Zootaxa,New Species of Anillinus Casey (Carabidae: Trechinae

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 spermathecae. 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 during 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 morphologically 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 patterns. 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 relationships 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 specimens 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 parsimony 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 proposed 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 extension 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. PCR products were separated on an ethidium-bromide stained 2% agarose gel, and the desired bands were extracted with the aid of a Zimoclean Gel DNA Recovery Kit (Zimo Research, Orange, CA). All reagents used, except those specifically mentioned were from Amresco (Solon, OH). The isolated DNA fragments were directly sequenced in the Molecular Medicine Laboratory at Louisiana State University, School of Veter- inary Medicine in both directions with the pair of primers used for amplifications. The overlapping sequences resulting from sense and antisense DNA strands were manually corrected and assembled using Chromas Pro. 1.34 software (http:/www.technelysium.com.au/ ChromasPro.html).

COI gene sequence-based phylogenetic analysis Eleven consensus sequences were obtained from langdoni-group species and one from Anillinus erwini Sokolov and Carlton representing a different species group (Sokolov et al. 2004). A COI gene sequence of Bembidion (Notaphus) nigripes (Kirby) was taken from GenBank (Accession Number DQ059789) for a dis- tant outgroup comparison (Table 2). These thirteen sequences were aligned using CLUSTAL X (Thompson et

NEW SPECIES AND PHYLOGEOGRAPHY OF ANILLINUS Zootaxa 1542 © 2007 Magnolia Press · 3 al. 1977) without additional changes, except for the B. nigripes sequence, which was trimmed from both ends to correspond to the amplified part of the gene. The resultant alignment was analyzed by neighbor joining (NJ), maximum parsimony (MP), and by maximum likelihood (ML) algorithms using PAUP* version 4.0b10. GTR+G and GTR+I models of nucleotide substitution were suggested as a best fit by likelihood ratio tests (hLTR) and by AIC criteria in Modeltest 3.7 (Posada and Crandall 1998), respectively. The GTR+I model was chosen because AIC approaches present several advantages over hLTR (Posada and Buckley 2004). The set- tings of the model [Lset Base=(0.3036 0.1370 0.1466) Nst=6 Rmat=(1.000 4.2576 4.9467 15.0305) Rates=equal Pinvar=0.6410] were used in all likelihood analyses. Bootstrap values for all tree-building methods were obtained from 100 resamplings. Trees were viewed with Tree-View, version 1.6.6. Sequence comparison in the form of a data matrix was performed by Kimura 2–parameter analysis (Kimura 1980) through PAUP* version 4.0b10.

Results

Diagnosis of the Anillinus langdoni species group

The smallest representatives of the genus belong to this group. Average size of included species 1.59–1.65 mm (individual size, 1.42–1.75mm). Diagnostic features separating the members of this group from all other species, besides size, include the following combination of characters: habitus markedly convex, ovoid; head and pronotum totally covered with a network of polygonal microsculpture; humeri form right angle with longitudinal axis of body; elytra with traces of 1–2 interneurs, lacking preapical sinuation; metafemora of males unmodified; median lobe of aedeagus evenly arcuate, not twisted; apex of median lobe not toothed, semicircular in form, not elongate and not separated from area of apical orifice by incision; armature of inner sack represented by dorsal sclerites only in form of strongly curved semicircular blades (~180° of circle) that are not elongate and do not extend beyond median lobe; spermatheca in shape of a question mark, weakly sclerotized; proximal part of cornu at least two times longer than distal part. Comments. At present this group includes four forest litter species: Anillinus langdoni Sokolov and Carl- ton, Anillinus daggyi Sokolov and Carlton, and two new species, described herein. Range of the group extends from south-eastern counties of Tennessee, at least from Monroe Co., west to south-western counties of North Carolina as far as Burke Co. in the east (Fig. 29). Morphologically, the most similar species to the langdoni–group is A. magazinensis Sokolov and Carlton, from Arkansas. It agrees well in most diagnostic characters, including aedeagal features, but differs in the form and striation of the elytra. Also, the virginiae–group of species (A. virginiae Jeannel, A. barberi Jeannel and, probably, A. balli Sokolov and Carlton) shares characters with species from the langdoni–complex. Because of lack of knowledge about aedeagal structure within the virginiae–group, evaluating the affinities of the two groups is not possible at present. The groups are allopatric, with the gap between ranges inhabited by Anillinus species from other lineages. Among extensively microsculptured species A. folkertsi Sokolov and Carlton is quite isolated and, based on aedeagal structure, is not closely related to species of the langdoni– group. Relationship of the group to A. indianae Jeannel remains uncertain because no specimens of this species are available for dissections and comparison. Anillinus nantahala Dajoz (2005), according to the description (head and pronotum totally covered by microsculpture), also belongs to the langdoni–group. We haven’t seen type material of this species, but have at hand representatives of the group from the type locality (Wayah Mt., Macon Co., NC). Unfortunately, the structure of the median lobe of our specimens does not match well with the drawing of the median lobe of A.

4 · Zootaxa 1542 © 2007 Magnolia Press SOKOLOV ET AL. FIGURES 1–2. Habitus of (1) Anillinus cieglerae sp.nov. (TN, Blount Co., White Oak Sink), and (2) A. pusillus sp.nov. (TN, Blount Co., Gregory Bald), dorsal aspect, males.

nantahala represented in the description. In fact, the illustration (median lobe with elongate apex and slightly curved dorsal sclerites) resembles the median lobe of species from other groups (A. cornelli Sokolov and Car- lton, A. docwatsoni Sokolov and Carlton and A. murrayae Sokolov and Carlton), widely distributed in counties along the North Carolina/South Carolina border. Until examination of the type of A. nantahala is possible, its position within the genus remains uncertain.

