relative frequency of sympatric species influences rates of...

Journal of Ecology

2008,

96

, 1198–1210 doi: 10.1111/j.1365-2745.2008.01434.x

© 2008 The Authors. Journal compilation © 2008 British Ecological Society

Blackwell Publishing Ltd

Relative frequency of sympatric species influences rates

of interspecific hybridization, seed production and

seedling performance in the uncommon

Eucalyptus

aggregata

David L. Field

1

*, David J. Ayre

2

, Robert J. Whelan

2

and Andrew G. Young

3

1

Department of Ecology and Evolutionary Biology, 25 Willcocks Street, Toronto, Ontario, Canada, M5S 3B2;

2

Institute for Conservation Biology, School of Biological Sciences, University of Wollongong, NSW 2522, Australia; and

3

CSIRO Plant Industry GPO Box 1600, ACT 2601, Australia

Summary

1.

Habitat fragmentation can alter the relative frequency of cross-compatible species within anarea, which can affect the levels of interspecific hybrid production and reduce the viability of smallpopulations through genetic and demographic swamping. For 18 populations of

Eucalyptus aggregata

,we examined the effects of absolute and relative population size (compared with its congeners

E. rubida

,

E. viminalis

and

E. dalrympleana

) on hybrid production, genetic diversity and subsequentseed production and seedling performance.

2.

Relative population size was strongly negatively correlated with rates of hybrid seed production,suggesting increased hybridization when the potential sources of interspecific pollen outnumber thesources of intraspecific pollen for

E. aggregata

trees.

3.

Genetic diversity was negatively correlated with relative population size which suggests thathybridization may maintain diversity lost through bottlenecks and drift following reductions inpopulation size. However, the presence of fertile hybrid adults, and introgressed leaf traits withinpopulations exhibiting high hybridization rates, suggests that small

E. aggregata

populations maybe vulnerable to genetic swamping by common congeners.

4.

Amongst an array of population parameters (population sizes, genetic diversity and inbreeding),seed production was only positively correlated with relative population size, whereby sites with lowrelative population sizes tended to produce fewer seed. This could be due to the action of pre-zygoticbarriers which removes inviable hybrid genotypes as levels of interspecific pollen flow increase.

5.

Germination and survivorship displayed a similar positive correlation with relative populationsize, suggesting post-zygotic hybrid breakdown may also contribute towards to demographicswamping of remnant populations.

6.

Synthesis

. Our results suggest that relative population size is an important parameter determiningrates of hybrid production, seed production and seedling performance. Furthermore, relative populationsize has stronger effects on population fecundity than absolute population size, genetic diversity andlevels of inbreeding. Relative population sizes > 0.5 (i.e. at least equal frequencies of parentals) may berequired to avoid the deleterious effects of genetic and demographic swamping on the viability of rare species.

Key-words:

fecundity, genetic diversity, genetic swamping, germination, habitat fragmentation,hybridization, inbreeding depression, introgression, pollen swamping, population size

Introduction

Habitat fragmentation, which can reduce the size andincrease the isolation of populations, is considered to be oneof the major threats to biological diversity and the long term

persistence of rare species (Young & Boyle 2000; Hobbs &Yates 2003). The genetic and demographic effects of habitatfragmentation on plant population viability are well-documented and include increased inbreeding (Dudash &Fenster 2000), loss of genetic diversity (Oostermeijer

et al

.1994), the disruption of plant

×

pollinator interactions(Mustajärvi

et al

. 2001) and reduced reproductive success

*Correspondence author. E-mail: [email protected]

Population size and hybridization in Eucalyptus

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© 2008 The Authors. Journal compilation © 2008 British Ecological Society,

Journal of Ecology

,

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, 1198–1210

(Cunningham 2000). For cross-compatible species, onepotential effect of habitat fragmentation is alteration of boththe rate and the spatial scale of interspecific hybridization.In some cases, this occurs through the anthropogenic intro-duction of foreign species within close proximity to compatiblenative species, which enables gene exchange to occur due tothe disruption of geographical barriers (Abbott

et al

. 2003).Increased hybrid production has also been observed in smallplant populations of sympatric congeners within fragmentedlandscapes (Potts & Wiltshire 1997; Butcher

et al

. 2005).However, few studies have empirically explored the extrinsicecological and population conditions influencing hybridproduction in fragmented landscapes. Such knowledge on theconditions promoting hybridization and the subsequenteffects on population fitness could be useful for predicting ratesof hybridization in fragmented landscapes and managingthe demographic and genetic viability of remnant populations.

Elevated rates of hybridization between congeners hasimportant implications for the demographic and geneticviability of small plant populations and ecological interactionswithin remnant populations (Ellstrand & Elam 1993). For arare species, increasing hybridization is expected to directlyinfluence the time to local extinction (Wolf

et al

. 2001) due toreductions in the number of pure-bred genotypes (demographicswamping) and the dilution of the gene-pool through intro-gression (genetic swamping) with the more abundant congeners(Levin

et al

. 1996). In several cases the demographic and geneticeffects of hybridization have been identified as a majorcause of plant population decline (Rieseberg & Gerber 1995;Levin

et al

. 1996; Rhymer & Simberloff 1996). Moreover, anincrease in the number of hybrids in plant populations canhave important implications for ecological communitiesthrough alteration of the composition of insect and birdcommunities (Whitham

et al

. 1999) and disruption ofparasite resistance in plant species (Fritz

et al

. 1999). Despitethe potential adverse effects, hybridization is also consideredto be a substantial adaptive force in the evolution of manyplant species (Rieseberg & Carney 1998; Barton 2001). In somecircumstances hybridization may be considered to be benefi-cial to small populations due to the influx of novel genotypes.This may counteract the deleterious effects of genetic driftand inbreeding (Savolainen & Kuittinen 2000) and impartnew adaptive variation required by remnant populations toadapt to changing environments (Ellstrand 2003). Consideringthe potential for both beneficial and adverse outcomes ofhybridization, knowledge on both the potential adverse declinesin population fitness and beneficial effects on genetic diversitywould be useful for managing remnant populations.

The distribution and size of the parent populations affectpollinator behaviour and subsequent plant mating patterns(Kunin 1997), and are therefore likely to be major factorsinfluencing hybrid production in remnant populations. Theabsolute and relative population sizes (i.e. relative frequenciesof hybridizing taxa) of parentals within a site can easily bealtered by habitat fragmentation, and may reflect preferentialclearing of one parental species when species exhibit landscapescale environmental preferences (e.g. for slopes or valleys).

Source-sink dynamics predict that these population parametersare probably important determinants of the amount of immi-grant/interspecific pollen received by populations (Ellstrand& Elam 1993). For example, hybridization rates would beexpected to be greater in small plant populations as they receivemore pollen than they export. Furthermore, reduced popula-tion size of a focal species relative to congeners would increasethe probability of interspecific pollen flow due to a greater rela-tive availability of conspecific pollen within the dispersal areaof pollinators (Ellstrand & Elam 1993). This is expected toresult in a decrease in pollinator fidelity, especially if the floralstructure of the hybridizing species are similar (Kunin 1993).

