CayanaBol.56(1);31-37, 1999 ISSN 0016-5301 BREEDING SYSTEMS OFTWO ENDEMIC RAINFOREST SPECIES IN SOUTHERN CHILE:AMOMYRTUSMELI(PHIL.) LEGR. ET KAUS. (MYRTACEAE) ANDLUZURIAGA POLYPHYLLA (HOOK.) MACBR. (PHILESIACEAE) SISTEMA REPRODUCTIVODEDOSESPECIESENDÉMICASDEL BOSQUELLUVIOSODELSURDECHILE: AMOMYRTUS MELI (PHIL.) LEGR. ETKAUS. (MYRTACEAE) KLUZURIAGA POLYPHYLLA (HOOK.) MACBR. (PHILESIACEAE) MaryT. KalinArroyo* & AnaMaríaHumana* ABSTRACT RESUMEN Experimental handself-andcross-pollinations, sponta- Se dan a conocer los resultados de pruebas de neous selfing triáis and emasculation tests to detect autopolinización manual,polinizacióncruzadamanual, compatibilitystatus,selfingcapacityandagamospermy, autopolinización automáticay emasculación, parala along with natural fruiting levéis were undertaken in detección del tipo de compatibilidad, autogamia y the rainforests ofChiloé, 42°S onAmomyrtusmeli agamospermiaenAmomynusmeli(Myrtaceae).especie (Myrtaceae), endemic to Chile and belonging to a delbosquelluvioso,endémicadeChile,ypertenecienteal genusendemic totempérate southem South America, génerosudamericanoaustralendémico,yenLuzuriaga andonLiizuriagapolyphylla(Philesiaceae),endemicto polyphylla (Philesiaceae),especieendémicaaChiley Chile and belongingtoasmall genus ofGondwanan pertenecienteaungénerodedistribuciónGondwanica. distribution.Amomyrtusmeli is genetically self-com- Amomyrtusmeliesgenéticamenteautocompatible.Lapro- patible and non-agamospermous. Natural pollination duccióndefrutosmediantelapolinizaciónnaturalesestadís- fruit set was statistically indistinguishable from hand ticamenteindistinguibledelniveldefructificaciónobtenido cross-pollination and hand self-poUination fruit set. porpolinizacióncruzadamanualyautopolinizuciónmanual. Thisisthesecondrecordofgeneticself-compatiblityin El registrodeautocompatibilidadgenéticaenA. meli Amomyrtus, confirming that theentire genus is self- constituyeel segundodeestesistemadereproducción compatible. Self-compatibility inAmomyrtuacontrasts paraelgénero,confirmandoquelaautocompatibilidades with presence ofgenetic self-incompatibility in other característicaanivel genérico. Laautocompatibilidad generaofMyrtaceae (LumaandMyrceugenia) in the genéticaen el géneroAmomyrtus contrastacon la températe rainforest ofsouthern South America. presenciade autoincompatibilidadgenéticaen otros Luzuriagapolyphyllaisstronglyself-incompatibleand génerosdelafamiliaMyrtaceae(LumaandMyrceugenia) non-agamospermous. Natural pollination fruit set was del bosque lluviosodel surdeSudamérica.Luzuriaga statisticallyindistinguishable from hand cross-pollina- polyphyllaesaltamenteautoincompatibleynopresenta tionfruitset,indicatingefficientpollinationintherain agamospermia. Laproducción defrutosmediantela forestofChiloé. Self-incompatibility in L polyphylla polinizaciónnaturalesestadísticamenteindistinguibleal constitutes a second report ofthis breeding system niveldefructificaciónobtenidomediantelapolinización amongSouthAmericanspeciesofthegenus,whereL. manualcruzada,indicandounsistemadepolinizaciónmuy radicanshas alsobeen reportedas self-incompatible, eficienteenestaespecieenlosbosquesdeChiloé. La andaddsanotherspeciestothegrowingiistofobligate- presenciadeautoincompatibilidadgenéticaenLpolyphylla lyoutbredspeciesamongwoodyandsemi-woodyele- constituyeelsegundoregistrodeestesistemaderepro- mentsintherainforestfloraofsouthemSouthAmerica. ducciónentre