Anillinus cieglerae Sokolov and Carlton sp.nov.

Holotype. Male labeled / Tennessee: Blount Co., GSMNP, West Prong Cmpg., 35°37.7’ N 83°42.2’ W, 550m, leaf litter sifting, 31 July 2004 J.Ciegler // HOLOTYPE, Anillinus cieglerae Sokolov and Carlton, des. 2007/. Deposited U.S. National Museum (USNM). Type locality. U.S.A, TN, Blount Co., Great Smoky Mountain National Park, West Prong Campground, 35°37.7’ N 83°42.2’ W. Paratypes (73). Eight males and 12 females with same data as holotype; 1 male labeled / Tennessee: Blount Co., GSMNP, West Prong Cmpg., 35°37.7’ N 83°42.3’ W, 520m. Forest litter 30 July 2004 J.Ciegler / ; 2 males labeled / Tennessee: Blount Co., GSMNP, West Prong Tr. nr. trlhd., 35°38.5’ N 83°41.5’ W, 430m, leaf litter sifting, 30 July 2004 J.Ciegler /; 1 male and 1 female labeled / Tennessee: Blount Co., GSMNP, Crooked Arm Ridg.Tr. at 35°36.7’ N 83°46.8’ W, 595m, leaf litter sifting, 31 July 2004 V.Bayless /; 2 males labeled / Tennessee: Blount Co., GSMNP, Rich Mt. Rd. 1.2mi from intc. with Cades Cove Loop Rd. Leaf litter sifting 28 July 2004 R.T.Allen /; 1 male labeled / Tennessee: Blount Co., GSMNP, Parson’s Branch Rd. nr. Gregory Ridge Trlhd., 35°33.8’ N 83°50.9’ W, 600m. Forest litter 28 July 2004 V.Bayless & C.Carlton /; 1

NEW SPECIES AND PHYLOGEOGRAPHY OF ANILLINUS Zootaxa 1542 © 2007 Magnolia Press · 5 FIGURES 3–6. Humeral outlines of (3) Anillinus cieglerae sp.nov.; (4) Anillinus pusillus sp.nov.; (5) A. langdoni Sokolov and Carlton; (6) A. daggyi Sokolov and Carlton, dorsal aspect, males.

male and 2 females labeled / U.S.A,TN, Blount Co., GSMNP, Cades Cove, 83°46’31” W 35°35’59” N / elv. 625m, forest litter berlese 22 March 2002 CECarlton, VLMoseley - these specimens were mentioned previously among the additional material in the description of A. langdoni (Sokolov et al., 2004)/; 1 male labeled / Tennessee: Blount Co., GSMNP, upper Russell Field Tr. at 35°34.08’ N 83°46.04’ W, 1280m, litter sifting, 13 Apr 2006 A.K. Tishechkin /; 1 male labeled / Tennessee: Blount Co., GSMNP, Gregory Ridge Tr. above Forge Creek Cmpgr. at 35°32.65’ N 83°50.15’ W, 830m, litter sifting, 12 Apr 2006 A.K. Tishechkin /; 3 males and 2 females labeled / TN, Blount Co., GSMNP, Middle Prong Trail at UTM 3944151 259955, 750m, berlese C.Ware 17 June 2006 /; 2 males and 5 females labeled / TN: Blount Co., GSMNP, White Oak Sink, 35°38’07” N 83°44’49” W, 620m, mixed forest, litter hand sifting Sokolov I.M. 16.06.2006/; 10 males and 3 females labeled / TN, Blount Co., GSMNP, right slope to Middle Prong Little R. nr junct. with Laurel Crk., 350m, u/ azalea bush, 35°39.451’ N 83°42.580’ W Sokolov I.M. 13 Sep 2006/; 2 males and 1 female labeled / TN, Blount Co., GSMNP, nr Middle Prong Little R., left slope, 35°39.006’ N 83°41.769’ W, 380m, mixed hardwood, hand litter sift. Sokolov I.M. 13 Sep 2006/; 2 males labeled / TN, Blount Co., GSMNP, nr Middle Prong Little R., left slope, 35°37.340’ N 83°40.800’ W, 530m, mixed hardwood, hand litter sift. Sokolov I.M. 12 Sep 2006/; 2 males and 1 female labeled / TN, Blount Co., GSMNP, Rich Mountain, nr Bull Cave, 35°38.694’ N 83°48.442’ W, 610m, mixed hardwood, hand litter sift. Sokolov I.M. 12 Sep 2006/; 3 males labeled / N Carolina: Swain Co., GSMNP, upper Eagle Creek Tr. at 35°33.03’ N 83°43.98’ W, 1165m. Litter sifting. 14 April 2006 A.K. Tishechkin/; 2 males and 2 females labeled / North Carolina: Swain Co., GSMNP, Jenkins Ridge Tr. ~1km from Appalachian Tr. 35°33’46” N 83°43’12” W, 1540m. Leaf litter sifting. 31 July 2004 A.K. Tishechkin /. Deposited Louisiana State Arthropod Museum (LSAM). Etymology. The specific epithet honors Janet Ciegler, avocational coleopterist, author, and regular volun- teer in the effort to document the beetle fauna of Great Smoky Mountains National Park. Description. Small to medium-sized for genus (ABL range 1.48–1.76mm, mean 1.65±0.081 mm, n=20). Habitus (Fig. 1) markedly convex, ovoid (WE/ABL 0.39±0.011), head normally proportioned for genus (WH/ WPm 0.72±0.022), pronotum narrow compared to elytra (WPm/WE 0.77±0.014). Body color dark, from dark brunneous to brunneorufous, appendages much lighter, testaceous. Microsculpture distinct across dorsal surface of head and pronotum. Pronotum moderately convex and transverse (WPm/LP 1.23±0.037), with margins rectilinear and moderately constricted posteriad (WPm/WPp 1.28±0.042). Anterior angles evident, slightly prominent. Posterior angles slightly obtuse (105–110°). Width between posterior angles greater than between anterior angles (WPa/ WPp 0.96±0.029). Elytra markedly convex, slightly depressed along suture, of normal length for genus (LE/ABL 0.59±0.022), with traces of 1–2 interneurs. Humeri (Fig. 3) moderately prominent, in outline nearly at right