Our understanding of hybrid promotion in fragmentedlandscapes is predominately based on theoretical models(Pederson

et al

. 1969) and extrapolations from distance anddensity studies which examined intraspecific pollen flow withinexperimental arrays (e.g. Ellstrand

et al

. 1989; Richards

et al

.1999). Although these studies suggest a negative correlationbetween hybrid production and both absolute and relativepopulation size, direct studies of natural hybrid systems aresparse. A number of studies have documented increasedhybridization or introgression in small populations (Rieseberg

et al

. 1989; Gallo

et al

. 1997; Kennington & James 1997; Burgess

et al

. 2005), but there have been no explicit attempts to examinethe rate of hybrid seed production across a range of absoluteand relative population sizes in a natural system. Testing thisrelationship in natural interspecific systems is importantbecause there is growing evidence of hybrid promotion infragmented landscapes (Potts & Wiltshire 1997; Buerkle

et al

.2003; Butcher

et al

. 2005) but empirical evidence that thesepopulation parameters are important in complex plantdistributions plants found in natural populations is limited.Related declines in demographic parameters would be expectedin populations with more frequent interspecific gene flow dueto the action of pre-zygotic barriers and hybrid breakdown onthe performance of hybrid genotypes (Rieseberg & Carney1998). However, the potential for hybridization to reduceimportant population demographic parameters such as plantfecundity and offspring performance remains untested. Thisinformation is vital in determining the processes promotinghybridization and the ability of remnant plant populations ofhybridizing species to persist and re-establish across fragmentedlandscapes.

The genus

Eucalyptus

(Myrtaceae) is a particularly goodsystem with which to explore hybrid promotion in fragmentedlandscapes. This is because many species are known tohybridize freely due to weak reproductive barriers (Griffin

et al

. 1988) and hybrid swarms have often been associatedwith the results of human activities such as habitat modificationand fragmentation (Potts

et al

. 2003; Butcher

et al

. 2005).

Eucalyptus

species are of high conservation priority as theyare a diverse and highly important vegetative component ofthe Australian continent (Hill 1994). However in moderntimes over half of the

Eucalyptus

forests have been cleared orhighly modified (Young

et al

. 1990), with many species nowinhabiting only small fragmented and disturbed remnantpopulations.

1200

D. L. Field

et al.

© 2008 The Authors. Journal compilation © 2008 British Ecological Society,

Journal of Ecology

,

96

, 1198–1210

The primary objective of this study was to examine therelationship between population size (absolute and relative)and hybrid seed production. This also provided the oppor-tunity to test the potential benefits of hybridization through themaintenance of genetic diversity. In addition, we examinedthe power of population sizes, hybridization rates and matingsystem parameters estimated from a current open-pollinatedseed cohort, to explain variation in seed production, seedgermination and seedling performance.

These questions were examined using the long-live treespecies

Eucalyptus aggregata

, which hybridizes with the morecommon sympatric species,

E. rubida

,

E. viminalis

(Field

et al.

2008) and possibly

E. dalrympleana.

The pollination systemof these

Eucalyptus

species is probably entomophilous due tofloral foraging by a diversity of insect species, in particularthe introduced Honeybee (

Apis mellifera

), and variousnative bees (

Leioproctus

Colletidae,

Lasioglossum/Homalictus

Halictidae). Populations of

E. aggregata

range from smallremnants where it is out-numbered by a number of morecommon species, through to large relatively intact woodlandpopulations where

E. aggregata

is numerically dominant. Thisrange provides a good opportunity to examine rates of hybridproduction in relation to absolute and relative populationsize. The pre-zygotic barriers and hybrid breakdown docu-mented in

Eucalyptus

(Potts

et al

. 2003) are expected to resultin reduced fecundity and seedling performance for popula-tions with more frequent interspecific gene flow. However,this association has not been tested in

Eucalyptus

or compared toother well-documented demographic and genetic consequencesof small population size.

We asked three specific questions: (i) Does the absolute andrelative population size of

E. aggregata

(compared to com-mon sympatric species) affect hybridization rates in progenyarrays? (ii) Do population sizes (absolute and relative) andhybridization rates affect genetic diversity and levels ofinbreeding in progeny arrays? (iii) Do hybridization rates(and parameters associated with hybrid production) have agreater affect on population fecundity and seedling perform-ance than population size, levels of inbreeding, outcrossingrates and genetic diversity?

Methods

STUDY

S ITES

AND

SEED

SAMPLING

This study was conducted on the southern and central tablelands ofNew South Wales, in South-Eastern Australia (Fig. 1). Eighteen mixedsites for

E. aggregata

were selected with populations ranging fromsmall road verge remnants dominated by compatible congenersthrough to larger woodland populations where

E. aggregata

isnumerically dominant (Table 1). Five reference populations of eachof

E. rubida

and

E. viminalis

were selected on the basis of high orcomplete dominance of the respective species and the absence ofputative adult hybrids. Reference populations of

E. aggregata

werenot used because previous work found hybrids were still recorded atthese sites and their inclusion made little difference to hybrid assign-ments (Field

et al.

2008). Reference populations of

E. dalrympleana

were also not used as this species co-occurred with

E. aggregata

at

Fig. 1. Map of the location of 18 Eucalyptus aggregata (black circles),five E. rubida (open circles), and five E. viminalis (grey circles) populationssampled for open pollinated seed for the assessment of hybridization rates.

Table 1. Site details, including the abbreviation code, geographiclocation and population sizes (APS, RPS) of three Eucalyptus speciessampled for open pollinated seed

Species/Population Code Location APS RPS

E. aggregataManar Creek MC 35°17′30″ S, 149°41′45″ E 1 0.25Willandra lane WL 35°07′20″ S, 149°36′00″ E 4 0.31Norongo NG 35°42′00″ S, 149°25′00″ E 6 0.21Sth Togganoggra ST 35°39′00″ S, 149°36′00″ E 7 0.15Medway MW 34°29′50″ S, 150°17′00″ E 15 0.75Mozart Road M 33°48′00″ S, 149°47′15″ E 30 0.75Rosevalley RS 35°39′00″ S, 149°25′30″E 30 0.33Berrima BR 34°29′13″ S, 150°18′50″ E 35 0.64Duck Flat DF 35°09′00″ S, 149°34′20″ E 42 0.51Grabben Gullen GG 34°32′00″ S, 149°23′00″ E 50 0.77Hollow H 35°40′00″ S, 149°27′00″ E 90 0.69Bendoura Sth BS 35°31′50″ S, 149°40′00″ E 80 0.67Reedy Creek RC 35°21′30″ S, 149°32′45″ E 100 0.83Neville N 33°42′00″ S, 149°12′00″ E 150 0.87Riverside RV 35°16′00″ S, 149°54′30″ E 200 0.95Fairview FV 33°24′00″ S, 150°04′00″ E 300 0.83Bendoura TSR BT 35°30′10″ S, 149°42′00″ E 400 0.75Captains Flat AP 33°21′00″ S, 150°05′30″ E 700 0.90

E. rubidaCaptains Flat CF 35°23′19″ S, 149°21′40″ E ~100 −Kings Hwy K 35°18′15″ S, 149°45′00″ E ~100 −Sth Captains Flat SCF 35°37′00″ S, 149°26′15″ E ~100 −Tarago T 35°10′00″ S, 149°39′45″ E ~100 −Reed Creek RE 34°21′00″S, 149°33′00″ E ~100 −

E. viminalisBallalaba B 35°34′16″ S, 149°37′45″ E ~100 −Faegans Creek FC 35°24′57″ S, 149°58′05″ E ~100 −Parker Gap PG 35°37′30″ S, 149°29′00″ E ~100 −Warri Bridge W 35°20′39″ S, 149°44′15″ E ~100 −Wattle Flat WF 33°09′00″ S, 149°41′00″ E ~100 −

APS, absolute population size reproductive adults; RPS, the relative population size of E. aggregata to congeners (see Methods).

