'U JOURNAL OF THE KENTUCKY ACADEIVIY OF SCIENCE Official Publication of the Academy Volume 73 Number 2 Fall 2012 The Kentucky Academy of Science Founded 8 May 1914 Governing Board 201 1 Elected Officers President: Dawn Anderson, Berea College/dawn_anderson@berea.edu President FAect: Cher) 1 Davis, Western Kentucky University/ehen kdavis@wku.edu Vice President: KC Russell, Northern Kentucky University/russellk@nku.edu Past President: Barbara Ramey, Eastern Kentuek) Universit)7barbara.ramey@eku.edu Secretary: Robert Kingsolver, Bellarmine Universit)/kingsolver@bellarmine.edu Treasurer: Kenneth Crawford, Western Kentucky University/kenneth.crawford@wku.edu Executive Director {ex officio): Jeanne Harris/executivedirector@kyscience.org Editor: JOURNAL {ex officio): Martin Matisoff, Kentucky Slate Universit)/martin. inatisoff@kysu.edu DivisiOxN and At-Large Representatives Biological Sciences (2011): Pamela Feldhoff, University of Louis^ ille School of Medieine/pwfeld01@ louisville.edu Biological Sciences (2011): Ronald Jones, P'astern Kentucky University; ron.jones@eku.edu Physical Sciences (2011): Eric Jerde, Morehead State Universit)/e.|erde@moreheadstate.edu Social 'ersit\7martin.matisoff@k) su.edu Abstract Editor: Robert J. Barney, West Virginia State Universit) / rbarney@wvstateu.edu Editorial Board: Susan Templeton, Kentucky State Universitv7susan.templeton@k)su.edu Claire Rinehart, Western Kentuek) University/claire. rinehart@wku.edu All manuscripts and correspondence concerning manuscripts should be addressed to the Editor (martin. matisoff@kysu. edu). The JOURNAL is indexed in BioOne, Cambridge Scientific Abstracts, Selected Water Resource Abstracts, State Acad- emies of Science Abstracts, and Zoological Record. Membership in the Academy is open to anyone with an interest in science. Interested parties can join online at www. kyscience.org where membership options and benefits are listed. Please contact the executive director at executivedirector® kyscicnce.org for additional information. The JOURNAL is made available as a PDF to all active members of the Academy (go to www.kentuckyscience.org). Hard copy subscriptions arc available. Subscription rates for nonmembers arc $50.00/ycar domestic, $60. 00/year for- eign. Back issues are $30.00 per volume. The JOURNAL is issued in spring and fall. Two issues comprise a volume. All other correspondence concerning memberships or subscriptions may be addressed to the Executive Director, Ken- tucky Academy of Science, P.O. Box 22579, Lexington, KY 40522-2579 or cxccutivcdirector@kysciencc.org. @ This paper meets the requirements of ANSI/NISO Z39. 48-1 992 (Permanence of Paper). INSTITUTIONAL AFFILIATES Bellarmine University Berea College Brescia University Centre College Eastern Kentucky University Georgetown College Kentucky State University Morehead State University Kentucky Community Members Midway College Murray State University Northern Kentucl^ University Spalding University Transylvania University University of Kentucky University of Louisville Western Kentucky University Technical College System Sustaining Member Campbellsville University Members Asbury University Pikeville College Kentucky Wesleyan College Thomas More College Lindsey Wilson College University of the Cuinberlaeds INDUSTRIAL AFFILIATES Honorary Patron— Lumins Associates Members Kentucky American Water Wood Hudson Cancer Research Laboratory Associate Members WKU Hoffman Environmental Research Institute WKU Crawford Hydrolo^ Lab FEB 2 1 2013 71 1?^. t.’^ ' ■• "’ . u-iivbt f nw-rtif W uihi /J K;^r n,» Hrru.>^i u i :f,\\ , * t, >.« i Ot-4 hMi' f .»• f ’ ^- • , ♦ ..■■.■»<... ■ Ki V !.'.»"■/■•• It \.^ i^felMl^Nijiill W'* ' ' • •**'“ ' '■*'- ■ * 'vt'U'w-nu *% I I I ij^iWiiiiiii I III ^ ' '»»■* MiEjkWi** * ♦ : ^Mi lK-.ii*»i' tM , -it - " 't f ^ ii'-.civti^ ’ r ■l•'.•■V'J^^ ,*tji!» -.itmk* Vr... IS.r-^.t.V.T H firr h-jifiA fc. - . b, ' t '^' i\ S ■ . I - I'.ii V'. ■« >itt***-V. i.- rr^^v'i'-vV ^ '*' i.l.~r- .. ... '■" ^■' •■' . . ‘.s #*. *^**'.“ tt< «tM A*:. ■‘. •T-'*' , ‘.ffc. ^ f».T(»AT*- ««T<*<»T*. <«»4r V50 iLtm^ik. 'i‘^>/'v%' >. ;.yr ■gy 3fc!si«ispn:<* e jr. rt*4 -‘b . 1 .. ... - ■t^U.StR ' *.*- f.j- -Sjli (Mh '.M > a- V- J. Ky. Acad. Sci. 73(2):73-76. 2012. Evaluation of the Tooth-wear and Replacement Method for Aging White-tailed Deer {Odocoileus virginianus) on the Blue Grass Army Depot, Madison County, Kentucky Charles L. Elliott^ Department of Biological Sciences, Eastern Kentucky University, Richmond, Kentucky 40475 and Thomas Edwards Kentucky Department of Fish and Wildlife Resources, #1 Sportsman’s Lane, Frankfort, Kentucky 40601 ABSTRACT We evaluated the reliability of tlie tootli-wear and replacement metliod for aging hunter-harvested deer on the Blue Grass Army Depot, Madison County, Kentuck)’. Eighty-seven hunter-har\^ested white-tailed deer were aged using die tooth-wear and replacement procedure and had an incisor toodi removed for aging via tiie cementum annuli metiiod. We found no significant difference (F < 0.05) in the distribution of ages (1.5 years, 2.5 years, 3.5 years, >4.5 years) assigned using either method for male (X- = 6.69) or femde (P = 6.85) deer. The majority (77%) of die toodi-wear; cementum pairs of age estimates were in agreement for deer in diis study. The tooth-wear and replacement technique tended to identify die age of female white-tailed deer >3.5 years of age incorrecdy. KEY WORDS: tooth-wear aging method, white-tailed deer, Odocoileus virginiantis INTRODUCTION To assure hunter harvest does not become an additive form of mortality in white-tailed deer {Odocoileus virginianus) populations, resource managers need to know the age distribution of the haiwest (Roseberry and Woolf 1991). Various methods have been used to determine the age of deer; e.g., antler beam diameter (Lueth 1963), body mass (Lueth 1963), eye lens weight (Connolly et ak 1969), incisor wear (Main and Owens 1995), hoof characteristics (Haugen and Speake 1958), molar tooth ratios (Robinette et ak 1957), mandibular tooth row length (Governo et ak 2006), cementum annuli (Asmus and Weck- erly 2011), dentine: enamel ratios (Meares et ak 2006) and tooth-wear and replacement [the “Severinghaus Method,” Severinghaus (1949)]. Although a regression formula de- picting the relationship of eye lens weight to the age of central Kentucky white-tailed was developed by Keller and Landry (1976), the most common aging procedure used by resource agencies in the region is the tooth- wear and replacement method. ' Corresponding author e-mail: Charles. Elliott@eku. edu The accuracy of the tooth-wear and replace- ment procedure has been assessed in a number of states [see review by Gee et ak (2002)], but investigations centered in the Southeast are limited [e.g., Mississippi (Hackett et ak 1979; Jacobson and Reiner 1989; Mitchell and Smith 1991)]. Patterns of tooth -wear can vary be- tween sexes of white-tailed deer (Van Deelen et ak 2000) and as the result of regional or local differences in soils and diet (Dimmick and Pelton 1994). Van Deelen et ak (2000) re- commended that tooth-wear and replacement aging, when used in managing a specific white- tailed deer population, should be calibrated witli local known-age specimens of both sexes. To our knowledge, an evaluation of the tooth-wear and replacement method for aging white-tailed deer in Kentucky has not been performed. The puipose of this study was to determine the reliability of the tooth-wear and replacement method for aging hunter-haiwest- ed deer on the Blue Grass Army Depot, Madison County, Kentucky. MATERIALS AND METHODS The Blue Grass Army Depot (BGAD) is a 5900 ha tract of land located 11 km south- southeast of Richmond, Kentucky. The Depot is primarily involved in activities associated 73 74 Journal of the Kentucky Academy of Science 73(2) Table 1. Summar)’ of age determination for 87 white- tiiiled deer {Odocoileus mrginianiis) on tlie Blue Grass Army Depot, Madison County, Kentucky. The age of each aniniiil was determined using two techniques; the tooth- wear and replacement procedure (Toothwear) and the cementum annuli (Cementum) method. Sample size is in parentheses. MALES FEMALES Toothwear Cementum Toothwear Cementum 1.5 VTS (34) 1 yr (26) 1.5 yrs (11) 1 >T (7) 2.5 >TS (28) 2 >TS (26) 2.5 yrs (5) 2 yrs (5) 3.5 yrs (5) 3 yrs (12 3.5 yrs (2) 3 )TS (1) >4.5 yrs (0) 4 yrs (3) >4.5 yrs (2) 7 yrs (2) 9 yrs (1) 4 )TS ( 1 ) 6 yrs ( 1 ) 10 )TS (1) 13 yrs (1) with the storage and maintenance of conven- tional and chemical munitions. As part of the Depot’s management of natural resources, white-tailed deer hunts are typically held each year. Because of security concerns, the hunt is highly regulated and all successful hunters are required to bring deer to a check station to be weighed and aged. Aging is performed by a Kentucky Department of Fish and Wildlife Resources (KDFWR) biologist who has expe- rience aging deer using the tooth wear and replacement method. During 1989 hunts, adult deer (no fawns) brought to the Depot check station were randomly selected for use in this study. Each deer was aged by the same KDFWR biologist using the tootli-wear and replacement method. Animals were assigned to an age categoiy (1.5 years, 2.5 years, 3.5 years, >4.5 years) and an incisor tooth removed. Incisors were aged (Matson’s Laboratoiy, Milltown, MX) using the cementum analysis technique (Dimmick and Pelton 1994). In a review of published literature, Asmus and Weckerly (2011) noted sectioned teeth of white-tailed deer from populations in the southeastern United States have been report- ed to contain cementum annuli that were difficult to interpret because the annuli often merged together and were poorly defined. Asmus and Weckerly (2011) found the preci- sion of the cementum analysis procedure varied among years and between sexes of mule deer (O. hemionus) from the same population. However, other researchers have reported the cementum analysis procedure to be more accurate than the tooth-wear and replacement technique for aging deer (Sauer 1971; Hamlin et al. 2000; Gee et al. 2002); we assumed that the ages determined by cementum analysis for deer analyzed in this study represented the animal’s true age. RESULTS Certainty codes supplied by Matson’s Lab- oratory indicated the amount of confidence in the accuracy of each cementum age estimate. The codes are defined as: (A) “Some tooth sections have a distinct annulus pattern and the result of age analysis is nearly certain;” (B) “There is histological evidence to support the reported cementum age. If error is present, it would be likely within the range given;” and (C) “There is little histological evidence to support the reported cementum age, which might be within the range given.” Of the 105 white-tailed deer incisors submitted, 15 were assigned certainty code B and 3 to code C; these were deleted from the analysis. Of the 87 deer teeth utilized in this study, 77% (n = 67) were from males; 23% (n = 20) from females (Table 1). There was no significant difference (F < 0.05) in the distribution of ages (1.5 years, 2.5 years, 3.5 years, >4.5 years) assigned using either age determination meth- od for male (X- = 6.69) or female (X^ = 6.85) deer. For the chi square analysis the following procedure was employed, animals in a partic- ular age categoiy determined via the tooth- wear procedure (e.g., 1.5 years) were the k)bser\^ed’ value; animals assigned an age via the cementum annuli procedure (e.g., 1 year) were considered the ‘expected’ value (all female deer identified as 4 years or older by the cementum annuli procedure were combined to form the ‘expected’ category). Accepting an age spread of 6 months, 77% of the tooth-wear: cementum pairs of age estimates were in agreement for white-tailed deer in this study (e.g., a deer assigned a tooth -wear age of 1.5 years and cementum annuli age of 2 years was considered in agreement, tooth-wear age of 2.5 years and cementum annuli age of 3 years was consid- ered in agreement, etc.). Of the 20 tooth- wear: cementum pairs of age estimates which varied by more than 6 months, the majority of the aging disagreements (70%; 6 males, 8 75 Aging Deer — Elliott and Edwards females) involved the tooth -wear assigned age being lower than the cementum annuli assigned age by an average of LI years for males; 4.0 years for females. DISCUSSION This study was site-specific, samples were obtained from deer in the same population, and tooth-wear ageing was performed by the same individual. As noted by Gee et ai. (2002) and Van Deelen (2000), these conditions should have minimized many of the variables (i.e., soil type habitat quality, range condition, variation between multiple individuals aging deer) that theoretically affect the accuracy of the tooth- wear and replacement aging technique. Using jaw bones from 98 known-aged white-tailed deer, Jacobson and Reiner (1989) had 55 deer biologists from southeast- ern states age the jaws using the tooth-wear and replacement method. The biologists correctly aged 96%, 87%, and 77% of the mandibles from fawns, yearlings, and 2.5 year old deer, respectively, but were correct only 25% of the time for animals ^3.5 years old (Jacobson and Reiner 1989). The tendency to incorrectly identify the age of white-tailed deer >3.5 years of age using the tooth-wear and replacement technique was evident in this study (especially in female deer) and has been reported by biologists from various states, e.g., Michigan (Ryel et al 1961), Maine (Gilbert and Stolt 1970), Texas (Cook and Hart 1979), Montana (Hamlin et al. 2000), Oklahoma (Gee et al 2002). Gee et al. (2002) reported ' that 34 white- tailed deer biologists from various southeastern states, when given 106 jaw bones or dental casts from known-aged deer, failed 60% of the time to correctly age deer >2 years old using the tooth-wear and replacement method. Gee et al. (2002) concluded that using the tooth- wear aging technique to determine ages of white-tailed deer beyond the general catego- ries of fawn, yearling, and adult are unfounded; and indicated a review of published data sets (i.e., 6 articles) using known-age deer support- ed their conclusion. In evaluating the accuracy of ages obtained for white-tailed deer, mule deer, and elk (Cervus elaphus) using the tooth -wear and replacement method and cementum analysis of incisors, Hamlin et al. (2000) reported the inaccuracy of ages assigned by the tooth-wear criteria, and the lack of compensation for the inaccuracy, are important considerations for even relatively simple tests of management practices. Nevertheless, they also noted the extra expense of obtaining age by the cemen- tum annuli method would not be warranted if a management question concerned only fawn, yearlings, 2-year old, and >3 year old age categories (Hamlin et al, 2000). Current aging protocol on the Blue Grass Army Depot requires reporting the age of harvested deer using the tooth-wear and replacement meth- od; but only within age categories of <6 months, 1.5 years, 2.5 years, and >3 years. Given the results of this study, we recom- mend the BGAD continue to use their current four categoiy tooth-wear aging meth- od; in which the maximum age assigned to a harvested deer is >3 years. However, we concur with Hamlin et al. (2000) who noted that should management needs ■ make it necessary to determine whether specific phy- sical or population parameters change signif- icantly with the age of deer in a population (like the deer herd on the BGAD); liaivested animals should be aged using the cementum annuli method. ACKNOWLEDGEMENTS We thank the Blue Grass Army Depot, and in particular the Depot's Land Management Office, for logistical and financial support. LITERATURE CITED Asnius, J., and F. W. Weckerly. 2011. Evaluating preci- sion of cementum annuli analysis foraging mule deer from southern California. J Wildlife Manage 75:1194- 1199. Connolly, G. E., M. L. DudinsM, and W, M. Longliurst. 1969. An improved age-lens weight regression for black-tailed deer and mule deer. J Wildlife Manage 33:701-704. Cook, R. L., and R. V. Hart. 1979. Ages assigned known- age Texas white-tailed deer: tootli wear versus cemen- tum analyses. Proc Ann Conf Southeastern Assoc Fish Wildlife Agencies 33:195-201. Dimmick, R. W., and M. R. Pelton. 1994. Criteria of sex and age. Pages 169-214 in T. A. Bookhout (ed). Research and Management Techniques for Wildlife and Habitats. The Wildlife Society, Betliesda, MD. Gee, K. L., J. H. Holman, M. K. Causey, A. N. Rossi, and J. B. Armstrong. 2002. Aging white-tailed deer by tooth replacement and wear: a critical evaluation of a 76 Journal of the Kentucky Academy of Science 73(2) time-honored technique. Wildlife Soc Bull 30:387- 393. Gilbert, F. F., and S. L. Stolt. 1970. Variability in Maine white-tailed deer by tootli wear characteristics. J Wildlife Manage 34:532-535. Governo, R. M., S. M. Shea, G. Somers, and S. S. Ditclikoff. 2006. Using mandibular tootli row lengtli to age yearling white-tailed deer. Wildlife Soc Bull 34:345-350. Hackett, E. J., D. C. Guynn, Jr., and H. A. Jacobson. 1979. Differences in age structure of white-tailed deer in Mississippi produced by two aging-techniques. Proc Ann Conf Southeastern Assoc Fish and Wildlife Agencies 33:25-29. Hamlin, K. L., D. F. Pac, C. A. Sime, R. M. DeSimone, and G. L. Dusek. 2000. Evaluating the accuracy of ages obtained by two methods for Montana ungulates. J Wildlife Manage 64:441-449. Haugen, A. O., and D. W. Speake. 1958. Determining age of young fawn white-tailed deer. J Wildlife Manage 22:319-321. Jacobson, J. A., and R. J. Reiner. 1989. Estimating age of white-tailed deer: tooth wear versus cementum annuli. Proc Ann Conf Soutlieastern Assoc Fish and Wildlife Agencies 42:286-291. Keller, C. J., and L. M. Landry. 1976. Eye lens weight as an age indicator of white-tailed deer in central Kentucky. Trans Kentucky Acad Sd 37:98-102. Lueth, F. X. 1963. A comparison of some aging techniques for Alabama deer. Proc Ann Conf Southeastern Assoc Game and Fish Commissioners 17:31-37. Main, M. B., and R, Owens. 1995. Estimating mule deer age from measurements of incisor wear. Northwest Nat 76:130-132. Meares, J. M., et al. (2006). A quantitative evaluation of the Severingliaus technique for estimating age of white- tailed deer. Proc Ann Conf Southeastern Assoc Fish and Wildlife Agencies 60:89-93. Mitchell, C. J., and W. P. Smitli. 1991. Reliability of techniques for determining age in southern white-tailed deer. J Tennessee Acad Sci 66:117-120. Robinette, W. L., D. A. Jones, G. Rogers, and J. S. Gasliwiler. 1957, Notes on tootli development and wear for Rocky Mountain mule deer. J Wildlife Manage 21:134-153. Roseberry, J. L., and A. Woolf. 1991. A comparative evaluation of techniques for analyzing white-tailed deer harvest data. Wildlife Monogr 117:1-59. Ryel, L. A., L. D. Fay, and R. C. Van Etten. 1961. Validity of age determination in Michigan deer. Michigan Acad Sci, Arts Lett 46:289-316. Sauer, P. R. 1971. Tootli sectioning vs. tootli wear for assigning age to white-tailed deer. Trans Nortlieast Fish Wildlife Conf 28:9-20. Severingliaus, C. W. 1949. Tootli development and wear as criteria of age in white-tailed deer. J Wildlife Manage 13:195-216. Van Deelen, T. R., K. M. Hollis, C. Anchor, and D. R. Etter. 2000. Sex affects age determination and wear of molariform teeth in white-tailed deer. J Wildlife Manage 64:1076-1083. J. Ky. Acad. Sd. 73(2):77-82. 2012. Occurrence of Three Leech Species (AneelMai Hirudinida) on Fishes in the Kentucky River Joseph E. Flotemersch^ aod Donald J. Klemm U.S. EPA, Office of Research and Development, National Exposure Research Laboratory (NERL), Ecological Exposure Research Division, 26 W. Martin Lutlier King Dr., Cincinnati, Ohio 45268 and William E. Moser Smithsonian Institution, National Museum of Natural History, Department of Invertebrate Zoology, Museum Support Center = MRC 534, 4210 Silver Hill Road, Suitland, Maryland 20746 ABSTRACT Leeches were collected from six fish species distributed among four of ten sites sampled. The leech species obsen^ed were Myzobdella reducta (Meyer 1940) and Myzohdella lugubris Leidy, 1851 of the family Piscicolidae and Flacohdella pediculata Hemingway, 1908 of the family Glossiphoniidae. Attachment locations for Myzobdella lugubris included various sites in tlie buccal cavity of green sunfish [Lepomis cyanellus (Rafinesque)], largemoutli bass [Microptems salmoides (Lacepede)], and spotted bass [Microptems punctulatus (Rafinesque)] and posterior to the eye of a largemoutli bass. Attachment locations for Myzobdella reducta included on the caudal and pelvic fins of logperch [Percina caprodes (Rafinesque)] and the caudal and anal fin of spotfin shiners [Cijprinella spiloptera (Cope)] - a new host record. Attachment location observed for Flacohdella pediadata was on the inside of the operculum of a freshwater drum [Aplodinotus grimniens (Rafinesque)]. This study established new host and attachment locations, the diverse presence of Myzobdella reducta on various fish species, and substantiated tlie high degree of host specificity of Placobdella pediculata for the freshwater drum. KEY WORDS; Hirudinida, host fish, attachment locations, new host record, Kentucky River INTRODUCTION Recent phylogenetic analyses have greatly enhanced our knowledge of the evolutionary relationships among leeches (Siddall et al. 2005; Williams and Burreson 2006). However, remaining limitations in distributional and life history data hamper our capacity for a more complete understanding, with substantial gaps in knowledge for the Commonwealth of Kentucky. Published accounts have docu- mented the host relationships for a few leech species in a limited geographic area (White and Crisp 1973; White 1974; Bauer and Branson 1975; Klemm 1985). During the course of a fish study by one of the authors (J. E. F.), specimens were examined for attached leeches and resulted in several new host relationships documented in this paper. ' To whom correspondence may be addressed. E-mail: floteiTierscli.joseph@epa.gov MATERIALS AND METHODS In the summer of 2000, electrofishing was conducted at 10 sites on the mainstem of the Kentucky River, Kentucky (Table 1; Figure 1). Electrofishing was conducted following the methods outlined in (Lazorchak et al. 2000), with the modification that 1000 m was elec- trofished on each bank of the river. Electrofisli- iiig was conducted in a downstream direction along the main-channel riparian habitat of each bank at a speed near or slightly exceeding the flow rate of the system if flow rates were low (Lazorchak et al 2000; Ohio EPA 1989; Reynolds 1983). All fish were identified, exam- ined for leeches, and tlien released, with the exception of representative vouchers specimens retained for laboratoiy identification. Voucher specimens were fixed and preserved with fonnaldeiiyde in the field and transported to the laboratoiy. During laboratoiy identification (principal keys: Etnier and Starnes 1993; Pflieger 1975; Stauffer et al. 1995; Trautman 1981), each fish specimen was carefully exam- ined for leeches. 77 78 Journai of the Kentucky Academy of Science 73(2) Table 1. Latitude and longitude of sites sampled on the Kentucky River. River River kilometer Site ID Longitude Latitude Kentucky 01 37.75 KOI -84.956850 38.465090 Kentucky 02 83.54 K02 -84.861230 38.317230 Kentucky 03 124.63 K03 -84.838510 38.096980 Kentucky 04 168.95 K04 -84.764900 37.881470 Kentucky 05 215.32 K05 -84.638560 37.774780 Kentucky 06 251.00 K06 -84.459340 37.840450 Kentucky 07 280.77 K07 -84.273170 37.914090 Kentucky 08 305.19 K08 -84.100150 37.858530 Kentucky 09 348.75 K09 -83.993850 37.709190 Kentucky 10 407.88 KIO -83.724100 37.567370 Leeches were relaxed in 5% ethanol (added dropwise in a vessel until the leech no longer reacted to a probe), fixed in 10% buffered formalin, preserved in 70% ethanol, and examined under a dissecting microscope. Voucher specimens of leeches were deposited in the Invertebrate Zoolo^ collections of the National Museum of Natural History, Smith- sonian Institution (accession no. 2057347). RESULTS A total of 3593 fish were collected during sampling; the majority of which were identi- fied and released live in the field. During field Figure 1. Collection sites on the main stem of the Kentucky River, identified by site ID. Leeches on Kentucky River Fish — Flotemersch et al. 79 Figure 2. Actinohdella pediculata attached to the inside surface of the operculum of a freshwater drum {Aplodinotus grunniens). processing, Placobdella pediculata Heming- way, 1908 was collected, and during laborato- ly identification of vouchered specimens, two additional leech species were collected - Mijzohdella luguhris Leidy, 1851 and Myzob- della reducta (Meyer 1940). Placobdella pediculata was collected from inside the operculum on the last gill arch of a freshwater drum [Aplodinotus grunniens (Ra- finesque)] at site KOI (Figure 2). Placobdella pediculata is not commonly encountered and found in the Great Lake and Mississippi River drainage systems (Klemm 1982, 1985). This leech species is host-specific to A. gmnniens, where it permanently attaches inside the operculum area (Klemm 1985; Bur 1994). Figure 3. Myzobdella luguhris attached inside the buccal cavity of a of a largemoutli bass {Microptems sdmokles). Figure 4. Piscicolaria reducta attached to the caudal fin of a spotfin shiner {Notropis spiloptenis) . Specimens of Myzobdella luguhris were collected from the buccal cavity of green sunfish [Lepomis cyanellus (Rafinesque)] and juvenile largemoutli bass [Microptems sab moides (Lacepede)] from site K02 and juve- nile spotted bass [Microptems punctulatus (Rafinesque)] from site K05 (Figure 3). Those of the spotted bass were consistently attached to the roof of the buccal cavity and difficult to detect. An additional specimen was collected posterior to the eye of a largemouth bass from site K03. Myzobdella luguhris is a common and widely-distributed opportunistic blood- feeding leech on fish (Klemm 1982, 1985). Any fish species occurring in North America is considered a potential host. Myzobdella reducta was collected from the caudal and anal fins of spotfin shiners (Figure 4) [Cyprinella spiloptera (Cope)] at sites K03 and K05, respectively, and on the caudal and pectoral fins of a logperch [Percina caprodes (Rafinesque)] at site K05. The spot- fin shiner (Cyprinella spiloptera) is a new host record for Myzobdella reducta. Myzobdella reducta is infrequently collected and has a scattered distribution in eastern North Amer- ica (Klemm 1982, 1985). It is also an oppor- tunistic blood-feeding leech on fish. The reported hosts of Myzobdella reducta are listed in Table 2. DISCUSSION Leeches are common in fresliwaters and generally more common in lentic than in lotic systems. As part of their normal life cycles, 80 Journal of the Kentucky Academy of Science 73(2) Table 2. Reported host fish of Myzobdella reducta (Meyer 1940). Fish species Common name References Ameiums melas (Rafinesque) Erimtjzon sucetta (Lacepede) Etheostoma hlennioides Rafinesque Etheostoma caertilemn Storer Etheostoma sagitta (Jordan and Swain) Etheostoma stigmaeum (Jordan) Etheostoma virgatmn (Jordan) Etheostoma zonale (Cope) Ictalunis punctMus (Rafinesque) Lepomis auritus (Linnaeus) Lepomis cijanellus Rafinesque Chaenohryttus gulosus (Cuvier in Cuvier and Valenciennes) Lepomis macrochirus Rafinesque Lepomis punctatus (Valenciennes in Cuvier and Valenciennes) Notemigonus crtjsoleucas (Mitcliill) Notropis atherinoides Rafinesque Cyprinella spiloptera (Cope) Percina aurantiaca (Cope) Percina caprodes (Rafinesque) Percina copelandi (Jordan) Percina evkles (Jordan and Copeland in Jordan) Percina macidata (Girard) Percina phoxocephala (Nelson) Percina sciera (Wain) Ponwxis nigromaculatus (Lesueiir ill Cuvier and Valenciennes) Tilapia aurea (Steindacliner) black bullhead lake cliubsucker greenside darter rainbow darter arrow darter speckled darter striped darter banded darter channel catfish redbreast sunfish green sunfish warmoutli Harms 1959, 1960 Price and Nadolny 1993 Bauer and Branson 1975; Bauer 1976; Murray et al. 1977 Bauer and Branson 1975; Erickson 1976; Kozel and Whittaker 1982 Klemm 1982 Bauer and Branson 1975 Bauer and Branson 1975 Bauer and Branson 1975; Erickson 1976, 1978 Harms 1959, I960; Nagel 1976; Wetzel 1982 Price and Nadolny 1993 Klemm 1972 Price and Nadolny 1993 bluegill Meyer 1946; Petty and Magnuson 1974 spotted sunfish Bootii and Aiiff 1978; Klemm 1985; Price and Nadolny 1993 golden shiner emerald shiner spotfin shiner tangerine darter logperch channel darter gilt darter Meyer 1954 White and Crisp 1973 This study Bauer 1976 White and Crisp 1973; Bauer and Branson 1975; Bauer 1976; White 1977; Schramm et al. 1981; Appy and Cone 1982; This study Bauer 1976 Bauer and Branson 1975; Bauer 1976; Erickson 1976 blackside darter Bauer and Branson 1975; Bauer 1976; Erickson 1976 slenderliead darter Meyer 1940, 1946; Page and Smith 1971; Erickson 1976 dusky darter Page and Smith 1971 black crappie Price and Nadolny 1993 blue tilapia Price and Nadolny 1993 many leeches parasitize a variety of hosts and feed on blood and body fluids. In doing so, they deprive their hosts of important nutrients (Sawyer, 1986). When an overabundance of leeches occurs in a system, it is generally considered to be an indicator of poor water quality, especially in lotic systems (McDonald et al 1990). Fish leeches (Glossiphonidae and Piscicoli- dae) commonly attach to various sites on the body of the host, including the pectoral, pelvic, dorsal, and caudal fins; the eyes; the interior of the gill chamber; the inside of the mouth cavity; and directly to the main body of the fish (as reviewed by Schulz et al. 2011). The presence of leeches in high numbers can negatively impact the recreational value of an aquatic system to local communities because people generally find them undesirable and view their presence on and in fish as an indicator of poor health. Opportunities to document the presence and extent of leeches on fish in freshwater systems are therefore warranted. Data presented in this study were derived from rapid examination of fish in the field supplemented with data collected during more thorough laboratory examination of vouchered fish specimens. As a result of this non^random study design, no estimates on the percentage of fish with leeches can be made from these data. Furthermore, even if it was possible to make these estimates, little is known about the expected occurrence rates of leeches in this system and it is, therefore, impossible to use these data to assess the condition of the Kentucky River. From a life history perspective, this study established new host and attachment loca- tions, the diverse presence of Myzobdella reducta on various fish species, and substan- tiated the high degree of host specificity of Leeches on Kentucky River Fish — Flotemersch et al. 81 Flacobdella pediculata for the freshwater drum. Given the paucity of work on the distribution and life histories of leeches in Kentucky, it is probable that additional research in the Kentucky River Basin and other basins in the Commonwealth of Ken- tucky would produce new host and distribu- tion records. As these records increase, the value of these organisms to serve as indicators of system condition will likewise increase. ACKNOWLEDGEMENTS The United States Environmental Protec- tion Agency through its office of Research and Development partially funded and collaborat- ed in the research described. It has been subjected to Agency review and approved for publication. Justicia Rhodus, Dynamac Cor- poration, provided editing and formatting services. LITERATURE CITED Appy, R. G., and D. K. Cone. 1982. Attachment of Mijzohddla lugubris (Hirudinea: Piscicolidae) to log- percli, Percina caprodes, and brown bullhead, Ictalurus nehulosus. T Am Microsc Soc 101:135-141. Bauer, B. H. 1976. Notes on the leeches found parasitizing some perciforme fishes in Tennessee. J Tennessee Acad Sci 51:9-10. Bauer, B. H., and B. A. Branson. 