha have narrow-enough pronota to allow curved, sickle-shaped mandibles of a large ant access to the pits, that seems much less likely for Oodinus, which are wide-bodied, oval carabids. Oodinus have a distinct ledge along the lateral edge of their bodies; to fit a mandible tip into the pit underneath this ledge an ant would need exceptionally curved mandibles. In general, ground-nesting ants are relatively rare in wet, near-shore, seasonally inundated habitats, and one would not expect large ants (ground-nesting or arboreal) with sufficiently curved mandibles in these near-shore environments (Philip S. Ward, pers. comm.). I have observed ants only rarely in the near-water habitats of subgenus Nothonepha, and not at all in the case of the four localities at which I have collected Bembidion tetrapholeon or the three localities at which I have found Bembidion germainianum. Furthermore, there is no evidence for any association between ants and these three carabid groups.

There is also evidence against the mesepisternal pits functioning as apodemes. As noted above, Nothonepha and Tachylopha do not have muscles attached to the internal walls of the mesepisternal pits. Spence (1982) reports that there are no conspicuous muscles that attach to the internal walls of the pits in Oodinus.

It is possible that the function is as a reservoir for the wax, although where the wax is produced is not evident. Spence (1982) states that the pit walls are perforated by numerous channels in Oodinus; these might be ducts for glandular secretions. I have not detected any large glands internally near the intrusions, although there is a thin layer of tissue in places on the inner surface of the structures. It is also possible that the wax is not produced near the intrusion; it might be produced elsewhere on the beetle’s body, and in fluid form flow into the pits. (As noted above, the wax is not evident in many ethyl acetate killed specimens, but rather in ethanol killed specimens, suggesting that the substance may precipitate in ethanol, but exist as a liquid otherwise.) More detailed histological work is needed to explore possible glandular sources.

Whatever the source of the wax, its function (if it has one) is unclear. It seems unlikely that it would be for retaining fungal spores (Grebennikov and Leschen 2010), as there is nothing known about these generalist predators that would suggest a benefit to the beetle to retain fungal spores. Wax as a defensive coating is plausible (Lawrence and Hlavac 1979), but more studies are needed to explore this and other possibilities.

Function of abdominal pits. In contrast to the mesepisternal pits, the abdominal pits in Bembidion (Nothonepha) evidently serve (in part) as apodemes, that is, as attachment points for muscles. However, the function of those muscles is unclear; in general the nature and function of muscles at the junction of the metathorax and abdomen in beetles is poorly known (Rolf Beutel, pers. comm. 2014). Even if the abdominal pits serve as apodemes, they may serve additional functions as well, perhaps related to the presence of wax that appears similar to that found in the mesepisternal pits.

Taxonomic treatment

Three clades of Bembidion comprise the South American fauna: subgenus Notaphus Dejean (32 species (Jeannel 1962; Toledano 2002; Toledano 2008)), subgenus Nothocys Jeannel (13 species (Toledano 2002; Toledano 2008)), and the Antiperyphanes Complex. Along with a number of lineages outside of South America, these all belong to one subclade of Bembidion, the Bembidion Series (Maddison 2012). Subgenus Notaphus is widespread and abundant throughout the New World, with over 50 species known from North America; in addition, seven species occur in the Old World (Lobl and Smetana 2003). Nothocys is restricted to southern South America, occurring in Chile, Argentina, Peru, and Bolivia (Toledano 2002; Toledano 2008). The largest of these clades is the Antiperyphanes Complex, with about 95 described species (Jeannel 1962; Maddison 2012; Toledano 2002; Toledano 2008), and many undescribed.

Antiperyphanes Complex

Members of the Antiperyphanes Complex are diverse in form (Figs 13, 5). There are no recognized derived morphological characteristics of the group, although the clade is moderately well supported by the concatenated DNA sequence data (Fig. 8, Table 3). Within the South American fauna, most species can be recognized by the lack of an N sclerite in the internal sac of the male genitalia (Toledano 2008). However, some members of the complex, including some species in subgenus Nothonepha, have a small sclerite that could be homologous to the N sclerite (Toledano 2008).

The Antiperyphanes Complex, as here classified, consists of at least five subgenera: Antiperyphanes, Chilioperyphus, Antiperyphus, Notholopha, Ecuadion, and Nothonepha. Each of these subgenera is briefly discussed below, with notes about their composition. Two other poorly known subgenera, Pseudotrepanes Jeannel and Notoperyphus Bonniard de Saludo, are likely members of this complex, but specimens will need to be examined to confirm their membership.

Subgenus Antiperyphanes Jeannel, 1962
Antiperyphanes Jeannel, 1962; type species Bembidium spinolai Solier, by original designation.
Plocamoperyphus Jeannel, 1962; type species Bembidium mandibulare Solier, by original designation. New synonymy.

This group contains at least 19 described species (Toledano 2002; Toledano 2008), and is characterized by males having an aedeagus lacking a brush sclerite, and with a very long flagellum (Maddison et al. 2013; Toledano 2008).