6 · Zootaxa 1542 © 2007 Magnolia Press SOKOLOV ET AL. FIGURES 7–10. Right protarsus and apex of tibia of (7) Anillinus cieglerae sp.nov., (8) Anillinus pusillus sp.nov., (9) A. langdoni Sokolov and Carlton, (10) A. daggyi Sokolov and Carlton, dorsal aspect, males. Setae omitted.

angle with longitudinal axis of body, dorsally moderately rounded, gradually enlarged to middle. Margins subparallel, slightly divergent in basal half, evenly rounded to apex, maximal width of elytra behind midpoint, approximately at 0.55–0.60 length. Elytra without subapical sinuation. Vestiture of elytra short (less than one- fourth length of discal setae). Prothoracic leg of males with strongly dilated tarsomere 1 (as in Fig. 7) and strongly swollen (as in Fig.11) profemur. Metafemora unmodified. Median lobe (Fig. 15a) evenly arcuate, with strongly enlarged, rounded apex and enlarged ventral margin with numerous poriferous canals. Canals absent on walls of median lobe itself. Dorsal copulatory sclerites semicircular, forming one large and one small curve. Base of copulatory sclerites slightly angulate, lacking distinctive sclerotized prolongations. Ventral sclerite and the spines of internal sac absent. Left paramere (Fig. 15b) slightly enlarged with four setae, apical longest. Right paramere (Fig. 15c) markedly enlarged and robust, with four equal setae. Spermatheca (Fig. 19) weakly sclerotized, shaped like a question mark, represented mostly by long and well-developed cornu with apical one-half sinuate. Distal part of cornu comparatively long, more than 1.5 times longer than its width. Ramus reduced and undifferentiated, nodulus short. Spermathecal duct more or less straight, without obvious coils. Stylomers and sternum IX as in Fig. 23. Distribution. Known from central and north-eastern parts of the Blount County, Tennessee, and adjacent parts of Swain County, North Carolina, within GSMNP. Habitat. Beetles were collected in litter of different types of forests: xeric oak-pine, mesic oak, cove, and northern hardwood forests from low to middle elevations of the Smoky Mountains (500–1500m). Differential diagnosis. Anillinus cieglerae is a member of a group of ovoid species with extensively developed microsculpture on foreparts of the body. It is the largest species of the langdoni–group. It is similar to A. langdoni in general outline, but the pronotum is proportionately less transverse. In comparison with A. pusillus the species has more oval elytra with maximal width further apically. Also, the proportion of the pronotum is different. The pronotum of Anillinus cieglerae appears more massive in comparison with the head. But these features are subtle, and more reliable identifications can be made by examination of males.

NEW SPECIES AND PHYLOGEOGRAPHY OF ANILLINUS Zootaxa 1542 © 2007 Magnolia Press · 7 FIGURES 11–14. Right profemur of (11) Anillinus cieglerae sp.nov., (12) Anillinus pusillus sp.nov., (13) A. langdoni Sokolov and Carlton, (14) A. daggyi Sokolov and Carlton, males.

Males of A. cieglerae can be distinguished from all other langdoni–group species by the following combination of characters: strongly swollen profemora, form of tarsomere 1 of protarsi, short left paramere, form of the median lobe and form of copulatory pieces. Females of the species externally are practically indistinguishable from the other species, though examination of the spermatheca may help to separate the species from others. Anillinus cieglerae occurs sympatrically with at least five species of Anillinus from different species groups and one species of Serranillus. In addition to the characters mentioned above to separate this species from other members of the langdoni-group, it can be distinguished from other Anillinus and Serranillus by the totally microsculptured foreparts of the body and by the smaller size.

Anillinus pusillus Sokolov and Carlton sp.nov.

