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A shift in nuclear state as the result of natural interspecific hybridization between two north american taxa of the basidiomycete complex Heterobasidion
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[Garbelotto M., Gonthier P., Warner R., Nicolotti G., Otrosina W., 2004.
Fungal Genetics and Biology, 41, pp. 1046-1051, DOI:
10.1016/j.fgb.2004.08.003]
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A shift in nuclear state as the result of natural interspecific
hybridization between two North American taxa of the
basidiomycete complex Heterobasidion.
Matteo G ARBELO TTO1, Paolo GO NTHIE R2, Ra che l WARNER1, Giovanni NICOLO TTI2, and William OTROSINA3
1
De part ment of Env ironm ent al Science, Poli cy and Man ag em ent -Ecosy st em Sci en ce Divis ion , Uni v ersi ty of Calif orni a, Ber kel ey , CA 9472 0, US A.
2 D epartm ent of Exp loitati on and Prot e ct ion of Agri c ultural and Forest ry
Res our c es -Pl ant Pat holog y, Uni v ersit y of To rino, Gruglia sco, I-10095, Ital y.
3
USDA Forest Service, Tree Root Biology Team, 320 Green St., Athens, GA 30602, USA. E-mail : m att eo @n at ure.b erk el ey.edu
A natu ral fi rst ge ne rati on hyb rid fu ngus s hows oblig at e inte rs pe ci fic het e rozyg osity
The nucl ear c onditio n o f a ra re n at ural hybrid b et we en two t axa o f t he
Het er obasidi on complex is inv est igate d. Hete robasid ion spe cies are known t o b e eit her h omok aryoti c (ha ploi d) or het ero karyot i c (n+n), but
het e roka ryo ns a re m ade up of bot h homo ka ryot ic a nd he te rokaryoti c sectors. T he natural hybrid appears to be eit he r a st ri ct he tero ka ryo n undergoi ng a prim ary homothalli c phas e or a dipl oid wi t h lim ited a bilit y to ex ch ange nu cl ei i n homoka ryo n-het e roka ryo n m atings. The na tural hybrid is ext rem ely s tabl e an d l ong -lived, sugge sting hybridi zation m ay pl ay an im port ant ro l e i n th e evoluti onary hist ory of t his fu ng al c ompl ex.
INTRODUCTION
Chan ge s in ploi dy are common in i nte rspe ci fic h yb rids ac ross all taxonomi c groups . The li st o f pol y ploid and an euploi d hybrid s is ext ens ive, i ncl udi ng exampl es from t he My cot a (S uz uki et al . 1986, Kuld au , Ts ai & Sc ha rdl 1 999) a nd the Oomycota (B rasi e r, Cooke & Dun can 1999 ). Often, unusu al ploi dy resul ts in i nfertilit y or genomi c inst abilit y.
The first nat ural hybrid hol ob asidiomyc et e report ed i n t he lit e rat ure was a cross b et ween two speci es of the Het erobasi dion anno sum s pe ci es com ple x (Ga rbelott o et al. 1996 a). H. annosum s ensu lat o is a wid esp re ad tre e patho gen com prising thre e spe cie s i n Eurasi a (Niemel ä & Ko rhonen 1998 ) and two spe ci es in Nort h Ame rica . Althoug h th e two No rth Ameri c an t ax a are awai ting fo rm al d esc ripti on at th e spe cie s level, t his taxonomi c c lassi fica tion is strongly supporte d by som e m orphologi cal tra its, m ati ng comp atibi lity, differentia l host speci fici ty, and num erous bi ochemi cal and DNA st udi es (C ha se & Ullric h 1 983, W orral l, Parm et er & Cobb 1983 , Ga rbelotto et al. 1993, Otrosin a et al . 1993, Johannesson &. Stenlid. 2003). In the me antim e, t he t wo t a xa are still refe rred t o as S a nd P interst erility grou ps (IS Gs ) based on the hosts i n which th es e two t axa were fi rst d es cri be d: spruc e and pin e (Korhonen 1978). Hybrids bet wee n the t wo No rth Am eri can t ax a ca n b e e asil y obt ain ed (20% suc ce ss ra te ) i n
the la bo rat ory (Harri ngton , Worrall & Ri zz o 1 989 ), but they app ear to be ra re in nature. A s ingl e hybrid SP genotype has be en di scovered i n nort he aste rn C ali fo rni a (Garbe lotto et al. 1996a). The hyb rid ha d col oni zed o ne Pond erosa pi ne tre e, t wo West e rn j unip ers, an d a l arge Po nd erosa pi ne stum p. A tot al of 16 i sol atio ns of t h e hybrid isol ate we re obt ained: t h e furt h est dist an ce bet ween t wo isol ations wa s 8 m, and base d on publi shed s pread rat e of t he fung us (Hodg es 19 69, Garbelott o et al. 1996 b), its lo ng evit y was esti mat ed to b e 5-25 ye ars.