1975. The leech Fiscicolaria reducta parasitizing some percid fishes. T Kentucky Acad Sci 36:18-19. Booth, L. C., and J. V. Aliff. 1978. Some metazoan parasites from north Georgia fishes. Georgia J Sci 36:147-152. Bur, M. T. 1994. Incidence of tlie leech Actinobdella pediculata on freshwater drum in Lake Erie. J Great Lakes Res 20:768-770. Erickson, J. E. 1976. New host and distribution records for Fiscicolaria reduct-a Meyer 1940. J Parasitol 62:409. Erickson, J. E. 1978. Parasites of the banded darter, EtheostowM zonale (Pisces: Percidae). J Parasitol 64:899. Etnier, D. A., and W. C. Starnes. 1993. The Fishes of Tennessee. The University of Tennessee Press, Knox- ville, TN. Harms, C. E. 1959. Checklist of parasites from catfislies of northeastern Kansas. T Kansas Acad Sci 62:262. Harms, C. E. 1960. Some parasites of catfislies from Kansas. J Parasitol 46:695-701. Klemm, D. J. 1972. The leeches (Annelida: Hirudinea) of Michigan. The Michigan Academician 4:405-444. Klemm, D. J. 1982. Leeches (Annelida: Hirudinea) of North America. E PA-600/3-82-025. U.S. EPA, Environ- mental Monitoring and Support Laboratory, Cincinnati, OH. Klemm, D. J. 1985. Freshwater leeches (Annelida: Hirudinea). Pages 70-173 in D. J. Klemm (ed). A guide to the freshwater Annelida (Polychaeta, Naidid and Tubificid Oligocliaeta, and Hirudinea) of Nortli America. Kendall/Hunt Publishing Company, Dubuque, lA, Kozel, T. R., and F. H. Whittaker. 1982. Ectoparasites of tlie rainbow darter, Etheosto'um- caemleum Storer, from Harrods Creek, Oldham County, Kentucky. Proc Helmintliolog Soc Washington 49:138-139. Lazorchak, J. M., B. H. Hill, D. K. Averill, D. V. Peck, and D. J. Klemm (eds). 2000. Environmental monitoring and assessment program - surface waters: field operations and methods for measuring tlie ecological condition of non-wadeable rivers and streams. U.S. EPA, Cincinnati, OH. Meyer, M. C. 1940. A revision of the leeches (Piscicoli- dae) living on fresh-water fishes of North America. T Am Microsc Soc 59:354-376. McDonald, B., W. Borden, and J. Latlirop. 1990. Citizen stream monitoring: A manual for Illinois. Illinois Department of Energy and Natural Resources, ILENR/RE-WR-90/18, Springfield, IL. Available at: http ://www.epa. gov/bioindicators/iitml/leeches .html. Meyer, M. C. 1946. Further notes on the leeches (Piscicolidae) living on fresh-water fishes of North America. T Am Microsc Soc 65:237-249. Meyer, M. C. 1954. The larger animal parasites of tlie fresh-water fishes of Maine. State of Maine Depart- ment of Inland Fisheries and Game. Fishery Research and Management Division Bulletin No. 1:1-88. Murray, A. B., W. S. Nance, and D. C. Tarter. 1977. The occurrence of the leech Fiscicolaria reducta on Etheostoma blennoides from West Virginia. T Am Microsc Soc 96:412. Nagel, M. 1976. New distribution records for pisci colid leeches in Oklalioma. J Parasitol 62:494-495. Ohio EPA. 1989. Biological criteria for the protection of aquatic life, Vol. Ill: Standardized biological field sampling and laboratory methods for assessing fish sampling and macroinvertebrate communities. Ohio EPA, Ecological Assessment Section, Division of Water Quality Planning and Assessment, Columbus, OH. Page, L. M., and P. W. Smitli. 1971. The life liistory of the slenderhead darter, Percina plwxocephala , in tlie Embarras River, Illinois. Illinois Nat Hist Surv Biol Notes 74:1-14. Petty, L. L., and J. J. Magnuson. 1974. Lympliocystis in age 0 bluegills (Lepomis macrochirus) relative to heated effluent in Lake Monona, Wisconsin. J Fisheries Res Board of Canada 31:1189-1193. Pflieger, W. L. 1975. The fishes of Missouri. Missouri Department of Conservation, Columbia, MO. Price, W. W., and J. V. Nadolny. 1993. Fiscicolaria reducta (Hirudinea: Piscicolidae) from fishes in a subtropical Florida stream. J Helmintliolog Soc Wash- ington 60:130-134. Reynolds, J. B. 1983. Electrofisliing. Pages 147-163 in L. A. Nielsen and D. L. Johnson (eds). Fisheries techniques. American Fisheries Society, Betliesda, MD. 82 Journal of the Kentucky Academy of Science 73(2) Schramm, J. C., C. H. Hardman, and D. C. Tarter. 1981. The occurrence of Mijzohdella lugubris and Piscicolaria reducta (Hirudinea: Piscicolidae) on fishes from West Virginia. T Am Microsc Soc 100:427—428. Schuiz, C. A., M. V. Thomas, S. Fitzgerald, and M. Faisal 2011. Leeches (Annelida: Hirudinida) parasitizing fish of Lake St. Clair, Michigan. USA Comp Parasitol 78:73-83. Siddall, M. E., R. B. Budinoff, and E. Borda. 2005. Phylogenetic evaluation of systeinatics and biogeogra- pliy of the leech family Glossiplioniidae. Iiivertebr Syst 19:105-112. Stauffer, J. R., Jr., J. M. Boltz, and L. R. White. 1995. The fishes of West Virginia. Acad Nat Sci Philadelphia, Philadelphia, PA. Trautman, M. B. 1981. The fishes of Ohio, 2nd ed. Ohio State University Press, Columbus, OH. Wetzel, M. J. 1982. Kansas leeches (Annelida: Hirudinea) with notes on distribution and ecology. IL Technical Publications of tlie State Biological Survey of Kansas, University of Kansas 12:105-111. White, G. E. 1974. Parasites of the common white sucker {Catostomm commersoni) from the Kentucky River drainage. T Am Microsc Soc 93:280-282. White, G. E. 1977. New distribution records of fish leeches in the Ohio River. J Parasitol 63:1138. White, G. E., and N. H. Crisp. 1973. The occurrence of four leeches (Hirudinea: Rliynchobdellida: Piscicoli- dae) on Kentucky river drainage fishes. T Kentucky Acad Sci 34:47-48. Williams, J. I., and E. M. Burreson. 2006. Phyiogeny of the fish leeches (Oligocliaeta, Hirudinida, Piscicolidae) based on nuclear and mitochondrial genes and morpholo^. Zooiogica Scripta 35:627-639. J. Ky. Acad. Sci. 73(2):83-89. 2012. Size Structure of Fagus grandifolia^ Liriodendron tulipifera^ and Celtis occidentalis Populations in a Wetland Forest in Campbell County, Kentucky Richard L. Boyce^ Department of Biological Sciences, Northern Kentucky University, Highland Heights, Kentucky 41099 ABSTRACT Size structure of a tree population not only reflects its past history but also how it is likely to change in tlie future. Three species, all located in a wetland forest in northern Kentucky near the Ohio River, were examined. This forest is part of a network of newly established permanent plots in North America, and tlie acquisition of baseline data for this network is critical. The Fagus grandifolia (American beech) population was the dominant species in a mature stand of approximately 1 ha, while the Liriodendron tulipifera (tulip- poplar) and Celtis occidentalis (hackberry) populations were located in an adjacent second-growth stand (also ca. 1 ha) that has developed over tlie past 50 years. F. grandifolia had a close fit to a power law function, but the deviations from the function suggest tliat the population is not currently reproducing fast enough to replace itself. L. tulipifera, on the other hand, had a bell-shaped size distribution, indicating that it is no longer reproducing and will decrease in importance over time. C. occidentalis had a very close fit to a power law function, suggesting it wall maintain itself at current levels in the future. Current F. grandifolia levels in the younger secondary stand suggest that it will increase its importance in the future and will come to resemble the population in tlie mature stand. KEY WORDS: American beech, tulip poplar, hackberry, diameter class, Campbell County, size structure INTRODUCTION The age structure of a plant population is not only a reflection of the history of the population but also indicates how the popu- lation is likely to change in the future, barring extreme stochastic events (Gurevitch et al. 2006). For many plants, especially perennials, age is either difficult to measure or is less important than size, or both. This is especially important for trees, since size is much better correlated with suiwivorship and reproduction than actual age. Although trees can be aged with an increment borer, diameter is much easier to measure, and so size structure is more frequently reported. Age or size (diameter) distribution of trees in stands is often used to tell how the stand originated. Even-aged stands usually show a bell-shaped curve of number of trees vs. diameter class, sometimes with a long left tail (log-normal distribution; Smith et al. 1997). The trees in these stands all germinated or were released at about the same time. Trees that exhibit this kind of pattern are often shade-intolerant; they require some kind of disturbance to begin growth, but they don’t ' Corresponding author e-mail: boycer@nku.edu regenerate under their own shade. Balanced uneven-aged stands, where regeneration is replacing mortality, show a reverse J-shaped (also called L-shaped) curve, with large numbers of small trees and gradually smaller numbers of larger trees (Smith et al. 1997). These kinds of trees are often shade-tolerant; they can regenerate under their own shade and thus can continue to regenerate as the stand develops. Many studies have found that Fagus grandifolia Ehrh. (American beech) often has this kind of distribution (Schmalzer 1988; Cho and Boerner 1991; Houle 1991; Busing 1998; Swanson and Vankat 2000; Woods 2000, 2004; Harcombe et al. 2002; Galbraith and Martin 2005; Ozier et al. 2006). In stands with mixed species, size structure distributions must be used carefully. Different species can become established at different times after a forest is disturbed and a new stand is established. Although each population may be essentially even-aged, i.e., established at the same time, in aggregate the stand structure may have the reverse J-shape of a uneven-aged stand (e.g., Shotola et al. 1992; Lowenstein et al. 2000; Harcombe et al. 2002). And this stand structure may continue into the future, even though species compo- sition changes. The histoiy and possible future 83 84 Journal of the Kentucky Academy of Science 73(2) of each species can only be determined by examining them individually. Leak (1975) showed that when the loga- rithm of number of trees in each age class is plotted against the logarithm of age, the shape of the resulting curves will indicate the state of the population. A straight line indicates a stationary or balanced uneven-age stand. A concave (reverse J- or L-shaped) curve indicates an increasing population, whereas a convex (bell-shaped) cui*ve indicates a de- creasing population that is no longer repro- ducing enough to replace itself. While age and diameter increase together monotonically, they are not always related linearly. Lorimer (1980) found that age and diameter are related in F. grandifolia by a power law function, while a quadratic equa- tion described Linodendron tulipifera L. (tulip poplar). When a power law function holds, as for beech, log(age) is linearly propor- tional to log(diameter), but witli a quadratic function, as for tulip poplar, log(age) is almost linearly proportional to untransformed diame- ter. When there is a linear relationship between age and diameter, as for Celtis laevigata Willd. (sugar hackberiy; Bariy and Kroll 1999), then the relationship between age and diameter is similar to that of a power law function [i.e., log(age) is proportional to log(diameter)]. Provided the age-diameter distribution of each species is understood, Leak’s (1975) method can be applied to diameter data to determine the current stmc- ture of the population and how it is likely to fare over time as the stand develops. The aim of this study was to determine the current size structure of three species found in a wetland forest in Campbell County, northern Kentucky: F. grandifolia (American beech), L. tulipifera (tulip-poplar), and Celtis occidentialis L. (hackberry). All three of these species have similar, widely-overlapping ranges in the eastern United States, but they vaiy considerably in their life histories and require- ments. Fagus grandifolia is a relatively long- lived and slow-growing mesophytic species that is very shade-tolerant. It can grow on poorly- drained (but not flooded) soils, and it is usually found on acidic soils. It attains some of its best growth on alluvial soils of the Ohio River valley. Liriodendron tidipifera is shade-intolerant, but its fast growth and longevity allows it to persist in mature stands. It achieves its best growth on moderately moist, well-drained, and loose- textured soils and, like F. grandifolia, some of its best growth is found in the Ohio Valley. Celtis occidentalis is a small- to medium- size tree of only moderate longevity that is intermediate to tolerant for shade. It is widely tolerant of soil and moisture conditions; it is both drought-resistant and tolerant of occa- sional flooding. It achieves its best growth on valley soils (Burns and Honkala 1990). A number of population size structure descrip- tions have been published for F. grandifolia (Leak 1975; Schmalzer 1988; Cho and Boerner 1991; Woods 2004; Galbraith and Martin 2005) and L. tulipifera (Clebsch and Busing 1989; Busing 1998; Galbraith and Martin 2005; Ozier et al. 2006), but I am aware of only one for C. occidentalis (Swan- son and Vankat 2000). The forests of the southwest Ohio-southeast Indiana-northern Kentucky region have long been of interest because their location near three different forest types, including the Fagus~Acer forests to their north, Quercus- Canja forests to their west, and mixed meso- phytic forests to their south and east (Braun 1950). Braun (1914, 1916) studied this area, which she called the Melbourne Forest. More recently, it was studied by Bryant (1987), Biyant and Held (2004), and Boyce et al. (2012). This wetland forest is now also part of tire Ecological Research as Education Network Pennanent Forest Plot Project (EREN PFPP; http://erenweb.org/projed/carbon-storage-project/). Two 400-irf pennanent plots were established in 2012. The goals of EREN PFPP are to examine long-tenn changes in forest ecosystems across tlie U.S. and Canada. This study has three objectives: 1) deter- mine what the current size structure indicates about the development of these forests; 2) determine how the these populations are likely to to change in the future, if the past and current conditions continue; and 3) establish baseline data against which to measure changes in size structure of these three contrasting species in the future. MATERIALS AND METHODS This study was carried out at the St. Anne Wetlands Research and Education Center (39° 2' 4" N, 84° 22' 24" W) in Melbourne, 85 Fagus, Liriodendron, and Celtis Size Structure — Boyce Kentucky, U=S=A. This 100-ha wetland forest is located in Campbell County near the Ohio River and supports an extensive system of ephemeral ponds and streams. The study site is described in more detail in Boyce et ah (2012). Data for this study were collected in September-October 2011 from the same two adjacent sites (Mature Stand and Developing Stand; separated by a stream) described in that study. The approximate location and boundaries of the two sites are shown in Figure 1; each is ca. 1 ha in area. One of the EREN PFPP plots is located in the Mature Stand site, and the other is located in the Developing Stand area. Soils are silt loams in the Avonburg, Newark, Rossmoyne, and Wheeling series (Weisenberger et al. 1973). Community structure was sampled with three parallel transects in each stand, each separated by 20 m. Along each transect, trees within 2.5 m of the transect were sampled for a distance of 40 m. Species and diameter at breast height (dbh) for trees with dbh >5 cm was determined. Density (ha^b and basal area (m^ ha~b were then calculated. Population structure was determined by censusing each area for particular species. F. grandifolia was sampled from the Mature Stand only, while L. tuUpifera and C. occF dentalis were sampled only from the Devel- oping Stand. Diameter was remeasured; in this census, individuals with dbh <5 cm were also tallied. Data were grouped into 10-cm size classes, e.g., >0-10 cm, >10-20 cm, etc., and the numbers for each size class were log- transformed. The midpoints of each size class were also log-transformed for F. grandifolia and C. occidentalism but not for L. tuUpifera ^ due to the different relations between size and age exliibited by these species. Data were then graphed and analyzed following the method of Leak (1975). RESULTS & DISCUSSION Although total basal area is similar for the two sites, density was much larger in in the Developing Stand, indicating that average tree size was smaller (Table 1). The Developing Stand also contained more species of trees. Within it, Celtis occidentalis had the greatest density, while Liriodendron tuUpifera had the greatest basal area. Within the Mature Stand, Fagus grandifolia had the greatest density and Figure 1. Approximate area of Mature Stand (on left) and Developing Stand (on right) in 1949 (A) and 2010 (B). Each stand is ~1 ha in size. Note that Mature Stand has complete canopy cover on both dates, while Developing Stand contains only scattered trees in 1949. The 1949 aerial photograph was obtained from USGS (2010), and the 2010 photograph was obtained from LINK- GIS (2012). the second largest basal area. While the two sites in this depression forest have similar elevations and are located next to each other, their histories are quite different. In 1949, the area of the Mature Stand had a completely closed canopy, while the area of the Develop- ing Stand contained only a few scattered trees (Figure 1). The older age of the mature forest is reflected in the larger average tree size. The F. grandifolia population in the Mature Stand shows declining numbers with increas- ing diameter class (Figure 2). While the fit between the power function (which appears as a straight line on a log-log plot) and the data is good (R^ = 0.85), the data distribution is slightly concave. This indicates that while F. grandifolia is still reproducing, it is not doing so at a rate that will maintain its current 86 Journal of the Kentucky Academy of Science 73(2) Table 1. Density and basal area of tree speeies in the Developing and Mature Stands. Data colleeted from trees with dbh S:5 cm only. Developing stand Mature stand Species Densit)' (ha'‘) Basal area (in- ha ‘) Density (ha 0 Basal area (irf ha 0 Acer negundo 16.7 0.58 - - Asimina triloba 33.3 0.11 - - Carija ovata 16.7 0.63 - - Celtis occidentalis 250.0 1.93 16.7 0.04 Corniis florida 16.7 0.06 - - Fagus grandifolia 116.7 1.70 150.0 14.20 Fraxinus aniericana 16.7 0.20 - - Li riodend ron tulipifera 216.7 28.42 16.7 6.98 Prunus serotina 33.3 7.40 - - Quercus palustris - - 33.3 16.15 Sassafras albidum - - 33.3 0.22 Ulmus rubra 100.0 0.93 16.7 0.04 Totals 816.7 41.96 266.7 37.62 population (Leak 1975). A number of other studies have found similar trends for F. grandifolia (Leak 1975; Cho and Boerner 1991; Woods 2004; Galbraith and Martin 2005). This species is quite long-lived, how- ever, and it has a much higher basal area and density than any other species in the Mature Stand, so it is likely to retain its dominance for some time. Lmodendron tulipifera clearly has a beU- shaped population structure (Figure 3), indicating it is no longer reproducing. Tliis is often seen in this species, at least in older stands (Clebsch and Busing 1989; Busing 1998; but see Schmalzer 1988; Galbraith and MaiUn 2005; Ozier et al. 2006). Wliile tliis species is not currently reproducing, it is quite long-lived, and so it is expected to retain its dominance in the Develop- ing Stand for some time. Successful regeneration could begin again if disturbance were to create gaps at of least 1.25-2.50 ha (Bums and Honkala 1990). Figure 2. Population structure plot for Fagus grandifolia in the Mature Stand. Axes are on logaritiimie scales; x-axis is dbh size-class mid point, and y-axis is number of trees in tlie size class. Data points are connected \Mth solid line segments. The dotted line is a power function fitted to tlie data points: No. Fagus = = 0.85. Fagus, Liriodendron, and Celtis Size Structure — Boyce 87 dbh (cm) Figure 3. Population structure plot for Liriodendron tulipifera in the Developing Stand. Y-axis is on a logarithmic scale; x-axis is dbh size-class mid point, and y-axis is number of trees in the size class. Data points are connected with solid line segments. Figure 4. Population structure plot for Celtis occidentalis in the Developing Stand. Axes are on logarithmic scales; x- axis is dbh size-class mid point, and y-axis is number of trees in the size class. Data points are connected with solid line segments. The dotted line is a power function fitted to the data points: No. Fagus = 172. = 0.94. 88 Journal of the Kentucky Academy of Science 73(2) Unlike F. grandifolia and L. tuliperifa, only one previous study on tlie population structure of C. occidentalis appears to have been pub- lished (Swanson and Vankat 2000), which showed a convex stmcture on a log-log scale, indicating an uneven-age stand that is increasing in importance. These same transfonnations were chosen for this study, based on tire relationship beh\^een age and diameter of the closely related C. laevigata (Barry and Kroll 1999). The data show a very close fit to the power law function (R^ = 0.94; Figure 4), indicating that C. occidentalis is replacing itself This is consistent with pre\4ous work showing that it is has moderate to good shade tolerance (Bums and Honkala 1990). Unlike the otlier two species in tliis study, it is relatively short-lived. These findings suggest that, in the future, C. occiden- talis is expected to retain a presence in the Developing Stand similar to that seen today. It is, however, much less important in the Mature Stand, so it is possible that shade cast by tire dense canopy of F. grandifolia-domiiva.ted forest may inliibit the regeneration of C. occidentalis. Fagus grandifolia basal area in the Devel- oping Stand is currently low, but it has the third-highest density, after C. occidentalis and L. tidipifera. Over time and in the absence of large gap-creating disturbances, F grandifolia is expected to increase its basal area in this stand and become dominant, as it currently is in the Mature Stand; a few large L. tidiperifa will remain, while C. occidentalis maintains a presence similar to or smaller than today. Thus, the Developing Stand is expected over the long term to become more like today’s Mature Stand, at least in the absence of major gap-forming disturbances. ACKNOWLEDGEMENTS Data for this study were collected by students in the Fall 2011 Plant Ecology course taught at Northern Kentucky University: Eliz- abeth Billiter, Joshua Blackaby, Michelle Bowl- ing, Tyler Broering, Molly Brown, Brittany Burk, Christine Clements, Crystal Courtney, Allen Davis, Gwylenn DesPres, Amanda Farley, Courtney Firth, Sarali Hamilton, Erin Hamm, Ashley Haws, Jodi March, Jessica Miller, Amanda Moermond, Patrick O’Heam, Payal Patel, Allison Rigger, and Alicia Sullivan. The Campbell Conservancy and St. Anne Convent kindly granted permission to us to use their land for this study. Tliis paper benefitted from a critical review by R. D. Durtsche. LITERATURE CITED Barry, D., and A. J. Kroll 1999. A ph)'tosociological description of a remnant bottomland hardwood forest in Denton County, Texas. Texas J Sci 51:309-316. Boyce, R. L., R. D. Durtsche, and S. L. Fugal. 2012. Impact of tlie invasive Lonicera maackii on stand transpiration and ecosystem hydrolo^ in a wetland forest. Biol Invasions 14:671-680. Braun, E. L. 1914. The physiograpliic ecolo^ of tlie Cincinnati region. Ph.D. Thesis, University of Cincinnati, Cincinnati, OH. Braun, E. L. 1916. The physiographic ecology of the Cincinnati region. Bull Ohio Biolog Surv 2:113-211. Braun, E. L. 1950. Deciduous forests of eastern North America. Hafner Publishing, Company, NY. Bryant, W. S. 1987. Structure and composition of the old- rowth forests of Hamilton County, Ohio and environs. Pages 317-324 in R. L. Hay, E. W. Woods and H. R. DeSelm (eds). Proceedings of the 6tli Central Hardwood Forest Conference, Nashville, TN. USDA Forest Seivice, Northern Research Station, Newtown Square, PA. Accessed on: 27 April 2012. Available at: http:/Av\^av. ncrs .fs .fed. us/pubs/cli/ch06/C Hvolume06page3 1 7.pdf Biy^ant, W. S., and M. E. Held. 2004. Forest vegetation in Hamilton County, Ohio: a cluster analysis and ordina- tion study. Pages 312-321 in D. A. Yaussy, D. M. Hix, R. P. Long and P. C. Goebel (eds). Proceedings of the 14th Centred Hardwoods Forest Conference, Wooster, OH. General Technicid Report NE-316. USDA Forest Service, Northeastern Research Station, Newtown Square, PA. Accessed on: 27 April 2012. Available at: http:/Av\\^v. fs . fed . us/ne/ ne\\4:own_square/publications/ technical_reports/pdfs/2004/3 1 6papers/BryantGTR3 1 6.pdf Burns, R. M., and B. H. Honkala. 