Included here are some species previously placed in subgenus Antiperyphus by Jeannel (1962): Bembidion hirtipes (Jeannel), Bembidion ringueleti (Jeannel), Bembidion rufoplagiatum Germain, Bembidion uniforme Csiki, and Bembidion parvum (Jeannel). Bembidium mandibulare Solier belongs to Antiperyphanes as well, and is nested well within it (Fig. 8); thus, subgenus Plocamoperyphus is a synonym of Antiperyphanes. There are no morphological characteristics of Bembidium mandibulare that would suggest it is not a member of Antiperyphanes; it shares all apomorphies of the group. As first reviser, I choose Antiperyphanes as the valid name of the group.

Antiperyphanes has two distinct clades, each very well supported: the Bembidion caoduroi group (clade j in Fig. 8; Fig. 1D), consisting (among the sampled species) of three large species from the northern Andes (Fig. 1D); (2) the remaining Antiperypanes (clade k in Fig. 8; Figs 1A–C, E). Each is supported by MLB=100 and PB=100 in the multi-gene analyses, and individually by support from five to seven genes (Table 3). As a whole, however, the monophyly of Antiperyphanes is only weakly supported by the combined analysis and analyses of four genes (Table 3).

Members of this subgenus are found at the edges of bodies of water. For example, Bembidion rufoplagiatum and Bembidion zanettii are common on gravel and cobble river shores, Bembidion ringueleti is found on gravel and sand shores of smaller creeks, and Bembidium mandibulare on the sand beaches of the Pacific Ocean in Chile and sand beaches of rivers in Argentina.

Subgenus Chilioperyphus Jeannel, 1962
Chilioperyphus Jeannel, 1962; type species Bembidium orregoi Germain, by original designation.

This subgenus contains two described species (Jeannel 1962; Maddison et al. 2013) (Fig. 1F) and at least three undescribed species. Males are characterized by having a brush sclerite, and by having an extremely elongate flagellum, so long that it can only fit within the median lobe through folding (Maddison et al. 2013). Members of this subgenus occur on steep sand or clay banks of rivers and creeks.

Subgenus Antiperyphus Jeannel, 1962
Antiperyphus Jeannel, 1962; type species Bembidium philippii Germain, by original designation.

As noted by Toledano (2008), Jeannel’s concept of Antiperyphus was polyphyletic, with at least Bembidion hirtipes, Bembidion ringueleti, Bembidion rufoplagiatum, and Bembidion uniforme Csiki belonging within Antiperyphanes. This is confirmed in part by my results (Fig. 8, Table 3). In addition, Bembidion engelhardti, Bembidion eburneonigrum, Bembidion tucumanum, and Bembidion germainianum are members of Nothonepha, not Antiperyphus. Of the species included in the subgenus by Jeannel (1962), this leaves only the type species, Bembidium philippii (Fig. 2A).

In addition, Bembidion peterseni Jensen-Haarup (1910), from Mendoza, Argentina, can tentatively be placed here. I have examined a male syntype (in ZMUC), and it is similar in appearance to Bembidium philippii, although with much deeper and longer elytral striae. It is not a member of Nothonepha (as it lacks mesepisternal pits), nor is it a member of Antiperyphanes (it has a brush sclerite, and does not have the long flagellum characteristic of Antiperyphanes). The internal sac of the male genitalia, although difficult to see because of the nature of the preparation, appears very similar to that of Bembidium philippii.

B. philippii is common on sand shores of rivers in the provinces of Neuquén and Chubut in Argentina; it also occurs in Chile.

Subgenus Notholopha Jeannel, 1962
Notholopha Jeannel, 1962; type species Bembidium punctigerum Solier, by original designation.
Pacmophena Jeannel, 1962; type species Bembidium scitulum Erichson, by original designation.

Notholopha consists of 11 described species (Toledano 2002; Toledano 2008), and several undescribed (two of which are sequenced here). These are small beetles with large, protruding eyes (Fig. 2B, C), with small flagella in the internal sac of the male genitalia, and with brush sclerites. They have the general appearance when running of a member of the Holarctic subgenus Bembidion. Some frequent habitats similar to those of subgenus Bembidion, including dry habitats far from water (Bembidion stricticolle), or upper banks of creeks (e.g., Bembidion sp. “Nahuelbuta”). Others occur at high elevation near small rivulets in open, alpine areas (e.g., Bembidion rugosellum and Bembidion melanopodum), or at the edges of snowfields (Bembidion sexfoveolatum).

Jeannel considered Pacmophena and Notholopha to be two subgenera within the genus Notholopha. As the characters that Jeannel used to distinguish the two are minor characters such as surface texture and antennal length, and as it appears that Pacmophena is paraphyletic with respect to the Notholopha (s. str.) (Fig. 7), I consider them synonymous, with Notholopha as the valid name.

Subgenus Ecuadion Moret & Toledano, 2002
Ecuadion Moret & Toledano, 2002; type species Bembidion fulvocinctum Bates, by original designation.

Ecuadion is the largest subgenus in the Antiperyphanes Complex, with over 50 described species (Maddison and Toledano 2012; Moret and Toledano 2002; Toledano 2008; Vigna Taglianti and Toledano 2008) and likely many undescribed. It occurs from Costa Rica south to the mountains near Mendoza, Argentina. There are no known exoskeletal synapomorphies of the group, but it is well-supported by the molecular data, with bootstrap support in six of the seven genes examined (Table 3).