Holotype. Male labeled / N Carolina: Swain Co., GSMNP, Twentymile Tr., 35°28.8’N 83°50.7’W. 690m. Leaf litter sifting. 28 July 2004 W.D.Merrit / / HOLOTYPE, Anillinus pusillus Sokolov and Carlton, des. 2007/. Deposited USNM. Type locality. U.S.A, NC, Swain Co., Great Smoky Mountains National Park, Twentymile Trail, 35°28.8’N 83°50.7’W. Paratypes (57). Nine males and 5 females with the same collection data labels as holotype; 4 males and 10 females labeled / N Carolina: Swain Co., GSMNP, Lost Cove Tr., 35°29.45’N 83°48.1’W. 685m. Leaf litter sifting. 8 May 2004 W.D.Merrit /; 1 male labeled / N Carolina: Swain Co., GSMNP, Appalach. Tr., just below Ekaneetlee Gap, 35°32.30’N 83°48.37’W. 1155m. Litter sifting. 12 April 2006 A. K. Tishechkin /; 6 males

FIGURES 15–18. Male aedeagus of (15) Anillinus cieglerae sp.nov., (TN, Blount Co., Middle Prong Little R.); (16) Anillinus pusillus sp.nov., (TN, Blount Co., Forge Creek); (17) A. langdoni Sokolov and Carlton (TN, Sevier Co., Albright Grove); (18) A. daggyi Sokolov and Carlton, (NC, Burke Co., Jacob Fork Section of South Mt.St.Pk); left lat- eral aspect. a–median lobe, b–left paramere, c–right paramere. Scale=100 µm.

labeled / N Carolina: Swain Co., GSMNP, upper Eagle Creek Tr. at 35°33.03’ N 83°43.98’ W, 1165m. Litter sifting. 14 April 2006 A.K. Tishechkin /; 5 males and 6 females labeled / Tennessee: Blount Co., GSMNP, lower Gregory Ridge Tr., 35°33.5’ N 83°50.5’W. elev. 630m. Leaf litter sifting 28 July 2004 A. Tishechkin /; 2 males and 2 females labeled / TN: Blount Co., GSMNP, Gregory Bold, W slope at 35°31.222’N 83°52.046’W, mixed forest, 1490m, berlese Gusarov V. 18.06.2006/; 5 males and 2 females labeled / TN, Blount Co., GSMNP, left slope to Forge Crk., nr Gregory Ridge Tr., 640m, 35°33.345’ N 83°50.398’ W, mixed hardwood, hand sift. Sokolov I.M. 13 Sep 2006 /. Deposited LSAM. Etymology. The specific epithet "pusillus" (Latin, “tiny, little, petty, insignificant") is an adjective refer- ring to the small size of the beetle. Description. Small to medium-sized for genus (ABL range 1.42–1.72mm, mean 1.60±0.087 mm, n=20). Habitus (Fig. 2) markedly convex, ovoid (WE/ABL 0.38±0.013), head normally proportioned for genus (WH/ WPm 0.74±0.020), pronotum narrow compared to elytra (WPm/WE 0.77±0.017). Body color brunneus to brunneorufous, appendages lighter, testaceous. Microsculpture distinct across dorsal surface of head and pronotum.

NEW SPECIES AND PHYLOGEOGRAPHY OF ANILLINUS Zootaxa 1542 © 2007 Magnolia Press · 9 FIGURES 19–22. Spermathecae of (19) Anillinus cieglerae sp.nov. (TN, Blount Co., White Oak Sink); (20) Anillinus pusillus sp.nov., (NC, Swain Co., Lost Cove Tr.); (21) A. langdoni Sokolov and Carlton (TN, Sevier Co., Little Pigeon R.); (22) A. daggyi Sokolov and Carlton (NC, Burke Co., 8 mi S Enola). Scale=50µm.

Pronotum moderately convex and transverse (WPm/LP 1.24±0.029), with margins rectilinear and slightly constricted posteriad (WPm/WPp 1.27±0.023). Anterior angles evident, slightly prominent. Posterior angles slightly obtuse (103–109°). Width between posterior angles greater than between anterior angles (WPa/WPp 0.96±0.027). Elytra markedly convex, slightly depressed along suture, of normal length for genus (LE/ABL 0.58±0.015), with traces of 1–2 interneurs. Humeri ( Fig. 4) moderately prominent, in outline forming right angle with longitudinal axis of body, slightly rounded, thence parallel towards middle of elytra. Margins subparallel in basal half, evenly rounded to apex at apical third of elytra, maximum width near middle, approximately at 0.45–50 of elytral length. Elytra without subapical sinuation. Vestiture of elytra short (less than one- fourth length of discal setae). Foreleg of males with slightly dilated tarsomere 1 (as in Fig. 8) and moderately swollen (as in Fig. 12) profemur. Metafemora unmodified. Median lobe (Fig. 16a) evenly arcuate, with normally proportioned rounded apex not enlarged along ventral margin and with few poriferous canals. No canals on walls of median lobe. Copulatory pieces consisting of semicircularly curved dorsal sclerites, together forming a single curve. Base of copulatory pieces forming one long sclerotized prolongation. Ventral sclerite and spines of internal sac absent. Left paramere (Fig. 16b) not enlarged, bearing four setae, apical seta longest. Right paramere (Fig. 16c) not enlarged or elongate, bearing four setae of equal length, spaced evenly across apex of paramere. Spermatheca (Fig. 20) weakly sclerotized, in the form of a question mark, represented mostly by long, well-developed curved cornu. Distal part of cornu short, length less than 1.5 X width. Ramus reduced and undifferentiated, nodulus short. Spermathecal duct more or less straight, without obvious coils. Stylomers and sternum IX as in Fig. 24. Distribution. Known from south-western Blount County, Tennessee, and from Swain County, North Carolina, adjacent to the state line with Tennessee to near the junction of Blount and Sevier Co., TN, within GSMNP. Habitat. Beetles were collected in litter of different types of forests: pine, xeric and mesic oak, cove and northern hardwoods from low to middle elevations of the Smoky Mountains (600–1400m).