1990. Silvics of Noiili America: 1. Conifers; 2. Hardwoods. Agricultural Hand- book 654. USDA Forest Sendee, Wasliington, DC. Accessed on: 27 Aprd 2012. Available at: http:/Av\nv.na. fs.fed.us/spfo/pubs/sil\dcs_manuaFtable_of_contents.htm. Busing, R. T. 1998. Composition, structure and diversity of cove forest stands in tlie Great Smoky Mountains; A patch dynamics perspective. J Veg Sci 9:881-890. Clio, D-S., and R. E. ]. Boerner. 1991. Canopy disturbance patterns and regeneration of Querciis species in two Ohio old-growih forests. Vegetatio 93:9-18. Clebsch, E. E. C., and R. T. Busing. 1989. Developing succession, gap dynamics, and community structure in a southern Appalachian cove forest. Ecol 70:728-735. Galbraith, S. L., and W. H. Martin. 2005. Three decades of overstoiy^ and species change in a mixed mesophytic forest in eastern Kentucky. Castanea 70:115-128. Gurevitch, J., S. M. Scheiner, and G. A. Eox. 2006. The ecology of plants, 2nd ed. Sinauer, Sunderland, MA. Harcombe, P. A, et al. 2002. Stand dynamics over 18 years in a southern mixed hardwood forest, Texas, USA. J Ecol 90:947-957. Fagus, Liriodendron, and Celtis Size Structure — Boyce 89 Houle, G. 1991. Regenerative traits of tree species in a deciduous forest of northeastern North America. Hokrctic Ecol 14:142-151. Leak, W. B. 1975. Age distribution in virgin red spruce and nortliern hardwoods. Ecol 56:1451-1454. LINK-GIS (Nordiem Kentucky Area Planning Commission). 2010. LINK-GIS j We're Al Connected. Accessed on: 27 April 2012. Available at: littp7Avww.linkgis.org^. Lorimer, C. 1980. Age structure and disturbance history of a soutliern Appalachian virgin forest. Ecol 61:1169- 1184. Lowenstein, E. F., P. S. Johnson, and H. E. Garett. 2000. Age and diameter structure of a managed uneven-aged oak forest. Canadian J Forest Res 30:1060-1070. Ozier, T. B., J. W. Groninger, and C. M. Ruffner, 2006. Community composition and structural changes in a managed Illinois Ozark Hills forest. Am Midland Natural 155:253-269. Schmalzer, P, A. 1998. Vegetation of the Obed River gorge system, Cumberland Plateau, Tennessee. Casta- nea 53:1-32. Shotola, S. J., G. T. Weaver, P. A. Robertson, and W. C. Ashby. 1992. Sugar maple invasion of an old-growth oak-liickory forest in southwestern Illinois. Am Midland Natural 127:125-138. Smitli, D. M., B. C. Larson, M. J. Kelty, and P. M. S. Ashton. 1997. The practice of silviculture: Applied forest ecolo^, 9tli ed. Wiley, New York. Swanson, A. M., and J. L. Vankat. 2000. Woody vegetation and vascular flora of an old-growtli mixed-mesopliytic forest in Southwestern Ohio. Castaeea 65:36-55. uses (United States Geological Survey). 2010. Eartli- Explorer. Full Display of AR1IP0000150127. Accessed on: 27 April 2012. Available at: http://eairtliexplorer.usgs. gov/form/metadatalookup/?coIlection_id = 4660&entity_ id= ARlIP0000150127&pageView= 1. Weisenberger, B. C., C. W. Dowell, T. R, Leathers, H. B. Odor, and J. J, Richardson. 1973. Soil survey of Boone, Campbell, and Keeton Counties, Kentucky. USDA Soil Conservation Service, Washington, DC. Woods, K. D. 2000. Long-term change and spatial pattern in a late-successional hemiock-nortliern hardwood forest. J Ecol 88:267-282. Woods, K. D. 2004. Intermediate disturbance in a late- successional hemlock-northern hardwood forest. J Ecol 92:464^76. J. Ky. Acad. Sci. 73(2):90-95. 2012. Evaluation of Cultivars of Sunflower (Helianthus annuus L.) and Selected Environments for Production of Cut Elowers Christopher G. Ferguson, Pavani G. Vuppalapati, Martin J. Stone, and Elmer Gray^ Department of Agriculture, Western Kentucky University, Bowling Green, Kentucky 42101 ABSTRACT Sunflower Helianthus annuus is native to tlie United States and was domesticated by American Indians many centuries ago. Sunflowers exliibit wide variation and are grown worldwide for multiple purposes, including cut flowers. The present cut flower study involved 23 cultivars, three local environments, and performances data included in seed company catalogs, for cut flower production in Central Kentucky. Plant development data included seedling emergence, stem diameter, days to flowering, height at flowering, number of heads (marketable and nonmarketable) per plant, head diameter, vase life, and personal preference. There were evidences of both genetic and environmental variations for the plant characteristics. Flowering heads were rated as pleasing by florists and laypersons. The number of heads produced by some combinations of cultivars and emironments would support profitable production. Further refinement is needed in the harv^esting schedule and handling to reduce frequency of nonmarketable heads, to extend vase life, and to permit shipping greater distances. The results indicated that cut flowers have potential as a niche crop for Central Kentucky. KEY WORDS: Sunflower, Helianthus annuus, cut flower. Western Kentucky Botanical Gardens, niche crop INTRODUGTION Sunflowers of the genus Helianthus are among the oldest, most widely grown, and taxonomically complex of all crops. More than 30 centuries ago, Indians in Southwest United States domesticated the native sunflower. Sunflower culture spread throughout the Indian Tribes of North America where the plants were widely used for food and nonfood purposes. Seeds were cracked and eaten like nuts or were ground and separated into flour and oil Nonfood uses included dyes, medi- cines, textiles, and construction materials. Early Spanish explorers introduced sunflow- ers into Europe where they spread into Russia reaching their zenith as a crop. In the latter part of the 19“‘ century, the sunflower was reintroduced into the United States for its recognized value as a silage crop and potential source of vegetable oil (Putt 1997). Helianthus includes 50 species that are native to the Americas and found in the United States. Taxonomic complexity has resulted from natural hybridization, difference in ploidy levels, and environmental interac- tions. Seiler and Rieseberg (1997) provided an extensive review of germplasm resources in sunflower. The annual species, H. annuus ' Corresponding author e-mail: elmer.gray@wku.edu (L.), includes plants variable for height, head diameter, number of days to flowering, photoperiod responses, and environmental adaptation. This wealth of diversity has enabled the development of cultivars for ornamental and cut flower uses. Worldwide distribution of sunflowers has been enhanced by the beauty and allure of the flowering heads. The cut flower industiy is the most recent extension of sunflower utilization. The ongoing goal of sunflower research at Western Kentucky University is to identify the geno- types and environmental conditions that are suitable for sunflower production in South Gentral Kentucky and that produce floral characteristics sought by area gardeners and florists. The present study objective was to observe the variability in cut flower related traits and to compare the relative contribu- tions of genetics and environments to that variation. MATERIALS AND METHODS Evaluation data were attained from three local experiments and from information pro- vided by the seed companies. In 2004, 18 sunflower cultivars were grown at Bowling Green (BGl). In 2011, 18 cultivars, including 13 from the 2004 study (Table 1), were grown at Bowling Green (BG2) and in Owensboro (OB), Kentucky. The design was a randomized 90 Sunflower Cut Flowers — Ferguson et at. 91 Table 1. Company sources for seeds of sunflower cultivars evaluated in different environments. Environments: BGl BG2 and OB' Cultivars Company seed sources" Moulin Rouge J J Ruby Eclipse V V Panache V Pacino Gold J Giant Greystripe .1 vs Velvet Queen J .1 Teddy Bear J J Apricot Twist V TS Peach Passion V V Valentine J V Sunrich Lemon J J Sunrich Orange J J Double Dandy V Sunbright .1 J Moonbright J Sunbeam J J Soraya .1 Ikarus J ST Solar Eclipse B Little Becka J Italian White PS Eull Sun Early Russian V EnWroiiments: BGl, Bowling Green 2004; BG2 and OB, Bowling Green and Owensboro 2011. ^ Seed companies: J-Johnny’s, V-^^esey's, \^S-Vic'tor)'’.s Seeds, ST-Swallow- tail, TS-Territorial Seed, B-Buq>ee, PS-Penya Seed. complete block with three replications. Each entry consisted of one lO^plant row with 30 cm spacing within the row and 60 cm spacing between rows. A complete fertilizer (N 19%, P2O5 19%, K2O 19%) was incoi*porated prior to seeding at the rate of 885 kg ha“f Weed control was obtained by a pre-emergence application of pendimethalin (1.12 kg A.l. ha~0 to control grass weeds, plus limited hand weeding. Although descriptive data provided by the seed companies reflected observations from different locations, the data were includ- ed in the evaluations (Table 2). The cultivars were selected to represent dwarf, normal, and large forms of H. annuus. Plant development data were collected from emergence through head harvest. Heading and flowering data were taken weekly during the flowering season. At each haiwest, all flowering heads were removed, counted, and classified as marketable or non-marketable. Classification of marketability was based on head deformity, pest damage, over maturity, or other undesir- able characteristics. Data were subjected to analysis of variance procedures to determine whether environ- ments differed significantly and to compare cultivar performance within environments. Linear correlation was used to compare cultivars for consistency in the expression of characteristics over environments (Steel and Torrie 1980). Consistency expression of a characteristic in different environments is evidence of a degree of genetic control. Cultivar popularity evaluations and vase life observations were based upon three freshly cut flowering heads placed in a vase contain- ing commercial strength 'Flora-life’ and water maintained at room temperature (approxi- mately 22°C). Individuals from the University and its greater community rated their personal preferences on a subjective scale. Vase life was based on the number of days to petal drop. RESULTS AND DISCUSSION Combinations of specific cultivars and environments and company sources of seeds are presented in Table 1. Comparisons of Table 2. Average and range of sunflower cultivars for selected criteria mfliin different environments. Criteria Environments' BGl Range- BG2 Range OB Range sc Range Emergence (%) NA^ 56 (13-87) 59 (23-93) NA Plants/cultivar NA 17 (4-26) 18 (7-28) NA Days to flowering (DTE) 58 (42-75) 62 (54-77) 60 (54-76) 78 (55-120) Height at flowering (m) 1.1 (0.6-L8) 1.5 (0.7-2.7) 1.8 (0.5-3.0) 1.4 (LO-2.0) Heads/plant 14 (1^0) 23 (1-62) 36 (1-101) NA Marketable heads/plant NA 10 (0-28) 27 (1-73) NA Non-marketable heads/plant NA 13 (0-34) 9 (0-28) NA Head diameter (cm) 7 (4-13) 8 (4-13) 8 (5-14) 13 (9-25) Stem diameter (cm) NA 2.7 (1-5) 3.1 (1-6) NA ' Environments; BGl. Bowling Green 2004; BG2 and OB, Bowling Green and Owensboro 2011, SG, Seed company information provided with cultivar seeds. ^ Range among cultivars. ^ NA, data not available. 92 Journal of the Kentucky Academy of Science 73(2) Table 3. Linear correlation coefficients (r) and coefficients of determination (r^ X 100) between sunflower cultivar performances in different environments. Environments^ Criteria" BGl vs. BG2 BGl vs. OB BGl vs. sc BG2 vs. OB BG2 vs. SC OB vs. SC r r r r‘ r r r ri r r r Plants/cultivar NA^ NA NA 0.62**^ 38 NA NA Days to flowering (DTF) 0.74** 55 0.76** 58 0.07 0 0.80** 64 0.29^^ 8 0.07^^ 0 Height at flowering 0.75** 56 0.76** 58 0.71** 50 0.96** 92 -0.34^’^ 12 -0.3Ps 10 Heads/plant 0.93** 86 0.74** 55 NA 0.89** 79 NA NA Marketable heads/plant NA NA NA 0.86** 74 NA NA Non-marketable heads/plant NA NA NA 0.96** 92 NA NA Head diameter 0.85** 72 0.76** 58 0.45^^ 20 0.82** 67 0.4P^ 17 0.05^^ 0 Stem diameter NA NA NA 0.63** 40 NA NA ‘ Environments; BGl, Bowling Green 2004; BG2 and OB, Bowling Green and Owensboro 201 1; SC, Seed company information provided with cultivar seeds. - KA, data not available. ^ KS and ** indicate non-significant (P > 0.05) and highly significant (F < 0.01); re.specbv'ely. environments for expression of a given trait were based upon cultivars common to the environments being compared. Averages and ranges in cultivar performance for the various criteria are included in Table 2. Linear correlations (r) and coefficients of determina- tion (r“) given in Table 3 indicate the level of consistency of cultivars in expression of a trait over the environments. Quantitative data on seedling emergence were obtained for BG2 and OB. Both environments had low emergence, but the averages (56 and 59%) and cultivar ranges (13-87 and 23-93%) were similar. In addition, the average number of plants in the two environments did not differ significantly (F > 0.05). The significant correlation (0.62) and magnitude of the coefficient determination (38%) indicated that both the environments and cultivars contributed to seedling emer- gence and plant survival. For the existing conditions, the environmental effects were greater than the genetic effects. It was obseiwed that heavy rainfall following planting resulted in soil crusting and, conse- quently, lowered seedling emergence. Gay, et al. (1991) reported that temperature is the most important environmental factor influ- encing germination of sunflowers and that the optimum is near 26°C. Germination is affect- ed by seed characteristics including dormancy and aging, moisture content, oil composition, seed size, and seed shape. In the present study, attempts to correct stand differences were unsuccessful because replants were overshadowed and rarely attained full devel- opment. A more effective approach would be planting at higher density and thinning to the desired stand. Days to flowering (DTF) did not differ significantly (F > 0.05) for BGl, BG2, and OB; however, these environments had signif- icantly lower DTF than those provided by seed companies (SG). Gorrelations for DTF were positive and highly significant for all comparisons except those involving SC which were non-significant (F > 0.05). These high correlations for DTF across environments support the results reported by Russell (1953) who observed correlation coefficients in the range of 0.86 to 0.91 for DTF. Average plant height at the time of flowering for cultivars in the three experi- ments and seed catalog data ranged from 1.1 to 1.8 m and was significantly lower for BGl than for the other environments. Skoric (1988), as cited by Seiler (1997), reported that stem length of commercial sunflowers ranged from 0.5 to 5.0 m with most cultivars included between 1.6 to 1.8 m. High correlations and determinations for cultivars over environments for both days to flowering and height at flowering were evidence of both genetic and environmental influences on flowering. Determination coef- ficients near 50% suggest that environmental and genetic effects were about equal, and that flowering in sunflowers is complex and variable. In a review of floral initiation, Seiler (1997) reviewed research results in which different genotypes responded as short-day, long-day, or day-neutral. Doyle (1975) report- ed that floral initiation was influenced more by temperature than by photoperiod. Sunflower Cut Flowers — Ferguson et al 93 Table 4. Floral characteristics of sunflower cultivars receiving high personal preference ratings as cut flowers. Cultivars (alphabetical) . Flower color Head' diameter (cm) Vase life' (days) Ikrarus light yellow 5 11 Litde Becka red/orange 5 11 Moulin Rouge deep burgundy 6 8 Sunbeam golden yellow 12 13 Valentine pale yellow 6 15 Velvet Queen burgundy red 7 13 ‘ Data are averages for BG2 and OB environments. Total number and marketable heads/plant were significantly (P < 0.05) higher in OB than in BGl and BG2, whereas non-marketable heads were more frequent in BG2 than in OB (P < 0.05). Non-marketable heads included cultivar-specific defomiities, pest induced inju- ries, and over-maturity. Some cultivars did not produce symmetrical heads desired by florists. Although sunflowers are susceptible to numer- ous insects and no pest control measures were applied, likely because the low intensity of sunflower production and isolation of environ- ments were not conducive to pest buildup. Over- maturity was the major factor in head deterio- ration, indicating that weekly harvest intervals were too long. Correlations of head production by cultivars over environments BGl vs. BG2, BGl vs. OB, BG2 vs. OB were highly significant (P < 0.01) for total heads. Marketable head production by cultivars was significantly corre- lated (P < 0.01) between BG2 and OB; the results were similar for non-mai'ketable heads. Average head diameter for cultivars in BGl, BG2, and OB ranged from 7 to 8 cm and were not significantly (P > 0.05) different, but appeared to differ from the 13 cm average diameter reported for the SC environment. Using a compilation of seed company infonna- tion was useful in the present study. However, new cultivars should be grown on small scale for observation on plant adaptation and head characteristics before making a large investment. Average stem diameter did not differ significantly between BG2 and OB; however, cultivars varied within the environments and that variation was significantly correlated between the two environments. Cultivars with larger stems usually had greater heights, fewer but larger heads, and occasional lodging. Tall robust plants were more difficult to harvest for cut flowers. Personal preferences for cultivars as cut flowers are presented in Table 4 and Figure 1. Cultivars receiving high ratings for BGl, BG2, and OB included more cultivars with the traditional yellow color; however, ‘Moulin Rouge,’ a highly preferred cultivar, had a deep burgundy color. With exception of ‘Sunbeam,’ the preferred cultivars had head diameters smaller than the average for all cultivars. Preferences appeared to be based upon overall appearance rather than specific color or head size. Vase life of cut flowers is critical to econom- ical production and marketing. The present cultivars exliibited vase life range of 1 to 15 days before post-harvest petal wilt occurred. Most of the cultivars had a vase life of 11 to 12 days when placed in a commercial strength solution of water and Flora-life at room temperature (approximately 22°C). In the harvest procedure only fully open heads were harvested leaving partially open heads for the next weekly haiwest. This delay consumed a portion of the vase life or resulted in overly mature heads that were classified as non- marketable. Study is needed to determine the earliest stage at which heads can be harvested without impairing normal development. Extension of vase life would enable cut flowers to be shipped greater distance and be enjoyed for longer periods. SUMMARY The results indicate that H. annuus sun- flowers were adapted to the soils and other environmental conditions of South Central Kentucky. Cultural practices applicable to commonly grown garden and field crops were suitable for sunflower production. Expressions of plant and floral characteristics were influ- enced by both cultivars and environments. Head production was sufficient to support an economical enterprise, but improvements in management practices are needed. The week- ly harvest schedule resulted in many heads becoming too mature and non-marketable. More frequent harvests would increase mar- ketable heads and extend shipping qualities and vase life. Cut flowers from the different cultivars were largely pleasing and acceptable by both lay and professional individuals. The most desirable characteristics of the plants included smaller heads and longer stems. These traits were exhibited by cultivars 94 Journal of the Kentucky Academy of Science 73(2) Little Becka Ikarus Moulin Rouge Sunbeam Valentine Velvet Queen Figure 1. Visual characteristics of sunflower cultivars receiving high personal preference ratings as cut flowers. included in the study; however, their expres- sion could be enhanced by higher planting densities and earlier head harvests. Collec- tively, the results indicate that sunflower cut flowers are potentially a profitable crop for Central Kentucky producers. ACKNOWLEDGEMENTS This study received ongoing support of Western Kentucky University through the Department of Agriculture for supplies and for personal assistance by William T. Willian, Roger L. Dennis, Yao Xue, and Joseph O. Reynolds, and through Graduate Studies and Research for graduate assistantship support. Sincere appreciation and thanks are expressed to the Western Kentucky Botanical Garden, Owensboro, Kentucky, for providing a study site and accommodations. LITERATURE CITED Doyle, A. D. 1975. Influence of temperature and day length on phenology of sunflowers in the field. Aust J Exp Agr Anim Husbandry 15:88-92. Gay, C., F. Corbineau, and D. Come. 1991. Effects of temperature and oxygen on seed germination and seedling growl:!! in sunflower {Helianthus annuus L.) Env Exp Bot 31:193-200. Putt, E. D. 1997. Early liistory of sunflower. Pages 1-19 in A. A. Schneider (ed). Sunflower technolog)^ and production. Am Soc Agron, Agronomy Series 35, Madison, Wl. Russell, W. A. 1953. A study of the inter-relationships of seed yield, oil content, and other agronomic characters with sunflower inbred lines and their top crosses. Can J Agr Sci 33:291-314. Sunflower Cut Flowers — Ferguson et al. 95 Seiler, G. J. 1983. Anatomy and morphology of sunflower. Pages 67-111 in A. A. Sclineiter (ed). Sunflower technology and production. Am Soc Agron Agronomy Series 35, Madison, WI, Seiler, G. J., and L. H, Rieseberg. 1997. Systematics, origin, and germplasm resources of the wild and domesticated sunflower. Pages 21-65 in A. A. Schneiter (ed) Sunflower technology and production. ASA, CSSA, and SSSA. Madison, WI. Skoric, D. 1988. Inside sunflower breeding. J Edible Oil Ind 25:1-90. Steel, R. G. D., and J. H. Torrie. 1980. Principles and procedures of statistics. Pages 272-277, 2nd ed. McGraw-Hill Book Company, NY. J. Ky. Acad. Sci. 73(2):96-100. 2012. Lady Beetle Composition and Abundance in Sweet Com Bordered by Pasture, Buckwheat or Sunflower Companion Plantings John D. Sedlacek/ Karen L, Friley, and Kirk W. Pomper College of Agriculture, Food Science and Sustainable Systems. CRS, Kentucky State University, Frankfort, KY 40601 ABSTRACT To determine the effect habitat management/border plantings have on beneficial insect diversity and abundance in agricultural crops, sweet corn, Zea mays L. ‘Garrison®’, was grown in replicated plots on Kentucky State University’s Agricultural Research and Demonstration Farm. Each 25 m X 12 m plot was bordered on each length by a 2 m wide border of un=mowed pasture, buckwheat {Fagopyrum esculentum Moench), or dwarf sunflower {Heliathus annuus L. var. ‘Big Smile’). Yellow sticky traps 15 cm X 15 cm were used to capture lady beetles weekly through antliesis. Pink lady beetle, Coleomegilla maculata (DeGeer); Asian lady beetle, Harmonia axyridis (Pallas); spotless lady beetle, Cyclonedo munda (Say); and seven spotted lady beetle, Coccinella septempunctata L. were caught in this study. The pink lady beetle was the most abundant species overall in all three borders and in the sweet corn plots wdtli 79% and 94%, respectively. There were greater numbers of pink lady beetles in buckwheat (P < 0.00001) and sunflower (P = 0.0064) borders than pasture borders. However, there were no differences among populations of any of the lady beetle species in any of tlie sweet corn plots. Pink lady beetles were more numerous in sweet corn bordered by buck'wheat (P ^ 0.05), sunflower (P < 0.0001) and pasture borders (P ^ 0.03) tlian in the borders themselves. KEY WORDS: Lady beetles, conservation biological control INTRODUCTION Reliance on insecticides for control of insect pests in sweet corn has caused concern for the development of insecticide resistance (Sparks 1981) and mortality of beneficial insects and other non-target arthropods (Musser and Shel- ton 2003), including honeybees foraging in intensively treated crops (Johnson et al. 2006). Other potential problems involve worker expo- sure to insecticides during mixing, application and commodity harvest, surface water and groundwater contamination, and the sustain- ability of conventional production systems (National Research Council 1989, 1996). Thus, there is a need for more ecologically sound pest management systems and decreased emphasis on insecticide applications. Many researchers have suggested that non- crop vegetation present in crop field margins can ser\^e as refuges and attract populations of predaceous arthropods and parasitoids, as well as increase the diversity of all arthropods (Dennis and Fry 1992). In most cases, predaceous arthropods such as ground bee- tles, syq3hid flies, and spiders have been studied in crop-border systems (van Emden ' Corresponding author e-mail:john. sedlacek@kysu.edu 1965; Pollard 1968; Altieri and Whitcomb 1980; Sunderland and Samu 2000). Farming practices utilizing ecologically based insect pest control offer ways to potentially lower input costs, decrease reli- ance on insecticides and nonrenewable re- sources, increase biodiversity, and obtain premium prices for agricultural commodities compared to those conventionally produced (Greene 2001). However, as farmers attempt to establish alternative agricultural entei'pris- es, a major constraint to the adoption of sustainable practices is the lack of research- backed information on ecologically based insect management and control strategies, especially for ear pests of sweet corn. Lady beetles have been recognized as important predators in agricultural systems. In general, lady beetles have been reported to be attracted to annual flowering plants in- cluding sunflower {Heliathus annuus L.) and the exotic buckwheat {Fagopyrum esculentum Moench) (Dufour 2000; Ambrosino et al. 2006; Adedipe and Park 2010; Woltz et al. 2012). However, additional information con- cerning lady beetles and other beneficial insects is needed in conjunction with border rows providing floral resources so that effec- tive and sustainable pest control options can 96 Lady Beetles in Sweet Com, Annual Borders — Sedlacek et al 97 Table 1. Species of lady beedes caught in buckwheat, sunflower and pasture borders during 2009 at tlie Kentucky State University Research and Demonstration Farm in Franklin County Kentucky. Pink lady beetle, Coleomegilla macidata (DeGeer) Asian lady beetle, Hamwnia axyridis (Pallas) Spotiess lady beetle, Cydoneda miinda (Say) Seven-spotted lady beetle, Coccinella septempunctata L. be identified and employed. Development of conservation biological control tactics such as habitat management may be expected to boost small farm income and benefit small farm communities. Thus, the objective of this research was to determine the effect habitat management/border planting has on lady beetle diversity and abundance in buckwheat, sunflower and pasture border rows and the sweet corn planted between pairs of each of these border types. MATERIALS AND METHODS Sweet corn, Zea mays ‘Garrison®', was planted on 19 June 2009 using conventional farming practices in 25 m X 12 m plots on Kentucky State University's Agricultural Re- search and Demonstration Farm in Franklin County, Kentucky. Each sweet com plot was bordered on each side of its length by a 2 m wide border of one of three treatments. The three treatments were 1) un-mowed pasture, 2) buckwheat (Fagoptjmm esculentum Moench) and 3) dwarf sunflower (Heliathus annuus L. var. ‘Big Smile'). Buckwheat borders were planted on 7 July using a Tye® Drill Planter (Lockney, TX). Sunflower borders were plant- ed on 7 and 8 July using an Earthway® Garden Push Planter (Bristol, IN). Pasture borders were mowed at the time of buckwheat and sunflower planting, but remained unmowed for the remainder of the study. Pasture borders were composed primarily of tall fescue, Festuca amndinacea (Scop.) Holub; johnsongrass, Sor- ghum halepense (L.); orchard grass, Dactylis glomerata L.; giant foxtail, Setaria faheri Herrm; and smooth pigweed, Amaranthus hybridus L. A randomized complete block design with five replicates was used and all plots were separated by 25 m. Yellow sticky traps 15 cm X 15 cm were used to capture lady beetles in August during sweet corn anthesis. Two traps were deployed at canopy height midway between the edges and equidistant from the ends of each border plot. Four traps were deployed in each sweet com plot at ear height, one in the center of each plot quadrant. Traps were changed weekly through anthesis. Sticky traps were placed individually in Ziploc® plastic bags, labeled, and transported to the laboratory for insect identification and enumeration. Population data for each lady beetle species were analyzed using ANOVA and Fisher's Protected LSD procedures in CoStat Statistical Software (CoHort Software 2006). Numbers of pink lady beetles, Coleomegilla maculata (DeGeer), were further analyzed according to habitat type using a ^-test using the same statistical software. Differences were consid- ered significant at F ^ 0.05. RESULTS AND DISGUSSION Pink lady beetle, Coleomegilla maculata; Asian lady beetle, Haniionia axyridis (Pallas); spotless lady beetle, Cydoneda munda (Say); and the seven spotted lady beetle, Coccinella septempunctata L. were the lady beetle species caught in this study (Table 1). The pink lady beetle was the most abundant species in all three border types and the sweet com plots with 79% and 94%, respec- tively. There were greater numbers of pink lady beetles in buckwheat than the pasture and sunflower borders on 14 August (F < 0.00001), and a greater number in both buckwheat and sunflower borders than pas- ture borders on 20 August (F = 0.0064) (Table 2). These results are similar to those reported by Woltz et al. (2012) for buckwheat and pasture strips bordering soybeans in southern Michigan. There were no differences in abundance of Asian lady beetle, spotless lady beetle or seven spotted lady beetles on any of the three sampling dates. Results of the present study contrast with those reported by Adedipe and Park (2010) where the Asian lady beetle was more attracted to sunflower in laboratory olfactometer studies than any of 8 other species of flowering plants. Unfortu- nately, they did not report the variety of sunflower used in their study that could conceivably have affected the results of their study. 