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1-1-2002 Investigating the Relationship Between Cryptococcus Fagisuga and Fagus Grandifolia in Great Smoky Mountains National Park
Ashley B. Morris University of Florida Randall L. Small University of Tennessee - Knoxville Mitchell B. Cruzan Portland State University
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Citation Details Morris A.B., R.L. Small, and M.B. Cruzan. 2002. Investigating the Relationship Between Cryptococcus Fagisuga and Fagus Grandifolia in Great Smoky Mountains National Park. Southeastern Naturalist 1:415-424.
This Article is brought to you for free and open access. It has been accepted for inclusion in Biology Faculty Publications and Presentations by an authorized administrator of PDXScholar. For more information, please contact [email protected]. 2002 SOUTIIE;\STERN NATURAWT 114):4 15--124
INVESTIGATING THE RELATIONSHJP BETWEEN CRYPTOCOCCUS FAG1SUGA A D FAGUS GRANDlFOLlA IN GREAT SMOKY MOUNTAINS NATIONAL PARK
A SIILEY B . M ORKIS " R ANDAll. L. SMALL 1. AND MITCIIELL B. CRUZAr\ 2.. I
AI3STRACT - The high elcvmioll beech ga ps of lhc Grc
INTRODUCTION
In recent years. populations of Fag/ls grclIItiijo/io Ehrh. (A mcricH n beech) ha e been deci mated ac ross the species' range due to an insect l11 ed ial ~ d fungal pa th ogen complex known us beech bark di sease (13BD). The insect component of lhis complex was acciden tally introduced into Nova ScotiH around 1890 by way of infected nursery stoc k. and has since prcad westward and southward to cover Illllch of the northern range of A merican beech (Ehrlich 1934. Houslon C\ 0 1. )979) (Fi g. I ). The woolly beech sc ale (CryplOc()cclfsfagi.w ga Lindinger) infects beech bark. w hich lhen provides u pathway ror infec tion by Nee/ria coccillea var./llgil/ata Lohman. Walso n. and Ayers or N. gal/igel/o Bresadola. IwO specie, of pmhogcnic fungi (Ehrlich 1934. Gavin and Pea rt 1993. I-I ouslon 1983). A recentllloiccular survey of N. cocdllea var.j"agiI/ClI(I indicates that ill1lay not be native to the United States , although the timing of its in ithll inlroduclion is uncert ain (Mahoney el al. 1999). The chronology of lhe disease has been well documenled (Ehrlich 1934. Houslon el " I. 1979. Jones 1986). A s this disease s pread~ lhroughout the range of A merica n beech, average mortality of beech w ithin a popu lation is estimated to be
I Dcpartment of B01 Kn oxvill e. TN 37996. I DepartlllcllI of Ei.:o logy ;"ind Evolulionnry Biology. ni ver... ilY ofTc nn es~ee. Knoxville. TN 37996. 416 Sml,hca.\Il'm Nt/tllmli," Vol.l , No. 4 tiS much a~ 85%. whi ch has been partially allributed to past logging praclices (I-I ouslon 1979). l-li slO ri call y. beech was nol con, idered a va lu able timber specie!i in northern hard woods 3nd was on en lef! behind in logged sland,. This has resulled in Ih e rise of beech as a major componenl in man y northern rOre~I.\. This overabu ndance or beech stems is thought to be a contributing fa clor in Ih e widespread dbpersal of beech bark di sease. Damage incurred by logging practices is thoughl to have pro duced a ni che ror C.f1l8isugo on otherwise hea lthy stems ( H o lJ ~ton 1982). Houslon ( 1983) reported Ihm allhough Il, ere are known predalors of Ihe beech scalc. th ei r effecti ve ness has been limited. Currentl y there is no kn own effecti ve tre<.ltmCIll for BBD. Potential for genetic resistance Re sistance to BBD in North America was tirsl studied by HOllston ( 1982). who inlroduced C. IagislI ga 0 111 0 apparently resiSla nl beech Irees in aftermath forests (0 test ~ u sce pti bili l y over a period of th ree years. On susceptible trees the im~ec t was able to complete it~ life cycle and produce massive amounts or eggs. Allcfllali vely. th