Population size and hybridization in Eucalyptus 1201

© 2008 The Authors. Journal compilation © 2008 British Ecological Society, Journal of Ecology, 96, 1198–1210

only one of the mixed sites and has little genetic differentiation fromE. viminalis and E. rubida (Cayzer 1993).

For each of the E. aggregata sites, two variables were estimated bycounts of mature reproductive individuals: (i) absolute populationsize of E. aggregata (APS), a count of mature E. aggregata individuals,(ii) relative population size of E. aggregata (RPS) a measure of therelative frequency of E. aggregata compared to the sympatric species,defined as the absolute population size of E. aggregata divided by thesum of the absolute population sizes of all compatible species at thesite [RPS = APS values for E. aggregata/(APS of E. aggregata + E.rubida + E. viminalis + E. dalrympleana)]. The relative populationsize ranged from 0.15 to 0.95, where a value of 0.50 indicates a sitewhere the frequency of E. aggregata individuals is equal to that ofthe sympatric species. The sites ranged from 50 × 50 m to 900 × 600 mwith the boundary selected on the basis of a buffer of at least 1 kmto the nearest E. aggregata or compatible congeners. For a few sites,compatible trees > 1 km from the site boundary may contribute tointerspecific pollen flow although they are not included in populationsize estimates. However their contribution is expected to be relativelysmall compared to trees within the sites because the majority ofpollen dispersal is expected to come from within the first 600 m oftarget trees (Barbour et al. 2005; Byrne et al. 2008).

Between November 2002 and January 2003, 50 to 200 open-pollinated capsules were collected from the canopies of one to 20haphazardly selected E. aggregata within each of the mixed populations,and from five trees at each of the E. rubida and E. viminalis referencepopulations. Trees of each parental species were selected on the basisof distinguishing morphological features including bud size andnumber, leaf size and bark persistence according to Brooker &Kleinig (1999). Capsules were dried for over 2 weeks at room tem-perature and the seed released was bulked within trees. A total of3120 seeds consisting of 20 to 30 seeds from each sampled tree wereindividually sown in a 33% river sand: 33% peat: 33% compost soilmix in separate pots (50 mm wide, 100 mm deep) in a randomisedblock design in an unheated glasshouse. These samples were used toexamine germination, seedling survivorship and performance, andestimate hybrid production rates at 13 of the E. aggregata populations.Due to low numbers of capsules, seed and germinated seedlings atthe remaining five E. aggregata populations, further seed were collectedand planted in a separate trial to estimate hybrid production. Due tothe offset timing of these plantings, these five populations wereexcluded from the germination, seedling survivorship and perform-ance analyses. The smallest mixed population (MC) which consistedof a single E. aggregata and four E. viminalis was removed from allfollowing regression analyses because the tree produced no hybridsand had high leverage, however it was used as a single data point toinfer the possible selfing capability of an isolated tree.

GENETIC ANALYSIS

A total of 2559 seedlings representing 15–25 seedlings per tree weregenotyped in order to identify hybrids, estimate hybrid productionrates for each population, and to determine population geneticdiversity and mating system parameters. Allozyme genotypes at sixloci Gpi-2, Gput, Pgm-1, Pgm-2, 6pgd-1, 6pgd-2 were assayed fromfresh leaf material of each seedling following the techniques ofMoran & Bell (1983). Previous work indicated that allele frequencieswere highly skewed or near diagnostic between E. aggregata andboth E. rubida and E. viminalis at the Pgm-2, 6pgd-2, and the Gpi-2locus (Field et al. 2008). The genotypes consisted of two data sets,the first a seedling data set from seedlings grown from seed arrays ofindividual trees that were classified on the basis of distinguishing

morphological traits of each parental species (Field et al. 2008), andthe second, an adult maternal genotype data set inferred from theprogeny arrays using the method of Brown & Allard (1970) in theMLTR program (Ritland 2002). The adult genotypes were used toidentify hybrid maternal parents. This data was used to detect hybridindividuals among the adult maternal genotypes, and progeny fromthese were removed from population estimates of hybrid production.

HYBRID IDENTIF ICATION

Hybrid adults and seedlings were identified from admixture proportions(q) using the Bayesian methods implemented in the program struc-

ture 2.1 (Pritchard et al. 2000; Falush et al. 2003). This method usespopulation allele frequencies to assign individuals (as represented bytheir multi-locus genotypes) to K groups/clusters by minimizingwithin group linkage-disequilibrium and simultaneously assumingwithin group Hardy Weinberg Equilibrium. In this way, individualscan be assigned to a single group (e.g. q1 = 0.99, q2 = 0.01) or jointlyto two or more groups if their multi-locus genotype indicatesadmixture due to hybridization (e.g. q1 = 0.5, q2 = 0.5). We usedstructure with the admixture model, no prior population information,a burn-in period of 20 000 generations and 200 000 MCMC’s(Monte Carlo Markov Chain) to calculate the admixture proportionand the 90% probability interval (default value recommended;Pritchard et al. 2000) for each individual with respect to the E.aggregata cluster. For all STRUCTURE analyses, two geneticgroups were assigned (K = 2), with E. aggregata in cluster 1 (q1) thecombined E. rubida and E. viminalis in cluster 2 (q2). This wasbecause E. aggregata was highly differentiated from both E. rubida(FST = 0.59, P < 0.01) and E. viminalis (FST = 0.58, P < 0.01). How-ever the latter two species were relatively poorly differentiated at theseallozyme loci (FST = 0.13, P < 0.01) (Field et al. 2008). Consideringthe low differentiation between E. dalrympleana and E. viminalispreviously described at many of these allozyme loci (Cayzer 1993),and the close taxonomic relationship with E. rubida we assumed thatE. dalrympleana would similarly group into cluster 2.

Critical thresholds of admixture proportions in the E. aggregatacluster (q1) were used to assign seedlings from pure-bred maternaltrees and adults into one of the following genomic groups; (i) pure-bred E. aggregata for q1 > 0.9, (ii) pure-bred E. rubida or E. viminalisfor q1 < 0.1; (iii) F1 hybrid (E. aggregata × E. rubida/E. viminalis/E. dalrympleana) for q1 0.7 to 0.3; (iv) backcross hybrid (generationunknown) q1 0.7 to 0.9 or q1 0.1 to 0.3. We grouped the differenthybrid combinations together due to the low genetic differentiationbetween E. rubida and E. viminalis, however we roughly separatedthe cross-type on the basis of morphology (Field et al. 2008) and oncongener species present at the each particular site. The admixturethresholds for each of the four groups were selected on the basis ofsimulated mating among pure-bred and hybrid individuals (Fieldet al. 2008). Previous modelling work by Boecklen & Howard (1997)would suggest that the assignment of hybrid classes with the numberof markers in our study should be sufficient as a coarse classificationof pure-breds, F1 and backcrossed hybrids. However, hybrid produc-tion is probably underestimated due to the difficulty in distinguishingmore advanced backcrosses from pure-bred individuals if these existwithin E. aggregata populations. Therefore, the classification ofhybrid classes is used here cautiously, especially for the adult popula-tions, as some F1 hybrids and pure-bred individuals may be moreadvanced backcrosses.