98 Journal of the Kentucky Academy of Science 73(2) Table 2. Average number of lady beetles caught in buckwheat, sunflower and pasture border rows. Date Treatment Pink“ lady beetle Asian lady beetle Spotless lady beetle Sev'en spotted lady beetle August 14 Buckwheat 8.5 a 0.35a 0.55a 0.05a Sunflower 1.75b 0.30a 1.15a 0.05a Pasture 2.85b 0,15a 0.85a 0.00a N 5 5 5 5 F value 16.36 0.90 0.74 0.49 P \ alue 0.00001*** 0.41 0.48 0.62 August 20 Buckwheat 6.55a 0.20a 0.35a 0.1a Sunflower 5.65a 0.35a 0.40a Oa Pasture 1.8b 0.20a 0.25a Oa N 5 5 5 5 F value 5.57 0.64 0.26 2.07 P value 0.0064** 0.52 0.76 0.14 August 27 Bucksvheat 4.0a 0.0a 0.1a 0.0a Sunflower 3.05a 0.1a 0.3a 0.0a Pasture 3.75a 0.05a 0.2a 0.05a N 5 5 5 5 F value 0.32 1.07 0.98 0.99 P value 0.73 0.35 0.38 0.37 Means followed by different letters are significantly different. Pink lady beetles were the dominant species in sweet corn bordered by buckwheat, sunflower and pasture borders. However, there were no differences among populations of any of the lady beetle species in any of the sweet corn plots bordered by any of the three border types (Table 3). Pink lady beetles were significantly more numerous in sweet corn bordered by buck- wheat than in buckwheat borders on 20 August (F < 0.05) (Table 4). On 14 August pink lady beetles were more numerous in sweet corn bordered by sunflower than sunflower borders (F < 0.00) (Table 4). Sweet corn bordered by pasture borders had signif- icantly greater numbers of pink lady beetles than pasture borders on 14 August (F = 0.03) and 20 August (F < 0.00) (Table 4). There was a trend toward higher numbers in sweet corn on all three sampling dates for all border types. Interestingly, Woltz et al. (2012) did not find higher numbers of any lady beetle species in soybean plots bordered by buckwheat in southern Michigan. Table 3. Average number of lady beetles caught in sweet corn bordered by buckwheat, sunflower and pasture border rows.^ Date Treatment Pink lady beetle Asian lady beetle Spotless lady beetle Seven spotted lady beetle August 14 Corn- B uckAvheat 8.7a 0.35a 0.65a 0.05a Corn-Sunflower 6.8a 0.25a 0.70a 0.00a Corn-Pasture 8.5a 0.15a 0.40a 0.00a N 5 5 5 5 F value 1,01 0.77 0.35 0.99 P value 0.7 0.47 0.71 0.37 August 20 Cor n- B uck^vheat 12.6a 0.0a 0.1a 0 Corn-Sunflower 12.0a 0.0a 0.05a 0 Corn-Pasture 13.5a 0.05a 0.2a 0 N 5 5 5 5 F value 0.17 0.99 1.32 - P value 0.84 0.37 0.27 - August 27 Corn- B uck'wheat 5.6a Oa 0.1a 0 Corn-Sunflower 6.4a Oa 0.2a 0 Corn-Pasture 6.5a 0.05a 0.05a 0 N 5 5 5 5 F value 0.26 0.99 1.68 - P value 0.78 0.37 0.19 - “ Mean.s followed by tlie .same letter.s are not .significantly different. Lady Beetles in Sweet Com, Annual Borders — Sedlacek et al 99 Table 4. Average number of pink lady beetles caught in buckwheat, sunflower or pasture border rows compared with tlie numbers caught in sweet corn bordered by each border type. 8/14 8/20 s/27 Com/Buckwlieat Buckwheat Com/Buckvi'Iieat Buck-wheat Com/Buckw'heat Buckw'heat Mean 8.70 8.50 12.55 6.55 5.55 4.00 N 5 5 5 5 5 5 P(T < = t) two-tail 0.94 0.05* 0.46 Corn/Sunflower Sunflower Com/Sunfiower Sunflower Coni/Simflower Sunflower Mean 6.75 1.75 12.00 5.65 6.40 3.05 N 5 5 5 5 5 5 P(T < = t) two-tail 0.00*** 0.08 0.12 Coin/Pasture Pasture Com/Pasture Pasture Coin/Pastiire Pasture Mean 8.50 2.85 13.50 1.80 6.55 3.75 N 5 5 5 5.00 5 5 P(T < = t) two-tail 0.03** 0.00*** 0.25 * P < 0.05, ** F < 0.03, ***F < 0.01. Kernel damage was not quantified; however there appeared to be no reduction in damage to sweet corn ears. Additional field experi- ments should be performed in which flower- ing of buckwheat and sunflower occurs before, and not the beginning of, the onset of sweet corn anthesis. Interplanting the buckwheat and sunflower in narrow strips within the sweet corn plots should also be investigated. In conclusion, die pink lady beetle was the most abundant species in all three border types and the sweet com plots. Buckwheat and sunflower borders contained more pink lady beetles than pasture borders and pink lady beetles in sweet com were more abundant than in borders. Based on these observations it was concluded that buckv/heat, sunflower and pas- ture border rows have little influence on lady beetle populations in sweet com when planted in the manner described in this study. Field experiments using other species of flowering plants, for example native perennials, should be performed. In addition, planting strategies to influence longer flowering periods of border plants could enhance the abundance of lady beetles, other predatory insects, and parasitoids in sweet com when ear pests are present. ACKNOWLEDGEMENTS This research was supported by Evans Allen Grant no. KYX.10-08-44P awarded by the National Institutes of Food and Agriculture. Syngenta Seed provided the sweet com seed for this research. Rachel Hayden assisted in identifying lady beetles. Anonymous reviewers read an early draft of this manuscript and offered helpful suggestions. Mention of pro- prietary products does not constitute endorse- ment. This manuscript is approved for publi- cation by the Director of the Kentucky State University Agricultural Experiment Station as manuscript number KYSU 000010. LITERATURE CITED Adedipe, F., and Y-LPark. 2010. Visual and olfactory preference of Hamwnia axijridis (Coleoptera: Cocci- nellidae) adults to various companion plants. J Asia Pacific Entomol 13:319-323. Altieri, M. A., and W. H. Whitcomb. 1980. Weed manipulation for insect pest management in corn. Environ Manag 4:483-489. Ambrosino, M. D., J. M. Uina, P. C. Jepson, and S. D. Wratten. 2006. Relative frequencies of visits to selected iiisectaiy plants by predatory lioverflies (Diptera:Syr- pliidae), other beneficial insects and herbivores. Environ Entomol 35:394-400. CoHort Software. 2006. CoStat Version 6311. Monterey, CA. Dennis, P., and G. L. A. Fry. 1992. Field margins; Can they enhance natural enemy population densities and general artliropod diversity on familand? Agric Ecosyst Environ 40:95-115. Dufour, R. 2000. Famiscaping to enhance biological control. Pest Management Systems Guide. ATTRA 40 p. Green, C. R. 2001. U.S. organic farming emerges in the 1990s: Adoption of certified systems. US DA Agric Inform Bull. No. 770. Johnson, R. M., Z. Wen, M. A. Schuler, and M. R. Berenbaum, 2006. Mediation of pyretliroid insecticide 100 Journal of the Kentucky Academy of Science 73(2) toxicit}' to honey bees (H)nnenoptera:Apidae) by e)tochrome P450 monooxygenases. J Econ Entomol 99:1046-1050. Musser, F. R., and A. M. Shelton. 2003. Bt sweet corn and selective insecticides: Impacts on pests and predators. J Econ Entomol 96:71-80. National Research Council. 1989. Alternative agriculture. National Academy Press, Washington DC. National Research Council. 1996. Biologically based pest management: New solutions for a new centur)-. Nationd Academy Press, Washington DC. Pollard, E. 1968. Hedges. III. The effect of removal of the bottom flora of a hawthorn hedgerow on the Carabidae. J Appl Ecol 5:125-139. Sparks, T. C. 1981. Development of insecticide resistance in Heliothis zea and Heliothis virescens in North America. Bull Entomol Soc Am 27:186-192. Sunderland, K., and F. Samu. 2000. Effects of agricultural diversification on the abundance, distribution, and pest control potential of spiders: A re\dew. Entomol Exp Appl 95:1-13. van Emden, H. F. 1965. The role of uncultivated land in tlie biology of crop pests and beneficicil insects. Sci Hort 17:121-136. Woltz, J. M., R. Isaacs, and D. B. Landis. 2012. Landscape structure and habitat management differentially influ- ence insect natural enemies in an agricultural land- scape. Agric Ecosyst Emdron 152:40-49. J, Ky. Acad. Sci. 73(2): 101-109. 2012. Genetic Diversity in Kentucky Spicebush Populations using Simple Sequence Repeat Markers Re'Gie Smith, Kirk W. Pomper, Jeremiah D. Lowe, Jacob Botkins, and Sheri B. Crabtree Land Grant Program, Kentucky State University, Atwood Research Facility, Frankfort KY 40601-2355 ABSTRACT Spicebush {Lindera benzoin L.) is a small native shrub that grows in the moist, understory areas of Appalachia and has potential as a new niche crop for small fanners. Native Americans and early settlers used this plant traditionally as a tea, and used tlie berries for jam, to spice food, and possibly for health benefits. Native spicebush patches may serve an important role in forest ecosystems in terms of fruit production for animals, soil erosion control, and enhancing insect biodiversity. Spicebush may also serve to hold ecological niches by outcompeting invasive plants compared to those in unchallenged areas. Genetic diversity of native spicebush populations in Kentucky has not been examined. The objective of tliis study is to determine tlie genetic diversity in spicebush populations in Kentucky using simple sequence repeat (SSR) DNA marker systems. Leaf samples were collected from 120 spicebush plants in the forests at tlie Kentucky State University Environmental Education Center (EEC), at a location near the Kentucky River, and at Cove Spring Park. DNA was extracted using the DNAmite Plant Kit. Primers A7, A115, B105, B122, C4, and CIO were used to amplify SSR products tliat were separated witli a 3130 Applied Biosystems capillary electrophoresis system. The software program Power Marker was used to examine genetic relationships among genotypes. The SSR markers generated showed high genetic variation among tlie spicebush genotypes witli tlie observed heterozygosity at 0.78. A number of selections witli unique genotypes should be sampled and propagated for study in the KSU germplasm collection for potential cultivar development. KEY WORDS: Lindera benzoin (L.), Microsatellites, Simple Sequence Repeat (SSR), Perennial shrub, Spicebush, Genetic Diversity INTRODUCTION Spicebush {Lindera benzoin L.) is a large, highly branched shrub or small tree. It is able to grow to over 12 feet tall and 15 feet wide. This plant is usually found as an uiiderstory plant in the forests of North America, especially in the eastern United States (Boyle 1980). Spicebush can be found near wetlands, v/oodlands, and in other shaded moist areas and in soils rich in limestone (Niesenbaum 1992). The leaves of the spicebush are one of the first to emerge in the spring or late winter compared to other plants. The leaves are obovate and alternate, and contribute to its name because of the aromatic spicy scent that comes from them as well as the berries and stems of the plant. Historically, the leaves of this shrub have been used for tea and medicine by Native Americans and early settlers (Anderson et al. 1992; Meuninck, 2007). The tea made from the leaves has been used to relieve pain, fever, arthritis, and breathing difficulties (Anderson et al. 1992; Meuninck 2007). The oil from the berries was sometimes used as a first aid treatment. Further research is needed to determine the effectiveness of these uses (Allison 2003). The berries have been used in jams, and are currently sold in combination with fruit such as blueberry and pawpaw to create flavors such as pawpaw spiceberry jam or blueberry spicebush jam. The berries have also been used as food flavoring by grinding the dried berries and using it as a seasoning on meat. The seeds, also known as drupes, are usually about 8 mm long, a bright cherry red color when mature, and contain a seed. The fruit matures in late summer or early fall (Tucker and Madarello 1994; Meuninch 2007). Inter- estingly, spicebush has no significant disease or pest problems, which allows it to have the potential to be a new niche crop for small farmers. Spicebush serves an important role in the ecosystems of forests. The fruit enhances biodiversity since it can be eaten by animals and insects, the root system in soils helps prevent soil erosion around streams and rivers, and biodiversity is enhanced from species that thrive specifically on spicebush such as the Spicebush Swallowtail (Papilio troilus) (Matlack 1994). With the unique food 101 102 Journal of the Kentucky Academy of Science 73(2) and medicinal qualities, as well as the impor- tance of this plant in the Kentucky ecosystem, spicebush is attractive to small farmers as a potential alternative crop, and as a plant for improving biodiversity in woodlands. Microsatellites, or simple sequence repeats (SSRs), are a marker of choice for genetic diversity estimates, genetic mapping, and DNA fingerprinting (Wiinsch and Hormaza 2002). SSRs are short (1-6 bp) tandem repeat DNA sequences flanked by unique, conseiwed DNA sequences. The relative random distri- bution of microsatellites in the genome, co- dominant inheritance, high level of reproduc- ibility and transportability across laboratories make these markers useful for assessing genetic diversity as well as fingerprinting (Kijas et al. 1995; Wiinsch and Hormaza 2002). SSR marker systems have been devel- oped for a number of fruit species including pawpaw [Asimina triloba (L.) (Pomper et al. 2010)], hazelnut [Conjlus avellana L. (Bassil et al. 2005)], blueberiy [Vacciniiim conpnho- sum L. (Bodies et al. 2006)], peach [Prunus persica (L.) Batsch; (Aranzana et al. 2002)], cheriy [Prunus cerasus L. (Cantini et al. 2001)], pear [Pijnis communis L. (Yamamoto et al. 2001)], and apple [Mains sylvestris (L.) Mill (Hokanson et al. 1998, 2001)]. Spicebush can be found in a range of different light environments (Veres and Pick- ett 1982). There have been several previous limited studies assessing genetic diversity of spicebush in eastern Pennsylvania (Edwards and Niesenbaum 2007; and Mooney et al. 2010). Edwards and Niesenbaum (2007) developed 12 polymoqihic SSR primer sets for examining genetic diversity in spicebush and Mooney et al. (2010) used these markers to examine genetic differences between spice- bush in sun and shade environments. Genetic diversity of spicebush populations in Ken- tucky has not been examined. The long term goals of this research are to understand the cycling and spread of native spicebush patches as a part of Kentucky State University’s mission and germplasm collection efforts in order to select genetically different varieties that can be grown by small farmers. Our objective was to determine the genetic diver- sity in spicebush populations in Kentucky using polymerase chain reaction (PGR) and SSR-DNA marker systems. Table 1. GPS coordinates for sample locations. Location Coordinates Cove Spring 1 38°13'08.39"N 84°53'31.80'W Cove Spring 2 38°13'1L24"N 84°50'43.44W EEC 1 38°21'38.45"N 85°00'44.07'W EEC 2 38°2r35.32"N 85°00'50.84'W Kentuck)^ River 1 38°08'19.10"N 84°51'58.9rW Kentucky River 2 38°08T8.irN 84°52'01.2rW MATERIALS AND METHODS Plant Material Leaf samples were collected from spice- bush plants in six spicebush populations in Kentucky using. Leaf samples were collected from spicebush plants in the forests at the Kentucky State University Environmental Education Center (EEC) in Henry County, Kentucky, Cove Spring Park in Frankfort, Kentucly, and a location near the Kentucky River near Frankfort, Kentucky (Table 1). Each population contained twenty individuals and two populations were sampled at each site. DNA Extraction DNA was extracted from the pawpaw leaves using the DN AMITE Plant Kit (The Gel Company, San Francisco, CA). About 1 ~ 2 cm“ of young leaf tissue was used. The DNA concentration and a 260/280 nm absorbance ratio were determined with GeneQuantTM pro RNA/DNA calculator (GE Healthcare, Piscataway, NJ). All samples were stored at — 80°C until needed. SSR-PCR Amplification The SSR-PCR amplification was performed with GoTaq Flexi DNA polymerase (Promega Co., Madison, WI). The reactions were set up follows: 4 pi of 5X colorless GoTaq Flexi buffer, 0.4 pi of 10 niM dNTPs solution, 2 pi of 25 inM MgCl2, 0.3 pi of 3 pM forward primer (fluorescence labeled with FAM) solution and 0.3 pi of 3 pM reverse primer (unlabeled) solution, 0.2 pi of 5 units/pl GoTaq DNA polymerase, 2 pi of diluted 1 ng/pl paviq)aw DNA, and 10.8 pi of ddH20 Simple Sequence Repeat Primer Sequences for the Six Loci Amplified with the Repeat Motif — Smith et al. 103 Table 2. The six loci tested, their forward and reverse sequences, and the repeat motif used in the SSR-PCR. Locus Primer sequence (5'-3') Repeat motif A7 FiAAAACGGATCAGATACTCCC R:GCAGCATTATTGGGTTAGTG (AC)j3 A115 F:AGGAGCTACCTCTGATTCTTGG R :TC AGCC AC ATCTC AATATC ATG (CT),9 + (AC)i3 BIOS F:AGAGGTCTTGACTTTGGGATAT R:GGATGGGTTATGGAGTGG (GA)„ B122 F:TGGTCAAGGAGAGATTCAAG R:GTGAGGGGAGTGTACTATGG (AG), 7 C4 TTGGGTGGAGTTGATGAG R:ACGGATTATTGACAGCCTT (GAA)i3 CIO F:TTGGTAAAGCCTGTTGTAAAAC R:GCCAATCATGTGACTATTGTC {AAG),5 to bring the total volume to 20 pi. Six primer pairs were selected and labeled with FAM or HEX for use in this study:sb-A7, sb-A115, sb- BIOS, sb“B122, sb-C4, and sb-ClO (Table 2). The PGR amplifications were performed using the Endurance Series TC-512 Thermal Cycler (Techne Inc., Burlington, NJ). The PCR program consisted of an initial period of 94°C for 3 min, followed with 30 cycles of 40 sec denaturation at 94°C, 40 sec annealing at 56°C, and 30 sec extension at 72°C, and a final extension period of 10 min at 68°C. The PCR results were then stored at 4°C until analysis. Products were separated with ABl 3130 Genetic Analyzer (Applied Biosystems Inc., Foster City, CA) with GeneScaiiTM 500 LIZTM as internal size standard. Individuals were genotyped with GeneMapper version 4.0 software (Applied Biosystems Inc., Foster City, CA). At least two replicate amplifications were subjected to electrophoresis and analysis for each primer set. Data Analysis PowerMarker (Version 3.25) (Liu and Muse 2005) was used to calculate the obseiwed number of alleles (nA), the observed hetero- zygosity (Ho), the expected heterozygosity (He) and polymorphic Information Index (PIC). The obseiwed heterozygosity was cal- culated as the number of heterozygous genotypes at a given locus divided by the number of genotypes present at the locus. Gene diversity was defined as the probability that two randomly chosen alleles from the population are different. PIC was an estimate of the probability that the parental origin of an allele can be determined from the marker locus genotype in any given offspring. The equation for Ho, He, and PIC are given in the PowerMarker software manual. Genetic dis- tance (D) between genotypes was computed as (I — proportion of shared alleles (Bowcock et al. 1994). Cluster analysis of distance data were used to generate a dendrogram based on the matrix of the distances using unweighted pair-group mean analysis (UPGMA). The level of genetic similarity among cultivars and advanced selections was also determined by Nei’s genetic distance (Nei 1978). RESULTS AND DISCUSSION All six of the SSR primer sets tested amplified SSR-PCR products in all popula- Table 3. The six loci tested, the number of genot)q)es, the number of alleles, the allele size, tlie expected heterozygocity, the observed heterozygocity, and the PIC for all loci tested. Marker Genot)'pe No. Allele No. Allele size (bp) H,. Ho PIC Alls 7S 30 160-248 0.92 0.85 0.91 A7 39 13 233-250 0.86 0.73 0.85 BIOS 87 27 238-292 0.93 0.88 0.93 B122 69 23 191-267 0.89 0.78 0.89 C4 38 13 222-247 0.87 0.60 0.86 GIO 42 17 133-230 0.87 0.82 0.85 Mean 58 21 133-292 0.89 0.78 0.88 104 Journal of the Kentucky Academy of Science 73(2) Figure 1. A dendrogram comparing genetic relationships of all the samples at all six patches at the three locations. Simple Sequence Repeat Primer Sequences for the Six Loci Amplified with the Repeat Motif — Smith et al. 105 Figure 2. A dendrogram comparing the genetic relationships between the six patches. tions examined. On average, each locus produced over 18 different alleles and 42 different genotypes. Allele size ranged from 133 to 292 base pairs and heterozygosity was high (He = 0.89; Table 3). When comparing populations, there was significant genetic diversity. The high average number of alleles per locus (21.0) is comparable to that in other woody perennial fruit and nut crop species such as 13.3 alleles per locus reported for hazelnut (Bassil et al. 2005) 12.1 for apple (Hokanson et al. 1998, 2001) and 9.1 for pear (Sisko 2009.). The He (0.89) in our study indicated high levels of genetic diversity among the spicebush populations examined. Edwards and Niesenbaum (2007) examined genetic diversity of 29 eastern Pennsylvania spicebush plants using 11 SSR primer pairs and found He ranging from 0.10 to 0.82, and alleles ranged from two to 16 per locus. Mooney et al. (2010) examined genetic diversity of 127 eastern Pennsylvania spice- bush plants using 12 SSR primer pairs and found He ranging from 0.61 to 0.65, and alleles ranged from 3.7 to 4.3 per locus. The higher Hg determined for spicebush popula- 106 Journal of the Kentucky Academy of Science 73(2) EEC 30 EEC 37 EEC 47 EEC 45 EEC 46 EEC 31 EEC 48 EEC 29 EEC 34 EEC 13 EEC 33 EEC 35 EEC 14 EEC 10 EEC 38 EEC 49 EEC 2 EEC 50 EEC 26 EEC 42 EEC 44 EEC 43 EEC 32 EEC 40 EEC 15 EEC 41 EEC 7 EEC 3 EEC 17 EEC 18 EEC 1 EEC 16 EEC 27 EEC 12 EEC 4 EEC 9 EEC 28 EEC 36 EEC 39 EEC 6 Figure 3. A dendrogram comparing the EEC location. tions in our study likely reflects the wider sampling of populations separated by greater distance and therefore a more precise estimate of genetic diversity of spicebush in the wild. UPGMA cluster analysis was used to gen- erate a dendrogram (Figures 1, 2, 3, and 4). UPGMA analysis indicated the EEC and KYR populations were easily separated. Figure 1 shows there was a significant amount of di- versity among the three locations. Figure 2 shows the genetic relationships between the six patches. The two Cove Spring patches Simple Sequence Repeat Primer Sequences for the Six Loci Amplified with the Repeat Motif — Smith et al 107 CSIO CS12 CS25 CS35 CS44 CSll CS13 CS45 CS37 CS7 CS40 CS31 CS34 CS38 CS39 CS23 CS9 CS27 CS36 CSS CS3 CS4 CS42 CS43 CS21 CS46 CS14 CS20 CS19 CS18 CS29 CS32 CS17 CS15 CS33 CS8 CS2 CS26 CS22 CS47 CS6 CS16 CS24 CS28 CS41 CS30 CSl 0.1 Figure 4. A dendrogram comparing the Cove Spring location. were genetically similar. Conversely, the EEC2 population was closer genetically to the KYR 2 population than to EECl; even though the two EEC populations were closer in proximity. Figure 3 shows the genetic relationships in the EEC populations. This population showed genetic diversity, but samples EEC31 and EEC48 and EEC29 and EEC34 were more similar. Figure 4 shows the genetic relationships in the Cove Spring populations. This population showed more similarities between samples than the 108 Journal of the Kentucky Academy of Science 73(2) — KY R 32 — KY R 35 KY R 40 — KY R 27 — KY R 23 — KYR48 — KY R 38 — KY R 39 - — ™ KY R 33 — KYR45 — KYR16 — KY R 29 — KYR46 — = KYR5 — KY R 37 — KYR7 — KYR17 — KY R 26 — KYR4 — ” KY R 34 — - KY R 20 — ™ KYR49 — KYR47 — KY R 50 — KY R 14 — KY R 30 — KYR31 — KYR 13 — KY R 22 — - KYR 9 — KYR 2 — KYR 3 — KYR 8 — — KY R 28 — = KY R 36 — KYR41 — KYR42 — KYR43 — KYR44 — KYR 10 — KYR 6 — KYRl — KYR 11 — KYR 15 — “ KYR 19 KY R 24 — KYR 21 0.1 Figure 5. A dendrogram eomparing the Kentiieky River location. EEC population. With the Cove Spring samples, CS22 and CS47, CS46 and CS21, CS38 and CS39, and CSIO and CS12 were similar. Eigure 5 shows the genetic relation- ships in the Kentucky River populations. Interestingly, with this population, three samples were similar, KYR 13, KYR22, and KYR9. Each of the three populations showed genetic similarities among samples. In conclusion, there was significant genetic diversity observed in the spicebush popula- tions examined. This is the first report of spicebush genetic diversity and relationships in Kentucky. The results of this study indicate 109 Simple Sequence Repeat Primer Sequences for the Six Loci Amplified with the Repeat Motif — Smith et al the six primers used should be useful in examining additional spicebush populations. For the establishment of spicebush varieties for small farmers in Kentucky, varieties with broad genetic diversity will be important for future insect and disease resistance when planted by farmers in larger monocultures that increase disease and pest pressure. Large fruit size v/ill also be important to growers for spicebush to become a successful crop for small farmers. Future studies will include looking at fruit size among different populations. A number of selections with unique genotypes will be sampled and propagated for study in the KSU germplasm collection for potential cultivar development. ACKNOWLEDGMENT This rese.arch was supported by Mclntire Stennis Grant Funding, accession number 0223840, project number KYX-POMPERMCIST. Kentuclq/ State University Experiment Station Number KYSU- 000008. LITERATURE CITED Mison, S. 2003. The Virtual Nature Trail at Penn State New Kensington. Accessed on 27 November 2011. Avail- able at:littp://www.psu.edii/dept/nkbiology/naturetraiI/ speciespages/spicebusli.litm Anderson, J. E., W. Ma, D. L. Smith, C-J. Chang, and J. L. McLaughlin. 1992, Biologically active Y-laetones and Metliylketoalkenes from Lindera benzoin. J Nat Prod 55:71-83. Aranzana, M. J., J. Carbo, and P. Arus. 2002. Microsat- ellite variability in peach {Primus persica (L.L Batsch)]: Cultivar identification, marker mutation, pedigree inferences, and population structure. Theor Appl Genet 106:1341-1352. Bassil, N. B., R. Botta, and S. A. Mehlenbacher. 2005. Microsatellite markers in hazelnut;isolation, character- ization, and cross-species amplification. J Amer Soc Hort Sci 130:543-549. Bodies, P., N. V. Bassil, and L. Rowland, 2006. Genetic diversity in the higlibusli blueberry evaluated with microsatellite markers. J Amer Soc Hort Sci 131:674— 686. Bowcock, A. M, et al. 1994. High resolution of human evolutionary trees with polymorphic microsatellites. Nature 368:455-457. Boyle, E. M. 1980. Vascular anatomy of the flower, seed, and fruit of Lindera benzoin. Bull Torrey Bot Club 107:409^17. Cantini, C., A. F. Lezzoni, W. F. Lamboy, M. Boritzki, and D. Stress. 