Beca us e of the im port an ce o f this si ngle nat ural hybrid geno type, we de cided t o loo k at i ts nu cl ear st at us in hyph ae a nd con idi a a nd at its abili ty to ex ch ange nuclei with hom okaryoti c isol ates. Nucl e ar mig ratio n bet we en hete rokary ons (n+ n) or bet wee n a h omokary on (n) and a het e roka ryo n (“Bul l er pheno me non” ) is a pot e ntia l me ch anism for the exchange o f ge net ic inform atio n among i ndivi duals of a popul atio n. Such migrati on ma y en ha nce th e est ablis hm ent of novel g en oty pe s and ge nom es (Garb elott o et a l. 19 99).
Pre vious studies have hi ghlig hte d some aspects of the nu cl ea r make-up of holob as idiom ycet e hyb rids obtain ed in t he laborat ory. In parti cul ar, Boi din and Lanqueti n (19 83 ) we re ab le to sho w that pa rent al nu cl ei of stab le hyb rids bet ween Di chos te reum dur um and D. sordul ent um behave d di ffe rentl y. W hil e only D. sordul ent u m n ucl ei were fo und in conidi a, only D. durum n ucl ei wo uld migrat e int o D. dur um h om ak aryons paired
with t h e hybrid. No D. sor dul entum m igrat ion o cc urred whe n th e h yb rid was paired wit h D. sord ule ntu m homoka ryons. It wa s al so obse rved t hat cult ures deri ve d from monosporous c onidi a an d b asi dios pores would event ual ly all form cl amp connect i ons , indi cat ing pri mary h omoth al li sm bet we en pa rental g enomes both c arri ed in the uni nu cl eat e prop agules.
Korhonen (197 8) s howed that coni di a of l abo rato ry h yb rids of H. a nnos um alway s b ore t he nu cl eus of one pa rent al s peci es, sug ge sting a tendency of hybrid genot yp es t o segreg at e nucl ei a nd revert to th e i ndi vi dual spe ci es. Ramsd al e & Ra yne r (199 4) report a simi lar phenom e non of nucl ear seg reg atio n in i ntras pe ci fic H. annosu m het e rok aryons comprised of t wo di st antly rel at ed g en otypes. The t end ency of unrelat ed nucl ei to segreg at e indivi dually, d efi ned as “genomi c con fli ct ,” was evaluat ed by looki ng at the nu cl ea r component o f con idi a an d hyph ae . A s trong posit ive correl atio n wa s found b et we en the propo rtion of u ninucl ea te c onidia pro du ce d by a het erok aryon a nd t he geneti c di sta nc e of t he p arent al nuc lei .
The exi ste nc e o f a natura l H. an nosu m hyb rid rai se s a numbe r of que stio ns. Will genomi c c on fli ct be st rongly expres sed i n this inters pe ci fi c hybrid, as is suggeste d by exp erime nts on labo rato ry hybrids? If so, how can H. annosu m h ybri ds su rvi ve ? How will hybri d hyph ae int eract wi t h homo ka ryoti c hyph ae i n som ati c inte ract ion s?
Beca us e the hy bri d g enot ype has functio nal genomes of bot h No rth Ameri c an spe ci es, will th at coexist en ce result in enhanc ed (nucl ei will migrat e into homokaryoti c g enot ypes o f both s peci es ), re stri ct ed (no migrati on i nto ei t he r sp eci es), o r i nte rm ediat e nu cl ea r t ransfe r?
