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Kongsbergia robisoni, n. sp. (Acari: Hydrachnidiae:Aturidae) from the Interior Highlands of North Americabased on morphology and molecular genetic analysisAndrea J. Radwell a , Ashley P.G. Dowling b , Ian M. Smith c & Vamsi Kaliki aa Department of Biological Sciences, University of Arkansas, 601 Science-EngineeringBuilding, Fayetteville, AR, 72701, USAb Department of Entomology, University of Arkansas, 319 Agricultural Building, Fayetteville,AR, 72701, USAc Canadian National Collection of Insects and Arachnids, Agriculture and Agri-Food Canada,K.W. Neatby Building, 960 Carling Avenue, Ottawa, ON, K1A 0C6, Canada

Available online: 16 Dec 2011

To cite this article: Andrea J. Radwell, Ashley P.G. Dowling, Ian M. Smith & Vamsi Kaliki (2011): Kongsbergia robisoni, n. sp.(Acari: Hydrachnidiae: Aturidae) from the Interior Highlands of North America based on morphology and molecular geneticanalysis, International Journal of Acarology, 37:sup1, 194-205

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International Journal of AcarologyVol. 37, Suppl. 1, 194–205

KONGSBERGIA ROBISONI, N. SP. (ACARI: HYDRACHNIDIAE: ATURIDAE) FROM THEINTERIOR HIGHLANDS OF NORTH AMERICA BASED ON MORPHOLOGY AND

MOLECULAR GENETIC ANALYSIS

Andrea J. Radwell1, Ashley P.G. Dowling2, Ian M. Smith3 and Vamsi Kaliki11. Department of Biological Sciences, University of Arkansas, 601 Science-Engineering Building, Fayetteville, AR

72701, USA (e-mails: radwell101@yahoo.com and vkkaliki@gmail.com); 2. Department of Entomology, Universityof Arkansas, 319 Agricultural Building, Fayetteville, AR 72701, USA

(e-mail: adowling@uark.edu); 3. Canadian National Collection of Insects and Arachnids, Agriculture and Agri-FoodCanada, K.W. Neatby Building, 960 Carling Avenue, Ottawa, ON K1A 0C6, Canada (e-mail: smithi@agr.gc.ca).

(Received 25 August 2010; accepted 8 December 2010)

ABSTRACT – A new water mite species, Kongsbergia robisoni, is described from the InteriorHighlands of North America based on morphological analysis supported by DNA sequence data.Populations from three ecoregions within the Interior Highlands were found to be morphologicallysimilar to one another and distinct from other known Kongsbergia species. To help to determinewhether subtle morphological differences between populations within the Interior Highlands shouldbe interpreted as variability within a single species or were indicative of species endemic to specificecoregions, mitochondrial cytochrome oxidase I sequences were analyzed. Three primary clades ofKongsbergia were supported by 100% bootstrap values and 1.0 Bayesian posterior probabilities. Thephylogenetic hypothesis indicated that specimens from the Interior Highlands, including specimensfrom the Ozark Highlands, Boston Mountains, and Ouachita Mountains, formed a clade that wasdifferentiated from various species of Kongsbergia from Appalachia (Great Smoky Mountains andSoutheast Tennessee).Key words – Water mites, Aturidae, Kongsbergia, new species, stream, cytochrome oxidase subunit I,Interior Highlands of the United States, Arkansas, Missouri.

INTRODUCTION

The water mite genus Kongsbergia includes over80 nominate species in five subgenera reported fromsurface and interstitial waters of rivers and streamsof North and South America, Europe, Asia, andAfrica (Viets, 1987; Cramer, 2000). In the New World,18 species have been described, including eight fromthe eastern and western regions of the United Statesand Canada (Habeeb, 1956, 1957a, b, 1961, 1974,1981). The purpose of this article is to describeadults of a new North American species, Kongsbergiarobisoni.

Populations of the new species described herewere collected from rivers and streams of the

Ouachita–Ozark Interior Highlands of the UnitedStates (Fig. 1). The region is a remnant of ancient,eroded mountains that were originally uplifted dur-ing the formation of the supercontinent, Pangaea,and was modified during the Pennsylvanian Period(320 million years ago) to produce the Ozark Plateauto the north and the folded Ouachita Mountains tothe south. Areas surrounding the Interior Highlandshave been periodically inundated by epicontinentalseas or subjected to more xeric conditions than thehighlands and flooded by meltwater from glaciers.The Interior Highlands, however, have remainedabove water throughout their history and morerecently served as an unglaciated refugium through-out the Pleistocene, although species distributions were

ISSN 0164-7954 print/ISSN 1945-3892 online© 2011 Taylor & Francis; printed 21 December 2011http://dx.doi.org/10.1080/01647954.2010.548404http://www.tandfonline.com

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Fig. 1. Location of the Interior Highlands and Great Smoky Mountains National Park. The specific ecoregionswithin the Interior Highlands from which specimens were collected for morphological and molecular comparisonare delineated.

influenced by climatic fluctuations associated withglacial cycles (King, 1973; Davis, 1983; Allen, 1990;Shepard and Burbrink, 2009).