e insect" failed to become establi shed on stems that Houston perceived to be resistant. Bused on th e~e triab. HouM on produced two hypotheses regarding the source of re~is tance in these trees: I) the in abilit y of overwintered in sec t ~ to complete th ei r Ii fe cycle on disease free trees may be a result or th e presence or some toxin or lack ofsolllc necessary chemical in the tree itself: 2) the complete lack of infestation on some trees ,Irld consiste nLl y low levels of infestation on others Illay be the result of ei ther a compl ete or partial analOmical barrier in lhese !HCms. In mOre recent wo rk. Houston and HOUSTO n ( 1994. _ Distribution or BBD Distribution of American . in U.s. Figure I. Di-: tribUlion of b~ech burk di se ase (BBD) in thc Unitcd Stales. The dblriblllion of Fag lls grondi/orin in the Unilcd SHIIC!'I is ind icated in lighl gmy. Counli es in wh il:h BBD has bee n documented arc in dark gray. The Greal Smoky Mountai n., r cp r e~elll lhe so ulhcrn - l1lo ~t occurrence of BBD. BOD dmu were providcd by Milflin Mac Kenzie. USFS . 2002 A.S, r>. lorri<.;, R.L Small. olld fl, U 3. CruJ:m ~17 2000) sill died a third hYPOIh esis: the inabili lY of Ih e scale in seC I to establ ish itscl f Oil some trees ma y be th e res lIlL of geneti c res istance. The pOlenlial for res istance hal> been reponed. all hough obse rved res istant trees are rarely record ed ( H o u ~lon 1983), Houston and H o u ~ ton (199-1) observed re,is",ncc in les' Ihan 1% of all beech Irecs lhal they sampled. They also noted th at rcsistun t SIems ort en occurred in disc rete clulllps or groupb. suggesting th e poss ibi lity of resistant clonal gcnOlypes. At leas t for F. sy l1 'alh:(J L .. rcsi'\ tance in planted orchards has been associated with genotype (Wainhouse and Dceble 1980). M ore recen tl y. Krnbel and Petcrcord (2000) found a corrcl:.1Iion be twee n beech scale infestation Hncl the hos t tree genotype in F. s),/l'{l/iCli. For F. grcllldijolia. no known relationship between genOtype and res istan ce has yel becn found . Houston and Houston ( 1994.2000) used isozymes in an allcmpt (0 identify resistant gcnotype:-, Cl nd found lhat in some instances individual '\ lcms sharing the sa me genotype could be ei ther infec ted or nOL infected. However. th ey did repon finding higher levels of ob!'crvcd hete rozygosity in !:> lI sceptiblc stands, suggesting some leve l of geneti c differentiation between ~ Ll sf.;e ptib l e and resislanrc trees (Houston and Houston 2000). It is import an t to no te Ih ~lI allozymc!oo often do not display adequate levels of varia tion to resolve ge neti c idcnti lic!ot in clonal popUlations (Cru zan 1998. Escarav"ge ET AL. 1998. Waycn" 1998). Whereas all oLymc studies arc coml11 onl y limited to less th an 10 poly morphic loci. studi e:-. lIsing markers based on DNA variation ca n include Illany limes that number of loci for the resolution of indi vidual gC llO types. In this study. we lIsed inter-~ illlpi c sequence repeilts (ISS Rs) to inves ti ga te the potential of geneti c res istance to beech bark di sease in "-. gralldijo/i(J in Grea l Smoky Mountains National Park (GRSM). We sampled geneti c variati on wi thin and among threc ~ it cs to es timate th e relationship be tween leve l or ~ca l c infcMalion and genotype in GRSM . Our re s ll lt ~ provide no evidence for genetic re:-. istan cc. but do suggest a relationship between stem diameter METHOOS Study system BBD was first nOlcd in GRSM in 1993. and since initial discovery, areas of nearl y complete :-o tand ll1ort:lli ty helve been loca ted (Blo.LUn 1995). We st or Clingman 's Dome. hi gh c l ev~lIi o n beech forests are l:ontil1uolls along the North Carolina/Tcnne:-.sec :-.tate bou ndary for sev eral miles (Blozan 