Introduction

While identifying beetles of the genus Bembidion Latreille that the senior author collected in Arkansas and Missouri some years ago, he came across a series of specimens of subgenus Trichoplataphus Netolitzky that keyed to Bembidion rolandi Fall using the latest revision of the group (Lindroth 1963). However, the collecting locality was far west of the known range of that species (Bousquet and Larochelle 1993). He contacted the junior author for his opinion on the species identification, which initiated a study that has resulted in the present work.

The purpose of this paper is to describe the new species that was discovered and provide a review of the North American species of the subgenus Trichoplataphus. The most recent revision of the subgenus was included in Lindroth’s treatment of the genus for the northern U.S. and Canada (Lindroth 1963), and this paper builds on that work by addition of the new species, and modification of Lindroth’s key.

It is with great pleasure that we dedicate this paper to Dr. Ross Bell. For one of us he has been a very generous, informative and helpful correspondent, for the other a mentor and close colleague. Both of us have benefited immeasurably by our interactions with him. This paper is a small contribution to the fauna of an area that has interested Ross since his youth, when he first collected the endemic Chlaenius viduus Horn in the Ozarks.

Methods

Approximately 580 specimens of Bembidion (Trichoplataphus) were examined as part of this review; no effort was made to do a complete survey of specimens in existing collections. Specimens were examined from the following collections; each collection listing begins with the codon used in the text.

CMNH Carnegie Museum of Natural History, Pittsburgh, PA, USA (Robert L. Davidson)

DAHC Drew A. Hildebrandt collection, Clinton, MS. USA

OSAC Oregon State Arthropod Collection, Oregon State University, Corvallis, OR (David R. Maddison)

TAMU Texas A&M University, College Station, TX, USA (Edward C. Riley)

Collecting methods. Specimens were collected by hand, by splashing or pouring water on gravel bars or by raking the gravel by hand during the day to dislodge the beetles hiding under the gravel; or at night when the beetles were out, actively moving about on the surface. During the day, raking the gravel by hand works best for collecting specimens of this subgenus because of their behavior when disturbed. Unlike members of most other Bembidiini, individuals of Trichoplataphus tend to run under the water and cling to stones, and thus they are more difficult to catch (Davidson 1978).

Specimens for morphological studies were killed and preserved in sawdust or woodchips to which ethyl acetate was added. Specimens for DNA sequencing were collected into 95% or 100% ethanol, with best results obtained if the abdomen was slightly separated from the rest of the body to allow better penetration.

Morphological methods. Methods for studying adult structures, and terms used, are given in Maddison (1993).

All measurements were made on dried, pinned or pointed specimens using an Olympus SZ6045 zoom stereo microscope with a calibrated ocular micrometer. Character examinations were made at magnifications ranging from 10x to 63x; all measurements were made at 30x. Standardized body length (SBL) was measured following the protocol of Kavanaugh (1979).

Taxon sampling for DNA studies. We sequenced DNA from 18 specimens of Bembidion (Trichoplataphus), including all known North American species, as well as Bembidion mimekara Toledano and Schmidt from China (Table 1). Preliminary analyses of multiple genes across Bembidion (Maddison, unpublished) indicate that the sampled Trichoplataphus form a clade, and that, among the species sampled, B. mimekara is the sister group to the North American species. DNA vouchers are housed in the David Maddison voucher collection at Oregon State University.