2001. DNA fingerprinting of tetrapioid cherry germplasm using sequence repeats. J Amer Soc Hort Sci 126:205-209. Edwards, M. J., and R. A. Niesenbaum. 2007. Eleven polymorphic microsatellite loci in Lindera benzoin Lauraceae. Molecular Ecology Notes 7:1302-1304. Hokanson, S. C., A. K. Szewc-McFadden, W. F. Lamboy, and J. R. McFerson. 1998. Microsatellite (SSR) markers reveal genetic identities, genetic diversity, and relation- ships in Malus X domestica Borkh. Theor Appl Genet 97:671-683. Hokanson, S. G., W. F. Lamboy, A. K. Szewc-McFadden, and J. R. McFerson. 2001. Microsatellite (SSR) variation in a collection of Malus (apple) species and hybrids. Eupliytica 118:281-294. Kijas, J. M. H., J. C. S. Fowler, and M. R. Thomas. 1995. An evaluation of sequence tagged microsatellite mark- ers for genetic analysis witliin citrus and related species. Genome 38:349-355. Li, Y-C., A. B. Korol, T. Fahinia, A. Belles, and E. Nevo. 2002. Microsatellites:genoniic distribution, putative functions and mutational mechanismsia review. Mol Ecol 11:2453-2465. Matlack, G. R. 1994. Vegetation dynamics of the forest edge:trends in space and successional time. J Ecol 82:113-123. Meuninck, J. 2007. Basic Essentials Edible Wild Plants and Useful Herbs, 3rd ed. Falcon Guides Publishing, Guilford, CT. Mooney, E., M. Edwards, and R. Niesenbaum. 2010. Genetic differentiation between sun and shade habitats in populations of Lindera benzoin L. Popul Ecol 52:417- 425. Niesenbaum, R. A. 1992. The effects of light environment on lierbivory and growth in the dioecious shrub Lindera benzoin (Lauraceae). Am Midi Nat 128:270-275. Pomper, K. W., J. D. Lowe, L. Lu, S. B. Crabtree, S. Dutta, K. Schneider, and J. Tidwell. 2010. Character- ization and identification of Pavq)aw (Asimina triloba) cultivars and advanced selections by simple sequence repeat markers. J Amer Soc Hort Sci 135:143-149. Sisko, M. J. 2009. Genetic relationships among Slovenian pears aassessed by molecular markers. J Amer Soc Hort Sci 134:97-108. Tucker, A. O., and M. J. Maciarelio. 1994. Spicebus {Lindera benzoin (L.) Blume var. benzoin Lauraceae):A tea, spice, and medicine. Econ Bot 48:333-336, Veres, J. S., and S. T. A. Pickett. 1982. Branching patterns of Lindera benzoin beneatli gaps and closed canopies. New Phytol 91:767-772. Wiinsch, A., and J. J. Hormaza. 2002. Cultivar identifica- tion and genetic fingerprinting of temperate fruit tree species using DNA markers. Eupliytica 125:59-67. Yamamoto, T., T. Kimura, Y. Sawamura, K. Kotobuld, Y. Ban, T. Hayaslii, and N. Matsuta. 2001. SSRs isolated from apple can identify polymorphism and genetic diversity in pear. Theor Appl Genet 102:865-870. J. Ky. Acad. Sci. 73(2);110-] 12. 2012. The Pawpaw Peduncle Borer, Talponia plummeriana Busck (Lepidoptera: Tortricidae): A Pest of Pawpaw Fruit John D. Sedlacek,^ Jeremiah D. Lowe, Kirk W. Pomper, Karen L. Friley, and Sheri B. Crabtree College of Agriculture, Food Science and Sustainable Systems. CRS, Kentucky State University, Frankfort, Kentucky 40601 ABSTRACT The pawpaw peduncle borer, Talponia plummeriana Busck (Lepidoptera: Torticidae), is a pest of pawpaw flowers often boring into the peduncle and causing flower drop. Here we document the first occurrence of this insect infesting ripe pawpaw fruit. Infested fruit that had been collected at tlie Kentucky State University Research and Demonstration Farm in Franklin County, Kentucky were dissected and small tan colored larvae with brown head capsules were discovered in the fruit. Pa\\y>aw peduncle borer adults were reared from fruit held at room temperature in the laborator)^. KEY WORDS: Asimina triloba fruit pest. Pawpaw peduncle borer INTRODUCTION The North American pawpaw, Asimina triloba (L.) Dunal is a tree-fruit in the early stages of commercial production. It has had few reported pest problems. Paw^3aws are native to mesic hardwood forests of 26 states ill the eastern United States, including Ken- tucky (Lagrange and Tramer 1985; Chester et al. 1995) and surrounding states (Rheinhardt and Rheinhardt 2000; Larimore et al. 2003). Its full range extends from northern Florida to southern Ontario (Canada) and as far west as eastern Nebraska (Krai 1960). This small, deciduous tree may attain a height of five to ten m and tends to be found in patches (Layne 1996). Pawpaws are often found growing as understoiy trees in the deep, rich fertile soils of river-bottom lands (Krai I960; Young and Yavitt 1987; Callaway 1990; Callaway 1993; Pomper et al. 2009). Pauyjaw trees flower from April through May in Kentucky and are pollinated by flies and beetles (Faegri and van der Fiji 1971). One recognized pest, the pawpaw peduncle borer, Talponia plmmneriana Busck, is a moth in the family Tortricidae. Pawpaw peduncle borer adults are approximately six mm in length with gray speckled wings and a wide copper band at the distal portion of the wings. The laiwae have been previously documented to feed only on pawpaw flower peduncles ' Corresponding autlior e-mail: iohn.sedlacek@k)^su.edu (Bratcli 2009). The larvae bore into the flower and peduncle eventually killing the flower. In the orchards of the Kentucky State University Pawpaw Research Program, a small number of unripe and ripe pawpaw fruit showed evidence of feeding activity from an unknown fruit borer (Figure 1) causing ex- tensive internal injury to the fruit (Pomper et al. 2008). Thus, the objective of this project was to determine the insect species responsi- ble for damage to the fruit of pawpaw. MATERIALS AND METHODS Ripe pawpaw fruit were collected in mid- August from those that had fallen from trees in orchards located at the Kentucky State University Research and Demonstration Earm located in Franklin County, KY. Three fruit were placed into each of two one-liter plastic vegetable crispers (six total fruit) and incubat- ed at room temperature in the laboratory until moths had emerged (Figure 2). We used the BugGuide Internet Identification Website (2010) and North American Moth Photogra- phers Group: Talponia plummeriana (2007) to identify adults. Digital photographs of adults were sent to Dr. Charles Covell, Florida Museum of Natural Histoiy, McGuire Center for Lepidoptera and Biodiversity, Gainesville, FL for identification confirmation. RESULTS AND DISCUSSION Approximately 10 individual pawpaw pedun- cle borer adults were reared from die fruit. Adults emerged from fmit were identified as no Pawpaw Peduncle Borer Infest Pawpaw Fruit — Sedlacek et al 111 Figure 1. Damaged pau^aw fruit and young pawpaw peduncle borer larva. the pawpaw peduncle borer (T. plmmmriana) (Figures 3, 4), Pawpaw peduncle borer moths have been previously collected in Henderson, Menifee, Powell, Wolfe, and Hart Counties, Kentucky (Kentucky Butterfly Net Database 2010; United States National Museum 2010). In most years, the damage is light (about one to five percent of fruit displaying damage). However, in some years there can be signih icant loss of flowers that can reduce fruit set (Jones et al. 1998). While pawpaw peduncle borer damage is usually light, it does have the potential to reduce fruit yields significantly by flower and fruit damage. The incidence and degree of damage in papaw fruit orchards to flowers and fruits by this species needs to be further investigated. Pawpaw pests may become of Figure 2. Damaged fruit witli puparia present. greater interest as the number of pawpaw plantings or monocultures increase and con- trol measures may need to be implemented. ACKNOWLEDGEMENTS This research was supported by Evans Allen Grant no. KYX.10-09-44P awarded by the National Institutes of Food and Agriculture. Dr. Charles Covell, Florida Museum of Natural Histoiy, McGuire Center for Lepi- doptera and Biodiversity, Gainesville, FL confirmed moth identifications. Anonymous reviewers read an early draft of this manu- script and offered helpful suggestions. This manuscript is approved for publication by the Director of the Kentucky State University Figure 3. Pawpaw peduncle borer adult. 112 Journal of the Kentucky Academy of Science 73(2) Figure 4. Pawpaw peduncle borer adults with U.S. quarter for scale. Agricultural Experiment Station as manu- script number KYSU 000011. LITERATURE CITED Bratch, A. 2009. Specialty Crop Profile: Pawpaw. Virginia Cooperative Extension Publication. 438-105. Avciilable at: http://pubs.ext.\4:.edu/438/438-105/438-105_pdf.pdf Bug Guide. 2010. Talponia plummeriana . Available at: http ://bugguide. net/node/view/ 1 07 394 Callaway, M. B. 1990. The pa\^q)aw {Asimina triloha). Kentuck)^ State University. Publications. CRS-HORTl- 90 IT. Callaway, M. B. 1993. Pav^qjaw {Asimina triloha): A “tropical” fruit for temperate climates. Pages 505-515 in J. Janick and J. E. Simon (eds). New Crops. Wiley Press, New York. Chester, E. W., S. M. Noel, J. M. Baskin, C. C. Baskin, and M. L. McReynolds. 1995. A phytosociological analysis of an old-gro\\4h upland wet woods on the Pennyroyal Plain, southcentral Kentucky'. Nat Areas J 15:297-307. Faegri, K., and L. van der Fiji. 1971. The principles of pollination ecology. Pergammon, New York. Jones, S. C., R. N. Peterson, T. A. Turner, K. W. Pomper, and D. R. Layne. 1998. Kentucky State University Pawpaw Planting Guide. Ext Bull PIB-003. Available at: http :/Avww. pawpaw . ky’s u . edu/pawp aw/ppg . htm Krai, R. 1960. A revision of Asimina and Deeringothamus (Annonaceae). Brittonia 12:233-277. Kentucky Butterfly Net Database. 2010. Available at: http://bioweb.wku.edu/facult)7marcus/map_out.asp? species =034 17 Lagrange, R. L., and E. J. Tramer. 1985. Geographic variation in size and reproductive success in the paw paw {Asimina triloba). Ohio J Sci 85:40-45. Larimore, R. L., D. T. Busemeyer, and J. E. Ebinger. 2003. Pawpaw, Asimina triloba (L.) Dunal (Annona- ceae), in the prairie peninsula of Illinois. Natl Areas J 23:356-361. Layne, D. R. 1996. The pawpaw triloba Dunal: A new fruit crop for Kentucky^ and the United States. HortScience 31:777-784. North American Moth Photographers Group: Talponia plummeriana. 2007. Available at: http://mothphotogra phersgroup.msstate.edu/species.phpPhodges = 34 1 7 Pomper, K. W., J. D. Lowe, L. Lu, S. B. Grabtree, and L. A. GoUins. 2009. Glonality of pawpaw {Asimina triloba) patches in Kentucky. J Kentucky Acad Sci 70:3-11. Pomper, K. W., S. B. Crabtree, D. R. Layne, R. N. Peterson, J. Masabni, and D. Wolfe. 2008. The Kentucky pa\\q)aw regional variety trial. J Amer Pomo- log Soc 62:58-69. Rheinhardt, M. C., and R. D. Rheinhardt. 2000. Canopy and woody^ subcanopy composition of wet hardwood flats in eastern North Carolina and southeastern Virginia. J Torrey Bot Soc 127:33-43. United States Nationd Museum. 2010. Available at: httpT/ www.butteilheScmdmotlis.org/species/Talponia-plummeriana Young, D. R., and J. B. Ya\dtt. 1987. Differences in leaf structure, chlorophyll, and nutrients for the understory tree Asimina triloba. Am J Bot 74:1487-1491. J. Ky. Acad. Sci. 73(2):113-121. 2012. Pawpaw Patch Genetic Diversity, and ClonaBty, and its Impact on the Establishment of Invasive Species in the Forest Understory Jacob Botkins, Kirk W. Pomper, Jeremiah D. Lowe, and Sheri Crabtree College of Agriculture, Food Science, and Sustainable Systems, Division of Agriculture and Natural Resources, Kentucky State University, Atwood Research Facility, Frankfort, Kentucky 40601-2355 ABSTRACT The pawpaw (Asitnina triloba) is a native understory tree of 26 states of tlie Eastern and Midwestern United States. Pawpaw's genetic diversity and clonality in relation to this tree's ability to compete witli local invasive species in Kentucky has not been examined. The objectives of tliis study were Ovo-fold; to determine tlie genetic diversity and clonality displayed in seven native pav^^aw patches located at the Kentucky State University Environmental Education Center (KSU-EEC), The Kentucky River, Cove Spring Park, and tlie KSU Research and Demonstration Farm in Franklin County using DNA microsatellite markers; and to determine if patches reduced the incidence of invasive species. Twenty- five trees from seven patches in the four different locations were sampled for genetic analysis. Leaf samples were extracted using the DNAinite Plant Extraction Kit and products from four microsatellite loci were analyzed using a 3130 Applied Biosystems Gene sequencer. String grids were created and invasive plants counted in tliree 5 meter squares in each of the patches and control plots outside of each patch. The number of plants for each invasive species within pawpaw patches was counted and compared to the control plots. Pauqiaw patches displayed high genetic diversity among populations with some clonal sections in patches using DNA microsatellite makers, but no totally clonal patches. Amur honeysuckle (Lonicera nwackii), garlic mustard {Alliaria petiolata), and winter creeper {Euonymus fortunei) were found in most locations; however, tliere was no significant difference in the incidence of invasive plants between the patches and the control plots. Pawpaw stem density and shading, among otlier factors may be important in the incidence of invasive plants witliin patches. KEY WORDS: Asitnina triloba (L) Dunal, Micro satellites, Simple Sequence Repeat (SSR), Pawpaw, Genetic Diversity, Forest Understory, Lonicera maackii, Amur Honeysuckle, Invasive Species, Kentucky State University, KSU INTRODUCTION Pawpaw [Asimina triloba (L) Dunal ] is an understoiy tree that is native to the Eastern and Midwestern United States. A. triloba can be observed in 26 states and ranges from northern Florida to southern Ontario, Cana- da, and as far west as Nebraska (Darrow 1975). Native pawpaw patches serve an important role in forest ecosystems and stream and rivers in terms of fruit production for animals, soil erosion control, enhancing insect biodiversity by bringing pawpaw exclu- sive species, and possibly resisting establish- ment of invasive plant species. Pa^vpaw is also in the early stages of commercial production. Unique native pawpaw germplasm could serve as future cultivars for growers or be used in KSU breeding efforts. Asimina triloba is a shade-loving understory tree which often forms dense patches in the understory of mixed mesophytic hardwood forests (Callaway 1990, 1993; Krai I960; Young and Yavitt 1987). In the wild fruit set tends to be low possibly resulting from a lack of pollinators (beetles and flies) or due to limited resources, such as low light levels (Wilson and Schemske 1980; Faegri and van der Piji 1971). When flowers are successfully produced and polli- nated, low light levels may impede the photosynthetic partitioning of ener^ to the fruit, reducing fruit production. Asimina triloba will regularly generate many rootsuckers, potentially producing large clonal patches from an original seed (Pomper et al 2010; Gould 1939). The propensity of individual pawpaw genotypes to form root- suckers, and therefore more clonal patches, may be a genetic trait. Rootsuckers usually emerge near the original tree; therefore, increased stem density near some original trees may shade other understory plants. Because A. triloba is considered a self- incompatible tree, it further contributes to the low fruit production displayed in the wild. In the event fruit is produced, the large size of the seed makes it easy for its dispersal via mammalian activity (Cypher and Cypher 113 114 Journal of the Kentucky Academy of Science 73(2) 1999). However, A. triloba seed often fall victim to desiccation (Geneve et al. 2003) and freezing temperatures (Pomper et al. 2000). Genetically diverse native tree seedlings are an important component of forests because current seedling stocks will contribute to future sumval, structure, composition, and economic value of the forest (Hutchinson and Vankat 1997). An invasive shrub, Amur honeysuckle {Lonicera imiackii), has become common throughout A. triloba^ native range. When L. maackii becomes established in the forest understoiy, it has a negative impact on native tree seedlings and herbs (Gould 1996; Collier 1997; Hutchinson and Vankat 1997; Trisel 1997). Lonicera imiackii was introduced to North America in 1897 (Luken and Thieret 1996) and has been widely used as an ornamental (Hutchinson and Vankat 1998). This plant has escaped cultivation and grows wild in at least 24 states east of the Mississippi River (Trisel 1997). Due to a lack of native coevolved biological controls, L. maackii often grows unhindered (Schierenbeck et al. 1994). In forest edges (Luken and Goessling 1995) and areas of noticeable canopy disturbance, L. maackii has become a wide-spread problem by occupying forest niches that were home to a range of native plants (Yost et al. 1991; Demars and Runkle 1992). A number of additional invasive species [garlic mustard {Alliana petiolata), winter creeper (Eiiompnus fortunei), and colts foot {Tussilago faifara)] are also commonly found in the Central Kentucky region and these species can also have a negative effect on native forest ecosystems. Lonicera maackii has demonstrated the capabilities to reduced suiwival, growth, and reproduction of native herbs and trees. Also it has a negative bearing on species diversity, richness, and abundance of native plant species (Hutchinson and Vankat 1997; Collier et al. 2002; Hartman and McCarthy 2004). The ability of L. nuiackii to invade native plant ecosystems may be linked to its physiological traits. Invasive plants typically have a high allocation of energy to reproduction, plastic responses to the environment (branch struc- ture), and primaiy productmty (Luken et al. 1995). Lonicera fruit ripening occurs in autumn when many native food sources of food are not available, thus large amounts of fruit/seed produced is scattered by birds (Ingold and Craycraft 1983). When high light levels are attained, primaiy productivity is high (Luken 1988; Luken et al. 1995). Lonicera maackii leaves are present for longer periods than native perennials (D. E. Trisel, unpublished data) allowing L. nuiackii to increase its net carbon gain (Harrington et al. 1989; Schierenbeck and Marshall 1993). Also some studies suggest that L. nuiackii may contain allelopathic chemicals which may inhibit native plant species from growing in its vicinity (Skulman et al. 2004; Doming and Cipollini 2006; Trisel 1997). If L. maackii does produce allelopathic chemicals they likely enter the soil from leaching from decaying leaf material or secretion from its roots (Inderjit and Duke 2003). Since both A. triloba and L. maackii hold the same niche in the forest understory (McEwan et al. 2009), it has been suggested that established A. triloba, along with other native understoiy species, could be used to prevent L. maackii s movement into the forest understoiy (Hall 2003). It is important for A. triloba to maintain a high level of genetic diversity for its genetic advancement and reduction to disease vulnerability. The ability of some pawpaw genotypes to form many rootsuckers and thereby large clonal patches, could allow some pawpaw patches to hold an ecological niche in the forest understory when challenged by L. maackii. Asimina species have large distribution ranges, which may enable them to retain significant genetic variation (Hamrick and Godt 1989; Lu et al. 2011). Asimina triloba is a diploid organism (Bowden 1948; Krai 1960) with protogynous and self-incompatible flowers (Wilson and Schemske 1980; Lu et al. 2011). However, because A. triloba is a clonal species genetic variation within populations may be low. Studies assessing genetic varia- tion within A. triloba patches using inter simple sequence repeat (ISSR) markers have found that at least half of the patches were not clonal, containing at least 2 genotypes (Pom- per et al. 2009b). Clonal pawpaw patches w/ith genotypes that favor rootsucker formation could be more effective in holding forest niches against invasive species. Marker sys- tems that have proven useful in the genetic 115 Impact of Pawpaw Genetic Diversity on Invasive Species — Botkins et al Table 1. GPS coordinates of ail patches sampled. Location Coordinates Cove Spring A 38'^ ’13T.82"N 84' 70' 7LirW Cove Spring B 38^ 13'7.06"N 84' 70' '3L44'W EEC C 38' ’2r38.21"N 85' 7A4.0r'W EEC D 38' ’2r35.96"N 85' 7'50.58'3¥ KSU Farm E 38' 7T2.70"N 84' 73' '2.8rW KSU Farm F 38' 7TL7rN 84' 73' 'L99'W Kentucky River G 38' ’8T9.10"N 84' 71' 79.30'W assessment of A. triloba include: minisatellite probes (Rogstad et al 1991), allozymes (Huang et al 1997, 1998), random amplified polymorphic DNA (Huang et al. 2000, 2003), amplified fragment length polymorphism (Wang et al 2005), intersimple sequence repeat (Pomper et al 2003), and simple sequence repeat (Pomper et al. 2010). Simple sequence repeats (SSRs), have become the marker of choice for genetic diversity assessment, genetic mapping, and DNA fingerprinting (Wiinsch and Hormaza 2002). SSRs are short (1-6 base pairs) tandem repeat DNA sequences flanked by unique, conserved DNA sequences. The relative random distribution of microsatellites in the genome, codominant inheritance, high level of reproducibility, and transportability across laboratories make these markers useful for assessing genetic diversity, as well as finger- printing (Kijas et al. 1995; Wiinsch and Hormaza 2002). Microsatellite markers have not been used to determining clonality or assess genetic diversity pawpaw patches. The objectives of this study were two-fold, to determine: 1) the genetic diversity and clonality displayed in seven native pawpaw patches located at the Kentuclq/ State Uni- versity Environmental Education Center, The Kentucky River, Cove Spring Park, and the KSU research and Demonstration Farm using DNA microsatellite markers; and 2) if patches, especially those that are clonal, reduced the incidence of invasive species. MATERIALS AND METHODS Plant Material Leaf samples were collected from a total of seven different A. triloba patches at five different locations surrounding Frankfort, Kentucky (Table 1. GPS coordinates and map). From each patch, 25 leaf samples were collected from different stems located along a line transect ran through the middle of the longest portion of the patch. Samples collected from each population were placed into individ- ual Ziploc bags with a tree identification number and placed into short term storage at 4°C until they were needed for DNA extraction. DNA Extraction DNA was extracted from the leaves using the DNAMITE Plant Kit (Gel CO., San Fran- cisco). Approximately 1-2 cm^ of leaf material was used. DNA concentration and the 260/ 280 nm absorbance ratio were determined using a GeneQuant™ pro RNA/DNA calcula- tor (GE Healthcare Biosciences, Piscataway, NJ). All samples were then stored at ““80°C for further use (Lu et al. 2011; Pomper et al 2010). SSR-Polymerase Chain Reaction (PGR) Amplification PCR amplification for the SSR markers was performed using GoTaq Flexi DNA polymer- ase (Promega, Madison, WI). The reaction ingredients were as follows: 4 mL of 5X colorless GoTaq Flexi buffer, 0.4 mL of 10 mM dNTPs solution, 2 mL of 25 mM MgClg, 0.3 mL of 30 mM forward primer (fluorescence labeled with FAM) solution, and 0.3 mL of 30 mM reverse primer (unlabeled) solution, 0.2 mL of 5 units/mL GoTaq DNA polymerase, 2 mL of diluted 1 ng mL“^ pawpaw DNA, and 10.8 mL of ddH20 to bring the total volume to 20 mL (Pomper et al. 2010). Six primers were chosen labeled with FAM/HEX for tliis study (Table 2). The PCR amplification was performed using an Applied Biosystems Gene Amp PCR System 9700 Techne thermal cycler (Carlsbad, CA). The program used for amplification was as follows: initial denaturation at a temperature of 94°C for 3 min, followed with 30 cycles of 40 sec denaturation at 94°C, 40 sec annealing at 56°C, a 30 sec extension at 72°C, and a final extension period of 10 min at 68°C. Amplified PCR products were stored at a temperature of 4°C until they were needed for analysis. Products were separated using the ABI 3130 Genetic Analyzer (Applied Biosystems) with Genescan'^'^ 500 LIZ'^'"' as the size standard. Samples were genotyped with GeneMapper (version 4.0; Applied Biosystems). 116 Journal of the Kentucky Academy of Science 73(2) Table 2. Locus, primer sequence, fluorescent label, and SSR motif for the 6 microsatellite loci in A. triloha. Locus Primer sequence Label Enriched motif Pp-B3 FoiAvard; GCGAAAACGAACATACCTC Reverse: CCTCCTCCACCACCACTAC FAM GA Pp-B103 Forward: ATGCCCCAACAGAGACTTC Reverse: GGATGAGACACTCGGCTTAC FAM GA Pp-B117 Fonvard: GCATTGGTGTTTAGAACTTCTC Reverse: GCACAACAAAAAAGGATAAGAC HEX GA Pp-G103 Fonvard: AGCCAAAATCAAGAAACC Reverse: CTGCTCAGGGTCACTACA FAM AAT Pp-G119 Foward: AAACCGTAGTAAAACCAGACAA Reverse: GGATAGGAAAACATGGTGATTA FAM ATT Pp-G124 Fonvard: GTAGCCAGGAGGAGATGAACT Reverse: GGTGATTGGATTGCCTAAAAT HEX AAT DNA Analysis Power Marker (version 3.25, Lui and Muse 2005) software was used to calculate the following statistics: major allele frequency (most common allele), number of genoty^ies, observed number of alleles (oa), expected heteroz)^gosity (He, expected number of het- erozygous genotypes at a given locus divided by the number of genotypes at the locus), and polymorphism information content (PIC, an estimate that the parental origin of an allele can be determined from the marker locus genotype in any given offspring). Pawpaw and Invasive Plant Incidence Each A. triloha population was measured for its length and width. Three 5X5 m (25 m^) plots (left, right, and middle) were outlined and the numbers of stems of A. triloha and L. maackii were counted. Control plots (5 m^) were marked upstream/downstream of similar habitat to the patch and A. triloha and L. maackii stems were counted. Additional invasive plant species [garlic mustard {Alliaria petiolata), winter creeper {Euonymiis fortti- net), and colts foot {Tussilago farfara)] were also counted if found in the A. triloha or L. nuiackii plots. Plant Stem Analysis The average of the number of stems of each species (pawpaw, honeysuckle, garlic mustard, and winter creeper) in each plot was calculat- ed. Averages were entered into JMP Statistical software (9.0.0 ®20I0 SAS Institute Inc.) and a matched pairs analysis was run to determine effect of pa\\q)aw on invasive plants. Data were analyzed using a paired t-test with a level of significance of 0.05. RESULTS Asimina triloha Genetics The pawpaw populations examined dis- played high levels of genetic diversity based on the SSR markers amplified. Of the six loci examined (Pp-B3, Pp-BI03, Pp-BII7, Pp- GI03, Pp-GII9, Pp-GI24), all yielded prod- ucts that could be used for analysis (Table 3). Of the 140 individual pawpaw trees sampled, and 6 loci examined, there were 51 genotypes and a total of 45 alleles amplified. There was great variation for each locus in terms of the Table 3. Major allele frequeney, genoUpe number, allele number, allele size (base pairs), gene diversit}^ heterozygosity, polvinoiphism information content (PIC), and SSR motif for all patches by loeus. Marker Major allele frequency Genot)pe No Allele No Allele size (bp) Gene diversit)' Heterozygosit)' PIC Motif Pp-G103 0.60 8.0 5.0 270-298 0.59 0.40 0.55 AAT Pp-G119 0.37 6.0 4.0 158-167 0.70 0.91 0.64 AAT Pp-B117 0.46 6.0 5.0 110-179 0.68 0.64 0.63 GA Pp-B3 0.21 8.0 8.0 175-195 0.85 0.86 0.83 GA Pp-B103 0.18 16.0 18.0 264-347 0.89 0.74 0.87 GA Pp-G124 0.47 7.0 5.0 192-204 0.63 0.38 0.56 AAT Mean 0.38 8.5 7.5 110-347 0.72 0.65 0.68 117 Impact of Pawpaw Genetic Diversity on Invasive Species — Botkins et al number of genotypes (6-16), number of alleles (4-18), the major allele frequency (0.18-0.60), and the allele size (110-347). All genotypes were separated using a UPGMA dendrogram, depicting the relation- ship between area, patch, and individual sampled (Figure 1). The dendrogram shows that all patches displayed at least two geno- types in each patch. After examining six loci, all of the A. triloba patches exhibited some degree of clonality to some degree ranging from two - thirteen genotypes. Because all of the patches sampled contained at least two genotypes, none were completely clonal. The patches KYR G and KSU F displayed only two genotypes per patch, respectively, indicating a high degree of clonality in these patches across the patch transect that was sampled. The dendrogram showed that A. triloba had a high genetic diversity between patches; based on the dendrogram break points, the patches are quite genetically diverse from each other. Cove Spring Park patch (CSP) B was of great interest. The CSP B patch was the most highly differentiated, containing thirteen genotypes and falling into two separate locations on the dendrogram. Conversely Kentucky State Uni- versity Research and Demonstration Farm patch KSU F and Kentucky River patch KYR G only contained two genotypes. The Envi- ronmental Education Center (EEC) C was the most genetically dissimilar, and EEC D and KSU F exhibited the most clonality of any of the other patches. Geography of the patches seems to have little to no effect on genetic similarity. Pawpaw and Invasive Plant Incidence A total of three different invasive species [garlic mustard (Alliaria petiolata), Amur honeysuckle, {Lonicera maackii), and winter creeper {Euomjmus fortunei)] were found in many of the plots sampled (Tables 5, 6). Unfortunately, pawpaw patches and neigh- boring control plots that were examined at the EEC locations did not contain honeysuckle plants and were not useful for this portion of the study; this area has so far escaped honeysuckle infestation. Due to similarity in niche L. maackii was of greatest interest to the study. None of the invasive plant densities were statistically different in the pawpaw plots from the control plots sampled. Three patches appeared to have fewer honeysuckle plants inside each pawpaw patch than outside the patch. A fourth patch had similar numbers of honeysuckle within and outside the patch. However, there were not significantly fewer honeysuckle plants in pawpaw patches. There was a strong trend for pavq)aw patches to contain larger populations of garlic mustard than control plots (P-value > 0.0512; Table 5). There was not a significant differ- ence between winter creeper incidences in pawpaw patches; however there appeared to be more plants inside patches KSU E and KSU F. DISCUSSION Genetics This is the first report of the assessment of genetic diversity and clonality of native pawpaw populations using the SSR method- ology. Asimina triloba is a plant species that is known to produce rootsuckers. In terms of genetic diversity, the expected heterozygosity He (0.65) was lower than reported in Pomper et al. 2010 for cultivated pawpaw varieties, but similar to results published by Lu et al. 2011 and polymorphism information content PIC (0.68) was equitable to both published results. Only two nucleotide repeat motifs were chosen for the experiment, GA (loci Pp-B3, Pp-B103, and Pp-B117) and AAT (loci Pp- G103, Pp-G119, Pp-G124). The GA motif had a major allele frequency of (0.29), number of genotypes (10.0) and He of (0.75). The AAT motif had a major allele frequency of (0.48), number of genotypes (7.0), and He of (0.56). Based on the loci examined in the study, none of the patches were completely clonal. Alleles per locus was 7.5 in the pawpaw patches as compared to other cultivated crops sampled such as 13.3 for hazelnut (Bassil et al. 2005) 12.1 for apple (Hokanson et al. 1998, 2001) and 9.1 for pear (Sisko 2009.). When compar- ing the results from this experiment to known data from Pomper et al, (2010) and Lu et al. (2011) the data was comparable. Ranges from their studies were: major allele frequency 0.38-0.48, genotype number 10.8-20, allele number 7.3-12, allele size 144-343, heterozy- gosity 0.66-0.7, and PIC 0.63-0.68. Results from this experiment were the following: major allele frequency 0.38, genotype number 118 Journal of the Kentucky Academy of Science 73(2) KYRG CSPB KSU E KSU F EECD CSPA CSPB EECC Figure 1. UPGMA dendrogram generated showing all indmduals sampled and highlighted to demark its respective patch. 