The US Environmental Protection Agency hasdivided the Interior Highlands into ecoregions toreflect differences in geology, physiography, vegetation,climate, soils, land use, biota, and hydrology (Fig. 1;Omernik, 1987). High species richness and signifi-cant numbers of taxa endemic to the Highlands orto specific ecoregions within the Highlands have beenreported in a wide range of aquatic and terrestrialtaxa including fish (Robison and Buchanan, 1988),crayfish (Bouchard and Robison, 1980), and insects(Allen, 1990). In this study, Kongsbergia specimenswere collected from the Ozark Highlands ecoregion, adissected limestone plateau with karst features, includ-ing caves, springs, and spring-fed streams; the BostonMountains ecoregion, a relatively undisturbed areacovered by oak-hickory forest with dissected sandstone

and shale; and the Ouachita Mountains ecoregion,made up of sharply defined east–west trending ridges,formed through erosion of compressed sedimentaryrock formations.

Specimens of the new species from these threeecoregions were found to share a number of mor-phological attributes that are not found in species ofKongsbergia from other regions of North America(see below). We noted differences among populationsfrom different ecoregions including intensity of colorpattern and the shape of the idiosoma, but these dif-ferences were subtle, suggesting that they representednatural variation. However, since species endemic toparticular ecoregions within the Interior Highlandshave been reported, we decided to investigate DNA-based information to decide whether these differencesshould be regarded as variability within a single speciesor were indicative of two or more species endemic tospecific ecoregions.

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The utility of the mitochondrial cytochromeoxidase subunit I (COI) for distinguishing amongclosely related species of birds, mammals, fish, andvarious arthropods has been demonstrated (Hebertet al., 2003; Savolainen et al., 2005). More specifically,COI has been shown to be suitable in acarologi-cal studies when low taxonomic levels are of interest(Cruickshank, 2002). COI sequence data have beenused to distinguish among putative species of thewater mite genus Unionicola (Ernsting et al., 2006)and to examine the taxonomic status of water mitespecies in the genus Hygrobates (Martin et al., 2010).We obtained partial COI for Kongsbergia specimensfrom three ecoregions within the Interior Highlands todetermine intraspecific genetic divergence among pop-ulations. Based on a combination of morphologicaland DNA sequence data, we present a single new watermite species, K. robisoni.

MATERIALS AND METHODS

Collection of specimens – Specimens of Kongs-bergia were collected from rivers and streams represen-tative of the Ozark Highlands, Boston Mountains, andOuachita Mountains. In addition to these specimens,we examined Kongsbergia collected from rivers andstreams before this study from the Interior Highlandsand other regions in North America.

Specimens were obtained by digging and stirringsubstrata in rocky riffles and gravel bars to depthsranging from 25 to 50 cm. Dislodged organisms anddetritus were collected in 250 µm mesh nets and passedthrough a set of sieves to remove coarse debris. Theresidual organisms and fine silt were placed in whitephotographic trays half filled with water and miteswere collected using pipettes as they moved about.For morphological study, specimens were preserved inmodified Koenike’s solution, also known as GAW (seeSmith et al., 2010).

Specimens to be used for molecular genetic studywere preserved in 99% ethanol. These specimens were

used to confirm that COI was capable of distinguishingamong populations that appear to be morphologicallydifferent. Specimens of the genus Aturus were alsocollected and used as an outlier group.

In the list of type specimens, the names of col-lectors are abbreviated as follows: Andrea J. Radwellas AJR, Ian M. Smith as IMS, Vamsi Kaliki as VK,Roger D. Mitchell as RDM, David R. Cook as DRC,Betty G. Crump as BGC, and Henry W. Robison asHWR.

Morphological analysis – Specimens preservedin GAW were cleared in 10% KOH, dissected, andmounted on microscope slides in glycerin jelly. Theywere examined using a Nikon Labophot II micro-scope and illustrated with the aid of a drawing tubeattachment.

In the description, measurements are expressedas ranges in micrometers (µm) with measurements ofthe holotype shown in parentheses. Measurement dataare included for all illustrated structures, and scale barsare included. We follow the terminology of Smith et al.(2010). The following abbreviations are used for thesegments of the appendages: Tr for trochanter, Fe forfemur, BFe for basifemur, TFe for telofemur, Ge forgenu, Ti for tibia, and Ta for tarsus. The width of thedorsal shield is measured at the pair of glandulariaposterior to the postocular setae, and the width of theventral shield includes projections covering insertionsof legs IV. Pedipalp height does not include ventralprojections.