1995). Thi s area i, likely Ihe most suscepl ible 10 BBD due to high dcnsitie::. or individual :-; within populations anti rclaLi vc prox imity of popu lations to one anoth er. and is rcared to be th e site of origin of BBD illlhe park (Blozan 1995) . Evidence s ll gges l ~ Ihut BBD is most preva lent in moist. shaded a re~ I S, Hnd thal larger trees in higher densities appear to be more sust.:ep tihle (GClv in :.II1d PCH rt 1993). In 418 SoIl//lea,'Ilt!rtI NOlI/mUsl Vol. I. No.4 addiLion , high incidence of BBD has bee n reported for lower elevaLion areas where heml oc k (7:"'&{I cal/adel/sis (L.) Carr) is common (likely due 10 high shad e and moi sture regimes) sugges ting that BBO is not limited to hi gher elevaLion (B lozan 1995). However, the high densiLY of sLe ms and consistently moist conditions at higher elevation sites in GRSM create ideal co ndilions for BBD infesLaLi on and spread (Blozan 1995). Sa mpling Methods Della were collected from three sit es, se lected with th e ass ist:lIl ce or GRSM slafr (Fi g. 2), Two of Ihese siles, Bal sa m Mountain. and Double Sprin g Gap, are above 1500 J11 in elevation and arc representatives of "beech gap" forests, which are located within topographic gaps in areas otherwise dominaLed by spruce-fir foresis. The Ihird site, Ihe Chimneys. occurs below 1100 In and is associated with TSlIga clIIwdellsis, Ace ,. rubrulII L.. A. saccharum Marsh.. H ale.sia c(lroNua L.. Aesclllus oculIIdrll Marsh.. and Conms alfemijolia L. Becau se stem densities va ry greally bel ween high elev3Iion and low elevalion siles. differenl .. Sim"hn! locations • e,l,"" N ORS'" hfmnclary N R Figure 2. Sampling l oc~lli o n s for Fllgll.f gralld((o/ia in Gre'll Smoky Mounwin s. National Park. 2002 A.B. Morrilo. R.L. Sm'lll. and M .B. ( ru/:III 419 sampling strategies were applied to th ese twO areas. AI th e Chimney::, . a 10m x 20 m 1'1 01 wus established. wilhin whi ch Iwelve. I m'subplol' we re ra ndoml y selec ted lI sin g a grid system and a random number ge nerator. In each subplOl, all F. gramli/o/ia stem::, were sampled. At Babam Mount ain and Doubl e Spring Ga p. a 90 m transect W;IS estab· li shed. along whi ch samples were coll ected al 10 m interval s. AI each sampling interval th e fi ve closest siems were sam pled nnd all sampled stems were mapped re lati ve 10 plm bound ari es. Disease illreCli on was assessed by estimating th e percent cover of scale on lh e lrunk or ell ch lrce (0 = none. I =< 25%. 2 =2 6 - 75%. 3 = >75%). Slem diameler wus record ed ror all su mpl ed Siems. regardless of size class, For stems greater than J.5 III tall. diameter at breast height (DSH) was recorded. For stems less than 1. 5 III tall. stem diametcr \\I,lS measured at ground level u'\ ing di al calipers. Leaf malerial w a ~ co l lected fro m each individual. placed ill se parate 1.5- I1lL microcenlrifugc tu bes and stored on icc while in the fi eld . Upon returning to th e lab. all sampl es we re snap fro zen in liquid nitrogen and stored at -7{rC. I)NA Extraction Total genomi c DNA W~l S eX lnlctcd fro m each sample using a proto col "dapled from Ed ward s el ai. ( 199 1) and modified by Marlin and Cru za n (1999). Leaf malerial wa.. ground in 100 I11L or eX lracli on buffer (200 mM Tris HCI pH 7.5. 