To provi de t he ba sis for an swering t he se ques tions, we con duct ed s ev e ra l studi es to ad dress : (1)- t he fre qu en cy of S, P, and hybrid SP hypha e in the thall us o f the nat ura l hyb rid; (2 )- th e fre quency of un inu cl e at e c oni dia in the natura l hyb rid; (3)- t he frequ en cy of re cov ery of S, P , o r o f hyb ri d gen om es from conidia; (4)- t he abil ity of th e natural hybri d to het e roka ryoti z e S an d P homoka ryons through di -mo n m atin g.
MATERI AL S AND METHODS
Cha racterizatio n of h yp ha e i n t he t hal l us
Hyb rid isol at e SP40 0 was grown on c ell oph an e overl aid on malt ext rac t agar (0.125 % malt, 1.5% agar) a t ro om temp erat ure. Aft e r 9-12 days , indivi dual hyphae we re s ub cult ured from hyphal tips vi e we d at 60X mag nifi c atio n. Twe nty hyphal tips were coll e cte d from ea ch of 5 sub cult ures. All 100 resulti ng cult ures were analyzed for pres en ce/a bs en ce of cl amps by d irect observa tion of hypha e on the underside of pl at es at 320X magni fi cat i on. Th e ISG of each cultu re was
also det erm ined by tax on-sp ecifi c competiti ve-priming polymeras e c hai n re action (TSC P-PCR ) (Ga rbelott o et al. 1 996a ).
Nucl ea r co ndi tion of coni dia fr om hyb rid and no n-hybr id i sol at es
Three s ets of a ppro ximat el y 200 conidi a ea ch, we re harve s ted from the hybrid S P is ol ate, o ne h omokary oti c S i sola te, a nd o ne he teroka ryot ic S isol ate Th e ha rvest was cond uc ted by pouri ng wa te r over a 10-da y-old col ony gro wi ng o n malt ext ract a ga r. The two S i sol at es L2.8.R1 an d L2.7.R5 (Lassen National Fores t) we re chose n for compa ris on bec au se they h ad been coll ec ted in t he s am e re gi on of no rtheast ern C ali fo rni a as the hybri d i solate an d had been c ha racte ri ze d by mating t est s and R APD analys es (Ga rbelot t o et al. 1999). Th e suspens ion was filt ere d throug h che ese cl oth and a dropl et wa s then place d on a microscope sl ide previo usl y overl ai d wit h pol yly sin e-D (Mol.Wt.= 450 .000). The suspens ion was t he n ai r-drie d, rehydrat ed, and st ai ne d with 4’, 6-di ami 6-din o-2-phenyli ndol e (DAP I (1 µg/ml)) for 10 min. Afte r a rins e wi th di still ed water, t he s lides were exami ned under 400 X magn ifi ca tion usin g an Olympus Axiop hot mi cros cop e an d a fluores cent light sourc e, and freq ue nc y of u ninucl eat e co nidi a fo r ea c h isol at e was q uantifi ed and com pa red usin g Z t e sts.
I SG a nd n ucl ear co ndi tio n of si ngl e conidial i sol ates
Thi rty i ndivi dual conidio phores were se le cte d from t he h yb rid ge notype for isolations o f si ngl e coni dia. C oni dia were coll ect ed by a st eril e ne edl e from the cl ub-l ike top of ea ch conid iop hore a nd put into a n Eppend orf t ub e c ont aini ng 1 ml o f st eril e wat er in whi ch a drop of Twe en 20 vi scous l iqui d (Sigma, St Louis , M O) was previousl y added. Tub es were imme dia tel y vortex ed, a nd t he susp ensi on was added to malt ext rac t agar i n Pet ri dishe s (5 cm di am ) onc e the t emperatu re of the medium re ached a bout 45 °C, but befo re solidi fi catio n. Th us, coni dia we re burie d in the medi a at di ffere nt d ept hs and ge rmin at ed at di ffe rent t imes, allowing for ea sy is olati on of sin gl e co nidi a at 320X ma gni ficati on. Te n ge rminati ng conidi a pe r coni di ophore were s ele ct ed an d is ol at ed. Pres en ce or abse nc e of cla mp s was as sess ed for eac h of th e 3 00 resul t ing cult ures . ISG det e rmin atio n was obtai ned th rou gh TSC P-PCR and by R FLPs of t he ITS fragm ent by t he endonucl eas e Cfo I as desc ribed i n Garbelott o e t al. (19 96a).