lasespecies sudamericanasdel género, dondeL. radicansfuerecientementedocumentadacomo Keywords:Amomyrtusmeli.Luzuriagapolyphylla, genéticamenteautoincompatible.Conelpresentetrabajo Myrtaceae, Philesiaceae. genetic self-compatibility, seagregaotraespeciegenéticamenteincompatibleala genetic self-incompatibility, endemic speciesofChile, listacrecientedeespeciesexógamasentreloselementos rainforest.Chile. leñososysemileñososdelosbosqueslluviososdelsurde Sudamérica. Palabras claves: Amomyrtus meli. Luzuriaga Departamento de Biología. Facultad de Ciencias. polyphylla.Myrtaceae,Philesiaceae,autocompatibilidad UniversidaddeChile.Casilla653,Santiago,Chile.E- genética,autoincompatibilidadgenética,especiesendémi- mail:[email protected] casdeChile,bosquelluvioso.Chile. 31 CayanaBot.56(1), 1999 INTRODUCTION with large solitary flowers containing small amounts of néctar and with fleshy fruits. The question as to whether genetic self- Luzuriagaisasmallmonocotyledonousgenasof incompatibility is basal to the angiosperms and Gondwanan distribution containing 4 species or has evolved on several different occasions (Rodríguez & Marticorena, 1987; Arroyo & within different angiosperm Hneages is still an Leuenberger, 1988; Arroyo et al., 1996). openone(Welleretal., 1995).Theanswertothis Luzuriagapolyphylla is endemic to Chile where crucialquestioninangiospermevolutionrequires itoccursfrom0-800melevationinRegionsVIII better knowledge ofthe distribution of self- to IX (Rodríguez & Marticorena, 1987), being incompatibilityatthegenericandfamiharlevéis. especially abundant in rainforest on the Coast Particularlycritical,arestudiesinwoodyspecies, RangefromValdiviasoath.Riverosetal. (1996) vines and long-lived perennial species where recently reported genetic self-incompatibility in self-incompatibility tends to be more frequent Luzuriaga radicans based on work in the (Arroyo&Squeo, 1990aandb;Arroyo&Uslar, Valdivian rainforest, 40°S, Chile. Nothing is 1993). However, because species in these Ufe knownaboutthebreedingsystemofL.parviflora forms must usually be studied in field popula- (Hook. f.) R. et P. distributed in New Zealand tions, they tend to garner less attention. In this (Rodríguez&Marticorena, 1987). paperwe report the results ofexperimental tests to detect genetic self-incompatibility in Amomyrtus meli (Phil.) Legr. et Kaus. MATERIALSANDMETHODS (Myrtaceae) and Liizuriagapolyphylla (Hook.) Macbr.(Philesiaceae)inhabitingtherainforestsof Tests on Amomyrtus meli were conducted Chiloé, Chile. In both ofthese two small genera. daringNovember, 1997attheEstaciónBiológica other species have already been tested for self- Senda Darwin , Chiloé (42°S). Tests on incompatibility(Riverosetal., 1996). Luzuriagapolyphylla were conducted over the Amomyrtus meli (Phil.) Legr. et Kaus. is a period October 1997- January 1998 at the same large rainforest tree to 20 m tall occuning from locality. Here both species occur in perhumid AraucotoChiloé.ThegenusAmomyrtus(Burret) rainforest zone (AiToyo etal, 1996) along with Legr. et Kaus, (Myrtaceae), comprising two Podocarpus nubigena, Saxegotliaea conspicua, species, is endemic to southern South America Weinmcmniatrichosperma,Caldcluviapaniculata, (Arroyo etai, 1996). Amomyrtus luma (Mol.) Myrceugeniaplanipesand