Ecuadion falls into two distinct clades among the sampled species: (1) the Bembidion chimborazonum group (clade m in Fig. 8), consisting mostly of larger, long-legged species (Figs 3A, B); (2) remaining Ecuadion (clade n in Fig. 8), consisting of mostly smaller species with shorter appendages (Figs 3C–F). The Bembidion chimborazonum group is supported by all genes examined (Table 3); support for the complementary clade is not quite as strong, with the clearest evidence coming from ribosomal genes (Table 3).

Unlike most Bembidion, this subgenus has radiated in habitats away from water. Some occur in leaf litter in cloud forests (e.g., Bembidion andersoni, Bembidion georgeballi, Bembidion onorei, Bembidion sp. “Papallacta”; Fig. 4D), in habitats that would typically be occupied by the genus Trechus Clairville in North America. A number of species are found in open, high-elevation grasslands (e.g., Bembidion chimborazonum, Bembidion guamani, Bembidion humboldti; Fig. 4C). Some species occur on the upper banks of creek shores (e.g., Bembidion sanctaemarthae, Bembidion ricei); others are found on clay or silt cliffs (e.g., Bembidion agonoides, Bembidion walterrossii).

Subgenus Nothonepha Jeannel, 1962
Nothonepha Jeannel, 1962; type species Bembidium baptisatum Csiki (=Bembidion lonae Jensen-Haarup), by original designation.

As here defined, the subgenus Nothonepha includes all species of the Antiperyphanes Complex possessing mesepisternal pits. Seven described species belong to Nothonepha:

Bembidion lonae Jensen-Haarup, 1910 (Fig. 5D)

Bembidion pallideguttula Jensen-Haarup, 1910

Bembidion eburneonigrum Germain, 1906 (Fig. 5E)

Bembidion engelhardti Jensen-Haarup, 1910

Bembidion engelhardti engelhardti Jensen-Haarup, 1910 (Fig. 5G)

Bembidion engelhardti rayoda Toledano, 2008

Bembidion tucumanum (Jeannel, 1962) (Fig. 5F)

Bembidion germainianum Toledano, 2002 (Fig. 5C)

Bembidion tetrapholeon Maddison, sp. n. (Figs 5A, B)

Four of these species (Bembidion germainianum, Bembidion tucumanum, Bembidion engelhardti, and Bembidion eburneonigrum) were formerly placed in subgenus Antiperyphus. In addition, there are at least three undescribed species (including Bembidion sp. nr. lonae, sequenced here). The species figured by Toledano (2008) as Bembidion germainianum is an undescribed species related to Bembidion germainianum. A revision of the subgenus is in preparation (Roig-Juñent and Maddison).

Bembidion (Nothonepha) tetrapholeon sp. n.


Figs 5A, B, 9A, 15, 16, 17A, 18

male (IADIZA), with 3 labels: “Argentina: Neuquén: Arroyo / Queñi at Lago Queñi, 830m, / 40.1575°S, 71.721°W, / 10-11.ii.2007. DRM 07.035. / D.R. Maddison, S.A.Roig”, “David R. Maddison / DNA2356 / DNA Voucher” [printed on pale green paper], and “HOLOTYPE / Bembidion / tetrapholeon / David R. Maddison” [printed on red paper]. Genitalia in glycerine in small plastic vial beneath specimen; extracted DNA stored separately. GenBank accession numbers for DNA sequences of the holotype are KJ653049 (28S), KJ653145 (COI), KJ653112 (CAD), KJ653181 (Topo), KJ653215 (wg), and KJ653082 (ArgK).


Total of 244, in IADIZA, MACN, MNNC, OSAC, MNHN, BMNH, EMEC, CTVR, and CMNH, from “Argentina: Neuquén: Arroyo / Queñi at Lago Queñi, 830m, / 40.1575°S, 71.721°W, / 10–11.ii.2007. DRM 07.035. / D.R. Maddison, S.A.Roig” [135 exx.], Argentina: Neuquén: Rio Pichi / Traful nr Lago Traful, 810m, / 40.4867°S, 71.5958°W, / 12.ii.2007. DRM 07.039. / D.R. Maddison, S.A.Roig” [95 exx], “Argentina: Chubut: Rio / Azul at Lago Puelo, 200m / 42.0929°S, 71.6244°W / 13.ii.2007. DRM 07.044. / D.R. Maddison” [12 exx], “Argentina: Chubut: Rio / Azul at Lago Puelo, 200m / 42.0929°S, 71.6244°W, / 13.ii.2007. DRM 07.045. / S.A.Roig, D.R. Maddison” [1 exx].

Additional material examined.

CHILE: Reg. X, Chiloé: Rio Puntra at rt 5, 55m, 42.1661°S, 73.7256°W, 19.i.2006. DRM 06.075. D.R. Maddison [5 exx, OSAC, MNNC]. CHILE: Region X, Rio Pullinque at Puente Huanehue, 8 km NE Panguipulli. 16 Jan 2002, 39.6162°S, 72.2286°W, 1590 ft. W. D. Shepard [2 exx, OSAC].

Additional identified material.

The following specimens have been examined by Luca Toledano and confirmed to belong to this species (based upon photographs we have shared). CHILE: Reg. X, Los Lagos, P.N. Vicente Péres Rosales, Petrohué, Lago Todos los Santos, 190m, mouth Rio El Caulle, 41.0924°S, 72.3950°W. 5.i.2014. L. Toledano, R. Olivieri, J.P. Morales. [1 ex, CTVR]; CHILE: Region XI, Parque Nat. Rio Simpson, H. Franz [4 exx, NHMW]; CHILE: Reg. X, I. Chiloé, R. Punta, 31.i.1986. M. Spies. [1 ex, USNM].