Fo r furthe r analyses, we cho se on e S isol at e (230-3 Plumas National Forest), one P is ol ate (T338 Modoc National Forest), t he hybri d genotype, and 15 singl e conidi al i sol at es. All s el e cte d i sol at es came fro m nort h ea st ern Cali forni a, a nd s ingl e con idi al i sol at es were rand omly s el ect e d from di ffe rent c oni diop ho res o f t he hy brid i sol at e. W e perfo rm ed RFLP
analys es o f t wo a ddit ion al n ucl e ar lo ci a nd s equence an aly sis of on e mitoc hondrial lo cus as fol l ows. Se gm ent s of th e nucl ear el on gati on fa ct or1-α and the he at stress p rot ein 80-1 loci were ampli fied using th e prim e r sets elongation factor 1-α forward/reverse (Johannesson & Stenlid 2003) and heat stress protein 80-1 forward/reverse (Johannesson, Johannesson &Stenlid 2000), re sp ec tively. R FLPs were obt ained by doubl e di ge stio n o f eac h a mpli con ; eac h enzym e m ad e o ne uni qu e cut of e ither the S- o r P- t yp e fra gm ent.
Sna BI an d Bsi EI were used on the elongati on fac tor 1- α , whil e Hind III and Bgl II were used on t he he at st ress p rotei n 8 0-1 ampli co n. All
enzym es were ma nufac ture d b y New England BioLabs Inc. (Beverly, MA) and use d ac co rding to ma nufacturer’s inst ructi ons. Di gests were
el ect ropho res ed at 50V/ cm on 3% Meta Phor agaro se (C ambrex, East Rut herfo rd, NJ). As S- and P-t yp e mit o ch ondria l ATPase-6 se quences were to o si mil ar to design an RFLP strat e gy as abov e, we amplifi ed this re gion us ing th e p ri mer s et ATP6-2/3 (Kretzer & Bruns 1999). PC R products were cl ea ne d the n s eque nc e d o n an ABI 3 100 c apill ary sequencer
(Appli ed Bios yst ems, Fost e r Cit y, C A) fo llowi ng standard p rotocols pro vid ed by the m an ufactu rer. S equences from e ach of th e 1 6 hyb rid isol ate s, of the S IS G isola te 230-3, and of t he P ISG isol at e T338 we re aligned and c hromat ograms for e ach were st udi ed for the p res en ce o f ambi guous or do ubl e peaks as i ndi cators of se qu en ce h et eroz ygosit y.
RAPD fing erp ri nt s of the sam e sub set of S, P, hyb rid, a nd h ybri d singl e con idi al i sol at es were obtai ne d using p rimers ATG5 (Ga rbelott o et al. 1993 ) and M13 (St e nlid, Karl sson & Hö gberg 199 4). RAPD pro fil es we re el ect ropho res ed at 50V/ cm on 1.7 % Met aPho r ag aros e (C a mbrex, Eas t Rut herfo rd, NJ).
Nucl ea r mi grati o n i n di-mon m ati ng wi t h t he hybrid isol ate
Fo ur homokary oti c S and 6 hom ok aryotic P t est ers were sel e cte d for matin g test s. All t est ers and the hybrid i sol at e were from nort he rn Cali forni a. Hyb rid i sola te SP400 wa s pa ired with all t est e rs in dupli c ate . Myceli al plugs were pla ced on m alt ext ract agar 1 cm apart and in cu ba te d in th e dark . Aft er 9– 12 days, pres en ce/ absen ce of cl am ps wa s dete rmi ne d as des c ri bed for t he ori ginal m ati ng plat es , above. Furthermore , a sub cult ure wa s t ak en from the t es ters’ col oni es at 1 c m from t he interact ion zon e and an aly ze d a ft er 9-12 day s fo r the pre se nc e of cl amps. One S and one P het eroka ryon we re also ma ted with the te st er homok aryons as a cont rol for the abili ty o f t he t est ers to be het e roka ryoti z ed .
Hyb rid SP4 00 a nd singl e co nidi al isolat es deri ved from it d ispl ayed the foll owi ng feat ures in this stu dy:
.