10 · Zootaxa 1542 © 2007 Magnolia Press SOKOLOV ET AL. FIGURES 23–26. Right stylomers and sternum IX of (23) Anillinus cieglerae sp.nov., (TN, Blount Co., White Oak Sink); (24) Anillinus pusillus sp.nov., (NC, Swain Co., Lost Cove Tr.); (25) A. langdoni Sokolov and Carlton (TN, Sevier Co., Little Pigeon R.); (26) A. daggyi Sokolov and Carlton (NC, Burke Co., 8 mi S Enola). Scale=100µm.

Differential diagnosis. Anillinus pusillus is an ovoid species with complete, extensive microsculpture on foreparts of the body. The species differs from A. langdoni and A. cieglerae by its subparallel body form. Also, males of Anillinus pusillus can be distinguished from all other species in the langdoni–group by the form of the aedeagus. Females of this species are indistinguishable from A. langdoni, though locality data are helpful in separating these allopatric species. Females of A. pusillus can be distinguished from A. cieglerae by examination of spermatheca. Anillinus pusillus occurs sympatrically with at least three species of Anillinus of different lineages. From other Anillinus species outside the langdoni–group it can be distinguished by the extensively microsculptured foreparts of the body and by the small size.

Key for identification of males of the A. langdoni–species group

This key will allow determination of langdoni–group species in the southern Appalachian Mountains. This area was not covered adequately in the previous key of Sokolov et al. (2004) for this group because the diversity and distributional limits were not fully understood. Non–langdoni-group species can be identified using the earlier key.

1. Both head and pronotum totally covered with polygonal microsculpture...... 2 1’. At least head with areas with effaced microsculpture, occupying both frons and vertex OR only frons between frontal furrows, OR with small paramedian patches of effaced microsculpture on each side of the vertex ...... (other Anillinus species) 2. Beetles from Appalachian Mountains of Tennessee, North Carolina, Virginia, and Maryland ...... 3 2’. Beetles from areas outside Appalachian Mountains (Kentucky, Indiana, Arkansas, Oklahoma) ...... (other Anillinus species) 3. Smaller: 1.41–1.74mm. Beetles from southern Appalachian Mountains (North Carolina and Tennessee )...... 4 (langdoni-species group) 3’. Larger: 1.64–1.98mm. Beetles from the middle Appalachian Mountains (Virginia, Maryland) ...... (virginiae-species group) 4. Males with right paramere short and wide (Fig. 15c,18c). Median lobe with enlarged apex and ventral margin (Fig. 15a,18a) ...... 5 4’. Males with right paramere elongated and narrow (Fig. 16c,17c). Median lobe with apex and ventral mar-

NEW SPECIES AND PHYLOGEOGRAPHY OF ANILLINUS Zootaxa 1542 © 2007 Magnolia Press · 11 gin of normal form (Fig. 16a,17a) ...... 6 5. Beetles from Burke Co., North Carolina. Walls of median lobe with poriferous canals (Fig. 18a). Humeri (Fig. 6), male protarsus (Fig. 10), male profemur (Fig. 14), aedeagus (Fig. 18a), spermathecae (Fig. 22), and stylomers (Fig. 26) as illustrated...... Anillinus daggyi Sokolov and Carlton 5’. Beetles from Great Smoky Mountains National Park. Poriferous canals restricted to the apex and ventral margin of median lobe (Fig. 15a). Humeri (Fig. 3), male protarsus (Fig. 7), male profemur (Fig. 11), aedeagus (Fig. 15a), spermathecae (Fig. 19), stylomers (Fig. 23) as shown ...... Anillinus cieglerae new species 6. Beetles from Swain Co., North Carolina or from south-western parts of Blount Co., Tennessee (west of Thunderhead Mountain). Dorsal sclerites with single, long prolongation basally (Fig. 16a). Humeri (Fig. 4), male protarsus (Fig. 8), male profemur (Fig. 12), aedeagus (Fig. 16a), spermathecae (Fig. 20), stylomers (Fig. 24) as illustrated ...... Anillinus pusillus new species 6’. Beetles from Sevier Co. and adjacent parts of Blount Co., Tennessee. Dorsal sclerites with three, approximately equal length prolongations basally (Fig. 17a). Humeri (Fig. 5), male protarsus (Fig. 9), male profemur (Fig. 13), aedeagus (Fig. 17a), spermathecae (Fig. 21), stylomers (Fig. 25) as illustrated...... Anillinus langdoni Sokolov and Carlton

Morphological phylogenetic analysis

Species of the langdoni-group do not exhibit external morphological characters (at least under 100x magnifi- cation) that can be used for cladistic analysis of within group relationships. Therefore, we could use only aedeagal structures (Table 1). All scored characters were informative and the program generated a single most parsimonious tree (Fig. 27). The topology was as predicted, based on a visual analysis. Species with enlarged median lobes paired with each other, with respect to species with simple aedeagi (see Figs. 15–18). Accord- ingly, A. langdoni and A. pusillus, with smaller and simpler aedeagi (presumed ancestral), paired into one clade, while A.cieglerae and A. daggyi, with enlarged aedeagi (presumed derived), paired into another. Nota- bly, the A.cieglerae + A. daggyi branch was supported by a greater number of derived characters than the A. langdoni + A. pusillus branch (Fig. 27).

TABLE 1. Data matrix of morphological characters used in the cladstic analysis of Anillinus langdoni–group.