250 111M NaC!. 25 mM EDTA. 0.5% SDS ) ror 15 seconds using disposu ble grinders. All additional 400 mL or ex tracti on burrer was ad ded to thi s mi xture and ground again . Each sam ple wa th en centrifuged al max imum speed for 2 minulc!'I. Foll ow ing ccnlri ru gati on. 400 mL of supcrn Dala efficiency Varying number. of loci (I through 24) were rand om ly rcsampled to infer Ihe effects of incre~l s ing the number of" markers on the numbe r of gene ts detected. The number of genets was calculnled for one thouS~l nd replicates of ench dtlla set size and these data were used to exam ine the relationship bel ween number of loci in the data set and the number of genets detected. We as~umed thm an asymptotic curve (i.e., the number of new genets delCCled approaches zero as the number or loci in the drll:t sci is rcuehed) would indicate thal adding ndclitionul loci wou ld not result in an appreciabl e change in th e total number of genets detected. Genetic ,'ariation and cstillmtes of resistance Genetic structure was anulYl..ed by calculati ng till! number or r:Ullets. number of' genets. and number or polymorphic loci as::.ayed. Ellstrand and Roose's (1987) prop0rlion distinguishable (PO) was used to esti mate clonal diversity wi thin sites. Due to the small number of clone~ detected, statistical ana l ysi~ of the relationship between genotype and level or infection was not possible. Thus, thi s relationship was qualiHl tively assessed. The hypothesis that scale coverage differs with ~te ll1 diameter was tested using the row lllean scores lC SI statisti c (Q~) as calculated using SAS" so ftware (SAS Institute, Inc., 1999). The restlits of thi'\ (eM were graphi call y vi5.ua lized by constructin g a mosa ic plot using JMP" ,oflware (SAS Institute. Inc .. I 989-2002). Gene diversity wi thin and among popuimiolls was estimated usin g the program Tools for Population Gcnetic Analyses (TFPGA. ve rsion 1.3. Miller 1997). which handles both eodominant and dominant marker data. TFPGA uses Weir and Cockerham's (1984) methods for ca lctl lat ing Wrigh" ~ F statistics where 'I" is the HllloulH of variati on explained Table I , Sampling loc;ltions in Grea t Smoky MounlUin~ National Park fGRS M ). Three ~ ite s were sampled for gcnelic varialitln ;md clonal SlruClure usi ng ISSRs. A 101;1\ of 85 lrcl!s we re sampled, Site names are followed by ahbrcvimions: eJevlllion indie-a les the approximale clcvl.ltion in melers: " = sample .. iLC (or number of famels): G = number of gencls: PO = Gin: P = perccnt polymorphic loci. As PO approaches 1.00. clonal Slruclltrc deCrCi.1 ~e:-.. Si te Elevation fI G PO P Chimneys Ion 19 8 0.42 45.83 Balsam Mouiliain 1524 30 22 0.73 83.33 J)(wblc Spring Gal? 1585 35 34 0.97 9 1.67 2l)02 I\ .B. roo lurrh. R.L. Small. and M.B. Crul.an .t21 by differentiation of populations. We performed bootstrapping over loci to obtain vu riance cs till1 a t e~ for lhese slalisti cs. Since we did not h ~lVe an estimatc or th e frequency of h eterozygt}tc~ in our data. HanJy- Weinberg eq uilibrium wa!ot as!ot umcci. RES ULTS AND DISCUSSION Th~ obj ec ti ve of this research was to id enti fy th e relati onship be· tween in cidence of C/:l'plOcocctlsp,!!i,l"Ugll and the ge notype and diam· eter of it s ho"1. Fa gus grlllIlJij"ofio. in GRSM . Due to loss or froze n leClf material becau se of fr\.!czcr fa ilure in the spring of 2000 and some generally problematic sa mples. th e actu al sample ~b-. c was not cqunl to the numbers of s::lIllple\ originally co ll ected. We a~sayed a tow l of 85 indi vidual s acro'\s the three ~i t cs studied. Us in g one ISSR primer. we identified 24 loci. 23 of which we re polymorphic. Locu" re sa mpling resu lt ed in nn asymptotic Clirve. indicating Ihat we had obtained a sufficient amount of loc i to detec t th e fl nl 011nt or v:ui;uion prese nt at th ese sites. Resu lts are ~ llnlll1ari led in Tabk I. The two hi gh elevation siles ( Dollble Spring Gap and Balsam MOll ",ain ) cx hibilcd very lill ie clona l structure (PD ;: 0.97 and 0.73, respec tive ly). indicating th al sland maintenance is dependent on seeu production. This i:-. in t.:ons i ~ t e nt with prc v i o u ~ hypothest='!ii thnl hi gh elevation beech gaps arc maintnined by cloll<.tl rr:prod uctio n. A lt ern ati ve ly. the low elevation site (th e Chim neys) W:J3 primarily clonal in nalUre lPD = 0.42). whi ch i~ mOSI likely a res ponse to mortality ca u~ed by beech bark disease. We found no evidencc fo r genetic resistance. with cioncma tes cx pre s~ in g all levels: or infection (from 0 LU 3) by C. jtlgi.