1- All 100 hy ph al ti p su bc ultu res were c lamp ed and het e ro zygous for S- and P-sp eci fi c m a rkers bas ed on T SC P-PCR result s: all had the ITS t ype of both spe ci es.
2- Nu cle i in coni di a stai ne d wit h DAPI were cl early dis cernib le and could be e asil y count ed. Tabl e 1 s umm ariz es t he resu lts obt aine d i n thi s s tud y. The m ajo rit y of c oni dia p rod uced by th e SP hybri d an d S het erokaryons were uninu cle at e, whil e the hom ok aryoti c S isol ate ha d a nucl ear di stribut ion s ke wed towards bi nucl eat e conidi a. Dist ri butio n patt e rns of number of conid ia from the th ree isol at es were signifi cantly different from one anot her. Z val ues for t he t hree possibl e c ompa ri sons ranged bet we en 2.4 an d 7 .26 and i ndi ca ted si gni fi cant differe nc es at P=0.05.
3- All 3 00 co nidi a were het e rozygous for the S - and P- linked mark ers as det e rmin ed by TSC P-PCR re sults and ITS R F LPs. R FL Ps of th e t wo add ition al and unrel at ed nu cl ear lo ci , el ong atio n fact or1-α a nd he at st ress prot ei n 80-1, cou ld diffe rent i at e b etwe en S a nd P isol at es . The hyb rid gen otype and all 15 rando mly s el ect ed singl e c onidial i sola tes were het e rozyg ous for S a nd P m arkers (Fi g.1; Tabl e 2 ). Ad ditio nally, a tot al of 21 fra gme nts (500 to 1600 bp in s ize, dat a not s hown ) we re rel i abl y
pro du ce d by th e M1 3 (13 ba nds ) a nd the ATG5 (8 bands ) R APD prim ers. The S P40 0 isol at e an d its 1 5 si ngl e conid ial is ol ates had id ent ical pro fil es with bot h prim e rs t est ed. The two unrel ated S a nd P isolates c ould be easily differenti at ed from one a nothe r and from isolat e SP400 a nd its con idi a.
Sequ en ce ali gnment and chrom atogram a nal yses of a 581 bp port ion of t he mitoc hondrial ATPa se-6 l oc us identi fi ed 9 base sub stitut ions be twe en t he S and t he P isol at e empl oyed in th e st udy. Th e hyb ri d isol a te and all of the hybrid single c onidi al isola tes ran do mly s el ected for t his analysis ha d una mbiguous S sequ en ces, witho ut a ny t rac e of h et eroz ygot ic patt ern. All seq ue nc es a re de pos ited i n GenB an k wit h a cc ession numb ers AY560330-AY5603 46.
4- Homokaryoti c te sters we re not he te roka ryot ized by th e SP hyb rid, but het e roka ryoti z ati on of the t est ers was succ ess ful when t he y we re mate d with S a nd P het erokaryons.
DI SCUSSIO N
The t hallus of hybrid isol at e SP400 c ompris e d onl y hyp ha e that we re het e rozyg oti c fo r S - and P-li nk ed marke rs (e .g. altern ativel y fi xe d polym orphism s i n the ITS regi on). Thi s resul t is in c ont rast with fi ndin gs
from nat ural Het erob asidi on he tero ka ry on s of eit he r North Am eri can t axa, in whi ch t hall i a re al ways comp rise d of both homokaryoti c and het e roka ryoti c hyphal co mpart me nts (Hansen et al . 199 3). The d at a suggest that all hyphae in the hybrid t hall us must b ear nucl ei of bo th spe ci es. Th e abs e nce o f homokaryoti c hypha e wa s confirmed by t he obs ervat ion t hat all subcultu res from singl e hyphal tips were cl amp ed. Arti fi ci al Dic host er eum hybrids also h av e be en report ed t o b e always cl amped (La nqueti n and Boi din, 198 3). Obl igat e h et erok aryosis, dipl oidy, or poliploidy o f hyp hal cells mus t be i nvoked to expl ain t he resul ts of the analys is.