Type locality.

Argentina: Neuquén: Arroyo Queñi at Lago Queñi, 830m, 40.1575°S, 71.7210°W. The habitat at the type locality is a cobble, gravel, and coarse sand river shore (Fig. 6D); the river is cold and has crystal-clear water. In the same habitat members of the genus Bembidarenas Erwin are abundant, as is Bembidion (Antiperyphanes) rufoplagiatum.

Derivation of specific epithet.

From the Greek “tetra”, meaning “four”, and “pholeon”, meaning “pit”, referring to the four prominent pits visible on the underside of adults.


A large, sleek, shiny Bembidion, with an unusual body form (Figs 5A, B) of narrow forebody and large elytra. With its shape, color, and smoothness it is one of the most distinctive Bembidion species in South America, and no other known species is likely to be confused with it; it is more reminiscent of some species in New Zealand, e.g., Bembidion (Zeplataphus) dehiscens Broun (Lindroth 1976).

Length (4.7–5.7 mm, with most specimens above 5.0 mm). Color piceous (Fig. 5A), with legs and antennae in some specimens slightly paler, and with a few specimens having a large orange spot just in front of the elytral apices (Fig. 5B).

Head with shallow and parallel frontal furrows.

Pronotum narrow, cordate, with hind angles flaring outward (Fig. 15). Very smooth, without punctures, and with a linear basolateral foveae; without distinct carina at hind angle. Lateral bead of pronotum not complete, not reaching front angle of prothorax and only in some specimens reaching the hind angle. One midlateral and one basolateral seta on each side.

Figure 15.

Pronotum of Bembidion tetrapholeon, DRM voucher V100781. Scale bar 0.1 mm.

Each elytron with two discal setae (ed3 and ed5); ed3 in third stria. Elytral striae with prominent punctures in their basal half, but with striae 2–7 absent in about the hind 40% or more of the elytra, such that the posterior discal seta, ed5, is in a region without striae. Striae 7 absent in many specimens. In many specimens the striae are effaced anteriorly, especially striae 2 and 3. Lateral bead of elytron effaced anteriorly, not extended onto shoulder (Fig. 16A), similar to that of Bembidion (Nothonepha) lonae (Fig. 16B), but unlike most other Bembidion (e.g., Bembidion (Nothonepha) germainianum, Fig. 16C).

Figure 16.

Humeral region of left elytron and posterior corner of pronotum of three Bembidion (Nothonepha) species. A Bembidion tetrapholeon, DRM voucher V100781 B Bembidion lonae, DRM voucher V100786 C Bembidion germainianum, DRM voucher V100782. Arrows show the anterior end of the lateral elytral groove and bead. Scale bar 0.1 mm.

Mesothorax with prominent pits in the mesepisternum (Fig. 9A), which appear internally as large intrusions that touch in the middle (Figs 10A, C, E). Smaller pits are present ventrally at the junction of abdominal segments II and III (Fig. 13A), which are evident internally as knob-like intrusions (Fig. 14A).

Hind wings full.

Microsculpture absent from entire dorsal surface of the body except for the cervical region of the head, labrum, and faintly on the clypeus; the beetles are thus brilliantly shiny. Microsculpture absent from most of the ventral surface as well, with the most notable microsculpture being on the undersurface of the head.

Aedeagus with nearly straight ventral margin, tip of variable width (Fig. 17). Prominent brush sclerite, and with flagellum not clearly evident from the left side. There is no evident correlation between aedeagal structure and presence or absence of orange spots on the elytra (compare Figs 17A, B to Figs 17C, D).

Figure 17.

Male aedeagus of Bembidion tetrapholeon. A Black form, Chile: Region X, Rio Pullinque at Puente Huanehue, 8 km NE Panguipulli, DRM voucher DNA1752 B Black form, Chile: Reg. X, Chiloé: Rio Puntra at route 5, DRM voucher DNA2236 C Orange-spotted form, Argentina: Neuquén: Rio Pichi Traful nr Lago Traful, DRM voucher DNA2564 D Orange-spotted form, Argentina: Neuquén: Rio Pichi Traful nr Lago Traful, DRM voucher DNA2555. Scale bar 0.1 mm.

Morphological variation.

The most noted variation is in color of the elytra. Of the 257 specimens examined (including the six specimens identified by Luca Toledano), 245 have uniformly piceous elytra (Fig. 5A); the remaining 12 have a large, diffuse orange spot occupying most of the posterior third of the elytra (Fig. 5B). Ten of these orange-spotted specimens are from the three localities in Argentina, with at least one orange-spotted specimen from each locality, and two of the orange-spotted specimens are from Chile. In most of orange-spotted specimens, the posterior discal seta (ed5) is in the orange region, but immediately around the seta is a small dark spot. No other aspect of morphological or molecular variation was observed to be correlated with presence or absence of the orange spot.

DNA sequence variation.

As noted above under Results, there was minor variation present in COI and the wingless gene, and no variation in the other genes studied.

Habitat and seasonality.