8.5, allele number 7.5, allele size 110-347, heterozygosity 0.65, and PIC 0.68. All patches showed clonality to some degree and KSU F and KYR G only showed 2 genotypes for all 20 stems sampled. When comparing Table 4 to Figure 1 it becomes clear that EEC C had the lower density of pawpaw stems per plot (11), with a similar number of pawpaw stems in the Impact of Pawpaw Genetic Diversity on Invasive Species — Botkins et al. 119 Table 4. Pa'W'paw stem density, patch lengtli, and number of genotypes found by patch. Patch Pau'paw stem density (stems/m=) Patch length (ft) #of Geiiot)'pes CSP A 0.85 72 7 CSP B 0.95 183 13 EEC C 0.44 82 5 EEC D 0.87 116 5 KSU E 0.99 103 6 KSU F 0.99 75 2 KYR G 0.65 70 2 other plots. Based on the data gathered from the experiment there is not a statistically significant relationship between A. triloba genetic diversity and any of the factors examined. Invasive Species Plots From the data obtained, it is difficult to determine if there is a negative relationship between the presence of A. triloba and L. maacMi. While KSU F and CSP A showed a trend that would support the hypothesis, the low incidence of honeysuckle inside and outside four of the patches do not provide enough additional information to reject or accept our hypothesis. Patch KSU F was highly clonal and CSP A did have a large clonal portion of the patch. KYR G was highly clonal, but not statistically different from the control to support the hypothesis that pres- ence of A. triloba will have a negative impact on L. maackii. Additional patches at different locations and higher honeysuckle pressure will be required to further test our hypothesis. It was interesting that garlic mustard had higher incidence in most pawpaw patches; protection by patches could be important in supporting the life cycle of this plant. No correlation could be identified between density of paw- paw stems, number of genotypes per patch or patch length (Table 4). Other variables could also be important to the incidence of invasive plants in pawpaw patches. All patches except KYR G, which grew beside the Keiitucty River, were located along stream banks. All the patches except the EEC patches (not seen in the table) were located on a steep bank; however KYR G and CSP A were much steeper than the others. All patches were found in the forest uiiderstoiy, although no light level readings were recorded during the progression of the season or during patch sampling to determine if light could have been a contributing factor. Just the presence of A. triloba was not enough to deter L. maackii occurrence. Additional fac- tors such as soil, light, and A. triloba canopy shading need to be considered when deter- mining correlations between A. triloba and L. maackii. Within the parameters of the experiment only stem numbers were counted, however there may be other factors that may help one understand the relationship. Examining stem diameter, stem height, leaf biomass, soil samples, light levels, water availability, etc. may reveal additional variables that influence pawpaw patch density and invasive plant incidence. Since L. maackii s foliage is present earlier in the spring and later into the autumn compared to A. triloba, it should be investi- gated whether periods of high light penetra- tion are able to sustain L. maackii throughout the season. Also, L. maackii may use allelop- athy, therefore its effects on A. triloba should be examined. It is worth noting that CSP B contained colt's foot in higher density than the control, but this was the only patch containing the species. In conclusion, the pawpaw populations in Kentucky examined displayed high levels of genetic diversity based on the DAN microsat- eilite markers amplified. All patches showed clonality to some degree in transects of patches. Three patches appeared to have fewer honeysuckle plants inside each pawpaw patch than outside the patch. A fourth patch had similar numbers of honeysuckle within Table 5. Invasive plant population, by species, in plots witli, and without pawpaws.* Count (plaiits/plot) ± SE Treatment Garlic mustard Honeysuckle Winter creeper Control 798.3 ± 230.7 8.5 ± 3.1 18.5 ± 9.8 Pawpaw 1123.8 ± 219.0 3.6 ± 1.3 19.3 ± 10.9 ‘ No significant difference detected between treatments. 120 Journal of the Kentucky Academy of Science 73(2) Table 6. Number of Pa\^^aws, Honeysuckle, Garlic mustard, and Winter creeper by treatment and location. Location Treatment Pawpaw Honeysuckle Garlic mustard Winter creeper CSP A Control 0.0 16.3 2750.0 0.0 Pawpaw 21.3 1.7 3083.3 0.0 CSP B Control 0.0 0.0 125.0 6.7 Pawpaw 23.7 1.7 250.0 0.0 EEC C Control 0.0 0.0 541.7 0.0 Pa\\^aw 15.7 0.0 391.7 0.0 EEC D Control 0.0 0.0 105.0 0.0 Pawpaw 21.6 0.0 93.3 0.0 KSU E Control 0.0 6.3 666.7 16.7 Pawpaw 24.7 11.0 1583.3 33.3 KSU F Control 0.0 34.7 291.7 6.3 Pawpaw 24.7 11.0 750.0 10.4 KYR G Control 0.0 2.0 1108.3 100.0 Pawpaw 16.3 0.0 875.0 91.7 and outside the patch. However, there were not fewer honeysuckle plants in pawpaw patches than control plots. Additional patches at different locations and higher honey suckle pressure and possibly different sampling approaches will be required to further test whether pawpaw patches reduce the inci- dence of honeysuckle bushes. ACKNOWLEDGMENT This research was supported by NIFA Mclntire-Stennis project funding project KYX-POMPERMCISf. Kentucky State Uni- versity Experiment Station Number KYSU- 000009. 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Cultivar identifica- tion and genetic fingerprinting of temperate fruit tree species using DNA markers. Euph)4ica 125:59-67. Yost, S. E., S. Antenen, and G. Hartvigsen. 1991. The Vegetation of the Wave Hill natural area, Bronx, New York. Bull Torrey Bot Club 188:312-325. Young, D. R., and J. B. Yavitt. 1987. Differences in leaf structure, chlorophyll, and nutrients for the understory tree Asimina triloba. Amer J Bot 74:1487-1491. J. Kv. Acad. Sci. 73(2):122-134. 2012. Abstracts of Some Papers Presented at the 2010 Annual Meeting of the Kentucky Academy of Science Edited by Robert J. Barney AGRICULTURE Evaluation of Sunflower {Helianthus annuus L.) Cultivars for Cut Flowers. CHRISTOPHER G. FERGU- SON MARTIN J. STONE and ELMER GRAY*, Department of Agriculture, Western Kentucky University, Bowling Green, KY 42101. The sunflower (Helianthus annuus L.), an ancient cultivated crop for oilseed production, more recently has gained acceptance as a cut flower in tlie florist industiy. Objectives of the present study were to sim^ey avmlable sunflower cultivars for adaptability to Kentucky growing conditions and for suitability to local florist markets. In 2011, sunflower cultivar trials were conducted at Bowling Green (36.93 N, 86.47 W) and Owensboro (37.78 N, 87.14 W) Kentucky. The randomized complete block design included 18 diverse cultivars and tliree replications. An experimental unit consisted of 10 plants of each cultivar spaced 30 X 60 cm. apart. Data were collected on seedling emergence, days to flowering, plant height, head diameter, stem diameter, branching, petal color, pollen production, and vase life. Tv4ce weekly harvests of flowering heads began 29 July and continued through mid-September resulting in 9 and 10 har\^ests in Bowling Green and Owensboro; respectively. Flowering heads were evaluated by both lay and professional groups. The data on plant and head characteristics are being stratified to determine the most adapted cultivars for growing in Kentucky and the ones most acceptable by tlie florist iiidustiy\ Effect of TiLssel Removal for Baby Corn Production in Kentucky. CHRISTOPHER G. FERGUSON*, ZHENG WANG, MARTIN STONE, and ELMER GRAY, Depart- ment of Agriculture, Western Kentuck)^ University, Bowling Green, KY 42101-3576. Baby corn (Zea mays L.) consists of unfertilized young ears harvested at silk emergence. The 2011 study was a culmination of four successive years of production and evaluation of baby corn at Western Kentucky University (36.93 N, 86.47 W). The objective was to compare tlie effect of tassel removal on baby corn (BC) production on four cultivars, two field (‘N77P-3000GT’, ‘N68B-3000GT’) and two sweet (‘Silver Queen’, ‘Peaches N Cream’) corns. Results indicated that tassel removal gave significant increases (P < 0.01) of BC ears across han^ests (H) and cultivars; however, the effect was not consistent over treatments. For harvests, the difference due to detasseling was highly significant (P < 0.01) for HI, significant (P < 0.05) for H2 and H3, but not significant (P > 0.05) for H4. For cultivars, numerical values were higher for detasseled than non-detasseled treatments in the first three har\'ests for each cultivar, but significant (P < 0.05) only for Peaches N Cream. Quality of BC from both tassel treatments decreased in H3 and H4. Further study is needed to determine economic returns of detasseling BC. Developing a Biofilter for Remediation of Pesticide Residues in Runoff Water. GEORGE ANTONIOUS, College of Agriculture, Food Science, and Sustainable Systems, Department of Plant and Soil Sciences, Ken- tucky State Universit)s Frankfort, KY 40601. Bioremediation is tlie use of Living organisms, primarily microorganisms, to degrade environmental contaminants into less toxic fonns. Nine biobeds (ground cartty filled witli a mixture of composted organic matter, topsoil, and a surface grass) were established at Kentucky State University research farm (Franklin County, KY) to study tlie impact of tliis practice on reducing surface mnoff water contamination by residues of diniethazone and trifluralin herbicides arising from an agricultural field. Biofilter systems were installed at tlie bottom of tlie slope of specially designed runofl plots to examine herbicides retention and degradation before enter- ing streams and rivers. In addition to biofilter systems, tliree soil management practices: i) municipal sewage sludge (SS), ii) SS mixed witli yard waste compost (SS + YW), and iii) no- mulch rototilled bare soil (NM used for comparison puiposes) were used to monitor the impact of soil amendments on herbicide residues in soil following natural rainfall events. Organic amendments increased soil organic matter content and herbicide residues retained in soil following rainfall events. Biofilters installed in NM soil reduced dimetliazone and trifluralin by 84 and 82%, respectively, in runoff water tliat would have been trans- ported down tlie land slope of agricultural fields and contaminate natural w^ater resources. Biobeds installed in SS and SS + Y\^^ treatments reduced dimetliazone by 65 and 46% and trifluralin by 52 and 79%, respectively. The use of biofilters in on-fanii bioremediation of pesticide residues in surface runoff water might prorfde a potential solution to contaminated runoff and seepage water arising from agriculturtil production operations. Evaluation of Constructed Wetlands for Nitrate and Phosphorus Removal. ERIC T. TURLEY* and GEORGE F. ANTONIOUS, College of Agriculture, Food Science, and Sustainable Systems, Department of Plant and Soil Sciences, Kentucky State University, Frankfort, KY 40601. Constructed wetlands are designed and utilized to reduce or eliminate tlie effect of agrochemicals on w^ater quality. The use of agrochemiciils, such as fertilizers, requires practices for remediation of these environmental contaminants. At Kentucky State University- Research Fami, tw^elve constructed wetland microcosms were 122 2010 KAS Meeting Abstracts 123 established. Six microcosms were filled witli river gravel and six with grade #2 limestone. Sweet flag plants, Acorns calamus, were planted in six microcosms (nine plants in tliree river gravel microcosms and nine plants in three limestone microcosms). The plants were observed for growtli and performance in the two types of rocks. Water samples were collected at regular time inter\^als to monitor die performance of the microcosms. Improvement was noted in orthophosphate, nitrate, ammonia, and dissolved oxygen concentrations. The results revealed that micro- cosms containing limestone, widi and widiout sweet flag, had a 23% reduction of orthophosphate ions compared to microcosms widi river gravel with and witiiout sweet flag. The NO3-N content in microcosms containing limestone and planted with sweet flag was reduced by 42% compared to microcosms containing river gravel and planted i\'itli sweet flag. Microcosms containing either limestone or river gravel and sweet flag had increased dissolved oxygen content over microcosms containing eitiier limestone or river gravel and no plants. The NH3-N content in all microcosms was reduced to near immeasurable amounts. Half-lives of Endosulfan Isomers on Field Treated Vegetables. KYLA ROSS*', GEORGE ANTONIOUS', and TEJINDER KOCHHAR^ 'College of Agriculture, Food Science, and Sustainable Systems, and ^Department of Biology, Carver Hall, Kentucky State University, Frankfort, KY 40601. Endosulfan 3 EC, a mixture of a- and j3-stereo isomers, was sprayed on field-growm pepper and melon plants at tlie recommended rate of 0.44 kg A. I. acre^'. Plant tissue samples (leaves and fruits) were collected 1 h to 30 days foUomng spraying and anal)^ed for endosulfan isomers. Anal)^is of samples was accomplished using a gas chromatograph (GC) equipped witli a mass detector in total ion mode. The results indicated the fomiation of endosulfan sulfate as tlie major metabolite of endosulfan sulfite and die relatively liigher persistence of die P-isomer as compared to die a-isonier. The initial total residues (a- and P-isomers plus endosulfan sulfate) were liigher on leaves dian on fniits. On pepper and melon fruits, die a-isonier, wliich is die more toxic to mammals, dissipated faster (Ty2 = 1-22 and 0.95 d, respectively) dian die less toxic P-isomer (Tiy2 = 3.0 and 2.5 d, respectively). These results confirm die greater loss of die a-isonier compared to die P-isomer, wliich can ultimately impact endosulfan dissi- pation in die environment. The liigher initial residues of endosulfan on pepper leaves should be considered of great importance for timing field operations and die safe entry of harvesters due to die liigh mammalian toxicity of endosulfan. Antioxidants and Heavy Metals Content of Hot Pepper. MCKENZIE JOHNSON*', GEORGE ANTONIOUS', and TEJINDER KOCHHAR^ 'CoUege of Agriculture, Food Science, and Sustainable Systems, and ^Department of Biolo^, Carver Hall, Kentucky State University, Frankfort, KY 40601. Hot pepper accessions that strongly accumulate heavy metals in their edible portions should be regarded as potential source of heavy metal contamination in the food supply. Phenols, ascorbic acid, capsaicin, and P-carotene are some of the classes of naturally occurring compounds having antioxidants activity in hot pepper. However, elevated concentration of heavy metals in hot pepper fruits could expose consumers to potentially hazardous chemicals. The main objectives of diis investigation were to: i) to select candidate accessions of hot pepper having high concentrations of phytochemicals for use as parents in breeding for these antioxidant compounds, and ii) assess if hot pepper genotypes that contciin great concentrations of capsaicin are also hea\y metals (Cd, Cr, Ni, Pb, Zn, Cu, Mo) accumulators. Seeds of hot pepper {Capsicum chineme) were collected from Belize, Brazil, Colombia, Ecuador, Mexico, Peru, Puerto Rico, and U.S. and planted in a silty-loam soil. Fruits of PI- 640900 (USA) contained the greatest concentration of capsaicin (1.52 mg g"' fruit) and dihydrocapsaicin (1.16 mg g“' fresh fruit), while total major capsaicinoids (capsaicin and dihydrocapsaicin) in tlie fruits of PI-438648 (Mexico) averaged 2 mg g"' fruit. PI- 152452 (Brazil) and PI-360726 (Ecuador) contained tlie greatest concentra- tions of ascorbic acid (1.2 and 1.1 mg g ' fruit, respectively). While PI-438648 (Mexico) contained the greatest concentration of total phenols contents (349 jig g ' fruit), PI-355817 (Ecuador) contained tlie greatest concentration of tycarotene among tlie other 63 acces- sions tested. Variability of these traits might be utilized via plant breeding approaches for their value-added health- promoting characteristics. An Update on the KSU Pawpaw Breeding Program. KIRK W. POMPER*, SHERI B. CRABTREE, and JEREMIAH D. LOWE, College of Agriculture, Food Science, and Sustainable Systems, Kentucky State Uni- versity, Frankfort, KY 40601-2355. The North American pa\\q)aw [Asimina triloba (L.) Dunal] is a tree fruit native to the eastern United States, which is in tlie early stages of domestication. Pawpaw fruit have fresh market appeal for farmers’ markets, community supported agriculture, and organic markets, as well as processing potential for frozen pulp production. New high yielding cultivars with excellent fruit quality would assist in tlie development of a pa\\q)aw industry. Kentucky State University (KSU) serves as the National Clonal Germ- plasm Repository for paviqiaw, and germplasm evaluation is an important research priority. Pawpaw germplasm has been screened for superior fruiting characteristics and trial as new pawpaw varieties. Pawpaw genot)qies in the KSU repositor)^ orchards under evaluation are from crosses of current pawpaw varieties or are open pollinated seedlings from a range of genetic sources. Some selections that produced high yields and excellent fruit quality have been selected for clonal propagation (budding onto rootstock) and field trials. Selections that have shown excellent fruit quality and yields include Hi4-1, Hi7-5, H3-120, G4-21, G4-25, G5-23, G6-120, G9-I09, and G9- 111. Most of the pawpaw advanced selections had similar 124 Journal of the Kentucky Academy of Science 73(2) budding success and \dgor to controls and are in field trials. When the genetic diversity of KSU advanced selections were evaluated using Simple Sequence Repeat DNA markers, these selections displayed significant genetic diversity compared to pa\\q)aw cultivars recently released by the PawPaw Foundation breeding effort. KSU advanced selections contain unique pawpaw germplasm that should enhance the genetic base of cultivars if these selections are released to the public. Ethanol Production Potential from Pawpaw Fruit Agricultural Waste. BRANDON K. MAY*, MICHAEL BOMFORD, KIRK W. POMPER, JON CAMBRON, and TONY SILWRNAIL, College of Agriculture, Food Science, and Sustainable Systems, Kentuck)' State Uni- versity, Frankfort, KY 40601-2355. The finite nature and rate of depletion of fossil fuels has prompted discussion into the production of biofuels to supplement our national energy demands. Government mandates diveiting corn or other grain products into cellulosic etlianol production has caused speculation into consequences of a reduced grain supply leading to increased food and livestock costs. Globally, studies are also being conducted to examine the potential useable sugars and starches derived from agriculturcd waste products ranging from olive mill waste, corn stover, peanut, and fruit waste. The pavip)aw {Asimina triloba (L.) Dunal] is a native tree fmit and is a new high-value fruit crop in Kentuclqx Pa\^q)aw fRiit have fresh market appeal for farmers’ markets, community supported agriculture, and organic markets, as well as processing potential for frozen pulp production. After haivesting of the pawqiaw fmit many poorer quality fmit remain unused in the orchard. Seed is a byproduct of pulp extraction of tlie poor quality fmit; the seed is valuable to nurseries, often selling for $10 per pound. Pulp waste from seed extraction has potential for ethanol production. In initial experiments \ritli pawpaw pulp waste from seed extraction, the extracted pulp sugar contents were found to be as high as 19° Brix or 1 12.3 g/L and once processed would yield 10.3% alcohol by volume. Using metliods devised by UC Davis for sugar to ethanol conversion, biofuel produc- tion potential of the pa\\paw fruit appears promising from tliis agricultural waste. The potentid for etlianol production and extraction methods for pawpaw pulp will be discussed. Prime-Ark®45 and Prime-Jan® Primocane Fruiting Blackberry Production Grown Under Organic Gulture in Kentucky. JEREMIAH D. LOWE*', KIRK W. POMPER', SHERI B. GRABTREE', JOHN R. GLARK^ and JOHN G. STRANGk 'Gollege of Agriculture, Food Science, and Sustainable Systems, Kentuck)^ State Uni- versity, Frankfort, KY 40601-2355, -Fruit Gulture & Breeding, 316 Plant Science Bldg. University of Arkansas, Fayetteville, AR 72701, and '^Department of Horticulture, N-318 Agricultural Sciences North, University of Ken- tucky, Lexington, KY 40546. Primocane-fruiting blackberries are attractive to Ken- tuck)^ growers because they can be grown organically and have the potential to produce a niche-market crop from late summer until frost. Therefore, locally produced fruit from primocane blackberr)^ selections can be harvested from July until usually October, provdding fruit for sale at farmers’ markets, community supported agriculture, and organic markets. In June 2010, a blackberry trial was planted at the KSU Research and Demonstration Farm on tlie certified organic land. The planting contained four replicate blocks each of the selections of Prime-Jan®, a primocane-fruiting selection from tlie University of Arkansas, and Prime-Ark®45, a newly released primo- cane-fruiting blackberry. Plants were arranged in a completely randomized design, with four replicate plots each containing five plants of each selection or cultivar (total of 20 plants of each selection or cultivar) in 10-foot plots. This tried was managed v^dth organic practices following the National Organic Program standards. A combination of cultivation, hand weeding, and straw mulch was used for weed control. Drip irrigation was used as needed. Ripe fruit were han^ested from the plants twice weekly, Monday and Thursday, from July until October 2011. Harvest period, tyeld, and berry^ weight were recorded for all selections. Prime- Ark®45 had higher yield and greater berry size as compared to Prime-Jan®. Warm summer temperatures in 201 1 appeared to reduced fruit set on Prime-Jan® as compared to Prime-Ark®45. Prime-Ark®45 appears to be a desirable cultivar well suited to Kentucky growing conditions. Kentucky State University Pawpaw Processing: An Update. SHERI B. GRABTREE*, KIRK W. POM- PER, and JEREMIAH D. LOWE, Gollege of Agriculture, Food Science, and Sustainable Sy^stems, Kentucky State University, Frankfort, KY 40601. The pawpaw {Asimina triloba) is tlie largest tree fruit native to the United States and is being grown on a small scale commercially in Kentucky^ and surrounding states. Pawqiaws produce unique fruit with creamy yellow-orange flesh and a flavor resembling a blend of mango, banana, and pineapple. The fruit’s short shelf life and difficulty mth posthaiwest handling and storage are impediments to commercial development. Marketing frozen fruit pulp as a value-added product could be one solution to this problem. However, pawpaw fruit pulp extraction is labor-intensive and made more difficult by the row of large inedible seeds in the fruit, and valuable pulp may^ be lost through ineffective extraction methods. The objective of this study was to determine if processed pulp recovery^ rate differs by pa\\q)aw cultivar. Three sets of five ripe fruit each of five pawpaw cultivars (KSU-Atu^ood, Mitchell, PA-Golden, Sunflower, and Susquehanna) were selected. Fruit were processed using a modified Roma Sauce Maker with a grape spiral and squasli/pumpkin screen to separate seeds from pulp and macerate the pulp. Individual fruit and seed weights were measured to determine percent seed. Processed pulp recovery rate varied significantly among cultivars, with Susquehanna, KSU-Atwood, and Sunflower having a greater percentage 2010 KAS Meeting Abstracts 125 of pulp recovered from fruit than Mitchell. Susquehanna, KSU- Atwood, and Sunflower had higher fruit weights tlian PA-Golden or Mitchell. With large fruit weights, a high rate of processed pulp recovery, and high fruit: seed ratio for more efficient processing, the cultivars KSU- Atwood, Susquehanna, and Sunflower are good choices for Kentucky pavtq)aw growers. Beneficial Insects Associated wtli Fall Established Native Perennial Plant Borders. JERMAINE DUNI- GAN*, JOHN D. SEDLACEK, and KAREN L. FRILEY, Kentucky State University, Frankfort, KY 40601. Native perennial plants are ideal for use in sustainable landscapes and are beneficial to the environment because tliey create habitat for wildlife species including beneficial insects. Attracting beneficial insects using a farmscaping approach can be important in trying to establish sustainable methods of insect pest management in vegetable and