The numbe r of uni nu cl eat e con idi a was l owest (32 %) i n th e homokaryoti c S is ol at e, in term edi at e (58% ) in th e S het erokaryon , and hig he st (70% ) for the SP hy brid. Nucl e ar dist ri bution pa ttern s in c oni d ia of th e S homok aryon and het erokaryon fel l withi n the range of val ue s publi shed by Ramsd al e and R ayne r (199 4) fo r homokaryo ns a nd het erokaryons re sp ec tively. The hybri d i sol at e s ho wed a p erc en tage o f uni nu cle at e con idi a gre at er th an th at obt ai ne d from the S ho moka ryo n an d het e roka ryo n an d larg er than th at reported for any is ol ate in previ ous studi es (R ams da le & Ra yn er 1994). Th es e dat a could be i nt erpret ed as the re sult of va ryi ng l evels of genomi c confli ct (R am sdale & R ayne r 1994). Genomi c confli ct wo uld be nil in coni di a gene rat ed by S ha ploid hy ph ae in which o nly one geno me is pres ent , result ing in la rge r numbers of bi -
and mult i- nu cl e at e conidi a. Confli ct wo uld be int erm edi at e i n S intras pe ci fi c het erokaryons in whi ch t wo diffe rent genomes bel ongi ng to the same t axon co exist in th e sam e c el l, result ing i n th e product ion of larger number o f uninu cl eat e conid ia. Confl ict would be hi ghe st in the inters pe ci fi c SP hyb rid in whi ch genome s fro m two dif fe rent tax a coexi st in the sam e ce ll, re sultin g in th e produ ctio n of th e l a rg est numb er of uninu cl e at e c oni dia . Ou r further an alysis allows us to reject t his hypot hesis fo r the hy brid thall us (s ee b elo w).
Altho ugh t he maj ority of DAPI st aine d co nidi a from t he hybrid we re uninu cl e at e, all cult ures gene rat ed from t hem were cl amp ed SP h yb ri ds as det e rmin ed per TSC P-PCR an d ITS R FLPs . Thes e res ults were c onfirme d by R FLP an aly se s of the elongation fa ctor 1-α and the heat st re ss p rot ein 80-1. Fo r both loci, th e enti re s ubs et of 1 5 si ngl e coni dial isolat es ra ndom ly s el ec ted from the hybri d culture dis pl ayed h et eroz y gous (e.g . S-P) RFLP pat te rns. In c ont rast, only th e ATPas e-6 s equenc e as so ci ate d with t h e S t axon wa s detect ed in all hybrid i sol at es. Thi s is in a gre em ent with t he rep ort ed uniparenta l migrati o n of the mitochondrio n durin g hybridi zation (Ols on & St enlid, 2001 ). RAPD profil es al so showe d that singl e conidi al isol at es were undi stin guishab le both from on e ano ther and from the pa rent al hy brid isol at e. Thus, i n the ca se of th e hy brid i sol ate , hi gh n umbe rs o f uni nucle at e c oni dia c annot be expl ained by the p res en ce of e xt rem e g en omi c co nflic t result ing in an in cre ased p rod uct ion of
uninu cl e at e conidi a, but rath er by a diploid or a pol yploid stat e o f the uninu cl e at e conidi al cells . Alth ough we did not try to det ermine genome size o f DAPI stai ne d co nidi a, no o bvio us di ffere nc e in si ze o f nu cl ei cou ld be det e ct ed when com pa ring nucl ei from an S h omok aryon, an S het e roka ryo n, and t he hy brid SP40 0. W e sugg es t t ha t ou r in abilit y to vi su ally dis c rimin at e nucl ea r size may be indi cat iv e o f simpl e diploi dy.
Our resul ts differ from th os e of L anqu et in and Boi din (1983 ) be caus e in our stu dy, bot h t he cod ominant nu cl e ar RFLPs and t he d om ina nt R APD mark ers i ndi cat ed m arkers fro mbot h pa rent al nu cl ei were pres ent in a ll con idi a. In Di choster eu m sordul entum x durum hy brids, only c oni dia be aring D. sor dul ent um nuclei could be det e cted. L anque tin and Boi din suggest ed t hat , in t he Dic host er eu m hybrid s us ed in t hei r tests, such re sults coul d be exp lai ned by pre ferenti al mig ratio n of the D.
sordulentum nu cl eus in the co nidi a or by fail ed g ermination of c oni dia be aring th e D. durum nucle us. During the exe cuti on of our stud y, we obs erved th at c oni di a o f all nucl ea r conditi ons were abl e to ge rmin at e. The dat a p rese nt ed i n this study, thus, are not lik ely to be expl ained by la ck of g ermi nation of unin ucle at e co nidi a.