At all four localities where habitat data were recorded, Bembidion tetrapholeon specimens were found on cobble, gravel, and coarse sand shores of clear, fast-flowing rivers (Fig. 6D), from 55 m elevation (Rio Puntra, Isla Grande de Chiloé, Chile) to 830 m elevation (Arroyo Queñi, Neuquén, Argentina). These shorelines lack vascular plants. The beetles occur close to the water, most within 1 m. Specimens have been found in January and February.

Geographic distribution.

In southern Argentina and Chile (Fig. 18). In Argentina this species has been found in Neuquén and Chubut, and in Chile from Regions X and XI.

Figure 18.

Geographic distribution of Bembidion tetrapholeon. Circles filled in black are based upon specimens I have examined and that have been sequenced; circles filled in gray are based upon specimens identified by Luca Toledano. Five cities are included as landmarks.

Relationship to other Bembidion.

Bembidion tetrapholeon is a member of subgenus Nothonepha, as strongly supported by DNA sequences (Table 3) and the presence of shared, derived mesepisternal pits. Bembidion tetrapholeon appears to be the sister of remaining Bembidion (Nothonepha) (Figs 7, 8). Four genes support this placement (CAD, wg, ArgK, and 18S; Table 3), although the support is weak or moderate in single-gene analyses.

Concluding remark

when the same organ appears in several members of the same class, especially if in members having very different habits of life, we may attribute its presence to inheritance from a common ancestor.” (Darwin 1859)

In groups such as beetles, in which a preponderance of lineages split without later reticulation, the evolutionary tree at the core of life’s history yields hierarchical patterns in the distributions of characteristics. Any particular lineage in the tree, if separated long enough or with a high enough rate of evolution, will leave in the bodies of its descendants marks of its existence. The echoes from that deep historical well can reverberate down through later lineages in the form of signals scattered throughout the genomes. The repeated patterns of these branch markers both in the DNA and on the bodies of organisms are among the most compelling signs of the existence of the tree of life, and provide to us evidence about its shape. On occasion the clades thereby revealed are so unexpected that it is only with multiple independent markers, all showing the same pattern, that we can confidently accept the existence of the clade. Nothonepha is such a clade.

Many of us who study the diversity of life, and see the hierarchical patterns of organismal traits, are steeped in evidence of the existence of a genetic tree of life, so much so that we perhaps take it for granted (I often do). I think about the evidence about the tree’s shape, but much less so evidence about its existence. In encountering the first evidence of Nothonepha, my belief in the tree-like structure of beetle genetic history was challenged, as the data made little sense in that light. As newly sequenced genes added to the evidence, my acceptance of the clade increased. The struggle was fully resolved when the mesothoracic pits came to light; this harmonizing of the morphological data with the molecular not only instilled a firm belief in this clade, but also a simple confirmation of the tree itself.


I am most grateful to Sergio Roig-Juñent, who arranged the collecting expedition that yielded most known specimens of Bembidion tetrapholeon, and most of the other Nothonepha studied; he was also a very welcome companion on the trip.

I am very thankful to all those who helped with a collecting expedition to Ecuador during which most sequenced members of subgenus Ecuadion were collected. Mauricio Vega arranged many details of the trip, including relevant collecting and export permits; Wayne Maddison, Marco Reyes, and Mauricio Vega accompanied the author into the field and helped collect the specimens. I am also grateful to Reserva Yanacocha, Pichincha, Ecuador, for permission to collect on their lands. The Ecuadorian Ministry of the Environment and the Museum of Zoology of the Pontificia Universidad Católica de Ecuador assisted with permits.

I would also like to thank Elizabeth Arias and Kipling Will for organizing and providing excellent logistical support, including permit acquisition, for field work in Chile. I am also grateful for their companionship during my first experience with South American Bembidion.

Some key specimens were provided by others, and I am thankful for their contributions; without a network of collectors, a work such as this would not be possible. In particular, I would like to thank William D. Shepard (for the Rio Pullinque specimen of Bembidion tetrapholeon), Wendy Moore (Bembidion tucumanum), Karl M. Kjer (for South African Tachylopha), Geoff B. Monteith (for Australian Tachylopha), Caroline S. Chaboo (for South African Sphaerotachys), and James M. Pflug (for Oodinus from Texas).

I am also grateful to Luca Toledano, both for his review of the manuscript, and for the information he provided about specimens of Bembidion tetrapholeon he has examined from Chile. My thanks to Wayne Maddison and an anonymous reviewer for their comments on the manuscript. Thanks as well to myrmecologists Alex Wild and Philip Ward for discussing the ants that might live in the same habitats as Nothonepha, Tachylopha, and Oodinus.

This project was made possible by funds provided by the University of Arizona, the Harold E. and Leona M. Rice Endowment Fund at Oregon State University, and National Sciences Foundation grant EF-0531754. National Sciences Foundation DEB-0445413, to Elizabeth Arias and Kipling Will, provided funds that helped support field work in Chile. The expedition to South Africa that yielded the Elaphropus (Sphaerotachys) I studied was supported by a Hellman Postdoctoral Fellowship (to C.S. Chaboo).