Tepualiastipularisas Legr. etKaus., the second species, is a shrub or the main tree species. Opaque-white-tlowered tree occurring in humid forest habitats from Luzuriagapolyphyllagrowsonlivingtrunksabout MauletoAyseninChile,andNeuquéntoChubut 1-2 m fromground level. Annual precipitation at in the Andes of Argentina. Both species of Pudeto. located cióse to Senda Darwin is 1.809 Amomyrtus have large white flowers with copi- mm; meanannual temperatureis 11°C (Hajek& ous pollen, and fleshy black fruits (Landrum, DiCastri, 1975). 1988).FlowersofA. lumaareknowntobevisited Toinvestígatethebreedingsystem,handself- by Hymenoptera (Halictidae) and Díptera pollinations, hand cross-pollinations, spontaneous (Syrphidae) (Riveros eta/., 1991). Although the selfing triáis and emasculations without further ovarles of these species contain numerous pollination were conducted on plants growing in ovales, matare fruits normally contain 1-3 large the field previously bagged at the flower bud seeds,4-6mmlong,thesebeinglargerinA. meli. stageinwhiteorgandypollinationbags.Eachtest Riveros et al. (1996) recently reported the pres- was conducted on several different individuáis ence genetic self-compatibility in Amomyrtus growing at the same location. Pollinations on luma based on work in the Valdivian rainforest, Luzuriagapolyphylla were spread over a period 40°S, Chile. It is of special interest, thus, to of 15 days; those onAmomyrtus meli over 20 determine whether self-incompability character- days. Over these same periods, to evalúate the izesA.meli.Amomyrtusmeli,unliketheA. luma, efficiency ofthe pollination system, additional isstrictlyendemictoChile. unbagged flowers on both species were marked Luzuriagapolyphylla (Hook.) Macbr. is a andassessedforfruitsetsoastocomparethelevel high-climbing semi-woody epiphytic species offruiting undernatural conditions ofpollination 32 Breedingsystemsot'twoendemicrainloicsispccics:Arrovo,M.T.K.&A.M.HUMANA tothatobtainedunderhandcross-pollination. For than spontaneous self-pollination fruit set (G,^^, = self-pollination. poUen from the same lloweror 5.415: p<().05). The ISI (measure ofdegree of from other ñowers on the same individual was self-incompalibilily lluctualingbetweenOand I:O iised. For cross-pollination, fresh pollen was =full self-inconipalibility: 1 =fullcompatibilily - collected from individuáis located distant from see Ruiz & Arroyo, 1978) for-4. meli is 0.50, thetestplant, andtransported immediatelytothe which iswellabovethevaluéof0.2u.sedbycon- receptivestigmasofbaggedtlowers.Eachflower vention todifferentiate between .self-incompatible was poliinated on three sepárate occasions so as andself-compatiblespecies(c.f. Arroyo&Squeo, to increase the likelihood ofencountering the 1990b).Amomyrtusmelithus isclassedas aself- stigmas at their máximum period ofreceptivity compatible species, although clearly, the level of and ofobtaining physiologically máxima! fruit compatibilityisonlymodérate. and seed sets. All poliinations werecairied in the Only3.5%ofthehandself-pollinatedtlowers earlyaftemoonwhentemperaaireswerewarmerixnd ofLiizuriagu polyphylla produced fruit (Table thetlowerswerefullyopen. Followingmarking,all II). Inthespontaneouslyselfingtriáis,notlowers manipuiated tlowers were immediately rebagged. producedfruits, as was thecase foremasculated