fruit crops. Some researchers have suggested that non-crop vegetation such as grasses and floral strips planted in crop field margins can enhance predaceous arthropod and parasitoid populations. There- fore, the objective of this study was to compare several insect groups colonizing newly established perennial borders vs. non-mowed mixed grass/weedy pasture borders. This study was conducted at tiie Kentucky State University Research and Demonstration Farm in Franklin County, KY. Sixteen plant species, including big bluestem (Andropogon gerardii), thimbleweed (Anemone virgini- ana). New England aster (Aster novae-anglica), side-oats grama (Bouteloua curtipendula) , purple coneflower (Ech- inacea purpurea), gray-headed coneflower (Ratihida pinnata), rattlesnake master (Enjgium yuccifoUum), common boneset (Eupatorium perfoUatum), blue lobelia (Lobelia siphilitica), bee balm (Monarch fistulosa), switcligrass (Panicum virgatum), foxglove beardtongue (Penstemon digitalis), hairy beardtongue (Penstemon hirsutus), slender mountain mint (Pycantheum tennuifo- Uum), little bluestem (Schizacharium scoparimn), and prairie dropseed (Sporobolus heterolepis), were estab- lished in 25 m X 2 m border rows replicated three times. Insects were sampled using four 15 cm X 15 cm sticky traps mounted to tobacco sticks in each border row. Lady beetles, solitary bees in tlie genus Agapostemon, soldier beetles (Chauliognathus pensylvanicus), and green lace- wings (Chrysops sp.) were caught in higher numbers in tlie native perennial border rows than the pasture borders. Wifl Mowing of Primocane-fruiting Blackberries Affect Fruit Ripening? KAREN L. FRILEY*, JOHN D. SEDLA- CEK, KIRK W. POMPER, JEREMIAH D. LOWE, MICHAEL K. BOMFORD, SHERI B. CRABTREE, MARQUITA L. GRAYSON-HOLT, CHRISTOPHER M. WALES, and RACHEL S. HAYDEN, Atwood Research Facility, Kentucky State University, Frankfort, KY 40601. Blackberry acreage has increased in Kentucky, while demand still often exceeds supply. The need for sustainable production practices is important for small and limited resource farmers as well as organic producers. Primocane-fruiting blackberries will set on both the overwintered canes as well as the current season primocanes. These varieties vAll produce two crops per pear - the regular summer crop on tlie floricanes and then a later crop on the primocanes. Trime Jim®’ and Trime Jan®’, which are two commercially available primocane- fruiting varieties, were used in this study. Three replicates of each of the two varieties were initially mowed to ground level 6 April, while tlie second mowing occurred on 24 June. Fruit ripening in Prime Jim plots began to drop off in late August, while fruit ripening remained constant throughout the season in Prime Jan plots. Stink Bug Species in Organic Blackberries. MAR- QUITA L. GRAYSON-HOLT*, JOHN D. SEDLACEK, KAREN L. FRILEY, KIRK W. POMPER, JEREMIAH D. LOWE, MICHAEL K. BOMFORD, SHERI B. CRABTREE, CHRISTOPHER M. WALES, and RA- CHEL S. HAYDEN, Atwood Research Facility, Kentucky State University, Frankfort, KY 40601. Stink bugs (Hemiptera: Pentatomidae) are pests of blackberries in Kentucky. These insects insert their beak into drupelets to extract tiie juice and may also leave a foul odor and taste. Consumer demand for damage-free produce means tiiat growers must use safe and effective management tactics for insect pests. Organic blackberry growers require sustainable and environmentally sound production methods to manage tliese insects. Spring- mowing of primocanes, on primocane fruiting blackberry varieties could avoid stink bug attack and delay fruit set. In 2011, three replicate plots of each of two varieties, Trime-Jim®’ or Trime Jan®’ were initially mowed to ground level on 6 April. Three replicate plots of each variety were then mowed a second time on 24 June. Stink bugs were sampled weekly using Florida Stink Bug Traps and hand collection methods. Stink bugs were found across treatments during the 2011 sampling period which extended from 11 July until 29 September. Five stink bug species were identified during the period of fruit ripening in the planting. The green stink bug was the most abundant, followed by rice stink bug and then brown, tvnce stabbed and one-spotted stink bugs at 53%, 16%, 11%, 11% and 11%, respectively. Both hand collection of stink bugs and the use of the Florida Stink Bug Traps resulted in the capture of stink bugs. Altiiough hand collecting required more time, more than twice as many stink bugs were captured compared to tlie stink bug trap, at 68% and 32%, respectively. Does a Metliyl Salicylate-based Lure Attract Lady Beetles to Blackberries? JUSTINA RIDDICK*, JOHN D. SEDLACEK, KAREN L. FRILEY, and JOY BIRIKE, Atwood Research Facility, Kentucky State University, Frankfort, KY 4(H)1. Kentucky produces approximately 45 ha of blackberries for a total value of $1,000,000 annually. Demand for locally grown and damage-free blackberries usually 126 Journal of the Kentucky Academy of Science 73(2) exceeds the supply. Developing more sustainable produc- tion metliods, including tlie use of beneficiiil insect attractants, such as a methyl salicylate-based lure, is important for tlie success of sniiill and limited resource farmers. Eight blackberr)^ plots, including six collabora- tors, were located in Franklin, Fayette, Scott and Shelby counties in Kentuck)-’. Three plots were certified organic and the other five plots had no pesticides applied. Four sticky traps and posts were placed in all plots and two PredaLure® lures were placed in each of tlie PredaLure plots. Sticky traps were collected weekly for tw^elve weeks, placed in labeled ziplock bags and taken to the laboratory where lady beetles were identified using an illuminated magnifier. Total number per species and average number per trap were then cxilculated. Pink lady beetle, Coleo- megilla maculata; seven-spotted lady beetle, Coccinella sepfempunctata; Asian lady beetle, Hannonia oxtjridis; parenthesis lady beetle, Hippodamia parenthesis; spotless lady beetle, Ctjcloneda niunda; orange-spotted lady beetle, Brachicantha ursina; tudce-stabbed lady beetle, Chilocoris stigma; and mildew eating lady beetle, PsijUobora vigitinmculata were caught in PredaLure baited sites. Parenthesis and seven-spotted lady beetles were not caught in non baited sites. PredaLure plots had more pink lady beetles, while non PredaLure plots had more Asian, spotless, and mildew-eating lady beetles. Lady Beetles Associated with Sweet Corn Bordered by Pasture, Buckwheat or Sunflower Borders. JOHN D. SEDLACEK*, KAREN L. FRILEY, MARQUITA L. GRAYSON-HOLT, CHRISTOPHER M. WALES, and RACHEL S. HAYDEN, Atw'ood Research Facility, Kentuck)^ State University, Frankfort, KY 40601. Sweet corn, Zea mays ‘Garrison®’, was grown in replicated plots on Kentuck)^ State University’s Agricultural Research and Demonstration Farm in Franklin County, KY. Each 25 m X 12 m plot w^as bordered on each side of its lengdi by a 2 m wide border of unmow^ed pasture, buckwheat {Fagopymm esculentum), or dw^arf sunflower {Heliathus anniius var. Big Smile). A randomized complete block design replicating each treatment five times was used and all plots w^ere separated by 25 m. Yellow^ sticky traps (15 cm X 15 cm) were used to capture lady beetles. Tw^o traps were deployed at canopy height between tlie edges and equidistant from the ends of each border. Four traps were deployed in each sweet corn plot, one in the center of each plot quadrant. Traps were changed weekly through anthesis. Stick)^ traps were placed indixidually in ziplock plastic bags, labeled, and transported to the laboratory for insect identification and enumeration. Pink lady beetle, Coleome- gilla maculata; Asian lady beetle, Harmonia axyridis; spotless lady beetle, Cy cloned a 7nunda; and seven spotted lady beetle, Coccinella septempunctata were caught in tliis study. The pink lady beetle was tlie most abundant species in aU three border t)pes and tlie sweet coni plots with 79% and 94% of tlie lady beetles caught in the borders and sweet corn, respectively. Pink lady beetle numbers decreased in buckwiieat from 14 August through 27 August, but increased markedly in sweet corn from 14 August to 20 August, potentitilly indicating movement into the sweet corn. Economics Systems, Role of Government, and Agricul- ture. STEPHEN A. KING, Department of Agriculture, Western Kentucky University, Bowling Green, KY 42101, Stephen.King2@wku.edu. The role of government in U.S. agriculture is in great part defined by the Farm Bill. The current U.S. Farm Bill is referred to as tlie “Food, Conseiv^ation, and Energy Act of 2008” and its provisions have far reaching impacts over a broad range of constituents. It is set to expire in tlie year 2012, at wiiicli time a new Fann Bill is expected to be enacted. In addition to traditional commodity programs diat directly impact the decisions of fanners, the legislation impacts the development of organic agriculture and biofuels markets, conser\^ation of natural resources, nutrition and rural housing programs, agricultural research, among odier areas. As a society’, how do we decide w4iat should and should not be included in die Fann Bill? Do we have any set of criteria for deciding the role of government in agriculture? How' does current and past fann legislation influence die economic system of die U.S.? The work presented addressed these questions. It has been hypotiiesized that our socio- economic values influence die political process and thus die legislation that is developed and in turn the role of government in agriculture and therefore die economic system. In general the research suggests diat current and past agricultural legislation moves our economic system toward the direction of centrally planned capitalism, that is agriculturiil resources are predominately ovned by die private sector but their allocation is strongly influence by governmentiil policy. A set of criteria were proposed for guiding the role of government in agriculture. Characteristics of and Reasons Why Farmers Choose Off-farm Work. CAITLIN N. CARTER, Department of Agriculture, Western Kentucky University, Bowling Green, KY 42101. caitlin.carter472@topper.wku.edu. In recent years, farmers in die United States have been seeking off-farm work to supplement fami operations income. In many cases, income prmaded by off-farm work is die largest component of die farm household income. PreUous research by the United States Depaitmeiit of Agriculture - Economic Research Service suggests diat die extent to wliich producers rely on off-farm income is dependent upon fami size and die type of enterprise. Results of diis study reveal the most prominent characteristics of diose farmers who choose off-farm work, die reasons why diey choose off-farm work, and die extent of income diat off- farm work provides to various categories of fann households. ANTHROPOLOGY AND SOCIOLOGY AcciJturation and Body Weight Status of Chinese Immigrants in Kentucky. CECIL BUTLER*, LINGYU HUANG, and CHANGZHENG WANG, Human Nutrition Program, Kentucky State University, Frankfort, KY 40601. Traditional Chinese diets are rich in vegetables and fruits and obesity is less prevident among Chinese people. 2010 KAS Meeting Abstracts 127 Immigrants adapt to American diets and behavior patterns. The objective of this study was to assess the acculturation and body weight status of Chinese immigrants in Kentucky. Tliirty Chinese American immigrants were recruited to participate in the study at a large community event. The subjects were asked to fill out a questionnaire before tliey were given a free analysis of their body composition (body fat %) with a Tanita TBF-521 body composition analyzer. Body mass indexes were calculated from the body weight and height measured on-site. 84% of the participants were udtliin nonnal body weight range with only 10.5% in tlie overweight and 5% in the obese category. Close to 50% of them speak Chinese and English about the same, but 28% speak mostly English. 46% of tlieni read better in Cliinese and speak mostly Chinese at home, but another 46% read better in English and speak mostly English at home. Only 38% speak Chinese only witli friends, 53% speak only English or mostly English to friends. 30% think in mostly Chinese, 23% tliink in Chinese and English about the same, but 45% think in mostly English. 54% watch TV mostly in English witli 30% do so mostly in Chinese. Over 73% listen to radio mostly in English witli none listening to radio in Chinese. 54% identify tliemselves as Cliinese American with 23% identifying with Chinese or American. 38% have mostly Chinese friends and 46% have some non- Chinese friends. 85% eitiier agree or strongly agree witli tlie statement that “I tliink of myself as being U.S. American.” 69% are proud or very proud of their Cliinese background. 69% eat mostly Chinese foods. 58% celebrate Chinese holidays most of the time. In conclusion, tliere are different degrees of acculturation among Chinese immi- grants but the effect on their body weight status was not clear due to the limited number of subjects in tliis study. Body Weight Perception and Willingness to Adopt Healtliy Eating and Activity Behaviors among Kentucky Adults. ERICA COLEMAN*, LINGYU HUANG, CECIL BUTLER, and CHANGZHENG WANG, Human Nutri- tion Program, Kentucky State University, Frankfort, KY 40601. Visitors to the 2011 Kentucky State Fair were recruited to fill out a questionnaire before they w^ere given a free analysis of tlieir body composition (body fat %) witli a Tanita TBF-521 body composition analyzer. 60% of overweight men considered themselves nomial and 77% of obese men considered tliemselves only overweight. 15% of normal weight women considered themselves over- weight, but only 21% of oveiweiglit women considered themselves normal and 51% of obese women put tliemselves in tlie overweight category. 80% of the participants would choose vegetables or fruits and nuts for snacks but 30% of tlie obese group would choose chips for a snack. 70% of die participants would leam to prepare vegetable dishes on their own but only 14% would do so by attending free workshops. 72% of participants were willing to add physical activities to their daily life such as walking but only 10% were willing to join a free club for exercise and 10% of the obese is willing to pay for an exercise program. 60% of the obese group was willing to cut soft drinks and 50% of tlie normal weight and overweight individuals were willing to drink water only. In conclusion, self-perception of body weight tended to lower tlie severity of weight problems in both men and women. Furtliermore, self-perception of body weight status could affect die willingness to adopt healthy eating and activity behaviors. BOTANY Effect of Natural Plant (Cocos nticifera) Derived Oil on Ulcerative Colitis in a Murine Model. PRANAV CHAN- DRA*, and NILESH SHARMA, Ogden College of Science and Engineering, Department of Biolo^ (TCNW), West- ern Kentucky University, Bowling Green, KY 42101. Ulcerative colitis (UC) is a chronic disease of the colon or large intestine that causes inflammation and ulceration (tiny open sores) of tlie inner lining of tlie colon and rectum. Ulcerative colitis can occur in all areas of the colon. In patients with ulcerative colitis, the body’s immune system over-reacts and body mistakes food, bacteria, or other internal materials in the colon for an invading substance and it signals die immune system to attack the material, thus irritating the colon. This irritation triggers a flare of ulcerative colitis symptoms likewise bloody, pus or mucus filled stools, diarrhea, cramping, abdominal pain and bloating. Highest incidences are seen in the United States, Canada, the United Kingdom and Scandinavia. Since die etiology of UC remains unclear, successful treatment strategies targeting large sections of affected population have not been found. UC is currently treated with medications tliat include a combination of anti-inflammatory, immunosuppressive and antibiotic drugs widi limited remission and significant episodes of side effects; often patients become refractory and seek an alternative therapy. Lack of efficacious drugs to treat patients with different forms of inflammatory bowel disease underscores need for the development of a new and effective alternative therapy. Currently, the role of saturated fatty acids on human health is being revisited, and this issue is drawing significant attention specifically in inflammatory and metabolic disorders. Effects of medium-chain saturated fatty acids (MCFAs) - like lauiic and caprylic acid- have been little studied, and thus drawing much attention. Natural coconut {Cocos nucifera) oil is a rich source of MCFA, main constituent being lauric acid: a 12C-cliain of fatty acids. Traditionally, coconut oil has been used as cooking oil in several parts of India and other Asian countries. Lauric acid converts to the fatty acid monolaurin in our body and has adverse effects on several microorganisms including bacteria, fungi, yeast and enveloped viruses. Lauric acid is one of the main components of human breast milk, and boosts immune system of children during infancy. Limited knowledge of inflammatoiy^ conditions coupled with a narrow range of therapeutic options necessitates investi- gating tlie role of natural products. Therefore, the present study focuses on the anti-inflammatory role of natural 128 Journal of the Kentucky Academy of Science 73(2) fatt)’ acids derived from Cocos nucifera in the murine model of ulcerative eolitis. Genetic Diversity in Kentucky Spicebush Populations Using Simple Sequence Repeat Markers. RE’GIE SMITH*, KIRK W. POMPER, JEREMIAH D. LOWE, JACOB BOTKINS, and SHERI B. CRABTREE, College of Agriculture, Food Science, and Sustainable Systems, Kentueky State University, Frankfort, KY 40601-2355. Spicebush {Lindera benzoin L.) is an aromatie small native shrub that grows in the moist, understory areas of Appalachia and has potential as a new niche crop for small farmers. Native Americans and early settlers used this plant traditionally as a tea. The berries ean be used for jam and spieing of foods, and may have health benefits including antioxidant compounds. Native spicebush pateh- es also can ser\^e an important role in forest ecosystems in tenns of fruit production for aniiUcils, soil erosion control, and enhancing insect biodiversity. Spicebush may ser\^e to hold ecological niches by outeompeting invasive plants compared to those in unchcillenged areas. Genetie diversity of native spicebush populations in Kentucky has not been examined. The objective of this study is to determine the genetic diversity in spicebush populations in Kentueky using simple sequence repeat (SSR) DNA marker systems. Leaf samples were collected from 20 spicebush plants in the forests at the Kentucky State University Environment^ Education Center (EEC) and at a location near the Kentueky River. DNA was extracted using the DN Amite Plant Kit. Primers A7, AI15, BIOS, and BI22 were used to amplify SSR products that were separated with a 3130 Applied Biosystems capillary electrophoresis system. The software program Power Marker was used to examine genetic relationships among genot)q)es. The SSR markers generated showed genetic variation among the spicebush genot}q)es. A number of selections w4th unique genot)q)es will be sampled and propagated for study in the KSU germplasm collection for potential eultivar development. Pawpaw Patch Genetic Diversity’ and Clonality and its Impact on the Establishment of Invasive Species in the Forest Understoiy. JACOB BOTKINS*, KIRK W. POM- PER, JEREMIAH d' LOWT:, and SHERI B. CRABTREE, College of AgriciJture, Food Science, and Sustainable Systems, Kentucky State University, Frankfort, KY 40601-2355. The pawpaw {Asimino triloba) is a native understor)^ tree of 25 states of the east and micKvest United States. Pawpaw’s ability to compete with local invasive species in Kentucky has not been examined. The objectives of this study were two-fold: to determine the genetic diversity and clonality displayed in seven native pawpaw patehes located at the Kentucky State University Environmental Education Center (KSU-EEC), the Kentucly’ River, Cove Spring Park, and the KSU Research and Demonstration Farm in Franklin County, using microsatellite markers; and to determine if patches reduced the incidence of invasive species. Tw^enty-five trees from seven patches in the four different locations w^ere sampled for genetic analysis. Leaf samples were extracted using the DN Amite Plant Extraction Kit and products from four microsatellite loci w^ere analyzed using a 3130 Applied Biosystems capillary electrophoresis system. String grids were created and invasive plants counted in three 10-meter squares in each of the patches and control plots outside of each patch. The number of plants for each invasive species wdthin pawq)aw patches was counted and compared to a control plots. Pawpaw patches displayed high genetic diversity among popula- tions. Japanese honeysuckle {Lonicera japonica), garlic mustard {Alliaria petiolata), wdnter creeper {Euonynius foi'funei), and colts foot {Tussilago farfara) were found in most locations; however, there w^as no significant difference in the incidence of invasive plants between the patehes and the control plots. Pawpaw stem density may be important in the incidenee of invasive plants wdthin patches. CHEMISTRY Synthesis of Homoleptic Nickel (II) Complexes and Examination of their Coordination D)mamics in Solu- tion. LAURA BISHOP*', DAVINDER KUMARk CRAIG A. GRAPPERHAUSk and CHRISTOPHER S. MUL- LINS', 'Division of Natural Science, Campbells\dUe University’, Campbellsvdlle, KY 42718, and ^Department of Chemistr)’, University of LouisvdUe, Louisville, KY 40292. In this study, w^e have begun to examine the structural dynamics for a group of homoleptic nickel (H) complex- es. All of the tridentate ligands have one fairly acidic proton attached to an oxygen or nitrogen donor atom that ligates the metal in the first eoordination sphere. The ligand 2-(scilicylideneamino)- 1-hydroxyethane ( H2-S AL- AHE) has been studied extensively for a variety^ of applications, including the s)’nthesis of single-molecule Octahedral structure Square-planar structure 2010 KAS Meeting Abstracts 129 magnets derived from cluster compounds. A previous publication of the homoleptic Ni(II) complex of this ligand reported tlie single-cr)^stal X-ray structure, wherein the nickel ion was found to be octaliedrally coordinated by two of the ligand molecules. Our recent studies with this complex suggest that the complex undergoes a coordination number change in solution to give a four-coordinate nickel complex. This complex has been found to give green crystals reminiscent of tlie octaliedral structure upon reciy^stallization. Future work will utilize several spectro- scopic techniques such as UV-Visible absorption, EPR, and NMR, etc. in order to study the fluxional nature of these complexes in solution. COMPUTER AND INFORMATION SCIENCES Statistical Analysis of Microarray Gene Expression Data from a Mouse Model of Toxoplasmosis. SHRIKANT PAWAR*, CHERYL D. DAVIS, and CLAIRE A. RINEHART, Department of Biology, Bioinfomratics and Information Science Center, Western Kentucky University, Bowling Green, KY 42101. Toxoplasmosis, caused by the protozoan parasite, Toxoplasma gondii, is a major cause of morbidity and mortality in patients with AIDS and an important cause of miscarriage, stillbirth and congenital disease in newborns. Previous studies have provided evidence that dietary supplementation with vitamin E and selenium is harmful during experimental toxoplasmosis in mice, whereas a diet deficient in vitamin E and selenium results in decreased numbers of tissue cysts in the brain and dramatically reduced brain pathology. The overall goal of the present study was to determine the impact of dietary supplementation with antioxidants on gene expression in the brains of non-infected mice and in mice infected with T. gondii using microarray analysis. RNA was isolated from the brains of C57BL/6 mice, and an Agilent Oligo Whole Mouse Genome Microarray (Agilent Technologies, Inc.) was performed. A total of 48 chips were normalized by Z ratios and the Data Driven Harr Fisch Normalization methods. Differen- tially expressed genes were identified by applying thresholds to identify significant values and the results were compared between the normalization methods. These differentially expressed genes and their respec- tive fold change ratios were used in Ingenuity Path- way Analysis (IPA) software to analyze the pathways involved with these genes. The identified pathways associated with differentially expressed genes are very important in determining the impact of dietary supple- mentation with antioxidants on gene expression in the brains of mice infected with T. gondii and specific alterations of those pathways can help us in reducing the harmful effects of the same in future. Support from the National Center for Research Resources NIH Grant Number 2 P20 RR-16481 and from the WKU Bioinfor- matics and Information Science Center is gratefully acknowledged. ECOLOGY AND ENVIRONMENTAL SCIENCE Assessing Kentucky State University’s Recycling Pro- gram. RE’GIE SMITH*, RODNEY RIPBERGER, BRANDAN BURFICT, DUSTIN HODGES, and JOHN D. SEDLACEK, Masters of Environmental Studies Program, Kentucky State University, Frankfort, KY 40601. The purpose of this study was to conduct a recycling audit of six buildings on Kentucky State University’s (KSU) campus and to conduct a survey of the attitudes, perceptions and knowledge of faculty, staff and students about recycling on campus. A six-week audit was conducted of waste from tv^o academic buildings, one administrative building, the student center, and one male and one female dormitory for a total of six campus buildings. Recycling bins were located in the six buildings and their locations were documented on maps. We collected a total of 815 lbs of waste; of the waste tliat was collected 361 lbs (44%) could have been recycled. Thirty six percent of the recyclables was plastic, 28% was paper and 24% was cardboard. The survey revealed that students recycle less compared to tlie faculty and tlie staff The faculty use the recycling bins an average of 1.32 times per day, staff use the bins an average of 1.47 times per day, and the students use the bins an average of 0.66 times per day. The survey respondents’ answers showed that they believe recycling is important and almost 97% are willing to help KSU recycle more. In fact, 81% of tlie respondents recycled prior to life at KSU, and only 3% consider themselves to not be “green,” or to not take actions to promote a heiilthy environment. The census of students, faculty, and staff provided data that will help make recommendations for the KSU recycling program. Winter Management of an Invasive Species, Garlic Mustard, Alliana petiolata, in Wooded Habitat. JACOB BOTKINS*, RUSSELL WILLIAMS, ADAM GER- UGHTY, and JOHN D. SEDLACEK, Masters of Emdronmental Studies Program, Kentucky State Univer- sity, FrankTort, KY 40601. Garlic mustard, Alliaria petiolata, is a biennial cool- season plant growing 0.5-1 ni tall. It is an aggressive competitor for resources excluding native plants from their habitats. This plant is shade tolerant allowing it to invade mature woodlands, where it shades out native understory flora and produces allelopathic compounds inhibiting seed germination of other species. It is threatening the federally endangered Braun’s rockcress {Arabis perstellata) known only from Franklin, Owen and Henry counties. The objective of this research was to quantify two methods of winter management of garlic mustard at tlie Julian Savanna State Nature Preserve in Franklin County, Kentucky. Hand removal, a 2% glyphosate solution and untreated control treatments were used. Plots were 1 m^ in area. A thatching rake was used to hand weed while a hand held 0.5-liter sprayer 130 Journal of the Kentucky Academy of Science 73(2) was used to apply glypliosate to each plot. A digital camera was used to photograph each plot 1.5 ni overhead before plot treatment on 16 February. Each plot was photo- graphed nine weeks after treatment and weed control quantified using tlie