Characters Species MLA* MLD* MLV* MLC* PF* DSF* DSP* DSB* A. magazinensis** 00000000 A. langdoni 00000101 A. pusillus 01000101 A. cieglerae 10101110 A. daggyi 10111100

* for acronyms–see Material and Methods; **outgroup

Phylogenetic analysis based on DNA sequences

Twelve novel sequences of a fragment of COI gene, ranging in size from 700 to 780 nucleotides, were obtained, nine of which were non-reduntant. Those nine were deposited into GenBank (for Accession numbers see Table 2).

12 · Zootaxa 1542 © 2007 Magnolia Press SOKOLOV ET AL. FIGURE 27. Single most parsimonious tree of langdoni–group with characters supporting each clade (all characters transformed to presumed derived states) (L=8; CI=1.0; RI=1.0). DSB–basal part of dorsal sclerites, length of prolongations; DSF–form of dorsal sclerites, their curvature; DSP– contour of dorsal sclerites, number of parts; PF–form of right paramere; MLA–shape of apex of median lobe; MLC–distribution of poriferous canals; MLD–direction of ventral margin of median lobe; MLV–state of ventral margin of median lobe.

Pairwise genetic distance among the investigated haplotypes of Anillinus and Bembidion species obtained by Kimura 2–parameter analysis (Table 3) showed 0–2% of dissimilarity among haplotypes of the same species. Interspecific variation within the langdoni-group ranged from 4.2 to 6.8%. Sequence divergence among species of langdoni-group and A. erwini was as high as 8.0–9.3%. All sequences of Anillinus spp. differed from Bembidion sequences by 12.8–14.3%. Within the langdoni-group the lowest dissimilarity was between A. pusillus and A. cieglerae (4.2–4.9%), and between A. pusillus and A. daggyi (4.9–5.1%). The highest was between A. langdoni and A. cieglerae (5.4–6.8%). Sequence divergence among other species of the group ranged from 5.2 to 6.2% (Table 3). Maximum likelihood (ML), maximum parsimony (MP) and neighbor joining (NJ) algorithms applied to the alignment revealed identical overall topologies of trees rooted with B. nigripes (Fig.28). The bootstrap supports for species-level clades were 100%. All species of the langdoni-group were united as a single clade (ML–70, MP–87, and NJ–99% of bootstrap support) with respect to A. erwini. Both new species, A. cieglerae and A. pusillus, formed a dichotomy (ML–55, MP–62, and NJ–61% bootstrap support) as a sister clade with A. daggyi, though with low (<50%) support.

NEW SPECIES AND PHYLOGEOGRAPHY OF ANILLINUS Zootaxa 1542 © 2007 Magnolia Press · 13 FIGURE 28. Maximum likelihood tree resulting from analysis of COI gene sequences in langdoni–group, and rooted with Bembidion. Bootstrap values (>50%) are indicated at nodes of major clades. The first/ second/ third numbers are from maximum likelihood/ maximum parsimony/ neighbor joining analyses, respectively, which all revealed an identical topology. Replicates=100 in all analyses. Bnig-Bembidion nigripes, for Anillinus codes–see Table 2.

Discussion

Geographical distribution. The area of distribution of the langdoni-group has been expanded from Monroe Co., TN in the west to Burke Co., NC in the East. The range of the species includes three mountain ranges. Two of them, Great Smoky and Nantahala Mountains, are adjacent to each other, while the third, South Moun- tains, is located on the border between the Mountain and Piedmont physiographic provinces of North Carolina some distance from the other two (Fig. 29). The taxonomy and distribution of Anillinus sp. in the Nantahala Mountains is still unclear, so we exclude it from this discussion. Anillinus daggyi, from the South Mountains, is obviously disjunct with respect to the others. At present no species of the group are known from the wide zone between 81°50’W and 83°15’W longitude. Three species within GSMNP (A. langdoni, A. cieglerae, and A. pusillus) represent a trio of parapatric species. The range of A. langdoni (Fig. 30, squares) forms a zone of contact with the range of A. cieglerae along the sources and upper parts of the Middle Prong Little River valley (Fig. 31a). The range of A. cieglerae (Fig. 30, circles) abuts the range of A. pusillus along Forge Creek (Fig. 31b) and just below the Thunderhead Mountain summit on the North Carolina slope above Eagle Creek (Fig. 31c). Anillinus pusillus occupies the most southern parts of the Tennessee and North Carolina slopes of the Appalachian Main Ridge within GSMNP (Fig. 30, triangles). The extreme western parts of GSMNP are not thoroughly investigated, so precise information about the relative distribution of these species in that area is not yet available. Obviously, the ranges of A. cieglerae and A. pusillus cross the Appalachian Trail and extend into the North Carolina, and the range of A. langdoni does not. A potential explanation for the apparent fine scale distributions of these three species, and possible insight into their divergence, may derive from ecological preferences of the species. Our data suggest that the fir-spruce community is the only type of the forest

Species Working Sex Locality Haplotype Acc. No code Anillinus langdoni 1 Alang01 female GSMNP, The Sinks A EF460872 A.langdoni 1 Alang02 male GSMNP, The Sinks B EF460871 A.langdoni 1 Alang03 female GSMNP, The Sinks C EF460873 A.langdoni 1 Alang04 male GSMNP, Sams Creek C 3 A.langdoni 1 Alang05 female GSMNP, Sams Creek C 3 A.erwini 1 Aerw male VA, Smyth Co., Mt. Rogers A EF460870 A.pusillus 1 Apus01 female GSMNP, Forge Creek A EF460869 A.pusillus 1 Apus02 male GSMNP, Forge Creek A 4 A.cieglerae 1 Acieg01 female GSMNP, Bull Cave A EF460865 A.cieglerae 1 Acieg02 male GSMNP, Bull Cave B EF460866 A.daggyi 1 Adag01 male NC, Burke Co., South Mts.SP A EF460867 A.daggyi 1 Adag02 female NC, Burke Co., South Mts.SP B EF460868 Bembidion nigripes 2 Bnig n/a n/a DQ059789

1novel sequences; 2sequence obtained through GenBank; 3sequence is identical to EF460873; 4 sequence is identical to EF460869.