Considering these caveats for hybrid classification we used threeapproaches to estimate hybridization rates for populations. First, a‘mean F1 hybrid production’ was calculated as the mean percentage

1202 D. L. Field et al.


of F1 hybrid progeny across pure-bred families within each population.A second estimate was also calculated across pure-bred families, butwith the percentage of F1 and backcrossed hybrids combined. Athird estimate of hybridization rate was calculated by the ‘meanadmixture’ of q1 as an alternative measure that has no thresholds forassignments.

HYBRIDIZATION RATES AND POPULATION CHARACTERISTICS

Univariate nonlinear regressions were used to test the relationshipbetween the two predictor variables, APS (log transformed) and RPSwith the three response variables; (i) mean F1 hybrid production, (ii)mean F1 and backcross hybrid production, and (iii) mean admixture.A variety of nonlinear models were examined to fit the data including,negative exponential, power, Gaussian and Weibull (genstat v 8).A leverage analysis was used to identify outlying data points thatexhibited disproportionate influence on the relationship (genstat v8) and the analyses were run with and without outliers to examinetheir influence on the relationships. This analysis was also conductedseparately for E. aggregata populations that were sympatric withE. rubida and those sympatric with E. viminalis. Populations withboth E. viminalis and E. rubida were classed as sympatric withE. viminalis due to this species’ greater dominance at those sites.Seedlings from maternal parents identified as hybrids or backcrosseswere excluded from the above analyses as we were only interested inhybrid production from pure-bred E. aggregata.

GENETIC DIVERSITY, ALLELE FREQUENCIES AND OUTCROSSING RATES

Genetic diversity measures were estimated for each populationseparately, including the percentage of polymorphic loci (P), meannumber of alleles (A), allelic richness (Ar), unbiased genetic diversity(He) and the inbreeding coefficient ( f ), averaged across loci using theprogram fstat (Goudet 1995). Allelic richness was estimated foreach locus separately with rarefaction to control for variable samplesizes between populations (Goudet 1995), and presented as anaverage across loci. Multi-locus outcrossing rates (tm) for eachpopulation were estimated with maximum-likelihood proceduresimplemented in the program MLTR (Ritland 2002) using 1000bootstraps. In order to examine the influence of population size andhybridization rates on genetic diversity, the APS, RPS and theaverage hybridization rate of each population was compared withthe average Ar and He and tm using nonlinear regressions fitted by apower function with a negative exponent (y = αx–β).

SEED PRODUCTION, GERMINATION, SURVIVAL AND SEEDLING PERFORMANCE

The following counts and measurements were made for seed arraysfrom each E. aggregata tree sampled: (i) seed weight (of 20 randomlyselected seed), (ii) number of seeds (weighed by dividing seed numberby capsule number), (iii) percentage weight of seed compared tototal capsule contents, (iv) total debris weight (excluding seed) as apercentage of capsule weight. Fertile seeds were clearly distinguishableunder low magnification from the remaining capsule contents whichconsisted of sterile particles of chaff and aborted seed. Viable seedwere dark and filled while chaff and aborted seed were pale and flat.A preliminary trial comparing the germination of putatively fertileand aborted seeds, placed in petri dishes on wet filter paper, indicated

that none of the seed identified as aborted were viable whereas> 50% of apparently fertile seed germination.

Starting one week after the 3120 seeds were planted, observationswere made every 3 days for 6 weeks, recording when individuals ger-minated. Eleven months after planting, the number of seedlings thatsurvived was recorded, and two traits were scored as surrogates ofindividual performance/vigour on all surviving seedlings (n = 1028)from E. aggregata mothers. These were plant height, measured to thetip of the apical bud on the main stem, and total number of leaf pairs(expanded and un-expanded).

Separate linear regressions provided the best fit (genstat v 8) todata used to examine the relationship between each of the sevenparameters (APS, RPS, H%, Ar, He, f, tm) and population averages(across families) of each of the seed production characteristics (seedweight, seed number, seed weight percentage, debris weight percentage),germination and survival rates and seedling performance (height,leaf pairs). Seed numbers, weight and germination were also combinedinto a single measurement of the number of viable seeds per 10 g ofcapsule contents (e.g. Burrows 2000). Population RV was removedfrom regression analyses because it exhibited consistently lower seedproduction, the percentage weight of seeds, germination, survival,and seedling height in comparison to the other large populations.This may be due to a past bottleneck or unusually high inbreeding,as this population previously exhibited the lowest outcrossing rates,despite large population size. This experimental design does notexplicitly control for the effects of inbreeding and genetic variationon seed production and early seedling performance parameters, butrather explores the amount of variation explained by each popula-tion and mating system parameter.

Results

HYBRID DETECTION

Of the 1838 seedlings from pure-bred E. aggregata, the majority1752 (89%) had a high affinity to the E. aggregata clusterq1 > 0.9 (Fig. 2a). Implementing the critical thresholds of q1

for individual classifications indicated 89% were pure-bred,4.4% were F1 hybrids, and 6.3% were hybrid backcrosses. Ahigher proportion of seedlings from pure-bred E. rubida andE. viminalis had a high affinity to cluster 2 due to q1 < 0.1(Fig. 2b,c). Applying the classification thresholds to the 361seedlings from E. viminalis indicated 92% were pure-bred,1.6% were F1 hybrids and 6.4% were hybrid backcrosses. Ofthe 343 seedlings from E. rubida 98.6% were pure-bred, zeroF1 hybrids and 1.4% were backcrossed hybrids.

The proportion of hybrid offspring in progeny arrays fromall of the pure-bred E. aggregata trees sampled (n = 124)ranged from 0% to 44.4% and hybrids were present in progenyarrays at 15 of 18 sites (83.3%). The average hybridization rateacross all E. aggregata populations was 4.2% for (F1) hybridsonly and 8.9% for F1 hybrids and backcrosses. Within eachpopulation the percentage of hybrids in progeny arrays forindividual trees varied markedly (e.g. site M, 0% to 5.4%, ST,10% to 44.4%; Table 2). The mean percentage of hybrid progenyper population ranged from zero to 24.4% for F1 hybrids andup to 31% for F1 and backcross hybrids (Table 2). At the foursmallest population that produced hybrids (WL, NG, ST,MW) all trees sampled produced some hybrid offspring



Fig. 2. Ranked admixture proportions (q1) of allozyme genotypes of seedlings from the open-pollinated progeny arrays of pure-bred(a) E. aggregata, (b) E. viminalis and (c) E. rubida. Grey bars indicate the 90% posterior probability intervals. Dashed lines indicate admixturethresholds used to classify individuals into each of four genomic groups including (i) pure-bred E. aggregata, (ii) E. viminalis/E. rubida,(iii) F1 hybrids and (iv) hybrid backcrosses.