All specimens and slides are deposited inthe Canadian National Collection of Insects andArachnids (CNC), K.W. Neatby Building, CentralExperimental Farm, Ottawa, Ontario, Canada.

Molecular analysis – DNA extraction, amplifi-cation, and sequencing – Whole genomic DNA wasextracted from individual specimens using the QiagenDNeasy Tissue Kit (Qiagen Inc., Valencia, CA)(Dowling and O’Connor, 2010). The COI was ampli-fied in 25 µl reactions using LCOI and HCOI fromFolmer et al. (1994) modified to include M13 tailsas indicated in Table 1. Each reaction contained the

Table 1. List and sequence of primers HCO1 and LCO1 that wereused in the initial polymerase chain reaction (PCR).

Primer name Sequence (5!–3!)

M13rev (F) CAGGAAACAGCTATGACCM13(-21)R TGTAAAACGACGGCCATHCO1 TAAACTTCAGGGTGACCAAAAAATCALCO1 GGTCAACAAATCATAAAGATATTGG

Note: M13 forward (F) and reverse (R) were used in the subsequent reamplifi-cations.

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Fig. 2. Results of the parsimony and Bayesian analyses resulted in the same phylogenetic hypothesis depictedhere. Branch lengths correspond to the amount of genetic divergence between cytochrome oxidase subunit I (COI)sequences. Bayesian posterior probabilities are placed above branches and parsimony bootstrap values are placedbeneath. VK numbers correspond to the data presented in Table 2.

following reagents and amounts: 12.5 µl of 10% tre-halose, 4.0 µl of dH2O, 2.5 µl of 10 " polymerasechain reaction (PCR) buffer, 1.25 µl of 50 mM MgCl2,0.25 µM of Primer 1, 0.25 µM of Primer 2, 0.125 µlof 10 mM dNTP, 0.12 of Platinum Taq polymerase

(Invitrogen, Carlsbad, CA), and 4 µl of templateDNA.

The reaction conditions for PCR were as follows:initial denaturation at 95#C for 3 min followed by 40cycles of denaturation at 95#C for 45 s, annealing at

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53#C for 30 s, and elongation at 72#C for 70 s, with afinal elongation step of 10 min after completion of allcycles.

Three microliters of each PCR product was runthrough a 1.5% agarose test gel to visualize the purityand quantity. The electrophoresis was run for 20 min at85 V and 400 mA. All positive samples were preparedfor gel extraction. The entire sample was visualized ona 2% low melting point agarose gel and the target bandwas excised from the gel with a sterilized scalpel. Gelextraction was completed with a Qiagen MinElute GelExtraction Kit with slight modifications to protocolstherein, including elimination of the isopropanol stepafter initial dilution of the gel and addition of only 700µl buffer PE to ensure ample room in the MinElutecolumns for both the buffer and the dissolved gel.Final elution was performed with 25 µl of dH2O addeddirectly to the membrane. Three microliters of purifiedproduct was visualized on a 1.5% gel to ensure thatconcentrations were high enough for sequencing. If thebands failed to show adequate brightness, the productwas reamplified using the M13 primers (Table 1), fol-lowed by gel extraction and purification. The reactionconditions for reamplification were as follows: initialdenaturation at 95#C for 3 min followed by 35 cyclesof denaturation at 95#C for 30 s, annealing at 53#Cfor 30 s, and elongation at 72#C for 1 min, with afinal elongation step of 10 min after completion of allcycles.

Sequencing of amplified samples was per-formed using MACROGEN USA (http://www.macrogenusa.com/). The resulting sequences wereanalyzed using DNASTAR© LASERGENE SEQMAN(Madison, WI, USA) software, and forward andreverse sequences were reconciled. BLAST searches inGenBank were made to ensure that sequences werenot contaminated.

Molecular analysis – sequence alignment andphylogenetic analysis – Sequences were aligned withCLUSTAL X (Thompson et al., 1997). The resultingalignment was visualized using BIOEDIT (Hall, 1999)and manually checked for errors. Aligned sequenceswere subjected to phylogenetic analysis using parsi-mony and Bayesian methods.

Parsimony analysis was implemented usingPAUP$4.0b10 (Swofford, 2002) with parsimonyinformative characters treated as unordered andunweighted. The entire data set was subjected to10,000 random addition replicates and tree bisection–reconnection branch swapping. Branch support wasdetermined through 10,000 bootstrap pseudorepli-cates, each with 100 random additions and treebisection–reconnections. Supports were labeled onthe appropriate branches of the final phylogenetichypothesis (Fig. 2 and Table 2).

