Increased Inbreeding and Inter-Species Gene Flow in Remnant

Conservation Genetics (2005) 6:213–226 Springer 2005 DOI 10.1007/s10592-004-7830-x

Increased inbreeding and inter-species gene ﬂow in remnant populations of the rare Eucalyptus benthamii

P.A. Butcher*, , A.K. Skinner & C.A. Gardiner CSIRO Forestry and Forest Products, GPO Box E4008, Kingston ACT 2602, Australia (*Corresponding author: Phone: +61 8 9480 3648; Fax: +61 8 9480 3641; E-mail: [email protected]; Current address: Kings Park and Botanic Garden, Fraser Avenue, West Perth WA 6005, Australia)

Received 11 May 2004; accepted 1 August 2004

Key words: endangered species, Eucalyptus benthamii, genetic diversity, habitat fragmentation, hybridisation, mating system, microsatellites

Abstract Eucalyptus benthamii Maiden & Cambage is a forest tree of interest for conservation and plantation forestry. It is vulnerable to extinction, occurring on the alluvial floodplains of the Nepean River and its tributaries, south-west of Sydney, Australia. These floodplains were largely cleared of native vegetation for agriculture by the mid-1800s. Flooding of the Cox Valley for Sydney’s water supply further decreased the species distribution. The species is now confined to one population of approximately 6500 trees in the Kedumba valley and three remnant populations on the Nepean River at Bents Basin (about 300 trees), Wallacia (nine trees) and Camden (about 30 trees). Genetic analysis of the four populations using microsatellite markers revealed significant divergence among all populations, despite the Bents Basin, Wallacia and Camden remnants being separated by distances of only a few kilometres. Trees in these populations have been estimated to range from 35 to 200 years old, suggesting genetic divergence among populations occurred prior to land clearing. To investigate the impact of fragmentation on the next generation, outcrossing rates were estimated from 41 families. While no direct relationship was found between population size and outcrossing rates, fragmentation and the isolation of trees appears to have resulted in higher levels of selfing and biparental inbreeding in seed collected from the Camden and Wallacia remnants. There was also evidence from seedling morphology that inter-species gene flow increased with fragmentation since 20% of the progeny from Camden and 30% of the progeny from Wallacia were hybrids. Seed viability and germination rates were significantly lower in the remnant populations, reducing their value as seed sources for regeneration and plantation forestry. To maintain the genetic integrity of the remnant populations, germplasm should be sourced from the local area. Outcrossed, non- hybrid seed could be produced by controlled pollination in ex-situ conservation stands or by using seedling morphology and microsatellites to screen seedlings from the remnant populations.