NIH ImageJ program (U.S. National Institutes of Health). There was a 22% and 24% increase in garlic mustard and purple deadnettle foliage in glypliosate treated and hand weeded plots, respectively. There was >722% increase in garlic mustard and purple deadnettle coverage in untreated plots. Thus, a single application of glypliosate in mid-February or hand weeding/surface tilling reduces, but does not eliminate, garlic mustard and purple deadnettle in wooded areas. White Tailed Deer in Frankfort, Kentucky: Population Assessment and Implications for the Community. JON CAMERON*, TERRELL HOLDER, MARK RASCHE, KIAH RODRIGUEZ, MIKE WARD, and JOHN D. SEDLACEK, Masters of Environmental Studies Pro- gram, Kentucky State University, Frankfort, KY 40601. A spotlighting assessment of the population of white- tailed deer was conducted in five Frankfort city parks. Two to four replications were done on each park. Cove Spring Park’s population was estimated at 24, Capitol View Park - 77, Fort Hill Park - 76, East Frankfort Park - 6, and Juniper Hills Park - 0. Based on a calculated estimate of deer/niF, Cove Springs and East Frankfort densities fell within the range of expected density based on mean densities of adjacent counties; Capitol View and Fort Hill did not. This could be because surveys of Capitol View and Fort Hill were not accurate or the densities are in fact much higher tlian expected in this region. To supplement tlie population survey, we did an informal assessment of the forest understory in three of tlie surveyed parks and looked at deer-vehicle collisions as a proxy for density. Understor)^ vegetation was limited to a small number of species dominated by bush honeysuckle {Lonicera spp.) and very few tree saplings of any species were observed. In Fort Hill Park, the understoiy^ was essentially non-existent. There were 851 deer- vehicle collisions between 1 January 2001 and 31 December 2010, mostly occurring in October, November and December. The collision count over ten years, looking at only November, suggests a two or three year deer population cycle. The census combined with the informal vegetation assessment and incidence of deer-veliicle collisions in November suggests that deer in Frankfort may be approaching ecological carrying capacity. Citizen Awareness of Invasive Plant Species in Kentucky. JOHN D. SEDLACEK*, ADAM GER- UGHTY, JACOB BOTKINS, and RUSSELL WIL- LIAMS, Masters of Environmental Studies Program, and MARA MERLINO, and TIERRA FREEMAN, Psychology Department, Kentucky State University, Frankfort, KY 40601. Non-native invasive species are one of the primary threats to biodiversity. Public support for invasive species management programs is critical to the success of such projects. Additionally, understanding the public’s knowl- edge, attitudes and perceptions can assist with the development of outreach and educational activities. In order to assess the level of understanding of the invasive plant species threat, attitudes towards invasive species management and demographic factors influencing such attitudes, a questionnaire survey of 400 randomly selected members of the public in the greater Louisville, Lexington, Frankfort and Bowling Green metropolitan service areas was conducted. We developed a survey that determined an elementary level of awareness of invasive plants, people’s understanding of what is native vs. non- native, why these concepts matter, and if respondents are motivated to assist in invasive species removal. The survey was administered via Survey Monkey. Surprisingly, only 4.5% of die population invited to participate in tlie survey actually responded. Thus, drawing major conclusions from tlie data would not be advisable. However, the low response indicates that the vast majority of citizens are unaware of, or don’t care about, tlie potential economic or ecological consequences of invasive species establishment. We provided all survey information to the Kentuck)^ State Nature Preserves Commission who hopefully will be able to furtlier address educational issues concerning invasive species and how to better market tliose messages in Kentucky. GEOLOGY Nutrient and Fecal Microbe Assessment of the Water Quality of Tates Creek, Madison County, Kentuclqr KRISTOPHER H. CARROLL*, and WALTER S. BOROWSKI, Department of Geography and Geolo^, Eastern Kentucky University, Richmond, KY 40475. Tates Creek is a significant tributary to the Kentucky River that has shown high levels of microbial and nutrient pollution. We sampled the waters of Tates Creek comprehensively by occupying 25 stations along its 13- mile lengtli, collecting stream water at tlie confluence of major tributaries from its headwaters to the Kentucky River. Samples were collected four times between May and August 2011 during dr}^ periods as well as immediately after rainfall events. We measured ammonium (NH/), nitrate (NOs") and phosphate (P04“) concentrations using color- imetry. Microbial samples were measured for total colifomi and Escherichia coli using IDEXX Colilert-lS media. Background levels of NH/, NO3" and P04^ are t)q)ically '^0.2 ppm, 13 ppm, and 1.0 ppm, respectively. Nutrient concentrations generally increase during rainfall events, presumably because nutrients are flushed into the stream. Background counts of E. coli are typically 100 cfu/mL but microbe counts reached 1000-2419 cfu/mL immediately following rain events. A sewage treatment plant exists approximately two miles from the headwaters and notice- ably affects water quality. Nutrient concentration, espe- cially NH4'' and P04“, are markedly increased at the plant’s outflow. These nutrients then decrease steadily in concen- tration downstream to background levels. In contrast, fecal 2010 KAS Meeting Abstracts 131 microbe counts are high upstream from tlie plant, but fall to near- zero levels at its outflow, and then increase anew dov^mstream. The treatment plant went offline on 19 July 2011, so we wiU be able to assess any changes in water quality and stream health in tlie future. Suspended Sediment Concentration in the Brushy Creek Watershed, Kentucky. TYLER A. WADE*, and WALTER S. BOROWSKI, Department of Geography and Geology, Eastern Kentucky University, Richmond, KY 40475. Suspended sediment concentration (SSC) can be used as a proxy for environmental health of stream water. For example, large sediment loads can cause harm to aquatic life and are a mechanism for introducing and transporting fecal microbes. We measure SSG of the Brushy Creek watershed, located in Rockcastle, Pulaski, and Lincoln Counties, where tlie Eastern Kentuck)^ Environmental Research Institute (EK-ERI) has been conducting an assessment of the watershed. Two auto-sampling units were placed in Brushy Creek to collect water samples for determination of SSC. The units collect samples every 14 hours for a two-week period, then samples are retrieved for analysis, and new sample botdes are loaded into the auto samplers. Sediment sampling has been in progress since January 2011 and mil continue until November 2011. We measure sediment transport during dry, wet, and storm periods. Retrieved samples are brought to the laboratory where sediments are filtered and weighed to determine SSC. The SSC data have been evaluated along witli records of rainfall events, as recorded by the UK Agriculture weather station located in Somerset, KY. Due to operational difficulties with our water and sediment samplers, we have only collected intermittent data, however, rainfall events seem to be correlated with increased SSC. The Micro- and Macro- Faunal Diversity of a Devonian Dysaerobic Environment. LARRY TACKETT*, KARA WELLS, and CHARLES E. MASON, Department of Earth and Space Sciences, Morehead State University, Morehead, KY 40351. This study examined the fauna contained in the type section of tlie Three Lick Bed of the Ohio Shale (Upper Devonian), which is located in Rowan County, Kentuclqv The Three Lick Bed separates the underltyng Huron Member from the overlying Cleveland Member of the Ohio Shale. The unit is 3.42 meters thick and is composed of three greenish gray shale beds separated by two intervening black shale beds. The three greenish-gray shale units were hypothesized to be deposited under dysaerobic conditions and tlius tlie focus of tliis study. To date slightly over 200 kilograms of samples have been processed for macrofossils and 90 kilograms for microfos- sils. The samples were broken down using tlie kerosene technique and washed through a nested set of U. S. standard sieves, a #20 for macrofossils and a #100 for microfossils. The residue caught on the #100 sieve under went heavy liquid separation and both the hea\y and the light fractions were examined for microfossils. All picking, sorting, and identification of fossils were conducted under a binocular microscope. The results of this study support our hypotliesis that the greenish gray shale units of the Three Lick Bed were deposited in a dysaerobic emdron- ment. Evidence supporting this conclusion includes the following: 1) a low diversity macro invertebrate fauna of 15 species, 2) of the 532 specimens identified nearly all were juveniles, 3) tlie fauna was dominated by mollusks, 12 out of 15 species, and 4) all macro invertebrates except Lingula were preserved as pyretic internal molds. Overall, benthic foraminifera dominate tlie microfossil fauna in both diversity and abundance, followed by ostracodes in terms of abundance. The macrofossil fauna is dominated by amnionoids being the most diverse (with four species) and a low- spired gastropod being the most abundant (198/ 532). HEALTH SCIENCE Procedure for Preparing Purple Sweet Potato Powder. LINGYU HUANG*, CECIL BUTLER, and CHANGZ- HENG WANG, Human Nutrition Program, Kentucky State University, Frankfort, KY 40601. Purple sweet potato has health promoting properties. Puqile sweet potato powders currently on tlie market are made by grinding the raw material and sun-di*ying the precipitates, or grinding of sun-dried slices of tlie sweet potato. Recent research indicates that such processes lead to significant loss of antioxidants. Our objective was to develop a process that better preseives the antioxidants when tlie powders are produced. Purple sweet potatoes were obtained from a Noitli Carolina farm. The whole sweet potatoes were steam-cooked at 200°F for 45 min before they were skinned and mashed. The mashed material was dried in a forced air-drying oven at 60°C or 80°C. After dr)ang the materials were crushed and ground into powder in a Hobart grinder. Cooking of the whole sweet potato avoided tlie activation of enzymes so the damage of the antioxidants would be reduced. The drying process resulted in 71% loss of weight. The skin accounted for 4.5% of the total weight. The dr)4ng temperature was critical. At low temperature, tlie materials would spoil and mold would grow, rendering the materials useless. At high temperature (100°C), browning of the materials occurred potentially damaging the nutrients. Drying process did not significantly reduce the total phenolic content of the powder. PHYSIOLOGY AND BIOCHEMISTRY Regulation of EMT Proteins in Breast Cancer Cell Lines. MARY WIECHART*, JACKIE JANSEN, ARIELLE MARASLIGILLER, HILLARY RESTLE, SHANE MULVIHILL, STEFAN SIWKO, and JULIA CARTER, Wood Hudson Cancer Research Laborator)^ Newport, KY 41071. During 2011, 232,620 new breast cancer cases are predicted in the US and 39,970 breast cancer deaths. This 132 Journal of the Kentucky Academy of Science 73(2) high mortality rate is doe to tumor metastasis. To metastasize, breast cancer cells must undergo epithelial- mesench)mial transition (EMT), a process tliat disaggre- gates the epitlielium, reshapes it for movement, and requires biochemical re-programming. Slug and p21 activated kinase (Pak 1) are two proteins that are increased during EMT. Another protein, eukaryotic initiation factor 4E (eIF4E), is elevated in breast cancers. Elevated eIF4E function selectively enhances the trans- lation of niRNAs with long, highly structured untranslated regions (UTRs) such as Slug and Pak 1. We hypothesized that elevated eIF4E function in breast cancer cells may enhance translation of Slug and Pak 1, thereby promoting EMT. To test this h)q)othesis we examined protein expression in western blots of lysates from 6 breast cancer cell lines witli different estrogen, progesterone, and Her 2 receptor status. Since MDA 231 breast cancer cells expressed ail three proteins, are negative for all three receptors and are reported to be the most invasive breast cancer cell line, we used this cell line to determine if knock down of eIF4E by siRNA transfection would alter expression of tliese EMT associated proteins. We found a slight reduction in Slug expression in MDA 231 cells witli reduced eIF4e but a slight increase in Pak 1 expression. Although these data are preliiiiina.iy, they do not support our hypothesis that eIF4E regulates Slug and Pak 1 expression and possibly EMT in breast cancer. Two Gerniliiie Variants of the TGFj^Rl Gene are Associated witli Initiation, Progression and Clinical Outcome of Colorectal Cancer. HILLARY RESTLE*f SHANE MULVIHILL*k JONATHAN BENDER*, KE- \TN MURRAY’, JESSICA SHAW’, BRIANA VOGT', ROBERT SHIELDS’, BRUCE COLLIGAN', JAMES DEDDENSf LARRY DOUGLASS’, JAMES SCHAE- RER’, and JULIA CARTER’, ’Wood Hudson Cancer Research Laboratory, Newport, KY 41071, and ^University of Cincinnati, Cincinnati, OH 4.5221. Germline variations of transforming grovAh factor beta (TGFP) are associated with tumor initiation and progres- sion, especially in bladder, breast, ovarian, kidney, and lung cancer. Colorectal cancer (CRC) is the second leading cause of cancer-related deatlis in tlie United States. There vdll be over 140,000 people diagnosed with CRC tliis year and nearly 50,000 CRC related deatlis. CRC is frequently diagnosed in the later stages due to tiie non-specific symptoms in its early stages, furtlier empha- sizing the need for genetic biomarkers. Two germline variants in the TCF^Rl pathway were analyzed via capillary electrophoresis in 233 cases and 219 controls to determine if tlieir incidence affected the prevalence and stage of cancer. We hypothesized that these variants could be significant factors in predicting initiation, progression, and growth in CRC. We found that patients witli Int7G24A, a single nucleotide polymorphism in the intron 7/exon 7 boundary of the TGFI^Rl gene, had a significantly higher incidence of CRC as compared to non-cancer controls. TGFfJRl^GA, a nine base pair deletion in exon 1 of the TCF^Rl gene, was not associated with increased CRC incidence but was associated with adenoma patients tiiat did not progress to advanced CRC. This suggests tiiat TGFfiRl*6A may have a protective effect. Patients with carcinoma-in-situ (CIS) or CRC stages 1-4 showed a significantly increased incidence of tlie Int7G24A variant as compared to non-cancer controls and patients who never progressed beyond adenoma. This discoveiy indicates that Int7G24A could be a bioniarker for identifying patients at a higher risk for developing CRC. SCIENCE EDUCATION Sharing Ideas About Assessing Student Learning. JOHN G. SHIBER, Dmsion of Nursing, Biology & Allied Healtli, Big Sandy Community & Technical College, Prestonsburg, KY 416.53. An emerging national policy of holding post-secondary educators more strictly accountable for student learning has many scrambling to re-evaluate their teaching strategies and tlie assessment parameters they employ. It is an overwhelming challenge because, as colleges increasingly become like businesses and treat their students like clients, student attitudes toward learning are undermined by an equally strong if not stronger one of entitlement, irrespective of how much or little tliey apply themselves in tlieir studies. This paper discusses the consequent need for an increased number of parameters, besides testing, to help assess student learning in the sciences such as tliose shown in studies by this investigator to be beneficial: pre-/post-testing, class attendance, in- class writing assignments immediately after reading articles or watching Udeos on scientific topics, active individual involvement and course-appropriate extra credit opportunities in and beyond the classroom, end- of-semester student opinion questionnaire on course, etc. An argument for establishing continuity among teachers within each science discipline to follow tlie same assessment guidelines will also be presented. ZOOLOGY Measurement of Differential Acid Concentration Along tlie Developing Gastroietstinal Tract of Tadpoles with an Improved pH Microprobe. SARAH E. CROSS, and RICHARD D. DURTSCHE, Department of Biological Sciences, Noitliern Kentucky University, Highland Heights, KY 41099. Digestion and assimiiiation of foodstuffs in vertebrates is often dependent upon changes in tlie chemical environment along the gut depending on tlie level of food decomposition and the optimality of conditions for enzyme activity. Lov/ pH in the stomach can chemically breakdown food while activating pepsin protein enzymes. Other areas of the gut (e.g., colon) could show lower pH levels that would indicate plant fermentation and the release of volatile, short-chain, fatty acids. Measurements across developmental stages can document ontogenetic shifts in acid concentrations in the gut suggesting 2010 KAS Meeting Abstracts 133 upregulation of digestive activites within tlie gastrointesti- nal (GI) tract. As free living vertebrates, tadpoles (anuran larvae) undergo developmental changes in tlie formation of tlie GI tract, but they are also consuming foods as might an adult vertebrate. Functioned changes in digestive processes in tadpoles can tlierefore be responses to eitlier maturation of the system or a response to the varied foods consumed. Pre\4ous research in our lab has shown changes in pH across tlie GI tract, suggesting digestive processes similar to adult vertebrates. Our continued investigations of differ- ential acid concentrations along the gut has resulted in the design and fabrication of an improved pH microelectrode witli a built-in micro reference electrode. We have been testing tliis new microprobe on locally collected Green frog (Lithobates damitans) tadpoles, where after dissection, various regions of the GI tract were measured for changes in pH. In testing our solid-state microelectrodes with tip diameters of =10 pm against standard pH solutions, we maintained precision of = 0.95. Beyond mtDNA: Morphology and Nuclear Gene Flow Suggest Taxonomic Oversplitting in tlie Ringneck Snake Diadphis punctatus, FRANK M. FONTANELLA, De- partment of Biology and Chemistry, Moreliead State University, Moreliead, KY 40351. Being able to efficiently and accurately delimit species is one of the most basic and important aspects of biolog)^ because species are the fundamental unit of analysis in biogeograpliy, ecology, and conservation. This delimita- tion may be hampered by variation witliin and betv^een populations making it difficult to determine whether populations have evolved into independent evolutionary units. Recently there has been a resurgence in species delimitation beyond traditional morphological and mito- chondrial data that incorporates species distribution modeling and nuclear data to assess ecological divergence and levels of gene flow between populations. Using the Nortlieastern ringneck snake Diadophis punctatus ed- wardsii as a model, I expanded upon previous work by combining 24 external meristic characters from 300 museum samples, species distribution models generated from 19 climatic variables derived from 415 unique locality data points, and 10 microsatellite loci from 288 individuals, to test whether the mtDNA clades represent distinct evolutionaiy units. The PGA and CVA analysis of tlie meristic data failed to recover significant differences between tlie two mtDNA clades. Demographic analysis of the mtDNA data depicts rapid population expansion of the northern clade that corresponds to large areas of shared suitable habitat predicted by the species distribu- tion models. Likewise, admixture analysis of the micro- satellite data suggests high levels of nuclear gene flow between populations. When combined, these results suggest tliat the mtDNA clades are likely the result of historical divergence followed by contemporary gene flow. Moreover, this study highlights the importance of incorporating multiple lines of evidence for populations suspected of being cr)q)tic species. A Dissolved Oxygen Microprobe for Measuring Gut Anaerobic Fermentation in Developing Vertebrates. KATHERINE BACHMAN, KELSEY CARNAHAN, and RICHARD D. DURTSCHE, Department of Biolog- ical Sciences, Northern Kentucky University, Highland Heights, KY 41099. Food assimilation in organisms is important in the extraction of energy. Previous research by botli our lab and others suggest anurans are herbivorous and detriti- vorous feeders. The extent to which cellulose breakdown and fermentation occurs in the GI tract is unknown. Studies done on pH in the GI tract of Lithobates cknnitam has shovm two major drops in pH, in the stomach and hindgut. The use of a dissolved oxygen microelectrode to measure oxygen levels in the gut would give us an idea of possible locations of cellulose fermentation and the possible existence of a colon. Over the past year, our lab has been working on building a dissolved oxygen micro- electrode probe to measure these levels. Assembly of these probes is comprised of four main components: the catliode, anode, electrolyte solution and outer casing. The catliode is a glass fused solid-state capillary tube, witli a gold tip. The anode is silver vtre coated in chloride ions. The outer casing is a glass pipette witli a tip diameter of 10 microme- ters and gas peniieable membrane. The electrol}te is a mixed potassium chloride solution maintained at a high pH. These probes were calibrated witli a high degree of accuracy and precision with rapid response times using standard concentrations of dissolved ox)/geii in water. Regi’ession analyses against standard solutions have R^ values in the range of 95%. Gastrointestinal tract samples were obtained from Lithobates damitam and readings taken from 15 positions along the gut. Amphibian Population Dynamics of a Rejuvenating Brown Field. JAMES ‘MITCH’ MERCER, and RICH- ARD D. DURTSCHE, Department of Biological Scienc- es, Northern Kentucky University, Highland Heights, KY 41099. The Lafarge Gypsum Plant, located in Silver Grove, KY, includes both secondary growth forest consisting of varied hardwood species and open wetland habitats. Just south of the Ohio River, grassland between the secondary forest and the riverbank supports vernal ponds, inundated \Hth water during the spring. Approximately 5 cm of top soil exists due to the land that once served as a railroad yard, tlie underl)4ng soil being permeated witli rock. Determined a brown field during this stage development, chemicals due to industrial waste may remain on site despite rejuvenation efforts. Amphibians are especially susceptible to these environmental conditions as their moist skin facilitates a pletliora of life processes. The study was conducted to analyze the ability of the site to sustain wildlife post- rejuvenation efforts. A comparative non- impacted wetland site (St. Anne Wetlands) approximately 2 km away was added to compare species thriving at either location. Various field techniques led to the capture of several species approximately every other day for a span of 134 Journal of the Kentucky Academy of Science 73(2) two years (during months of activity) to understand tlie dynamics of populations at eitlier site. Species were marked to track migration between sites. Field sound recorders were implemented to track potentially unob- sen^ed species. Results suggest that tlie Lafarge site is able to sustain some reptile and amphibian species, however, several species, in particular salamanders, thrive at the St. Anne wetlands but have not settled at Lafarge. JUNIOR ACADEMY OF SCIENCE ENGINEERING Lubrication Efficiency of Oil Weights in Engines. GABRIEL L. M. WEBB-YEATES, Bowling Green High School, Bowling Green, KY 42101. Engine oils have different weights such as 5W-30 and lOW-30. Each oil weight has a different \dscosity and lubrication ability. High weight oil is more viscous. Clean oil should be less viscous than used oil. My hypothesis is that lower weight oils will lubricate better at cold temperatures. Clean oil should lubricate better tlian diity oil. The relative viscosity of different weights of oil from tlie same manufacturer increased for heavier weights. For tlie same weight of oil from different manufacturers, the relative viscosity is similar. Used engine oil was more Mscous. Lubrication ability was measured by putting a fixed volume of oil between two metal plates placed on a self-manufactured adjustable inclined plane. The height of the inclined plane when the top metal plate starts to slide or slip over tlie bottom plate was used to measure relative lubrication. Engine oil of the same weight from different companies was measured. Different weights of oils from tlie same company were measured. Botli clean and used oil of tlie same weight were measured to see if lubrication changes as the oil becomes dirty and used. The inclined plane worked well witli good consistency in the slip point height. For the same manufacturer, tlie lightweight 5W-30 oil lubricated better at cold tempera- tures tlian heavy weight oils. Clean oil lubricated better tlian dirty oil. Surprisingly, high mileage engine oil, which will stick to metal parts better, had a higher slip point tlian other oils of tlie same weight. J. Ky. Acad. Sci. 73(2): 135. 2012. Erratum; David D. Taylor was omitted as one of the contributors to Micropropagation, Cnjopreservation, and Outplanting of the Cumberland Sandwort Minuartia cumherlan- densis from the Fall 2011 issue of the Kentucky Academy of Science, 135 f ‘ ’ iC*'. \ ■ *<• u. 4’t/i i*>c»mf'*iif vcr* tM:Tjl«iitif i ' •*'»un iff't/i t*n<> -T. Viit lUlit 9^ .4c>iTf>rW* tin (i .;• KV'’ ' ' -VI* - t ’ u' |j) '> vr '^>e ||ipP|«s9-:iI- * ^ #t f '« ' '31^ -S ■ ,c<% i** >»■• -«|i *, * -^.> ■• 7^. ... ' » ■, f . •» i,' .C ■ ' ' .i :■■ ' Ji - '^*r f . ’- . ;‘. f ■ •: , .. > *4^ - 4» -ttt I 1 k:tii\ *‘**i*r# T‘.. ifA'* A -> ■, I. ■ , ■ ■ _ j4$-lSir • ii 4vii4ir«* • iSi(iii^tMi|iit|..tkU f/ h0,u lu*t wn ‘.stM'r Wt, ‘■»4l ' •».^ •V, J4u:.^^ ;a»iY^ ^ J f • ’ ) i ' ' I ► ■■' iil^ri. ' V.. ''>1' ,-i '*■.:■ s. niv iv(pi4fiv. -v;^ ’ '•»•. Wf J Klirth •►.:l“* "' fu fA0 ^4*4 fiMr liN- f‘^ ,«f,. i. f f ^%ifj ■^■■' ■ •■• '. f ;J^ ’ »• •Jii A' tv •f^, ■• f M r I t, Iwii .* .r*!*.! ♦'U.iit W.* ■ V'“i ‘ i SMITHSONIAN INSTITUTION LIBRARIES ^ 9088 01681 7173 CONTENTS Evaluation of the Tooth-wear and Replacement Method for Aging White-tailed Deer (Odocoileus virginianus) on the Blue Grass Army Depot, Madison County, Kentucky. Charles L. Elliott and Thomas Edwards 73 Occurrence of Three Leech Species (Annelida: Hirudinida) on Fishes in the Kentucky River. Joseph £. Flotemersch, Donald J. Klemm and William E. Moser 77 Size Structure of Fagus grandifolia, Liriodendron tulipifera, and Celtis occidentalis Populations in a Wetland Forest in Campbell County, Kentucky. Richard L. Boyce 83 Evaluation of Cultivars of Sunflower (Helianthus annuus L.) and Selected Environments for Production of Cut Flowers. Christopher G. Ferguson, Pavani G. Vuppalapati, Martin J. Stone, and Elmer Gray 90 Lady Beetle Composition and Abundance in Sweet Corn Bordered by Pasture, Buckwheat or Sunflower Companion Plantings. John D. Sedlacek, Karen L. Friley, and Kirk W. Pomper 96 Genetic Diversity in Kentucky Spicebush Populations using Simple Sequence Repeat Markers. Re'Gie Smith, Kirk W. Pomper, Jeremiah D. Lowe, Jacob Botkins, and Sheri B. Crabtree 101 The Pawpaw Peduncle Borer, Talponia plummeriana Busck (Lepidoptera: Tortricidae): A Pest of Pawpaw Fruit. John D. Sedlacek, Jeremiah D. Lowe, Kirk W. Pomper, Karen L. Friley, and Sheri B. Crabtree 110 Pawpaw Patch Genetic Diversity, and Clonality, and its Impact on the Establishment of Invasive Species in the Forest Understory. Jacob Botkins, Kirk W. Pomper, Jeremiah D. Lowe, and Sheri B. Crabtree 113 2010 ABSTRACTS 122 DAVID TAYLOR ERRATUM 135