Bayesian analysis was performed usingMRBAYES ver. 3.1.2 (Huelsenbeck and Ronquist,2001; Ronquist and Huelsenbeck, 2003). Because thestarting conditions for Bayesian analysis can affectthe probability of being trapped at a local optima(Huelsenbeck et al., 2002), two independent runsof four heated Markov chains were performed. Theappropriate substitution model was determined forthe data set by MRMODELTEST (Nylander, 2004).Relative fit on a neighbor-joining tree calculated fromJukes–Cantor corrected distances (Jukes and Cantor,1969) was tested for 56 different models of nucleotidesubstitution using the Akaike Information Criterion(Akaike, 1973, 1974). Parameters for site-specific rateheterogeneity (!-distributed rate parameter; Yang,1994) and for the proportion of invariant sites (I) werealso assessed to determine whether inclusion improvedthe fit of the model to the data. The model determinedto be the best fit was the one for which additionalparameters no longer significantly improved thelog-likelihood score. The HKY + I + ! model ofnucleotide substitution was specified and the analysiswas allowed to proceed for 10 million generations.Uniform interval priors were assumed for all baseparameters except base composition, which assumeda Dirichlet prior. Likelihood scores of each chainwere printed every 100 generations to monitor theruns and determine when stationarity had occurred.Burn-in, or the removal of the early generations dur-ing which likelihood scores fluctuate as stationarityis approached, was determined after the runs werecompleted. The remaining generations were used toproduce a majority-rule consensus tree of the results.

SYSTEMATICS

Family ATURIDAE Thor, 1900Subfamily ATURINAE Thor, 1900

Genus Kongsbergia Thor, 1899

Kongsbergia Thor, 1899: 1–2, Figs. 166–167.Kongsbergia Cook, 1974: 292, 359–360.Kongsbergia Viets, 1987: 412.Kongsbergia Cramer, 2000: 271–273.

Type species – Kongsbergia materna Thor, 1899.Monobasic.

Diagnosis (modified from Cook, 1974; Cramer,2000) – Character states of Aturidae, subfamilyAturinae. Dorsal and ventral shields completely sep-arate in both sexes. Idiosoma of males usually withouta cauda, rarely with a short cauda. Dorsal shieldbearing numerous (up to 12) pairs of glandularia;

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excretory pore usually borne on dorsal shield in bothsexes, rarely on a separate platelet in male. Ventralshield bearing three pairs of glandularia; coxal platesfused together with suture lines obliterated medially.Anterior coxal plates projecting beyond anterior edgeof dorsal shield; lateral projections of fourth coxaepartially covering insertions of legs IV. Fourth coxalplates lacking glandularia; genital acetabula numerousand arranged in rows on posterolateral edges of ven-tral shield. Pedipalp of males stockier than those offemales; segments variable in length; femur with orwithout ventral projections. Legs lacking swimmingsetae. Leg IV of males with distal segments highlymodified, basifemur often with a hook-like ventralprojection, tibia expanded and bearing one or twoblade-like setae ventrally. Tibia of legs I and II withor without thickened, curved setae.

Habitat – Rocky riffles and hyporheic gravels instreams.

Distribution – Temperate North America, CentralAmerica, and northern South America (Columbia) inthe New World and temperate Europe and Asia, andmost of Africa in the Old World.

Remarks – Cook (1974) recognized four subgen-era based on degree of caudal development, position ofexcretory pore, and presence or absence of thickenedsetae on the tibia of legs I and II.

Subgenus Kongsbergia

Diagnosis (modified from Cook, 1974) –Idiosoma without caudal development. Excretorypore borne on dorsal shield in both sexes. Tibia of legsI and II without thickened setae.

Habitat – As for genus.Distribution – As for genus.

Kongsbergia robisoni n. sp.(Figs. 3–9)

Description – Character states of subgenus Kongs-bergia.

ADULT MALE (Figs. 3–6) – Surface of idio-somal sclerites reticulate. Dorsal shield (Fig. 3) witha prominent rounded projection on anterior margin,two well-developed elevated areas, and a series ofridges posteriorly; variable in color but usually withblack anterior and posterolateral patches, often withbright orange medial area extending between blackpatches. Ventral shield (Fig. 4) with projections asso-ciated with insertions of legs IV well developed andbearing numerous genital acetabula in one or two rowsposterolaterally. Pedipalp (Fig. 5) with femur greatlyenlarged and bearing three ventral projections. Leg IV(Fig. 6) with tibia bearing large, thickened ventral seta