Table 2. Details of population size parameters (APS, RPS), sampling size of open-pollinated seedlings (n) and families (n − f ), from 18 E.aggregata populations sampled for assessment of mean hybridization rates. For each population, hybridization rates estimated as the meanadmixture proportion in the E. aggregata cluster (q1) and the mean percentage, standard error (± SE) and range for F1 hybrids only and F1

hybrids and backcrosses combined

Population APS RPS s† n n − f

Mean F1 hybrid % F1 hybrid + backcross %

H-families‡q1 Mean SE Range Mean SE Range

MC 1 0.25 v 24 1 0.99 0.0 (0.00) − 0.0 (0.00) 0 0WL 4 0.31 v 17 2 0.84 12.1 (0.02) 10–14.3 31.0 (0.11) 20–42.9 100NG 6 0.21 r 41 3 0.95 1.4 (0.01) 0–4.2 4.0 (0.04) 2–12.5 100ST 7 0.15 v 72 4 0.85 24.4 (0.08) 10–44.4 30.0 (0.10) 10–55.6 100MW 15 0.75 r 53 3 0.92 6.5 (0.07) 0–13.0 9.0 (0.04) 4.3–13.0 100M 30 0.75 r 83 5 0.94 1.1 (0.01) 0–5.3 7.0 (0.05) 0–26.7 60RS 30 0.33 d 90 10 0.86 10.1 (0.04) 0–25.0 24.0 (0.06) 0–57.1 90BR 35 0.64 r, v 59 5 0.95 1.4 (0.01) 0–5.6 5.0 (0.02) 0–11.1 80DF 42 0.51 r 134 10 0.94 2.8 (0.01) 0–6.7 7.0 (0.02) 0–13.3 90GG 50 0.77 r 67 6 0.94 1.2 (0.01) 0–5.9 5.0 (0.03) 0–13.3 50H 90 0.69 r 143 9 0.96 0.7 (0.01) 0–6.3 5.0 (0.01) 0–11.8 66BS 80 0.67 v 157 11 0.96 1.8 (0.01) 0–6.3 3.0 (0.01) 0–12.5 36RC 100 0.83 r 60 8 0.95 1.3 (0.01) 0–5.0 5.0 (0.05) 0–20.0 50N 150 0.87 r, v 59 5 0.93 0.0 (0.00) 0 0.0 (0.03) 0–16.7 60RV 200 0.95 r 77 5 0.96 0.0 (0.00) 0 0.0 (0.00) 0 0FV 300 0.83 r, v 198 16 0.94 0.8 (0.01) 0–8.3 8.0 (0.02) 0–26.7 56BT 400 0.75 r 255 20 0.95 3.3 (0.01) 0–15.4 6.0 (0.02) 0–30.0 50AP 700 0.90 v, r 249 17 0.91 5.4 (0.01) 0–14.3 12.0 (0.04) 0–53.8 58Mean 0.93 4.2 8.9

APS, absolute population size of E. aggregata; RPS, the relative population size of E. aggregata to congeners (see Methods).†Sympatric species at population, r = Eucalyptus rubida; v = E. viminalis; d = E. dalrympleana.‡Percentage of sampled families with hybrid progeny (F1 hybrid + backcross).



(100% H-families; Table 2). In contrast, for the largestpopulations that produced hybrids, fewer of the sampled treesproduced hybrid offspring (e.g. 50% population BT, 58%population AP; Table 2).

For adult plants putatively assigned as E. aggregata (140trees from 18 populations), classification based on theadmixture proportions resulted in 129 (92.1%) assigned aspure-bred E. aggregata, 8 adults as F1 hybrids (5.7%), and 3(2.1%) as hybrid backcrosses. Within reference populationsof the congeners, all 25 E. rubida trees were classified aspure-bred, and of the 25 E. viminalis trees, 1 (4%) was classifiedas a backcrossed hybrid.

POPULATION SIZES AND HYBRIDIZATION RATES

Nonlinear regressions of all E. aggregata sites showed that themean (F1) hybridization rate was moderately and negativelycorrelated with absolute population size (APS) (R2 = 0.35,P = 0.007; Fig. 3a) and strongly negatively correlated withrelative population size (RPS) (R2 = 0.59, P < 0.001; Fig. 3b),with the latter best modelled with a power function witha negative exponent (y = 1089.89x–1.47). When limiting theanalysis to E. aggregata sites sympatric with E. viminalis,there remained a strong and significant negative correlationbetween hybridization rates and RPS (R2 = 0.88, P = 0.004;

Fig. 3c) which was best modelled with a power function witha negative exponent (y = 730.80x–1.26). However, for thesesame sites we did not detect a significant correlation with APS(P = 0.09). Nonlinear regressions of all E. aggregata sitessympatric with E. rubida showed that the mean hybridizationrate was not significantly correlated with APS (P > 0.05) orRPS (P > 0.05; Fig. 3d). Using the mean admixture as analternative measure of hybridization rates revealed similartrends, with mean admixture negatively correlated with APS(R2 = 0.27, P = 0.02) and RPS (R2 = 0.41, P < 0.003).Regressions of absolute and relative population sizes ofE. aggregata combining both F1 and backcrossed hybrids,and run separately for sites sympatric with E. viminalis andE. rubida showed similar trends to the above analysis for allcomparisons (data not shown).

GENETIC DIVERSITY AND OUTCROSSING RATES

A number of genetic diversity measures displayed trends withpopulation parameters and hybridization rates. The meannumber of alleles (A) for E. aggregata ranged from 1.16 in asmall remnant population consisting of a single tree to 3.8 inone of the largest populations (Table 3). Adjusting fordifference in sample sizes, the allelic richness (Ar) displayedno relationship with APS (P > 0.05), but there was a linearrelationship with average hybridization rate (R2 = 0.29,

Fig. 3. Relationship between the average percentage of F1 hybridproduction within open-pollinated seed arrays from 17 E. aggregatapopulations, against the absolute population size of E. aggregataadults (APS) and the relative population size of E. aggregata adults toits congeners (RPS). Separate nonlinear regressions were used for; (a)APS for all populations, (b) RPS for all populations, (c) RPS forpopulations sympatric with E. viminalis, and (d) RPS for populationssympatric with E. rubida. Populations sympatric with E. viminalis areindicated with filled circles (�), E. rubida with open circles (�), andE. dalrympleana with open squares (�). Bars indicate standard errorsof the mean at each population. n.s. P > 0.05 (not significant);*P < 0.05.

Table 3. Adult population size parameters (APS, RPS), geneticdiversity estimates (P, A, f, Ar, He) and outcrossing rates (tm)estimated from six allozyme loci of progeny arrays collected fromEucalyptus aggregata populations (n = 18)

Population APS RPS P A Ar He f tm SE

MC 1 0.25 16 1.16 1.17 0.08 −0.80 − −WL 4 0.31 83 2.10 2.09 0.36 −0.16 1.02 0.09NG 6 0.21 83 3.00 2.45 0.33 0.01 1.05 0.06ST 7 0.15 100 3.50 2.67 0.47 0.01 0.94 0.17MW 15 0.75 100 2.70 2.11 0.34 0.07 0.99 0.15RS 30 0.75 100 3.80 2.08 0.29 0.23 0.71 0.13M 30 0.33 83 2.60 2.65 0.37 −0.04 0.89 0.20BR 35 0.64 83 2.50 1.96 0.28 0.05 1.11 0.14DF 42 0.51 100 3.60 2.19 0.28 0.09 0.98 0.08GG 50 0.77 83 2.50 2.02 0.29 0.16 0.80 0.20H 90 0.69 83 2.50 2.07 0.28 0.04 0.62 0.21BS 80 0.67 83 2.60 1.85 0.23 0.16 0.85 0.08RC 100 0.83 83 2.30 1.87 0.26 −0.06 0.98 0.07N 150 0.87 83 2.66 2.29 0.39 0.08 1.18 0.14RV 200 0.95 66 2.33 1.76 0.21 0.09 0.59 0.28FV 300 0.83 100 3.30 2.11 0.28 0.19 0.71 0.13BT 400 0.75 100 3.80 2.12 0.26 0.05 0.83 0.15AP 700 0.90 100 3.50 2.02 0.28 0.04 0.82 0.08Mean 89.00 2.90 2.14 0.31 0.06 0.89

APS, absolute population size of E. aggregata adults; RPS, the relative population size of E. aggregata to congeners.Genetic diversity estimates, P = number of polymorphic loci; A = mean number of alleles per locus; Ar = mean allelic richness; He = unbiased expected heterozygosity (gene diversity); f = inbreeding coefficient; tm = multi-locus outcrossing rate; SE = standard error of multi-locus outcrossing rate.



