Zootaxa 1542: 1–20 (2007) ISSN 1175-5326 (print edition) www.mapress.com/zootaxa/ ZOOTAXA Copyright © 2007 · Magnolia Press ISSN 1175-5334 (online edition) New species of Anillinus Casey (Carabidae: Trechinae: Bembidiini) from Great Smoky Mountains National Park, U.S.A. and phylogeography of the A. langdoni species group IGOR M. SOKOLOV1, YULIYA Y. SOKOLOVA2 & CHRISTOPHER E. CARLTON1 1Louisiana State Arthropod Museum, Department of Entomology, LSU Agricultural Center, Baton Rouge, Louisiana, 70803, USA. E-mail: [email protected]; [email protected] 2Laboratory for Insect Pathology, Department of Entomology, LSU Agricultural Center, Baton Rouge, Louisiana, 70803, USA. E-mail: [email protected] Abstract The Anillinus langdoni–species group is characterized and two new species are described, Anillinus cieglerae Sokolov and Carlton sp. nov. and A. pusillus Sokolov and Carlton sp. nov., both from Great Smoky Mountains National Park. The langdoni–group includes four species at present, three apparently endemic to the Great Smoky Mountains and adjacent mountains of western North Carolina/Tennessee, and a fourth from South Mountains of middle North Carolina. They are distinguished mainly using characters of the male genitalia and to a lesser extent, differences in shapes of female sper- mathecae. Phylogenetic analyses based on aedeagal morphology and COI gene sequences yielded conflicting results, with the later providing a phylogeny that was more parsimonious with expectations based on geographic distributions. Speciation within the group may derive from ecological constraints and altitudinal fluctuations of habitat corridors dur- ing past climate changes combined with the impact of local watersheds as fine scale isolating mechanisms. Key words: Coleoptera, Adephaga, Carabidae, Anillinus, South Appalachians, new species, taxonomy, identification key, COI gene sequences, phylogeography Introduction The genus Anillinus Casey is one of the most diverse genera of carabid beetles in the Southern Appalachian region of eastern United States. Species representing several distinct forms adapted to certain types of habitats inhabit different altitude zones. Localities within each zone may harbor up to three morphologically distinct lineages that presumably reflect a long and complicated history of speciation in the region. In the last review of the genus (Sokolov et al. 2004) morphological characters of these lineages were summarized. That paper provided a basis for character analysis within complexes of Anillinus species across the region. The Great Smoky Mountains of eastern Tennessee and western North Carolina, and Great Smoky Mountains National Park (GSMNP) in particular, is exceptionally important as an area of Anillinus species radiation. Five species of the genus have been described from GSMNP to date (l.c.), and each of these species represents a morpho- logically distinct lineage with putative relatives in other localities of the Southern Appalachian region. During the past three years of sampling of the litter fauna at the GSMNP, we have discovered two new species that are similar to Anillinus langdoni Sokolov and Carlton externally, including microsculpture pat- terns. In this paper we describe these species, provide a determination key for all extensively microsculptured species of Anillinus from the Southern Appalachians, and discuss the evolutionary history of the langdoni– species group as inferred from distributional data, morphology, and analysis of cytochrome oxidase I (COI) gene sequences. Accepted by I. Ribera: 23 May 2007; published: 6 Aug. 2007 1 The mitochondrial gene encoding COI has proven useful in assessing species status and evolutionary rela- tionships among different groups of Carabidae (Emerson et al. 2000; Kim et al. 2000; Clarke et al. 2001; Marek and Kavanaugh 2005; Zhang et al. 2005). The chosen region of the COI gene, from E3 to E6 structural regions (Lunt et al. 1996), is comparatively conserved, and therefore was particularly recommended for infer- ence of phylogenetic relationships among species and genera of insects (Zhang and Hewitt 1996). Material and methods Sampling, measurements, dissections, terms, and illustrations This study was based on examination of more than 200 specimens of Anillinus belonging to the langdoni– species group. Specimens were collected in Great Smoky Mountains National Park (GSMNP) using com- monly employed techniques, either Berlese funnels or hand sifting of forest litter. For DNA analyses speci- mens were stored in 100% ethanol. Verbatim label data are given for type specimens of all newly described species, with label breaks indicated by a slash (“/”). Type depositions are indicated under each species treat- ment. All specimens were measured electronically using a Leica Z16 APO microscope equipped with a Syn- croscopy AutoMontage photomicroscopy system (SYNCROSCOPY, Synoptics Ltd.). Measurements for vari- ous body parts are encoded as follows: ABL = apparent body length, from clypeus to apex of elytra; WH = width of head, at level of first orbital setae; WPm = maximal width