and with chaetotaxy of other segments as illustrated.Measurements (n = 6): dorsal shield length 310–348(346), width 270–290 (274); ventral shield length 350–386 (386), width 284–294 (288). Lengths of pedipalpsegments: Tr 21–24 (24), Fe 74–100 (92), Ge 28–33(33), Ti 85–98 (95), Ta 30–33 (30). Pedipalp femurheight 70–95 (86). Length of leg segments: ITr 40–48(43), IBFe 55–61 (60), ITFe 52–57 (57), IGe 59–65 (65),ITi 81–90 (89), ITa 88–99 (99), IITr 38–45 (45), IIBFe51–56 (56), IITFe 52–57 (55), IIGe 63–72 (66), IITi 83–90 (90), IITa 88–99 (99), IIITr 35–36 (36), IIIBFe 49–60(51), IIITFe 55–64 (62), IIIGe 75–82 (82), IIITi 89–98 (98), IIITa 98–108 (107), IVTr 69–75 (75), IVBFe86–95 (89), IVTFe 65–75 (74), IVGe 72–80 (79), IVTi76–85 (80), and IVTa 95–101 (100).

ADULT FEMALE (Figs. 7–9) – Idiosomal scle-rites similar to male in color and surface texture.Dorsal shield (Fig. 7) similar to that of male, butwider and lacking elevated medial and posterior areas.Ventral shield (Fig. 8) similar to that of male but withfewer acetabula. Pedipalp (Fig. 9) with tibia propor-tionally thicker and femur more slender than in male.Measurements (n = 6): dorsal shield length 348–368,width 278–298; ventral shield length 348–372, width276–296. Length of pedipalp segments: Tr 23–25, Fe65–69, Ge 28–32, Ti 99–110, Ta 29–32. Length of legsegments: ITr 28–38, IBFe 48–55, ITFe 43–49, IGe 52–56, ITi 68–73, ITa 70–80, IITr 25–38, IIBFe 48–55,IITFe 43–48, IIGe 52–59, IITi 65–73, IITa 75–87, IIITr28–37, IIIBFe 45–53, IIITFe 47–53, IIIGe 62–68, IIITi72–86, IIITa 80–90, IVTr 55–62, IVBFe 67–73, IVTFe54–62, IVGe 69–74, IVTi 80–88, and IVTa 88–92.

Etymology – Named for Henry W. Robison inrecognition of his numerous important contributionsto knowledge of the aquatic fauna of Arkansas.

Habitat – Gravel and sand deposits and moss-covered rocks associated with riffles of streams.

Distribution – Ouachita–Ozark Interior High-lands of the United States.

Types – HOLOTYPE (CNC) – Male adult(slide) collected from gravel and sand deposits of ariffle in Tomahawk Creek, Searcy County, Arkansas,USA, on 6 September 2006 by IMS.

PARATYPES (CNC) – USA: Arkansas: BooneCo.: Crooked Creek beside Rt. 7, 1.6 km south ofHarrison, 30 August 1949, RDM – three males, fourfemales (slides). Franklin Co.: Cass, Mulberry River atHigh Bank Canoe Launch off Rt. 215, 9.4 km west ofRoute 103, 9 September 2006, IMS – one female (slide)+ three males, seven females (GAW); 29 September2008, IMS – eight males, five females (GAW). HowardCo.: Cossatot River in State Park across from VisitorCenter, 13 June 2008, AJR and BGC – one male, onefemale (GAW). Johnson Co.: Mulberry River beside

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Figs. 3–6. Kongsbergia robisoni n. sp., adult male – 3. dorsal shield; 4. ventral shield; 5. pedipalp; 6. leg IV.

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Figs. 7–9. Kongsbergia robisoni n. sp., adult female – 7. dorsal shield; 8. ventral shield; 9. pedipalp.