P = 0.01) and a negative power relationship with RPS (R2 = 0.52,P < 0.01; y = 2.66x–0.0035). Similarly, genetic diversity (He)displayed a significant linear relationship with averagehybridization rate (R2 = 0.42, P = 0.01) and a negative powerrelationship with RPS (R2 = 0.42, P < 0.01; y = 0.42x–0.054),and there were no relationships with APS (P > 0.05).

The inbreeding coefficient ( f ) ranged from −0.16 to 0.23,with linear regressions indicating no relationship with eitherAPS, RPS or hybridization rates (P > 0.05). The majority ofE. aggregata populations were highly outcrossed with anaverage level of multi-locus outcrossing (tm) for E. aggregataof 0.91, with populations ranging from 0.59 to 1.2 (Table 3).However, no significant relationships were detected betweenoutcrossing rate and either APS or RPS (P > 0.05).

SEED PRODUCTION

Among populations of E. aggregata, there was a substantialvariation in several seed production parameters, with theaverage seed weight ranging from 4.3 to 7.2 mg and thenumber of seeds ranged from 0.8 to 2.8 seeds per capsule.There was evidence of substantial seed abortion as seed com-prised less than half the capsule contents by weight (16.9 to38.4%), with the remaining contents consisting of chaff andlikely aborted seed. Among seven population variables, onlyrelative population size was significantly correlated with seedproduction (Table 4). On average, populations with higherrelative population sizes (E. aggregata numerically dominant)tended to produce more seed (R2 = 0.29, P = 0.03; Fig. 4a)and seed comprised a greater percentage of the total weight ofcapsule contents (R2 = 0.30, P = 0.03; Fig. 4b). However,there were no significant linear relationships between anypopulation variables and either seed weight or debris weightpercentage (P > 0.05; Table 4). There was an almost significantnegative relationship between the inbreeding coefficientand seed weight percentage (R2 = 0.29, P = 0.06), howeverthere were no significant relationships between any seed

production variables and outcrossing rates (tm; P > 0.05), allelicrichness (Ar; P > 0.05), or genetic diversity (He; P > 0.05).

GERMINATION, SURVIVORSHIP AND SEEDLING PERFORMANCE

Seed germination, mortality and the number of viable seed(per 10 g) varied substantially between E. aggregata populations.Germination ranged from 41% at NG to 80% in populationM, survivorship from 51% at RS to 94% at MW, and thenumber of viable seed per 10 g of capsule contents rangedfrom a population mean of 11 660 ± 8246 at WL to 4520 ± 3040at RS. Among the seven population variables, only relativepopulation size was significantly correlated with seed germi-nation and seedling survival (Table 4). There were significantand positive linear relationships between RPS and germinationrates (R2 = 0.27, P = 0.02; Fig. 5a), seedling survival (R2 = 0.33,P = 0.02; Fig. 5b), and the number of viable seeds per 10 g(R2 = 0.28, P = 0.02; data not shown).

Table 4. Summary of separate linear regressions between population components and measures of seed production† and seedling performance.Seed production and seedling performance calculated from population averages (across n = 2 to 20 families) at each of 14 E. aggregatapopulations

Population component

Number of seeds

Seed weight %

Germination % Survival %

Plant height Leaf pairs

Slope R2 Slope R2 Slope R2 Slope R2 Slope R2 Slope R2

APS n.s. n.s. n.s. − n.s. n.s. n.s.RPS + 0.29* + 0.30* + 0.27* + 0.33** n.s. n.s.H % n.s. n.s. − 0.18(*) − 0.16(*) n.s. + 0.27*Ar n.s. n.s. n.s. n.s. n.s. + 0.20(*)He n.s. n.s. n.s. n.s. n.s. + 0.22*f n.s. − 0.22(*) n.s. n.s. − 0.29* n.s.tm n.s. n.s. n.s. n.s. n.s. n.s.

n.s. P > 0.1 (not significant), (*) P < 0.1, *P < 0.05, **P < 0.01.The direction of the slope (+/−) is given for pairs with significant terms. APS = absolute population size of adult E. aggregata; RPS = relative population size of E. aggregata compared to its congeners; H % = average percentage of F1 hybrid production in progeny arrays; (Ar) mean allelic richness; (He) unbiased expected heterozygosity (genetic diversity); f = inbreeding coefficient; tm = multi-locus outcrossing rate.†Additional measures of seed weight and seed debri % were not significantly related to any population component.

Fig. 4. Relationship between the relative population size of E.aggregata to its congeners (RPS) and the average seed production ofseed arrays (n = 13 populations). Linear regressions were fittedbetween RPS and; (a) the average number of seeds, (b) the averageweight of seed as a percentage of the weight of total capsule contents.*P < 0.05.



There was substantial variation in average measures ofseedling performance amongst populations. Seedling heightranged from 419 mm (RV) to 578 mm (RC), and the numberof leaf pairs ranged from 35.0 (FV) to 67.3 (ST). Only theinbreeding coefficient ( f ) was significantly related to plantheight (Table 4), because populations with more heterozy-gotes tended to have taller seedlings (R2 = 0.27, P = 0.02).Populations with higher hybridization rates (H%) and genediversity (He), tended to have more leaf pairs (H%, R2 = 0.27,P = 0.03; He, R

2 = 0.22, P = 0.05).

Discussion

Reductions in absolute and relative population size of plantspecies within an area can have implications for hybridizationrates and the risk of local extinction of many rare species. Ourresults indicated a consistent negative relationship betweenrelative population size and hybridization rates, and associ-ated declines in seed production, germination and seedlingsurvival when E. aggregata was less numerous than its con-geners. As a consequence of increased hybridization, bothdemographic and genetic swamping is likely to be occurringin E. aggregata sites with low relative population sizes (i.e. RS,WL, ST).

Some Eucalyptus species have a high rate of hybrid production(E. aggregata here 4.2–8.9%) compared to a number of othergenera (e.g. Iris (< 1%), Phlox (< 1%), Senecio (< 1%), andBrassica (1.5%)) (Rieseberg & Carney 1998), which may sug-gest weaker reproductive barriers exist in this genus. In com-parison to other Eucalyptus species, the average percentage ofhybridization for E. aggregata is similar to those described inE. argutifolia (6%; Kennington & James 1997). However, theyare greater than the rates in natural populations reported in areview of 13 Eucalyptus species, where F1 hybrid productionranged from 0.03% to 3.5% (Potts et al. 2003). Furthermore,the two largest E. aggregata populations exhibited hybridiza-tion rates (AP 5.4%; BT 3.3%) higher than those reported inthe largest population of E. argutifolia which similarly usedallozymes to identify hybrids (< 0.1%; Kennington & James1997). This could reflect weaker pre-mating barriers to

hybridization in E. aggregata or that the sampled populationsexhibited lower relative population sizes compared to Euca-lyptus populations sampled in earlier studies.