Baehr M (1988) A review of the Australian tachyine beetles of the subgenera Tachyura Motschoulsky and Sphaerotachys Muller, with special regard to the tropical fauna. (Insecta, Coleoptera, Carabidae, Bembidiinae). Spixiana 10: 225–269.
Bonniard de Saludo P (1969) Nouveaux Carabiques du Chili. Bulletin de la Société d’Histoire naturelle de Toulouse 105: 311–328.
Bousquet Y (1996) Taxonomic revision of nearctic, Mexican, and West Indian Oodini (Coleoptera: Carabidae). Canadian Entomologist 128: 443–537. doi: 10.4039/Ent128443-3
Bruneau de Miré P (1966) Révision des Tachylopha Motschulsky d’Afrique. Revue de Zoologie et de Botanique Africaines 73: 59–100.
Chakrabarty P, Warren M, Page LM, Baldwin CC (2013) GenSeq: An updated nomenclature and ranking for genetic sequences from type and non-type sources. ZooKeys 346: 29–41. doi: 10.3897/Zookeys.346.5753
Darriba D, Taboada GL, Doallo R, Posada D (2012) jModelTest 2: more models, new heuristics and parallel computing. Nature Methods 9: 772. doi: 10.1038/nmeth.2109
Darwin CR (1859) On the Origin of Species by Means of Natural Selection, or the Preservation of Favoured Races in the Struggle for Life. John Murray, London, 502 pp.
Erwin TL (1970) Unique Structures in Members of Tachys sensu lat. (Coleoptera: Carabidae). Pan-Pacific Entomologist 46: 231–232.
Erwin TL (1982) Small terrestrial ground-beetles of Central America (Carabidae: Bembidiina and Anillina). Proceedings of the California Academy of Sciences 42: 455–496.
Grebennikov VV (2008) How small you can go: Factors limiting body miniaturization in winged insects with a review of the pantropical genus Discheramocephalus and description of six new species of the smallest beetles (Pterygota: Coleoptera: Ptiliidae). European Journal of Entomology 105: 313–327. doi: 10.14411/eje.2008.039
Grebennikov VV (2009) Discheramocephalini, a new pantropical tribe of featherwing beetles (Coleoptera: Ptiliidae): description of new taxa and phylogenetic analysis. Systematic Entomology 34: 113–136. doi: 10.1111/J.1365-3113.2008.00444.x
Grebennikov VV, Leschen RAB (2010) External exoskeletal cavities in Coleoptera and their possible mycangial functions. Entomological Science 13: 81–98. doi: 10.1111/J.1479-8298.2009.00351.x
Green P (1999) Phrap. Version 0.990329. http://phrap.org
Green P, Ewing B (2002) Phred. Version 0.020425c. http://phrap.org
Guindon S, Gascuel O (2003) A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Systematic Biology 52: 696–704. doi: 10.1080/10635150390235520
Jeannel R (1962) Les Trechides de la Paléantarctide occidentale. In: Deboutteville CD, Rapoport E (Eds) Biologie de l’Amérique Australe, Études sur la Faune du Sol. Paris, 527–655.
Jensen-Haarup AC (1910) New species of Coleoptera from West Argentina. Deutsche Entomologische Zeitschrift 1910: 541–554.
Katoh K, Standley DM (2013) MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and Usability. Molecular Biology and Evolution 30: 772–780. doi: 10.1093/Molbev/Mst010
Lanfear R, Calcott B, Ho SYW, Guindon S (2012) PartitionFinder: combined selection of partitioning schemes and substitution models for phylogenetic analyses. Molecular Biology and Evolution 29: 1695–1701. doi: 10.1093/molbev/mss020
Lawrence JF, Hlavac TF (1979) Review of the Derodontidae (Coleoptera: Polyphaga) with new species from North America and Chile. The Coleopterists Bulletin 33: 369–414.
Lindroth CH (1976) Genus Bembidion Latreille (Coleoptera: Carabidae) in New Zealand: a revision. New Zealand Journal of Zoology 3: 161–198. doi: 10.1080/03014223.1976.9517912
Lobl I, Smetana A (2003) Catalogue of Palaearctic Coleoptera. Volume 1: Archostemata-Myxophaga-Adephaga. Apollo Books, Stenstrup, 819 pp.