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Rt 103 ca. 20 km north of Harmony, 30 June 1987,IMS – six males, seven females (slides) + three males,11 females (GAW). Montgomery Co.: Caddo Riverat Caddo Gap, AJR and HWR, 6 October 2007 –two males (slides); Willowbrook at Polk Road 42 andCounty Road 402 in Mena, 19 October 2007, AJR– one female (slide); Montgomery Creek at CrystalRecreation Area off Forest Road 177, 18 September2008, IMS – one male (GAW). Newton Co.: MillCreek at Marble Falls beside Rt. 7, 16 km southof Harrison, 1 September 1949, RDM – four males,one female (slides); Buffalo National River at Pruitt,20 July 2006, AJR – one male, four females (slides);Buffalo National River at Ponca, 26 July 2006, AJR– one male (slide); 3 October 2008, IMS – one female(GAW); 28 July 2009, AJR – one male (GAW). PolkCo.: Blaylock Creek at Bard Springs RecreationalArea, 24 July 2008, AJR and BGC – two males(slides); 12 March 2009, AJR and BGC – one female(GAW); 12 May 2009, AJR and BGC – one male(GAW); 11 August 2009, AJR – one male, one female(GAW); stream below Abernathy Spring beside Hwy8 near Big Fork, 5 October 2007, AJR and HWR– one female (slide); 23–26 September 2008, IMS –three males (GAW); East Saline Creek beside ForestRoad 38, 0.6 km north of Shady Lake RecreationArea, 24 September 2008, IMS – two males (GAW).Searcy Co.: Tomahawk Creek beside Tomahawk SlabRoad near Gilbert, 6 September 2006, IMS – twomales, three females (slides) + three males, one female(GAW); 27 September 2009, AJR and VK – onemale, one female (GAW). USA: Missouri: Barry Co.:Roaring River State Park 11 km south of Cassville,Roaring River, 26–27 August 1949, RDM – one male,four females (slides). Crawford Co.: Huzzah Creek atRed Bluff Campground east of Davisville, 26 June1987, IMS – two males, two females (slides). Dent Co.:Meramec River beside Rt. 19 ca. 18 km northeast ofSalem (Short Bend access), 1 July 1961, DRC – onefemale (GAW), 26 June 1987, IMS – four males, fourfemales (slides) + six males, seven females (GAW);22 November 2009, AJR – two females (GAW).Oregon Co.: Eleven Point River beside Rt. 19 north ofGreer, gravel in riffle, 28 June 1987, IMS – 10 males,11 females (slides) + three males, nine females (GAW);Eleven Point River at Cane Bluff access, 22 November2009, AJR – two males (GAW). Shannon Co.: AlleySpring State Park, Jacks Fork River, 13 July 1960,DRC – one male (slide); Round Spring State Park,Current River, 25 July 1960, DRC – six males, twofemales (slides) + five females (GAW); 30 June 1961,DRC – six males, three females (slides) + three females(GAW); 29 June 1987, IMS– one male, three females(slides) + 10 females (GAW). Wayne Co.: small streamnear Sam A. Baker State Park, 8 July 1960, DRC

– one female (GAW); St. Francis River at Sam A.Baker State Park, 27 June 1987, IMS – one male(GAW). Webster Co.: small stream 6 miles west ofMarshfield, 24 August 1949, RDM – one male (slide);James River 6 miles south of Marshfield, 24 August1949, RDM – one male (slide); Finley Creek 20 milessouth of Marshfield, 25 August 1949, RDM – 15 males,6 females, 3 deutonymphs (slides).

Remarks – K. robisoni appears to be related to theeastern North American species Kongsbergia reticu-lata Habeeb (1956) and Kongsbergia labyrintha Habeeb(1957a, 1974) based on synapomorphic topography ofthe dorsal shield with a large elevated hump anteriorlyand a strong declivity posteriorly. Males of K. robisonican readily be distinguished from those of all otherKongsbergia species known from North America bythe presence of a prominent rounded projection nearthe anterior margin of the dorsal shield and by thepresence of a medial ridge in the posterior declivity onthe dorsal shield.

Authorship of the new species should be cited asK. robisoni Radwell and Smith.

DISCUSSION

Based on morphological examination and anal-ysis, authors Radwell and Smith concluded that pop-ulations of K. robisoni from the three ecoregions inthe Interior Highlands differed from all other knownspecies of Kongsbergia and probably belonged to asingle species. However, variability in body shape andcolor pattern among these populations left open thepossibility that their apparent geographic separationmay have resulted in cryptic speciation. Analysis ofCOI data offered us an opportunity to investigate thispossibility further.

Sequence alignment of COI was straightforwardand resulted in an alignment containing no gaps and atotal of 694 characters. Parsimony analysis resulted ina single most parsimonious hypothesis (tree length =616, consistency index = 0.66, retention index = 0.73).Bayesian analysis resulted in the same topology as par-simony analysis (Fig. 2). Specimens and site locationsfrom which COI sequences were obtained are shown inTable 2. Three primary clades of Kongsbergia were allsupported by 100% bootstrap values and 1.0 Bayesianposterior probabilities. The phylogenetic hypothesisindicates that specimens from the Interior Highlands,including specimens from the Ozark Highlands,Boston Mountains, and Ouachita Mountains, form aclade separate from Kongsbergia in Appalachia (GreatSmoky Mountains and Southeast Tennessee). Geneticdistance based on the uncorrected p for Appalachia

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Kongsbergia sp.1–5 is high: Appalachia Kongsbergiasp.1 is 11.7% divergent from Appalachia Kongsbergiasp.2; Appalachia Kongsbergia sp.3 is 13.4% divergentfrom the node containing Appalachia Kongsbergiasp.4–5; Appalachia Kongsbergia sp.4 is 11.1% diver-gent from Appalachia Kongsbergia sp.5. Distanceswithin the Interior Highlands, excluding those fromthe Ouachita River (VK9, VK47, VK48, and VK49)are low and typical of intraspecies variation, rangingfrom 0.4% to 1.9%. Among the Interior Highlandsspecimens, those from the Ouachita River showed dis-tances ranging from 6.3% to 6.8% from other InteriorHighlands specimens. However, no obvious morpho-logical differences were found between specimens fromthe Ouachita River and those from other OuachitaMountain streams. As endemism of other aquatic taxain the Ouachita River has been reported (Fitzpatrick,1965; Hobbs, 1979; Mayden, 1985), further study ofthe population of Kongsbergia from this river is war-ranted, and we are not identifying it as K. robisoni atthis time. Aturus specimens demonstrated the expecteddivergence from Kongsbergia.