TABLE 3. Pairwise genetic distance between the investigated haplotypes of Anillinus and Bembidion species obtained by Kimura 2–parameter analysis.

12345678910111213 1 Alang03 - 2 Alang05 0.001 - 3 Alang04 0.001 0.000 - 4 Alang02 0.003 0.001 0.001 - 5 Alang01 0.011 0.010 0.009 0.008 - 6 Adag01 0.060 0.058 0.057 0.056 0.053 - 7 Adag02 0.060 0.058 0.059 0.062 0.055 0.011 - 8 Acieg01 0.063 0.062 0.059 0.056 0.054 0.053 0.057 - 9 Acieg02 0.068 0.067 0.063 0.062 0.059 0.052 0.052 0.012 - 10 Apus01 0.062 0.059 0.058 0.056 0.053 0.049 0.050 0.042 0.049 - 11 Apus02 0.061 0.059 0.058 0.057 0.053 0.049 0.048 0.043 0.049 0.000 - 12 Aerw 0.093 0.091 0.091 0.089 0.086 0.081 0.080 0.083 0.089 0.085 0.085 - 13 Bnig 0.142 0.143 0.141 0.138 0.138 0.134 0.141 0.128 0.135 0.137 0.137 0.137 -

* Bnig, B.nigripes; for Anillinus codes–see Tabl.2

NEW SPECIES AND PHYLOGEOGRAPHY OF ANILLINUS Zootaxa 1542 © 2007 Magnolia Press · 15 FIGURE 29. Geographical distribution of langdoni–group in the southern Appalachians. Counties of Tennessee and North Carolina species were recorded from are darkened. The easternmost dark area represents Burke Co. and the range of Anillinus daggyi. The map is Copyrighted (1995) by Ray Sterner, Johns Hopkins University Applied Physics Labora- tory and licensed by North Star Science and Technology, LLC.

FIGURE 30. Geographical distribution of langdoni–group within Great Smoky Mountains National Park. Open squares–A. langdoni; solid circles–A. cieglerae; open triangles–A. pusillus. 1–Thunderhead Mt.; 2–Clingmans Dome.

FIGURE 31. Geographical distribution of langdoni–group in NW part of Great Smoky Mountains National Park. Open squares–A. langdoni; solid circles–A. cieglerae; open triangles–A. pusillus. Gray outlined areas: a–upper parts of Middle Prong Little River, b–Forge Creek, c–Eagle Creek; cross –Thunderhead Mt. summit.

16 · Zootaxa 1542 © 2007 Magnolia Press SOKOLOV ET AL. FIGURE 32. Elevation records for species of langdoni–group projected on profile of Main Ridge Crest (north slope along the Appalachian Trail) within Great Smoky Mountains National Park. Open squares–A. langdoni; solid circles–A. cieglerae; open triangles–A. pusillus. Upper dotted line corresponds to maximum altitude of Clingmans Dome peak and the lower dotted line characterizes the low limits of fir (Abies fraseri)–spruce (Picea rubens) community distribution. that is not inhabited by langdoni–group species. The altitudinal distribution of the species along the Tennessee slope of the Main Ridge of GSMNP supports the importance of coniferous communities as barriers to dispersal of these species (Fig. 32). Most of the known range of A. langdoni extends through mid-elevations below the limits of spruce-fir forest. The species may cross the Appalachian Trail only at eastern and western extremes of its range, where a few gaps occur below the spruce-fir community’s distribution. In the eastern part of its range, the extensive valley of Big Creek probably blocks A. langdoni from the main divide. The sit- uation in western part of its range is obscure. No visual barriers preventing the dispersal of A langdoni into the North Carolina part of GSMNP are apparent. One reason for the absence of A. langdoni from the North Caro- lina slope might be related to the configuration of the contact zone between A. langdoni and A. cieglerae. The zone of contact between this pair of species does not reach the passes to the North Carolina slope, and these are occupied only by A. cieglerae. Possibly, the ranges of these species are controlled through competitive exclusion, but more fine scale distributional data are necessary to determine whether they are truly parapatric or if indeed a zone of overlap exists. The ranges of A. cieglerae and A. pusillus cover areas of GSMNP at elevations lower than spruce-fir communities, and also lower than that of A. langdoni. Both species’ ranges extend across the Main Ridge to the Tennessee and North Carolina slopes. Apparently, the range of A. cieglerae is limited by two drainages, the Middle Prong of Little River/Lynn Prong drainage in the east and Abrams Creek/Forge Creek drainage in the west (Fig. 31a and 31b). Both drainages have their origins on Thunderhead Mountain. Probably, these particular water routes played roles as effective barriers during langdoni–group speciation. The isolation and subsequent emergence of A. cieglerae as a distinctive species is probably related to the history of Thunderhead Mountain and its attendant drainages. Cladistic analysis. To our surprise, cladistic analysis based on morphological characters of the aedeagus did not support relationships inside the group inferred from the geographical distribution of species. Analysis of aedeageal structures demonstrated that the species with enlarged aedeagus (presumed derived character) are closer to each other than to any of the species with normally proportioned aedeagus (presumed ancestral character); i.e. that A. cieglerae is more closely related to A. daggyi than to A. pusillus or A. langdoni. Accord- ing to their geographical distributions, the three GSMNP species would be presumed to be more closely related to each other than to A. daggyi, inhabiting the remote South Mountains (Fig. 29). This discrepancy can be explained by the tendency of external aedeagal structures to become modified independently among different Anillinus lineages. These modifications were especially common among peripheral species away from the core of population complexes (“peripheral trend,” Sokolov et al. 2004). Thus, similar external modifications in the median lobes have likely occurred independently in A. cieglerae and A. daggyi, and artificially joined them into one clade in our analysis.