Maddison DR (1993) Systematics of the Holarctic beetle subgenus Bracteon and related Bembidion (Coleoptera: Carabidae). Bulletin of the Museum of Comparative Zoology 153: 143–299.
Maddison DR (2008) Systematics of the North American beetle subgenus Pseudoperyphus (Coleoptera: Carabidae: Bembidion) based upon morphological, chromosomal, and molecular data. Annals of Carnegie Museum 77: 147–193. doi: 10.2992/0097-4463-77.1.147
Maddison DR (2012) Phylogeny of Bembidion and related ground beetles (Coleoptera: Carabidae: Trechinae: Bembidiini: Bembidiina). Molecular Phylogenetics and Evolution 63: 533–576. doi: 10.1016/j.ympev.2012.01.015
Maddison DR, Baker MD, Ober KA (1999) Phylogeny of carabid beetles as inferred from 18S ribosomal DNA (Coleoptera: Carabidae). Systematic Entomology 24: 103–138. doi: 10.1046/j.1365-3113.1999.00088.x
Maddison DR, Maddison WP (2011a) Chromaseq: a Mesquite module for analyzing sequence chromatograms. Version 1.0. http://mesquiteproject.org/packages/chromaseq
Maddison WP, Maddison DR (2011b) Mesquite: a modular system for evolutionary analysis. Version 2.75. http://mesquiteproject.org
Maddison DR, Ober KA (2011) Phylogeny of minute carabid beetles and their relatives based upon DNA sequence data (Coleoptera, Carabidae, Trechitae). ZooKeys 147: 229–260. doi: 10.3897/zookeys.147.1871
Maddison DR, Toledano L (2012) A new species of Bembidion (Ecuadion) from Ecuador (Coleoptera, Carabidae, Bembidiini), with a key to members of the georgeballi species group. ZooKeys 249: 51–60. doi: 10.3897/zookeys.249.4149
Maddison DR, Toledano L, Sallenave S, Roig-Junent S (2013) Phylogenetic relationships of the South American ground beetle subgenus Chilioperyphus Jeannel (Coleoptera: Carabidae: Trechinae: Bembidiini: Bembidion Latreille). Zootaxa 3636: 547–560. doi: 10.11646/zootaxa.3636.4.3
Moret P, Toledano L (2002) Ecuadion, nouveau sous-genre de Bembidion Latreille, 1802 du páramo équatorien (Coleoptera, Carabidae, Bembidiini). Bollettino del Museo Civico di Storia Naturale di Venezia 53: 155–205.
Ober KA, Heider TN (2010) Phylogenetic diversification patterns and divergence times in ground beetles (Coleoptera: Carabidae: Harpalinae). BMC Evolutionary Biology. doi: 10.1186/1471-2148-10-262
Spence JR (1982) Taxonomic status, relationships, and biogeography of Anatrichus LeConte and Oodinus Motschulsky (Carabidae: Oodini). The Coleopterists Bulletin 36: 567–580.
Stamatakis A (2006) RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 22: 2688–2690. doi: 10.1093/bioinformatics/btl446
Swofford DL (2002) PAUP*. Phylogenetic Analysis Using Parsimony (*and Other Methods). Version 4.0b10
Thalmann O, Hebler J, Poinar HN, Pääbo S, Vigilant L (2004) Unreliable mtDNA data due to nuclear insertions: a cautionary tale from analysis of humans and other great apes. Molecular Ecology 13: 321–335. doi: 10.1046/j.1365-294X.2003.02070.x
Thiele H (1977) Carabid beetles in their environments: a study on habitat selection by adaptations in physiology and behaviour. Springer-Verlag, Berlin. doi: 10.1007/978-3-642-81154-8
Toledano L (2002) Nomenclatorial revision of the supraspecific taxa of Bembidiini s.str. of South America described by Jeannel (1962) and related taxa with some descriptions of the fauna of South America (Coleoptera: Carabidae). Koleopterologische Rundschau 72: 1–14.
Toledano L (2008) Systematic notes on the Bembidiina of the northern Andes with particular reference to the fauna of Ecuador (Coleoptera, Carabidae). Memoirs on Biodiversity 1: 81–130.
Vigna Taglianti A, Toledano L (2008) Bembidion (Ecuadion) agonoides sp. n. from Ecuador (Coleoptera, Carabidae, Bembidiina). Memoirs on Biodiversity 1: 77–80.
Appendix 1