The results of COI analysis clearly separateK. robisoni, n. sp. from Kongsbergia specimens sam-pled in the Appalachian Mountains and support theconclusion based on morphological analysis that itis a different species. Additionally, COI distances forspecimens within the Interior Highlands, with theexception of those from the Ouachita River, are lowand typical of intraspecies variation. On the otherhand, distances ranging from 11% to 13.4% betweenAppalachia Kongsbergia sp.1–5 (Fig. 2) fall well out-side the range typically identified as intraspecific vari-ation (Hebert et al., 2003), indicating the presence offive different species, some of which may be unde-scribed. Revisionary work is underway to clarify theidentities and relationships of these species.

ACKNOWLEDGMENTS

We thank Henry Robison and Betty Crumpfor encouraging and facilitating our exploration ofthe Ouachita Mountain ecoregion and for provid-ing essential advice on collection sites and assistanceboth in the field and the laboratory. Terri Phelan pro-vided expertise in map production. We also wish toacknowledge the financial support for this project fromthe Ouachita Division of the US Forest Service andthe Honors College of the University of Arkansas,Fayetteville. Finally, we appreciate the support we havereceived from Discovering Life in America, which wasprovided for our fieldwork in Great Smoky MountainsNational Park.

REFERENCES

Akaike, H. 1973. Information theory and an exten-sion of the maximum likelihood principle.pp. 267–281. Second International Symposiumon Information Theory; Akademiai Kiado,Budapest.

Akaike, H. 1974. A new look at the statistical modelidentification. IEEE Trans. Automat. Contr. 19:716–723.

Allen, R. T. 1990. Insect endemism in the InteriorHighlands of North America. Florida Entomol.73(4): 539–569.

Bouchard, R. W. and H. W. Robison. 1980. An inven-tory of the decapod crustaceans (shrimps andcrayfish) of Arkansas with a discussion of theirhabitats. Ark. Acad. Sci. Proc. 34: 1980:22–30.

Cook, D. R. 1974. Water mite genera and subgenera.Mem. Am. Entomol. Inst. 21: 860 pp.

Cramer, C. 2000. New species of the water mite genusKongsbergia (Acari: Hydrachnida: Aturidae)from Mexico. Internat. J. Acarol. 26: 271–278.

Cruickshank, R. H. 2002. Molecular markers for thephylogenetics of mites and ticks. Syst. Appl.Acarol. 7: 3–14.

Davis, M. B. 1983. Quaternary history of deciduousforest of eastern North America and Europe.Ann. Missouri Bot. Gard. 70: 550–563.

Dowling, A. P. G. and B. M. O’Connor. 2010.Phylogenetic relationships within the suborderDermanyssina (Acari: Parasitiformes) and a testof dermanyssoid monophyly. Internat. J. Acarol.36: 299–312.

Ernsting, B. R., D. D. Edwards, M. D. Vidrine, K. S.Myers and C. M. Harmon. 2006. Phylogeneticrelationships among species of the subgenusParasitatax (Acari: Unionicolidae: Unionicola)based on DNA sequence of the mitochondrialcytochrome oxidase I gene. Internat. J. Acarol.32(2): 195–202.

Fitzpatrick, Jr., J. F. 1965. A new subspecies ofthe crawfish Orconectes leptogonopodus from theOuachita River drainage in Arkansas. TulaneStud. Zool. 10(1): 57–79.

Folmer, O., M. Black, W. Hoeh, R. Lutz and R.Vrijenhoek. 1994. DNA primers for amplificationof mitochondrial cytochrome c oxidase subunit Ifrom diverse metazoan invertebrates. Mol. Mar.Biol. Biotechnol. 3: 294–299.

Habeeb, H. 1956. North America Hydrachnellae,Acari. XXXVIII. On three species ofKongsbergia. Leafl. Acad. Biol. 10: 1–2.

Habeeb, H. 1957a. New Hydrachnellae from NorthCarolina. Leafl. Acad. Biol. 15: 1–8.

Dow

nloa

ded

by [D

r And

rea

Radw

ell]

at 0

6:22

27

Dec

embe

r 201

1

Vol. 37, Suppl. 1 Internat. J. Acarol. 205

Habeeb, H. 1957b. North America Hydrachnellae,Acari. XLII–XLVII. Leafl. Acad. Biol. 16: 1–8.