DNA sequencing. Methods for obtaining DNA sequences are described in Maddison (2008). In brief, we obtained ca. 1000 bases of sequence data in the D1 through D3 domains of 28S ribosomal DNA (28S or 28S rDNA) and 650 to 750 bases of cytochrome oxidase I (COI). Fragments for these genes were amplified using the Polymerase Chain Reaction on an Eppendorf Mastercycler Thermal Cycler, using either Eppendorf Hotmaster Taq or TaKaRa Ex Taq and the basic protocols recommended by the manufacturers. Primers and details of the cycling reactions used are given in Maddison (2008). In particular, we used the primer pair LS58F and LS998R and the pair NLF184/21 and LS1041R to amplify and sequence 28S rDNA. For COI, two amplification and sequencing strategies were used: use of primer pairs B1490 and Bcoi2R (see Maddison 2008), or the LCO1490 and HCO2198 primers (Hebert et al. 2003). Amplified products were cleaned, quantified, and sequenced at the University of Arizona’s Genomic and Technology Core Facility using either a 3730 or 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 2009a; Maddison and Maddison 2009b), with subsequent modifications by Chromaseq and manual inspection. Multiple peaks at a single position in both reads were coded using IUPAC ambiguity codes.

Sequences have been deposited in GenBank with accession numbers JF800039 through JF800074.

Alignment. Alignment for both genes could be unambiguously determined, as there were no insertion or deletion events evident in the COI sequences, and only one in 28S, in which Bembidion mimekara has 2 bases not present in the other species.

Molecular phylogenetic analysis. Models of nucleotide evolution were chosen with the aid of ModelTest version 3.7 (Posada 2005). For 28S rDNA, the model chosen by the Akaike Information Criterion (AIC) was a General Time Reversible (GTR) rate matrix with a proportion of sites being invariant (the GTR + I model); for COI it was a GTR rate matrix with site variation following a gamma distribution (the GTR + G model).

Likelihood analyses of nucleotide data were conducted using Garli version 1.0.699 (Zwickl 2006). For each matrix, 1000 non-parametric bootstrap search replicates were conducted.

Results from molecular analyses

Specimens of each of the species have distinctive COI and 28S sequences (Fig. 1). Bembidion rolandi shows consistent differences from similar specimens from the Ozarks, which we are describing as Bembidion ozarkense. All specimens of Bembidion rolandi differ from all specimens of Bembidion ozarkense at 18 bases among the 766 sequenced sites in COI (2.3% divergent), as well as 2 of the 995 sites sampled of 28S rDNA (0.2%). These two species are reciprocally monophyletic in the inferred phylogenetic trees (Fig. 1).

Descriptions and identification of taxa Subgenus Trichoplataphus Netolitzky, 1914

The subgenus Trichoplataphus of the genus Bembidion (Coleoptera: Carabidae: Trechinae: Bembidiini) contains 19 described species in the Palaearctic Region (Toledano and Schmidt 2010), and four species reported from North America (Bousquet and Larochelle 1993; Lindroth 1963; Lorenz 2005).

Adult specimens of the subgenus are easily separated from most other groups in North America by the irregular scattering of setiferous punctures on the abdominal sterna (Lindroth 1963). The only other species in North America with adults that have extra setiferous punctures on the abdominal sterna is Bembidion hasti Sahlberg, in which the setae are arranged in a regular, transverse row on each sternum.

Members of Trichoplataphus are found on open, bare gravel bars and banks, sometimes mixed with clay or sand (Fig. 2; Larochelle and Larivière 2003). Although mostly confined to running waters, they have also been collected among gravel and pebbles on banks of islands in Lake Champlain (Davidson 1978). These beetles are macropterous and have been found to fly to light (Larochelle and Larivière 2003).

The North American species of Trichoplataphus are:

Bembidion ozarkense sp. n.

Bembidion rolandi Fall, 1922

Bembidion grandiceps Hayward, 1897

Bembidion fugax (LeConte, 1848)

Bembidion planum (Haldeman, 1843)

Identification of Species Using Morphological Data

Species of this subgenus are very difficult to tell apart using morphological features, as the known differences are very subtle. Future morphological studies may reveal better characters to separate them. In the meantime, the key we present below (a modified version of couplets 77 through 80 in Lindroth 1963) should help in identifying specimens to species. The reader is strongly encouraged to read carefully the descriptions in Lindroth (1963) and this paper; sometimes species can be separated on a suite of characters that in aggregate are more useful than in isolation. In the following key, “fig.” refers to figures from Lindroth (1963); “Fig.” refers to figures in this paper.