\·IIMa. Previo us work on F. syll'alicfl in Lower Saxony idelllified a correlution between beech scale infestation and th e genotype of the hos t Iree~ ba3ed on a sin gle '''''"Yllle locus ( Krabel and Pelercord 2000). However, C. /af,:isflga is an illl rotlu ccd sp\.!cics in th e Un it ed SlatC !ii. with its first introducti on dating to about 1890. One h y p o th e~ i s to exp luin the appal'· Cnt absence of genetic re sistance in P. grandi/olia is a lack of time for ~ lI c h 1.00 0 l 2 0.75 c ~ .g ~ & ~ c .= 0.50 (:g'" .. ~ ~ "' "' 0.25 0 0.00 I < 1.5 em 1.5 - 3.0 em > J.Ocm DBtl Figure 3. Mosaic plOl or Ihe rehui ol1ship between ~ l c m diameter of FaRfls grwult/oJia and leve l of infestati on by CrYPf(lcon'w; fagi~tlgll . This pial repre se nts the frequ encies of co rnbimllion s or tWo variab les. in thi s case stem di :l m Clef and sca le infestati on. Each rectangle represe nt s the jOint probabili ty of CI part ic lI i;lr combina ti on of th c.<;c (Wo variabl es. Th e shading key indi ca te" whic h t'olorcorrcspond .!l to eHch leve l of !loca le infestati on (0= none; I = < 25 %; 2 = 25- 75%: 3 = > 75%). In addition. the shading key ind icutes the over.t ll prob;:,bility of e'l(' h leve l of infec tion based on all d~ta co llec ted. 2002 A.B. Murri~. R.L. 5111:1.11 , and M.B. Crul:1Il 423 genetically identical l-i ICI1lS (persona l communicat ion, W. D. Ostrofsky). Thi :ot not only c h a n ge~ ~ta l1d ~ pecies compos ition by limiting openings for the recruitment of other specie:ot, but also makes th e stand Illorc suscep tible to fut ure BBD outbreak,. Because no genet ic res istance has yet been iden t i fied i n F. gralld(f(i/ia. management decisions are limited. A number of recolllmen dations have been made by prev ious rcsea n:hers. 1110 !beeches repre sen t the only hmd l1l :lsting species in th ese otherwise spru 'c-fir domi na ted fores ts. providing food for i.l large number of animal species. In order to protect th ese un ique cO llllllunities, more work is needed LO undcrswnd the potential roles of genetic rc~ i s t a n ce :lnd chcmical defense to BBD. W ork i:\ curre ntly underway to conti nU l! the sea rch ror genetic resbH3 ncc using several di ffercllI mo lecular techniques. A CKNOWLEOGEME TS The authors thank C. G. Morris. G. Wiggin~. G. Taylor. S. Datwylcr. and M. Gill:cndanner for an LITERATURE CITED BLOZAN. W. 1995. Dcndrol!cology of Americall beec h ~ I ands infe! of beech hark disc:l>ic on Ihe growth or A I11cril.:an beech (FClf.:IIS grtmdljoli(l). CClluu/itlll jOllrnal oj Furest Re.\·NIITIt 23: 15fl6- 1575. HOUSTON, D.B .• ilnd O.R. HOUSTON. 1994. Vari~lIion in A merican bt.!cch (Fagtu grandi/olia Ehrh.): i),o;~y m c analysb of genelic '-i lruclU rc in ~e l cctcd ..,tan d ~. Sih'ac Genclic:} 43:~77 -2X4. Sl)lIfhetlSlel'll N(/fllmli.H Vol. r, NO. 4 HOUSTON. D.B.. und D.R. HOUSTON. 2000. Allo7Yll1e genetic diver,ity among Fagus grclluJijoJia trees re~ i ~tnnt or susceptible 10 beech bark discu"c in natural population~ . Cnnadian Journ al of Forest Research 30:778-789. HO USTON. D.R. 1982. American beech re:-oi slililce 10 CrJptococclIsfagisugtl. IUFRO Beec h bark disease working party l:onfercncc proceedings. Harnden. CT. Pp 38-12. HOUSTON. D.R. 1983. Dcvelopmcnt:-. in bivlogic;tI control of be..:ch bark discnse. 10 International Co ngress of Plant ProtecLion , Brighton. Englu nd . Pp. 1035- 1041. HO USTON. D.R .. E.J. PARKER. R. PERRIN. and K.J . LANG. 1979. Bc