The uni qu e nucl e ar condition o f the n at ural hy bri d m ay a ffect n ucl e ar migrati on du ring di-m on mat ing. Homokaryoti c t est ers were n ot het e roka ryoti z ed by the hybri d is ol ate SP400, su gg estin g a ne w type of
interact ion between hybrid a nd ha ploi d myc eli a. In Het erobasidi on, two isol ate s can mate if a)- t hey have the sa me positi ve all el e at on e of five interst erility (IS) genes (h omog enic i nteracti on), a nd b)- they have di ffe rent mati ng a lleles at a singl e matin g loc us (hete roge ni c int eracti on) (Chas e & Ullri ch 1990). Het erobasi dio n indivi du als b el ongi ng to th e s ame spe ci es a ll sh are th e sam e po si tive al lele at one of th e two “p rimary” interst erility l oc i: fo r inst an ce all North Ame ri can S is ol ates have a ‘+’ all el e at t he S l ocus a nd a ‘-’ a llele at the P l ocus. In No rth America, abo ut 20% o f the isolate s also h av e a positiv e all ele i n one of t hree “sec ond ary ” loci (V1, V2, V3 ) all owing fo r int erspec ifi c mat ing . Resul ting hyb rids will b e het e rozygous at t he S and P loci, and homozygous for the ‘+’ all el e at on e o f t he thre e V l oci .
Het er obasidi on is a highl y out crossing org an ism, ch ara ct e rize d by a bi pol a r ma ting s yst e m and an extremely large num be r of mating al lel es (Chas e & Ull ri ch 1983, Garbel otto et a l. 1999 ). Thes e fe atures e xpl ain the hi gh su cc ess rat e o f het erokaryon-h om okaryon pai rings when isol at es com e from the s ame regi on. Th e percen tag e of fail ed mati ngs inc reas es when i sol at es from d istant loc at ions a re p aire d (Gonthi e r et al . 20 02 ). In ord er to explai n the lack o f su cc ess o f di -mon matings be tween th e h yb rid SP400 a nd homoka ryons from the sam e region, we su rmis e tha t in the case of t he hyb rid th allu s, he tero zy gosi ty at b oth the S and P int ersterilit y loci may hind er nucl e ar migrati on. When pai re d wi th homo ka ryo ns o f e ith er
ISG, th e c o-exi st en ce of bot h t he S and the P IS all el es m a y resu lt i n an het e rogeni c int era cti on and in fai led nucl ear mig ration. Th es e result s are in co ntrast with th ose by Lanq uet in a nd Boidi n (1983), who showed migrati on o nly of D. dur um nu cl eus when pai ri ng Dic hos ter eum hy brids with eit her D. durum or D. sordu lentum h apl oids.
While L anquet in an d Boi di n (1983) s ho wed independen ce of nuc lei in the hybrid thalli and in t he res ulting conid ia, our re sults from th e an alyses of indivi dual hyp ha l ti ps, uni nu cle at e co ni dia, and di-mon m ating al wa ys indi cat e co -segregati on of m arkers associ at ed with bot h paren tal s pe ci es . In pa rti cul ar, the a bse nc e of in de pe nd ent n uc lea r migrati on in di -m on matin g m akes a good a rgume nt in favor of eithe r a diplo id natu re or of a stri ct het erokaryos i s of t he nat ural hyb rid is ol ates. An alt e rnative hypot hesis may be that of prima ry homot hal lism res ulting in a modi ficati on vi a recombinati on at th e IS loci. A simil ar h ypothesis h as be en s uggest ed for Dichost ereum arti fici al hyb rids (Lan qu etin and B oidi n, 1983 ). Fl ow cyt ometri c studies wil l b e n ec es sary to di ffere nt iat e b etwe en thes e t wo vi abl e hy pothese s reg arding t he nucl ear condition of th e hybri d
Het er obasidi on t h all us. Nonethel ess , t he se cond hypothesis is so me what neg at ed by th e presenc e o f i de nti ca l R APD profi les amon g all isolat es gen era ted by si ngl e c onidi a.