CompletefruitmaturationinLitziiríagapolyphylla tlowers (Table II). In contrast, 58.5% ofhand in Chiloé requires around 8-9 months (Smith- poliinated cross-pollinated flowers produced Raniírez & Armesto, 1994). Long fruit matura- fruits.aproportion thatismuchhigherthanwith tion periods have also been described in L. hand self-pollination. These results give an ISI marginara (Arroyo & Leuenberger, 1988) and valué of0.026, which is well below the cut-off wouldappeartobecharacteristicofthegenus.In valué of0.2 and allow the conclusión ofa high order to avoid loss offruits in the experimental levelofgeneticself-incompatibilityinLuzuriaga crosses andothertestsduetoautumn andwinter polyphylla. stormdamage,weharvestedfruitsofthisspecies Natural fruitproductioninAmomyrtu.smeli in late summer as they began to mature. Fruit was modérate (Table III), but not significantly maturation in Amomyrtiis meli occurs over the different from that in any of the experimental period December to April (Smith-Ramírez & treatments (G^^. = 1.696: NS: (spontaneous self- Armesto, 1994). Forourexperiments,fruitswere ing); G^jj = 0.519; NS (hand self-pollination): harvestedinApril, 1998. Inbothspeciespollina- G^j. = 0.413, NS (hand cross pollination)). tion bags were retained on the plants until the Thereforethe fruits produced under natural con- timeoffruitcoUection,soastopreventfruitloss ditions ofpollination in Amomyrtus meli could through the action ofthe biotic dispersal agents havebeenderivedfromspontaneousintra-flower (Armesto et al., 1987) and facilítate capture of self-poIUination orgeitonogamy (self-pollination anymaturefruitsthatpotentiallycouldhavefallen as a result ofpollen transference among tlowers by the time offruit harvest. Fruits ofLuzwiaga of the same individual) or cross-pollination. polyphylla tend to fall easily with any distur- Mostly likely natural pollination fruit set is bance. derivedfromalltypesofpollination. Natural fruitproduction in unbagged tlowers ofLuzuriaga polyphylla was surprisingly high RESULTSANDDISCUSSION (Table IV) for a strongly self-incompatible species and not significantly different (G^| = Under hand self-pollination 13.5% ofthe 1.46;NS)fromthatobtainedunderhand-pollina- flowers ofAmomyrtus meli produced fruits, in tion in which all stigmas were artificially polii- comparison with 24.3'7f under hand cross-polli- nated, suggesting high efficiency ofthe natural nation. In the spontaneously selfingtriáis. 10.5% pollination system. Seed number per fruit was ofthe tested tlowers produced fruits (Table I). somewhat higher in naturally poliinated flowers Cross-pollinationandself-pollinationfruitsetfor than in hand cross-pollinated tlowers (t (d.f. = A. meli(Gjj =2.234; NS) were not significantly 80)=5.598:p<0.001)(TableIV).Thislastsitu- different, as wasthecaseforself-pollination and ation is commonly encountered in field pollina- spontaneous self-pollination (G^j = 0.269: NS). tiontests(c.f.Arroyo&Squeo, 1990a)where. in Cross-pollinationfruitsetwassignitlcantlyhigher spite of repeated poliinations. it is difficult to 33 CayanaBot.56(1),1999 assure the exact timing of máximum stigma representaron ofself-incompatibility and other receptivity. As an aside, it should be noted that outcrossing breeding systems such as dioecism mature fruits ofL. polyphylla are greenish at among woody species