Introduction population (Prober and Brown 1994). Long-term impacts of fragmentation can occur if increased Habitat fragmentation can have immediate and isolation alters patterns of gene flow, either by long-term effects on the abundance and diversity increasing gene flow within and among popula- of eucalypt ecosystems. The immediate effects re- tions (reviewed in Young et al. 1996), or reducing late to reductions in population size and declines gene flow in insect-pollinated species in genetic diversity, usually attributed to loss of (Cunningham 2000). Reduced gene flow may lead rare alleles as individuals are lost from the to increased inbreeding in eucalypts, most of which 214 have a mixed mating system. Adverse effects of inbreeding on seed production, viability, germination and growth have been reported in several eucalypts (Hodgson 1976; Griffin and Cotterill 1988; Hardner and Potts 1995) and this may, in turn, threaten the long-term viability of small, fragmented populations. Camden white gum (Eucalyptus benthamii Maiden and Cambage) is a long-lived, tall forest tree with a restricted distribution that is considered vulnerable to extinction (NSW National Parks and Wildlife Service 2003). The species is confined to the alluvial banks of the Nepean River and its tributaries, south-west of Sydney, Australia. These fertile riverflats, favoured for agricultural development, were mostly cleared by the 1840s (Benson et al. 1996). The extent and size of populations of E. benthamii was further reduced following con- struction of Warragamba Dam for Sydney’s water supply and the associated flooding of the Cox Valley. Reductions in the species distribution, to- Figure 1. Geographical distribution of Eucalyptus benthamii. gether with interest in the species for plantation Filled circles show sampling sites; hatched areas indicate the forestry (Swain 2001) have focused attention on species distribution prior to land clearing (after Benson 1985). the need to conserve what remains of the species genetic resources (NSW National Parks and range of eucalypts, including E. deanei, E. crebra Wildlife Service 2003). F. Muell., E. punctata DC., E squamosa Dean & Eucalyptus benthamii is currently known from Maiden and E. acmenoides Schauer; exotic species two main sites; about 6550 individuals in the Ke- are uncommon. dumba Valley, Blue Mountains National Park and The major threat to E. benthamii is poor about 300 individuals at Bents Basin, part of regeneration due to competition with introduced which is protected in a State Recreation Area species and altered flooding and fire regimes. (NSW National Parks and Wildlife Service 2003). Additional threats include the impact of habitat In addition, approximately 30 scattered individu- fragmentation on genetic diversity; both direct ef- als occur along the Nepean River, near Camden fects due to the loss of alleles as the number of and nine trees downstream of Bents Basin near individuals in a population declines, and indirect Wallacia. The remnant populations near Camden effects due to reduced gene flow within and among and Wallacia consist of small groups of up to five populations leading to increased inbreeding. Most trees and isolated trees. Trees in the Camden eucalypts are insect-pollinated with a mixed mating population are dispersed along the banks of the system where inbreeding can lead to significant Nepean River over a distance of about 5 km, declines in growth and survival (see Butcher and adjacent to cleared agricultural land. The Bents Williams 2001 for review). Declines in diversity may Basin stand is a relatively dense, pure stand with also limit a species ability to respond to changing the understorey dominated by introduced weed selection pressures (Frankel et al. 1995), thereby species. It is separated from the remnant popula- limiting its long-term survival. Inter-species gene tions at Camden and Wallacia by about 5 km flow poses an additional threat to E. benthamii, (Figure 1). Many of the trees in the remnant particularly hybridisation with E. viminalis which populations are in poor health, and standing dead occurs naturally in the Camden region and readily trees are common. Other eucalypts occurring in hybridises with eucalypts that are in the same tax- the remnant populations include E. elata Dehnh., onomic section of the genus (Griffin et al. 1988). E. deanei Maiden and E. viminalis Labill. The In this study the level of genetic diversity in Kedumba population includes a more diverse Kedumba and the three remnant populations was 215 compared using microsatellite markers. Genetic numbers in the Camden and Wallacia remnants divergence among the populations was examined were limited by the number of trees with seed to determine whether the three remnants form crops. In Kedumba, sampled trees were separated distinct genetic entities. As the time since forest by at least 100 m to minimise sampling of related fragmentation is less than the lifespan of individuals. It was not possible to follow this E. benthamii trees, analysis of the adult trees is strategy in the remnant populations due to the likely to reveal more about patterns of diversity limited number of individuals with seed crops. before clearing. The impacts of fragmentation may Capsules from each tree were stored separately be more evident in subsequent generations if iso- in calico bags, dried at 35 C for 2 days and seed lation of trees results in increased levels of extracted using a 600-lm sieve to separate seeds inbreeding with adverse effects on seed viability from chaff. The following measurements were and growth. Seed set and viability were therefore made on each capsule following methods described compared among populations. Outcrossing rates by Burrows (2000); weight of capsule contents were estimated from progeny arrays to provide an (seeds and chaff), number of seeds per capsule, indication of the possible consequences of frag- number of seeds per 10 g capsule contents and mentation on following generations. seed weight per capsule. To estimate seed viability, one to four replications of 50 seeds from each tree were sown into moist vermiculite in Petri dishes Methods and placed in growth cabinets at 25 C for 2 weeks. The number of germinants per dish was Population sampling recorded after 2 weeks. Twelve seedlings from each of 41 trees were grown on and leaves sampled Mature leaves were collected from 16 trees in for the mating system analysis. Seedlings with leaf Camden, 17 trees in Bents Basin, nine trees at morphology typical of E. viminalis were recorded Wallacia and 32 trees in the Kedumba Valley as putative hybrids. Eucalyptus benthamii seedling population in December 2001. All trees in the leaves are ovate and pruinose (blue-green) and Wallacia remnant population were sampled. These differ from those of E. viminalis which are lance- were located in urban parkland between the river olate and green (Brooker and Kleinig 1999). and the township. The number of trees that could be sampled in the Camden population was limited by height of the crowns and concerns that sam- Microsatellite analysis pling using firearms or climbing would damage the trees which were located in an urban area. Twelve DNA was extracted from adult leaves using a trees in the Camden population were inaccessible. modified CTAB procedure, described by Glaubitz The location of populations and species distribu- et al. (2001). DNA from a subsample of eight tion are shown in Figure 1. Leaves were also col- E. benthamii individuals was screened with 66 pairs lected from 20 randomly selected individuals in a of microsatellite primers that had been developed in seed orchard to allow comparison of the level of E. nitens (Byrne et al. 1996), E. grandis (Brondani genetic diversity in the orchard with that in the et al. 1998), E. sieberi (Glaubitz et al. 2001) and E. natural populations. The seed orchard was estab- globulus (http://www.ffp.csiro.au/tigr/molecular/eu- lished at Yarralumla, ACT, in 1998, using seed cmsps.html). Twenty-two primer pairs were selected from the Camden and Bents Basin populations. which gave clear amplification products at a single locus (Table 1). Nineteen of the 22 loci had been Seed sampling and testing mapped in either E. globulus (Thamarus et al. 2002) or E. grandis · E. urophylla pedigrees (Brondani Up to 100 mature, unopened capsules per tree et al. 1998) and had shown Mendelian inheritance. were collected from nine trees in the Camden Following screening of adult trees, seven of the population, 11 trees in Bents Basin, five trees in more variable loci were selected to estimate out- Wallacia and 16 trees in the Kedumba Valley crossing rates in progeny arrays from 41 families. population. These were a subset of the trees DNA was extracted from leaves of 2–3-month-old sampled to assess genetic diversity. Sampling seedlings using a FastDNA kit (BIO 101). 216