HYBRIDIZATION AND RELATIVE POPULATION SIZE

Our results indicate that reduction in population size mayfacilitate hybridization in the uncommon E. aggregata.Relative population size was strongly correlated with rates ofhybrid seed production for E. aggregata populations whenexamining all sites, and for those sites sympatric with E.viminalis. Importantly, these relationships were consistent foralternative methods of estimating hybridization rates with theinclusion of backcrossed hybrids with F1 hybrids or by usingmean admixture rate. For all tests, relative population sizeproved to be more informative than the absolute populationsize as a predictor of hybridization rates, which likely reflectsthe importance of relative population size as a measure of thepotential ratio of interspecific to intraspecific pollen. As such,pollen swamping is likely occurring within the sites with lowrelative population sizes (RS, WL, ST), because (i) the potentialsources of interspecific pollen (E. viminalis, E. dalrympleana,E. rubida) outnumber the sources of intraspecific pollen forE. aggregata trees, and (ii) the dominant insect foraging atthe flowers of these species was the generalist Honeybee (Apismellifera). The occurrence of pollen swamping within thesmall hybrid producing sites is further evident with all of thetrees sampled at the small sites (WL, NG, ST, MW) producinghybrid offspring, in contrast to the largest populations (FV,BT, AP) where 50% to 58% of the trees at these sites producedhybrids.

The increase in hybridization rates as relative populationsize declines follows theoretical model predictions (Pedersonet al. 1969) and is in line with empirical expectations from twosource-sink experimental arrays (Klinger et al. 1992; Richardset al. 1999). While few natural population studies have explicitlymeasured relative population size, similar patterns have beenobserved in Nothofagus, with higher hybridization rates forisolated trees surrounded by compatible species (hybrid rate;80%) compared to trees within dense conspecific stands (0%;Gallo et al. 1997; Marchelli & Gallo 2001). In an allozymestudy of E. argutifolia, the small populations exhibited thehighest hybridization rates in progeny arrays (47%; Kennington& James 1997). Similarly, estimates of hybrid rates withmorphological identification have reported increased hybrid-ization in progeny arrays from small populations comparedto large populations for E. agygdalina (small 14% vs. large 3%)and E. morrisbyi (small 17% vs. large 1.6%) (Potts & Wiltshire1997).

Considering the similar flower size and structure of each ofthe Eucalyptus species studied here, pollinator behaviour isunlikely to be a strong pre-mating barrier to interspecificpollen flow. Behavioural studies have found Honeybeesforage as generalists (Goulson 2003), exhibiting low fidelity inmixed species arrays, especially when co-occurring plantspecies have similar flower size and structure (Kunin 1993). Inother Eucalyptus species, evidence of past hybridization has

Fig. 5. Relationship between the relative population size of E.aggregata to its congeners (RPS) and germination and survivorshipof seed arrays (n = 14 populations). Linear regressions were fittedbetween RPS and; (a) percentage of seeds germinated, (b) thepercentage of seedlings survived to 11 months of age. *P < 0.05.



been identified from extensive chloroplast sharing acrossgeographic regions (McKinnon et al. 2001). In our study, thepresence of large hybrid adults and likely advanced backcrossingsuggests that hybridization probably predates the arrival ofthe introduced Honeybee to Australia in the early 19thCentury (Goulson 2003). Interestingly, some studies havereported less interplant movements by Honeybees comparedto other bee species (Goulson 2003), which suggests that theintroduction of Honeybees could alter patterns of interspecificpollen flow. Therefore, it would be interesting to examine theforaging behaviour of insect visitors to these Eucalyptusspecies to ascertain the importance of Honeybees and nativebees for current interspecific pollen dispersal patterns inremnant populations.

Correlations were detected between relative populationsize and hybrid production separately for sites sympatric withE. viminalis but not for sites with E. rubida. This could be duefine-scale effects of local plant density and plant size onpollinator behaviour and plant mating patterns (Thomson1981; Field et al. 2005), a lack of E. rubida sympatric siteswith lower relative population sizes (RPS < 0.5), or strongerpre-mating barriers to hybridization with E. rubida comparedto E. viminalis. The latter could be confirmed with experimentalcrosses, and if pre-mating barriers were stronger with E. rubidathen optimal conservation strategies would depend on whichspecies were present within a given area.

GENETIC DIVERSITY AND OUTCROSSING RATES

Absolute population size was not a good predictor of geneticdiversity in seed cohorts, suggesting that small E. aggregataremnants may not have undergone reductions in geneticdiversity following habitat fragmentation. However, thepositive correlation between hybrid production and two geneticdiversity measures (Ar and He) indicates that hybridizationcould be playing a role in maintaining genetic diversitythrough the introduction of new genetic material from sym-patric species. Considering the parallel negative correlationsdetected between relative population size and both geneticdiversity and hybrid production, elevated hybrid productionand introgression could be masking declines in genetic diversityin small remnants. Nevertheless, a lack of correlation betweenmeasures of population size and genetic diversity has beenfound in the majority of Eucalyptus studies (Potts & Wiltshire1997). In contrast to the traditional model of reduced geneticdiversity in small populations, Potts & Jackson (1986) arguethat in Eucalyptus, hybridization may play a role in maintaininghigh diversity in remnant populations. This has been suggestedas an explanation for high genetic diversity in small populationsof rare species such E. rameliana (Sampson et al. 1995) andE. benthamii (Butcher et al. 2005). While our study was basedon a limited number of loci (six), our findings suggest hybrid-ization can play an important role in remnant populations bynot only maintaining genetic diversity, but through increasingdiversity above levels observed in large populations.

Outcrossing rates in E. aggregata were moderate to highacross populations and are similar to rates described with

allozyme markers across a range of Eucalyptus species (Potts& Wiltshire 1997). Many of the populations with slightlydepressed outcrossing rates (e.g. M, H, FV, RV) also exhibiteddeficits in heterozygotes ( f > 0) indicating a departure frompanmictic mating and some level of inbreeding in the seedlingcohort. In contrast, the populations with the highest hybridrates exhibited random mating or a slight excess of heterozy-gotes (ST, WL, RS) that would be expected from the influx ofalleles from E. viminalis, E. rubida and E. dalrympleana. The lackof relationship between population size and either inbreeding( f ) or outcrossing rates (tm), suggests that even small popu-lations are able to avoid potentially deleterious inbreedingand maintain the output of high numbers of outcrossed prog-eny. While relationships between outcrossing rates and popula-tion size have been reported previously in Eucalyptus(Sampson et al. 1989; Hardner et al. 1996), as with our study,others have also found no relationship (Kennington & James1997; Butcher et al. 2005). The lack of relationship may bedue to variation in the frequency of male sterile individuals in apopulation (Ellis & Sedgley 1992b), or abortion of increasednumbers of selfed seeds in smaller populations (Kennington& James 1997). The maintenance of substantial numbers ofoutcrossed progeny is important for the viability of remnantpopulations as the risk of genetic swamping may be lesseneddue to hybrid offspring competing with potentially supe-rior outbred seedlings (Lopez et al. 2000).