in températe forests in maturityinChiloé(Aimestoetal,1987).Hoffmarm Chile (Arroyo & Uslar, 1993; Riveros et al., (1982) illustrates the fruits ofL. polyphylla as 1995, 1996;Castoretal, 1996)andaddsanother greenish-yellow. Rodríguez & Marticorena species to the long list of self-incompatible (1987) describe the fruits ofL. polyphylla as species in southern South American températe reddish-orange. Evidently more work is needed rainforests. Thepresenceofself-incompatibilityin to define fruit colours in the South American twoSouthAmericanspeciesofLuzuriagaraisesthe species ofLuzuriaga. The high level ofgenetic question about the remaining two species ofthe self-incompatibility demonstrated in Luzuriaga genus,andespeciallyNewZealandL.parviflora.In polyphyllahighlightsitsdependenceuponextemal view ofthe disjunct distribution ofthe genus in pollinating agents. Riveros etal. (1991) reported NewZealand and southem SouthAmerica, Infor- thatflowers ofL. radicans are visited by halictid mationonthebreedingsystemofthelatterspecies bees in Parque Nacional Puyehue, 40°S, and would be particularly welcome. Interestingly, and flowers ofL. marginata, aspecies closely related in contrast with what has been demonstrated in toLpolyphylla(Rodríguez&Marticorena, 1987), the Chilean flora (e.g. Arroyo & Squeo, 1990b; are reported to be sweetly fragrant (Arroyo & Arroyo&Uslai-, 1993;Riverosetal, 1995, 1996) Leuenberger, 1988), also suggesting bee pollina- self-incompatibility is known for a very limited tion. Unfortunately nothing is presently known number of species in the New Zealand flora, aboutthepollinationbiologyofL.polyphylla. We although as Webb & Kelly (1993) point out, the failedtoobservepollinatoractivityonLpolyphyl- sample size forNew Zealand is perhaps still too la while carrying out our crossing experiments. smallforanyconvincingconclusionsatthisstage. However, causal visits by Bombus dalhbomi to the very similar flowers of L. radicans were observed. Bombus dahlbomi is a common large ACKNOWLEDGMENTS bumblebee in the forests of Chiloé. Undoubtedly adetailed studyofthepollination Research supportedbyCONICYTGrantN°. mechanisms ofthe three Chilean species, taking 1950461 and an Endowed Chilean Presidential their wide latitudinal distributions into account, Science Appointment (CátedraPresidencial de wouldproveveryinteresting. Ciencias-1997)(MTKA). The results reported here in conjunction with earlier work by Riveros et al. (1996) on Amomyrtus lurna confirm that all extant species LITERATURECITED of the genus Amomyrtus are self-compatible. Armesto,J.J.; R. Rozzi,P,Miranda&C. Sabag. Thisisparticularlyinteresting, sinceself-incom- 1987. Plant/frugivore interactions in South patibility has been found in Myrceugenia and American températe forests. Revista Chilena Luma (Riveros et al, 1996). Little is known Hist.Nat.60:321-336. abouttherelativephylogeneticpositionsofthese Arroyo,M.T.K&F.Squeo.1990a.Ceneticself-incom- threegeneraofMyrtaceae.Thepresenceofself- pBaetnitbhialmiiteyllian(Stohleanaecnedaee)m.iCcayPanaat,agBootn.ia47n:5g1e-n5u5.s compatibility inAmomyrtus could be indicative Arroyo, M.T. K. & F. Squeo. 