NEW SPECIES AND PHYLOGEOGRAPHY OF ANILLINUS Zootaxa 1542 © 2007 Magnolia Press · 17 DNA–inferred phylogenetic analysis. Comparison of COI gene sequence similarity among the species of langdoni-group and consequent phylogenetic analyses unambiguously supported species status of the two new species. Indeed, interspecific variations between A. pusillus and A. cieglerae, as well as between each of them and other analyzed Anillinus spp., ranged between 4.2 and 9.3%, which greatly exceeded the observed intraspecific variations. In addition, all three methods of phylogenetic analysis produced trees with 100% support of branches leading to the clades corresponding to the new species (Fig. 28). The COI gene-based phylogenies, unlike the morphology-based cladistic analysis, but in accord with observed data based on geographical distributions, grouped A. pusillus and A. cieglerae together, demonstrating their closer relationship to each other than to A. daggyi. The exact position of A. daggyi within the langdoni–group is yet to be determined. Phylogenetic analysis also suggested a monophyletic origin of the A. langdoni–group by demonstrating robust (70–99%) support for the langdoni–clade. Based on tree topology and on the geographical ranges of these species we infer the following presumed scenario for speciation within the group. The distribution area of the langdoni–group common ancestor was split into two parts. The ancestor of A. langdoni occupied the western slopes and the common ancestor of other three species occupied the eastern slopes. Eventually, the eastern stock was fragmented to an unknown number of species, of which A. daggyi represents the most northern. The common A. pusillus–cieglerae ancestor inhabited eastern slopes to the south. Low altitudes of the southern part of the Smoky Mountains allowed occupation of adjacent areas of the western slope, but subsequent dispersal was impeded by rivers horizontally and coniferous forests vertically. Climatic fluctuations during the Pleistocene shifted boundaries of the coniferous zone up and down slope numerous times, eventually dissecting the range of the common ancestor of A. cieglerae and A. pusillus, and facilitating their subsequent divergence through localized allopatry. Such a scenario was proposed for divergence of different Trechus (Carabidae) species groups (Barr 1962). Recent works on Trechus by Donabauer (2005a, 2005b) and Geostiba (Staphylinidae) by Gusarov (2002) have further highlighted the influence of shifting ecological zones during the Pleistocene or prior in the evolution of the diverse endemic beetle fauna of the southern Appalachians (e. g. Trechus thunderheadensis Donabauer, 2005b). In summary, we suggest the following major events influenced the speciation within the langdoni-group: (1) fragmentation of the former ancestral range, in areas with predominantly deciduous forests; (2) partial extinction and fragmentation of the group’s ancestral range; and (3) subsequent speciation and dispersal that was constrained by physical (drainage patterns) and ecological (coniferous communities) barriers. This model can be tested only through analysis of additional material within the langdoni-group and other lineages of Anillinus from the southern Appalachians and through congruence analysis of other lineages of narrowly endemic southern Appalachian faunal elements that should have been affected in similar or complementary ways. Phylogenetic analysis of DNA sequences is in agreement with the hypothesis that apparent similarity in aedeagal structure between A. cieglerae and A. daggyi is the result of convergence, not inheritance from a common ancestor. So, from a phylogenetic perspective differences in the external morphology of the median lobe, at least within Anillinus, must be homologized with caution. Probably this statement is true for other groups of beetles in which differences in aedeagal structures sometimes reach unexpected diversity. In the context of the current study in the taxonomy of Anillinus, DNA data have proven useful as an independent “character system” (sensu Will and Rubinoff 2004), under the assumption that COI gene divergence reflects the evolutionary history of the species.

Acknowledgements

We are indebted to Vladimir Chouljenko (LSU School of Veterinary Medicine) for designing primers used in this research, and his help in establishing a successful amplification protocol. We also would like to thank

18 · Zootaxa 1542 © 2007 Magnolia Press SOKOLOV ET AL. Dmitri Chouljenko and Alexey Tishechkin (Entomology Dept LSU) for the sequence of A. erwini and James R. Fuxa (Laboratory for Insect Pathology, Entomology Dept LSU) for allowing us to use equipment for DNA isolation and amplification. This manuscript is published with the approval of the Director of the Louisiana Agricultural Experiment Station as manuscript number 07-216-0122. Support was provided by grants from Discover Life in America and the National Science Foundation (DEB–0516311, C. E. Carlton and V. L. Bay- less, Co-P.I.s).

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