Und erst andi ng t h e me chani sms regul ating n ucl e ar mig ratio n in the pres en ce of hy bri d gen otypes is ext rem ely im port ant. Evi de nc e sugg ests
Het er obasido n h et e rokaryons a re ca pabl e of exchanging nuc lei i n nature, allowing for a d ec oupli ng o f n ucl ea r mi grat i on and hy pha l growth (Garb elott o et al. 1 999). B as ed on th i s sc ena rio, invadin g nucl ei are cap abl e o f ta king ove r thalli de ve lop ed by the o rigin al nucl ei. Thi s mechanism may allow for a fu rth er est ablishm ent of h yb rid genomes. In the root p ath ogen Armilla ria, fo r inst an c e, di ploid n uc lei qui ckl y ov ert ake hap loid hy ph ae , all owin g fo r a qui c k nuc le ar re pla cem ent with out t he ne ed fo r fu rthe r cell ular g rowth (R iz zo & Harrin gto n 1 992 ).
Resul ts from this study ident ify a nove l nu cle a r conditi on for
Het er obasidi on. Ou r finding s also sugg ests int erspecifi c h ybri di zat ion may be a possible ev olutionary me chani s m through whi c h Ba sidiom yc et es may atta in di pl oi dy. Finally, thi s study indi cat es the na tural
Het er obasidi on hybri d is geneti cally ext rem ely st abl e, and the en tirety of its hyphae and mi t ospores are hyb rid i n n atu re as well . Instabilit y or tendency fo r se gregation of parent al nu cl ei has be en p reviously rep ort ed when stu dying hybrids obt ain ed i n the labo rato ry. The na tural hybri d studi ed he re is p roof that st abl e hybrid s also exis t. Th es e hy brids may be the on ly ones surv i ving in nature. Th e geneti c sta bilit y desc ri bed here suggest s hybridi zati on and inters peci fi c gen e fl ow m ay pl ay an imp ort ant rol e i n the evolut ion ary hi sto ry of t hi s s pecie s comp lex.
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Tabl e 1. R ecovery of u ninucl eat e, bi nu cl eat e, and multinucleate conidi a
from t hre e is ola tes o f Het eroba sidi on annosum.
Fu ng al Isol at e Put ati ve nuc lea r stat us ISGa Nb Uninucl eat e conidi a N % Binu cle at e conidi a N % Multinucl e at e conidi a N % L2.8.R1 Homo k. S 188 61 32 104 56 23 12 L2.7.R5 Het e rok . S 187 108 58 74 39 5 3 SP4 00 Het e rok . S P 194 135 70 55 28 4 2 a
ISG= In te rst e rility group b
Tabl e 2. R FLPs of t wo unlink ed DNA regions ampli fi ed by PCR,
di ffe renti at e bet ween the S an d th e P int e rste rility groups of the pat hogen
Het er obasidi on ann osum fro m nort heast ern C ali fo rni a. All hybri d co nidi a test ed had hete rozyg ous R F LPs at bot h lo ci.
DNA region
NA WC PISG
NA WC SISG
enzyme Sna BI Bsi EI
elongation factor 1-αααα fragment 1 size (bp) 363 313
fragment 2 size (bp) 85 135
total size uncut (bp) 448 448
enzyme Hind III Bgl II
heat stress protein 80-1 fragment 1 size (bp) 199 415
fragment 2 size (bp) 298 82
Figure 1. A 3% MetaPhor® agarose (FMC Corp., Rockland, ME) gel showing RFLP
fragments of the PCR product generated with elongation factor 1-α forward and reverse primers for SP hybrid, S ISG and P ISG isolates of Heterobasidion annosum from
California. PCR amplicon was digested with Sna BI and Bsi EI restriction enzymes. First and last lanes are molecular standards (100-bp ladder). Lanes 2-16 are randomly chosen single conidia isolates of the AWR400 SP hybrid genet. Lane 17 is a Californian P ISG isolate, and lane 18 is a Californian S ISG isolate. The 448 bp uncut product appears in each lane. The P ISG digest yielded 85 and 363 bp fragments, and the S ISG digest yielded 135 and 313, while digests of all AWR400 SP single conidia isolates show all four S and P ISG RFLP fragments.
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