1990b. Relationship ofa derived condition. The majority ofwoody betweenplantbreedingsystemsandpollination. speciesinthesouthemSouthAmericantempérate In Kawano,S. (ed.),BiologicalApproachesand rain forest are dioecious or genetically self- Evolutionary Trends in Plants, pages 205-227. winocoodmypaftoirbmlaet,ionassiinngdeeneedrailsitnhCehiclaese(Airnrooytohe&r ArroytAoecm,asdMei.mnTi.aaKtP.reemísp&sé,rPa.LtoeUnsdmloenad.ri.ter1r9a9n3e.anB-rteyepdeincglimsayts-e Uslar, 1993;Riverosetai, 1995, 1996;Castoret montane sclerophyllous forest in central Chile. al., 1996).Amomyrtusatthegenericandspecific Bot.J.Linn.Soc. 111:83-102. Lleuvzelu,rDiiseamcgolaenasrptlorylaaytnpiheoyxnlcleopaft,isoaenl.sf-pienccioemspawtiibtihlistoymien ArroyrCceooalo,alVtIitMEoeR.nmEsTphS.éiKrp&as.t;aAen.MrdMa..irneFRfgoaiirgovegnesaitrl,ofrls1io,9cr9ha6nA.oe.sfPsPhsypeotauñottagthleeeomomgszraSoa,fopuhttiLhch.e degreeofwoodiness,isconsistentwiththestrong America. InLawford, R.C.; P. Alaback&E.R. 34 Breedingsystenisoftwoendemicruint'orestspecies:Arrovo,M.T.K.&A.M.HUMANA Fuentes (eds.), High Latitude Riiint'oiests üt'the biologyinspeciesofthegcnusNoüuifugus.Environm. WestCoastoftheAmericas:Climate,Hydrology, E.\p.Bot. 35:519-524. Ecology, and Conservation, pages 134-172. RivERos. M.; A.M. Humana & M.T.K. Arroyo. Springer-Verlag.NewYork. 1996. Sistemasde reproducción en especiesdel Arroyo,S.C.&B.E.Leuenberger. 1988.Anoteon bosque valdiviano (40° Latitud Sur). Phyton Litzuriaga marginata (Philesiaceae) from (BuenosAires)58: 167-176, Patagonia.Herbertia44; 17-21. Rodríguez, R. & C. Marticorena. 1987. Las Castor. C;J. Cuevas.M.T.K Arroyo.Z. Rafh. R. especiesdel generoLuzíiriaga R. et P. Cayana, DODD& A. PeÑALOZA. 1996. IsAiistmcednis Bot.44:3-I5. chilensis(D.Don)Pie-Ser.etBizz.(Cupressaceae) RUIZ,Z.T.&M.T.K,Arroyo. 1978.Planlreproduc- from ChileandArgentina: monoeciousordioe- tive ecology ofa secondary deciduous tropical cious?RevistaChilenaHist.Nal.69:84-93. forestinVenezuela.Biolropica 10:221-230. Hajek,E. & F. Di Castri. 1975. Bioclimatologíade Smith-Ramirez, Z C. & J.J. Armesto. 1994. Chile. Dirección de Investigación, Universidad Floweringandfruitingpatternsinthetempérate CatólicadeChile,Santiago. rainforestofChiloé:ecologiesandclimaticcon- HOFFMANN, A. 1982. Flora Silvestre de Chile. Zona straints.J.Ecol.82:353-365. Austral. Ediciones FundaciónClaudioGay. 258 Webb.C.J.&D.Kelly. 1993,Thereproductivebiolo- pages. gyoftheNewZealandflora.TrendsEcol. Evol. Landrum,L.R. 1988.TheMyrtlefamily(Myrtaceae) 8:442-447. inChile.Proc.Calif.Acad.Sci.45:277-317. Weller, S.G.;M.J.Donoghue&D,Charlesworth RrvEROS,M.;A.M. Humana& D. Lanfranco. 1991. 1995.Theevolutionofself-incompatibilityinflow- ActividaddelospolinizadoresenelParqueNacional eringplants:Aphylogeneticapproach.In:Hoch,P, Puyehue,XRegión,Chile.MedioAmbiente11:5-12. & A.G. Stephenson (eds.). Experimental and Rtveros,M.;M.Paredes,E.Rosas,E.Cárdenas,J.J. MolecularApproachesto PlantBiosystemalics. Armesto&M.T.KArroyo. 1995.Reproductive 355-382pp.MissouriBotanicalCarden,St.Louis. Tablei.Resultsofcontrolledhandpollinations,spontaneousselfmgtriáisandemasculationtestsinAmoinyniismeli (Myrtaceae)intempératerainforestinChiloé. CayanaBot.56(1), 1999 TableII. ResultsofcontroUedhandpollinations, spontaneousselfingtriáis andemasculationtests inLiizuriaga polyphylla(Philesiaceae)intempératerainforestinChiloé. BreedingsyslemsoftwoL-ndemicrainlbrestspecies;Arkovo,M.T.K.&A.M.HUMANA 7¿1o Canal de AC^AU/L*I^NCHACAO Chacao Golfo de Ancud LINAO Estoción Biológica "Sendo Dorwin x^ ^Q CHILOE