Locality data for newly sequenced specimens

Locality data for Bembidion specimens newly sequenced for this study. Under “#” the D.R. Maddison DNA voucher number is listed.

# Locality
Bembidion agonoides 2675 ECUADOR: Napo: Vinillos, 4.1 km S Cosanga, 2090m, 0.6024°S, 77.8509°W
Bembidion andersoni 2651 ECUADOR: Pichincha: Reserva Yanacocha, start Andean Snipe Trail, 3540m, 0.1152°S, 78.5837°W
Bembidion chimborazonum 2659 ECUADOR: Pichincha: Paso de la Virgen, 4070m, 0.3331°S, 78.2025°W
Bembidion cotopaxi 2658 ECUADOR: Pichincha: Quebrada Lozada, on road to Res. Yanacocha, 3460m, 0.1105°S, 78.5642°W
Bembidion eburneonigrum 2204 CHILE: Reg. IX: Rio Allipén at route 119, 132m, 39.0164°S, 72.5045°W
Bembidion engelhardti 2334 ARGENTINA: Neuquén: Rio Salado at route 40, 725m, 38.2143°S, 70.0931°W
Bembidion georgeballi 2661 ECUADOR: Pichincha: Quebrada Lozada, on road to Res. Yanacocha, 3455m, 0.1105°S, 78.5642°W
Bembidion germainianum 2336 ARGENTINA: Neuquén: Rio Salado at route 40, 725m, 38.2143°S, 70.0931°W
Bembidion guamani 2660 ECUADOR: Pichincha: Paso de la Virgen, 4070m, 0.3331°S, 78.2025°W
Bembidion humboldti 2673 ECUADOR: Pichincha: Paso de la Virgen, 4070m, 0.3331°S, 78.2025°W
Bembidion jimburae 2674 ECUADOR: Napo: Rio Guango, 2730m, 0.3758°S, 78.0748°W, 26.x.2010
Bembidion neodelamarei 2342 ARGENTINA: Mendoza: Uspallata, 1880m, 32.5908°S, 69.3513°W, 25.ii.2007
Bembidion onorei 2678 ECUADOR: Pichincha: Quebrada Lozada, on road to Res. Yanacocha, 3455m, 0.1105°S, 78.5642°W
Bembidion paulinae paulinae 2783 ECUADOR: Pichincha: Quebrada Lozada, on road to Res. Yanacocha, 3455m, 0.1105°S, 78.5642°W
Bembidium philippii 2327 ARGENTINA: Neuquén: Rio Collón Curá ca 13 km S La Rinconada, 625m, 40.1015°S, 70.7545°W
Bembidion ricei 2653 ECUADOR: Napo: Rio Chalpi Grande, 2800m, 0.3645°S, 78.0852°W
Bembidion sanctaemarthae 2652 ECUADOR: Napo: Rio Chalpi Grande, 2780m, 0.3657°S, 78.0848°W
Bembidion sp. nr. caoduroi 2677 ECUADOR: Napo: Rio Cosanga at mouth of Rio Angenaro, 2185m, 0.6407°S, 77.9083°W
Bembidion stricticolle 2240 CHILE: Reg. IX: ca. 28 km E Melipeuco, 1262m, 38.83°S, 71.4038°W
Bembidion tetrapholeon 1752 CHILE: Region X, Rio Pullinque at Puente Huanehue, 8 km NE Panguipulli. 39°36’58’’S, 72°13’43’’W, 1590 ft.
Bembidion tetrapholeon 2236 CHILE: Reg. X, Chiloé: Rio Puntra at rt 5, 55m, 42.1661°S, 73.7256°W
Bembidion tetrapholeon 2356 ARGENTINA: Neuquén: Arroyo Queñi at Lago Queñi, 830m, 40.1575°S, 71.7210°W
Bembidion tetrapholeon 2357 ARGENTINA: Neuquén: Arroyo Queñi at Lago Queñi, 830m, 40.1575°S, 71.7210°W
Bembidion tetrapholeon 2555 ARGENTINA: Neuquén: Rip Pichi Traful nr Lago Traful, 810m, 40.4867°S, 71.5958°W
Bembidion tetrapholeon 2562 ARGENTINA: Neuquén: Arroyo Queñi at Lago Queñi, 830m, 40.1575°S, 71.7210°W
Bembidion tetrapholeon 2563 ARGENTINA: Chubut: Rio Azul at Lago Puelo, 200m, 42.0929°S, 71.6244°W
Bembidion tetrapholeon 2564 ARGENTINA: Neuquén: Rip Pichi Traful nr Lago Traful, 810m, 40.4867°S, 71.5958°W
Bembidion tetrapholeon 2565 ARGENTINA: Neuquén: Rip Pichi Traful nr Lago Traful, 810m, 40.4867°S, 71.5958°W
Bembidion tetrapholeon 2566 ARGENTINA: Neuquén: Arroyo Queñi at Lago Queñi, 830m, 40.1575°S, 71.7210°W
Bembidion tucumanum 1430 ARGENTINA: Santa Cruz District, Dept. of Deseado, Cañadón Minerales. 25 km S. of Caleta Olivia. 46.7146°S, 67.367°W. 25m.
Bembidion walterrossii 2650 ECUADOR: Napo: Vinillos, 4.1 km S Cosanga, 2090m, 0.6024°S, 77.8509°W
Bembidion (Ecuadion) sp. “Mendoza 2701 ARGENTINA: Mendoza: Reserva Villavicencio, 1540m, 32.5232°S, 68.9949°W
Bembidion (Ecuadion) sp. “Papallacta 2657 ECUADOR: Napo: Papallacta, 3315m, 0.3703°S, 78.1481°W
Bembidion (Notholopha) sp. “Nahuelbuta 2239 CHILE: Reg. IX: P.N. Nahuelbuta, 1090m, 37.8274°S, 73.0096°W
Supplementary material 1

NEXUS file containing DNA sequence matrices and maximum likelihood trees

Authors: David R. Maddison

Data type: NEXUS file with DNA data and trees

Explanation note: This is a NEXUS file containing the seven individual gene matrices as well as the two matrices of the concatenated genes. In addition, it contains the maximum likelihood trees for each matrix, and all of the bootstrap trees (2000 trees for each matrix). The file is formatted to be opened in Mesquite 2.75 or later.

Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.

Link: doi: 10.3897/zookeys.416.7706.app1

Supplementary material 2

NEXUS file containing DNA sequences for Bembidion tetrapholeon

Authors: David R. Maddison

Data type: NEXUS file with DNA data

Explanation note: This is a NEXUS file containing six matrices showing observed sequences for all of the specimens of Bembidion tetrapholeon sequenced. The file is formatted to be opened in Mesquite 2.75 or later.

Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.

Link: doi: 10.3897/zookeys.416.7706.app2

Supplementary material 3

Images of maximum likelihood trees

Authors: David R. Maddison

Data type: images of phylogenetic trees

Explanation note: This file shows the maximum likelihood trees for the concatenated, seven-gene matrices as well as each individual gene. Each figure is labeled to indicate the nature of the data analyzed for that tree.

Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.

Link: doi: 10.3897/zookeys.416.7706.app3

Supplementary material 4

Images of maximum likelihood bootstrap trees

Authors: David R. Maddison

Data type: images of phylogenetic trees

Explanation note: This file shows the maximum likelihood bootstrap trees for the concatenated, 7-gene matrices as well as each individual gene. Each figure is labeled to indicate the nature of the data analyzed for that tree. Numbers on each branch are the frequencies of that clade in the bootstrap replicates, expressed as a percentage.

Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.

Link: doi: 10.3897/zookeys.416.7706.app4