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 1–3) 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.

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.

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.

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.

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.

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.

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.

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.

Bembidion (Nothonepha) tetrapholeon sp. n.

http://zoobank.org/90188564-6B1E-4F0E-B411-5AD99047F716

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

Holotype

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).

Paratypes.

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.

Diagnosis.

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.

“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.