across pronotum; WPa = width across anterior angles of pronotum; WPp = width across posterior angles of pronotum; LP = length of pronotum from base to apex along midline; WE = width of elytra, at level of 2nd discal setae; LE = length of the elytra, from apex of scutellum to apex of left elytron. ABL measurements are given in mm; others are presented as eight ratios: mean widths–WH/WPm and WPm/We and body parts–WPa/WPp, WPm/WPp, WPm/LP, LE/ABL and WE/ABL. All values are given as mean ± standard deviation. Dissections of male genitalia were made using standard techniques as described by Sokolov et al. (2004). Female genitalia were dissected from abdomens of specimens previously softened in boiling water for 3–5 minutes. Contents of the abdomen were cleared using 10% KOH for 24 hrs to remove internal tissues then washed in hot water before examination. Terminology of female genitalia structures follows Maddison (1993) with only one difference. We call the ramus only the protruding area of attachment of the spermathecal gland. Other parts are defined as follows: the nodulus is the basal section of the spermatheca, near the attachment of the spermathecal duct; the cornu is the portion adjacent to the attachment of the spermathecal gland. In the species under consideration the area of attachment of the spermathecal gland is flat and not protruding, so we consider the ramus to be poorly developed. Photographs of the dorsal habitus of new species were taken with the AutoMontage system. Line draw- ings of selected body parts were made using a camera lucida on an Olympus BX 50 compound microscope. Maps of species distribution were generated using MapDisplay software (Great Smoky Mountains Map Dis- play, 2003, M. Kunze, U. S. National Park Service). Cladistic analysis Morphological characters of the external and internal aedeagal structures (Table 1) were coded and parsi- mony analysis was executed using PAUP* version 4.0b10 (Swofford 2002). External aedeagal characters: 1. Shape of apex of median lobe (MLA), 0–of normal proportions, width of apex equal or less than 1/4 width of aedeagus at position of apical parts of dorsal sclerites; 1–enlarged, width of apex equal to or greater than 1/3 width of aedeagus at position of apical parts of dorsal sclerites. 2. Direction of ventral margin of median lobe (MLD), 0–straight, 1–curved to apex. 2 · Zootaxa 1542 © 2007 Magnolia Press SOKOLOV ET AL. 3. Condition of ventral margin of median lobe (MLV), 0–of normal proportions, width of ventral margin equal to or less than 1/4 width of aedeagus at position of apical parts of dorsal sclerites; 1–enlarged, width of ventral margin equal to or greater than 1/3 width of aedeagus at the position of apical parts of dorsal sclerites. 4. Distribution of poriferous canals (MLC), 0–on apex and ventral margin only, 1–also on wall of median lobe. 5. Form of right paramere (PF), 0–elongate, 1–short. Internal aedeagal characters: 6. Form of dorsal sclerites, their curvature (DSF), 0–slightly curved, formed by sector of ~100°, 1– strongly curved, semicircular, formed by sector of ~180°. We homologized the dorsal sclerites of Anillinus species with the upper sclerotized platelets of Bembidion species, probably with the CH3–CH5 series pro- posed by Erwin and Kavanaugh (1981) for the B. erasum–group. 7. Contour of dorsal sclerites, number of parts (DSP), 0–as one curved piece, 1–as two curved pieces united at base. 8. Basal part of dorsal sclerites, length of prolongations (DSB), 0–short or absent, 1–elongate. In the parsimony analysis all characters were treated as unordered and unweighted. An exhaustive search option was utilized. Anillinus magazinensis Sokolov and Carlton was chosen as an outgroup because, in our opinion, it belongs to a different species group and exhibits more plesiomorphic aedeagal characters (Sokolov et al. 2004). DNA extraction, PCR amplification and sequencing Beetles were removed from alcohol and abdominal integuments were perforated. The whole insects were incubated in Proteinase K overnight at 55°C. Total DNA was extracted using DNeasy® Tissue kit (Qiagen Sciences, Maryland, USA) following manufacturer’s standard protocol for insects. Fragments of mitochondrial gene COI of approximately 750 bp were amplified by forward 5´GTA TTA GCA GGA GCT ATT AC 3´ (corresponding to UEA5 (Lunt et al. 1996), with slight modifications) and reverse 5´GAA ATT GTT GAT CCA ATA G 3´ primers. Two successive PCR reactions were run for each DNA sample, in which the product of the first reaction served as a DNA template for the second, using a Lab- systems thermocycler. The same program (initial denaturation of 3 minutes at 96°, followed by 35 cycles with denaturation of 15 sec at 94°, annealing of 30 sec at 55°, and extension of 60 sec at 72°, and the final exten- sion step of 7 min at 72°) and same concentrations of the reaction mix components (20 µM of each primer, 2/ 5µl of template, 10/25 µl of Failsafe PCR buffer E; and 0.2/0.5 µl of Failsafe PCR enzyme, Epicenter, Madi- son WI) were used in the 1st/ 2nd reactions with respective volumes of 20 and 50 µl.