Habeeb, H. 1961. Two new Hydrachnellae fromCalifornia. Leafl. Acad. Biol. 23: 1–2.

Habeeb, H. 1974. Notes on water-mites. IX. Leafl.Acad. Biol. 63, 1–2.

Habeeb, H. 1981. Another aquatic oribatid mite. Leafl.Acad. Biol. 77, 1–2.

Hall, T. A. 1999. BioEdit: a user-friendly biologicalsequence alignment editor and analysis programfor Windows 95/98/NT. Nucl. Acids Symp. Ser.41: 95–98.

Hebert, P. D. N., S. Ratnasingham and J. R. deWaard.2003. Barcoding animal life: cytochrome c oxi-dase submit I divergences among closely relatedspecies. Proc. R. Soc. Lond. B. 270(Suppl.):S96–S99.

Hobbs, Jr., H. H. 1979. A new crayfish fromthe Ouachita River Basin in Arkansas(Cambaridae:Decapoda). Proc. Biol. Soc.Wash. 92(4): 804–811.

Huelsenbeck, J. P., B. Larget, R. E. Miller and F.Ronquist. 2002. Potential applications and pit-falls of Bayesian inference of phylogeny. Syst.Biol. 51: 673–688.

Huelsenbeck, J. P. and F. Ronquist. 2001. MrBayes:Bayesian inference of phylogeny. Bioinformatics17: 754–755.

Jukes, T. H. and C. R. Cantor. 1969. Evolutionof protein molecules. pp. 2l–l32. In: Munro,H. N. (Ed.). Mammalian Protein Metabolism.Academic Press, New York.

King, J. E. 1973. Late Pleistocene palynology and bio-geography of the Western Missouri Ozarks. Ecol.Monogr. 43: 539–565.

Martin, P., M. Dabert and J. Dabert. 2010. Molecularevidence for species separation in the water miteHygrobates nigromaculatus Lebert, 1879 (Acari,Hydrachnidia): evolutionary consequences ofthe loss of larval parasitism. Aquat. Sci. 72:347–360.

Mayden, R. L. 1985. Biogeography of OuachitaHighland fishes. Southwest. Nat. 30(2): 195–211.

Nylander, J. A. A. 2004. MrModeltest v2. ProgramDistributed by the Author. Evolutionary BiologyCentre, Uppsala University, Sweden.

Omernik, J. M. 1987. Ecoregions of the conterminousUnited States. Ann. Assoc. Amer. Geog. 77(1):118–125.

Robison, H. W. and T. M. Buchanan. 1988. Fishesof Arkansas. University of Arkansas Press,Fayetteville, AR, USA. 536 pp.

Ronquist, F. and J. P. Huelsenbeck. 2003. MRBAYES3: Bayesian phylogenetic inference under mixedmodels. Bioinformatics 19: 1572–1574.

Savolainen, V., R. S. Cowan, A. P. Vogler, G. K.Roderick and R. Lane. 2005. Towards writing theencyclopaedia of life: an introduction to DNAbarcoding. Philos. Trans. R. Soc. B-Biol. Sci. 360:1805–1811.

Shepard, D. B. and F. T. Burbrink. 2009.Phylogeographic and demographic effects ofPleistocene climatic fluctuations on a montanesalamander, Plethodon fourchensis. Mol. Ecol.18: 2243–2262.

Smith, I. M., D. R. Cook and B. P. Smith. 2010.Water mites (Hydrachnidiae) and other arach-nids. Chapter 15, pp. 485–586. In: Thorpe, J. andA. Covich (Eds.). Ecology and Classification ofNorth American Freshwater Invertebrates. 3rded. Academic Press (Elsevier Inc.), New York,USA.

Swofford, D. L. 2002. PAUP$ 4.0b10: PhylogeneticAnalysis Using Parsimony ($and Other Methods)Version 4.0b10. Sinauer Associates, Sunderland,MA, USA.

Thompson, J. D., T. J. Gibson, F. Plewniak, F.Jeanmougin and D. G. Higgins. 1997. The ClustalX windows interface: flexible strategies for multi-ple sequence alignments aided by quality analysistools. Nucleic Acids Res. 24: 4876–4882.

Thor, S. 1899. En ny hydrachnide-slægt og andre nyearter fundne i Norge sommeren 1899. foreløbigmeddelelse. Kristiania (O. Norli), 1–5.

Viets, K. O. 1987. Die Milben des Süßwassers(Hydrachnellae und Halacaridae [part.], Acari).2. Katalog. Sonderbände des Naturwiss. Ver.Hamburg, 8: 1–1012.

Yang, Z. 1994. Maximum likelihood phylogenetic esti-mation from DNA sequences with variable ratesover sites: approximate methods. J. Mol. Evol. 39:105–111.

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