SEED PRODUCTION

Among an array of population parameters, relative populationsize of E. aggregata compared to its congeners had the strongestinfluence on seed production, with fewer seed set as relativepopulation size declined. The strong negative correlationbetween hybridization rates and relative population size,suggests that elevated interspecific pollen flow onto E. aggregatatrees may be responsible for depressed seed production. Whilethese species are clearly cross-compatible and the identificationof fertile hybrid adults at these sites indicates some proportionare viable, the declines observed in our study could be due tohybrid breakdown. Previous work in Eucalyptus indicatesthat in some cases, the success of interspecific pollinations canbe lower than intraspecific crosses (Griffin et al. 1988; Elliset al. 1991). Declines in hybrid seed production are oftenattributed to pre-zygotic barriers to interspecific pollen in theform of structural and physiological barriers in the style thatreduce the frequency of pollen tubes penetrating the ovule(Gore et al. 1990; Ellis et al. 1991). The role of hybrid breakdownin seed production in E. aggregata could be confirmed bycomparing the success of controlled crosses between E. aggregataand its congeners with outcrossed pollinations betweenE. aggregata individuals. If a higher number of hybrid polli-nations fail compared to outcrossed pollinations, this wouldsuggest that increased interspecific pollen flow followed byearly hybrid breakdown is responsible for declines in seedproduction as the relative population size decreases.

The lack of significant relationships between seed productionand levels of inbreeding, genetic diversity and absolute



population size are surprising since such relationships havebeen inferred in a number of systems (Oostermeijer et al.1994; Kunin 1997; Kery et al. 2000) including Eucalyptus(Burrows 2000, but see; Butcher et al. 2005). Declines in seedset with population size are often attributed to reductions inpollen quality (selfing) or pollen quantity due to overallreductions in the number of pollinators attracted to smallerpopulations. While these factors may play a role in our study,the high outcrossing rates within all populations except thesingle isolated tree (population MC) suggests pollinatorlimitation is unlikely a cause at these populations. Alter-natively, declines in seed set could be due to increased levels ofself pollen, but the production of substantial seed set throughselfing with single isolated trees (population MC) suggeststhat E. aggregata is to some extent self-compatible. However,variation in self-compatibility among individual trees hasbeen reported in Eucalyptus (Ellis & Sedgley 1992a) suggestingthat experimental pollinations may be required to determineif pollen quality is contributing to depressed seed productionfor small E. aggregata populations. In our case, it could bethat declines in seed set due to increased interspecific hybrid-ization and potential hybrid breakdown may be more severethan the effects of pollen quantity and quality on seed production.

GERMINATION, SEEDLING SURVIVAL AND PERFORMANCE

Germination and survivorship showed a similar negativecorrelation with relative population size which paralleled therelationship with seed production. Reduced germinationrates and survivorship in populations of low relative sizesuggests that these trees are producing a greater number ofinviable seeds and the remaining seedlings have poor vigour.Populations of E. aggregata out-numbered by congenerstended to have less than 5000 viable seeds per 10 g (exceptionWL) compared to over 8000 for most of the populations whenE. aggregata was dominant. Similar declines in viability havebeen detected for several other Eucalyptus species, for example,E. melliodora trees exhibited averages of 7162 viable seed (per10 g) for in large woodland populations which declined to3192 for isolated trees (Burrows 2000). Such declines in seedviability in Eucalyptus and other genera have been attributedto increased geitonogamous pollinator movements (Kunin1997) and subsequent depressed seedling growth and survivaldue to inbreeding depression (Borralho & Potts 1996;Hardner et al. 1996).

Declines in germination and survivorship for remnantpopulations of E. aggregata could be due to a number of factorsincluding post-zygotic selection against increased numbers ofhybrid individuals (Lopez et al. 2000), or increased deleteriousinbreeding (Borralho & Potts 1996). Considering relativepopulation size was the most consistent parameter related togermination and seedling survival, this indicates that increasesin the number of inviable hybrids could have an importantrole in reducing seedling viability and performance. Hybridbreakdown can be caused by a range of mechanisms includingthe deleterious and complementary action of genes (Orr

1995), cytoplasmic epistasis (Tiffin et al. 2001), and in latergenerations the disruption of co-adapted gene complexes(Rieseberg & Carney 1998). In Eucalyptus, reports havedescribed both slower germination and poorer survival ofhybrid individuals compared with out-bred individuals(Lopez et al. 2000). However there are also cases of hybridgermination rates as successful as those of intraspecific seed,at least in the first generation (Tibbits 1988; Ellis et al. 1991;Potts et al. 1992). In E. aggregata, the almost significantnegative correlation between hybrid production and seedlinggermination and survival suggests hybrid breakdown couldbe an important factor, and could be confirmed with experi-mental fitness trials of controlled crosses.

There were two trends in seedling performance: a negativecorrelation between height and inbreeding, and a positivecorrelation between leaf pairs, hybridization rates and geneticdiversity. The first trend is likely due to the effects of inbreedingdepression on plant growth, as a number of studies inEucalyptus have similarly reported declines in the fitness ofopen-pollinated progeny as outcrossing rates declined(Borralho & Potts 1996; Hardner et al. 1996), and there aredirect reports of depressed growth of inbred compared tooutbred progeny (Griffin & Cotterill 1988; Hardner & Potts1995; Lopez et al. 2000). The positive correlation between thenumber of leaf pairs and both hybridization rate and geneticdiversity is probably due to the effects of hybridizationand introgression from E. viminalis, which has significantlygreater numbers of leaf pairs than E. aggregata (Field et al.2008). This has important implications for the viability ofremnant populations and implies that E. aggregata populationsof small relative size have been subject to introgression ofE. viminalis traits.

IMPLICATIONS FOR CONSERVATION

These results demonstrate elevated levels of hybrid productionin fragmented populations, and that for some species pairs,the relative frequencies of the parental species can be a strongpredictor of hybridization rates. The negative correlationbetween relative population size and hybrid production, thesubstantial numbers of backcrossed hybrids and declines infecundity suggest that hybridization could represent a significantthreat to population viability through both demographicand genetic swamping. Despite the potential deleteriouseffects of demographic and genetic swamping, the potentialadaptive benefits of hybridization should not be ignored.The observed increases in genetic diversity in remnantpopulations through hybridization have the potential torapidly provide new multi-locus genotypes that may berequired by remnant populations to survive and re-establishin changing environments.

Our study suggests that the maintenance of pure-bred insitu populations or collection of mostly pure-bred ex situ seedfor reforestation should focus on preservation and samplingfrom higher relative population sizes > 0.5 (i.e. at least equalfrequencies of parentals). If seed are required to be collectedfrom sites with lower relative population sizes, our results



suggest twice as many capsules may need to be collected tooffset the losses due to depressed seed production, seedlinggermination and survival.

Acknowledgements

Authors thank Linda Broadhurst, Tony Brown, Leanne Cox, Robert Godfree,Liz Gregory, and Melinda Pickup for assistance. This work was conductedwhile (D.L.F.) was receiving a University of Wollongong Post-graduateResearch Award and ‘top-up’ scholarship from CSIRO Plant Industry.

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Received 30 January 2008; accepted 30 July 2008Handling Editor: John Pannell

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