Table 1. Repeat motifs, PCR conditions used for ampliﬁcation, allelic diversity and size range of alleles at the microsatellite loci scored in Eucalyptus benthamii. Linkage group refers to the chromosome on which each locus has been mapped. Loci in bold were used to assess outcrossing rates

Locus Repeat motif PCR conditions Linkage No. alleles Range of sizes (bp) groupb a TA (C) MgCl2 (mM)

Es076 (TC)19(AC)4ATA(CA)15 55 1.5 1 18 145–183

Es140 (GT)20(GA)8 55 1.5 5 10 114–151

Es157 (CT)16 55 1.5 6 12 100–126 c Eg18 (CTT)19 55 1.5 nm 26 169–268

Eg22 (GAA)9 55 1.0 nm 4 333–359

Eg26 (CTT)10 55 2.0 nm 5 257–272

Eg61 GAA9(GAT)9 55 1.5 2 13 308–326

Eg67 CTT8(CT)7(TC)5 55 1.5 5 18 159–198

Eg84 (CT)7(CTT)7 55 1.5 7 14 110–139

Eg86 (CTT)29 55 1.5 4 18 202–313

Eg91 (GAA)6 55 1.5 10 12 128–152

Eg96 (GAG)8 & (GAA)5 55 1.5 4 6 274–289

Eg99 (CTT)11 55 1.5 3 9 181–204

Eg126 (GAA)8 55 1.5 1 12 329–380

Eg128 (GAA)19 55 1.5 11 6 176–193

Eg134 (TC)10 & (CTT)10 55 1.5 7 15 259–306

En6 (GA)15 55 2.0 7 9 74–98

En16 (CAGA)3(GA)15 60 2.0 6 18 85–136

Embra4 (AG)23 55 1.75 E4 21 64–127

Embra6 (AG)19 55 1.5 7; E1 22 119–181

Embra10 (CCT)3(AG)14 55 1.75 E10 16 113–163

Embra11 (AG)4GG(AG)13 55 1.25 E1 23 87–173 a TA, annealing temperature. bLinkage group numbers refer to the E. globulus linkage map (Thamarus et al. 2002) except where denoted by E; these loci were mapped in a E. grandis · E. urophylla pedigree (Brondani et al. 1998). cnm, not mapped

For amplification of microsatellite loci, 20 ng leles were scored by two people, independently, to of template genomic DNA was amplified by PCR minimise possible scoring errors. with 10 mM Tris–HCl (pH 8.4), 25 mM KCl, 0.2 mg/ml of bovine serum albumin, and 0.2 mM Data analysis of each dNTP, 1–2 mM MgCl2, 0.2 lM of each primer and 0.5 unit Taq polymerase in 10-ll Seed production and viability reactions. Annealing temperatures and magnesium The weight of capsule contents (seed and chaff), chloride concentrations were varied to optimise number of seeds per capsule and seed weight per the amplification of each primer pair (Table 1). capsule were calculated for each tree and the Amplification cycles were 94 C for 2 min; 30 cy- population means compared by analysis of vari- cles of 94 C for 30 sec, 55 Cor60C for 30 sec ance and paired t-tests. A nested analysis based on and 72 C for 60 sec; and 72 C for 20 min. PCR a linear model was carried out using the REML products were separated by capillary statistical package (Robinson 1987) within GEN- electrophoresis using an ABI PRISM 310 Ge- STAT V and used to compare germination rates; netic Analyser (Applied Biosystems, Foster City accounting for differences in replication across CA, USA) and fragment sizes determined using families. Germination rates were then compared GenotyperTM software (Applied Biosystems). Al- among populations by analysis of variance. 217

Population genetic parameters that progeny were derived independently from a Genetic diversity of each population was com- panmictic pollen pool, the number of detectable pared based on the following measures; mean outcrossed progeny (i.e. progeny with an allele number of alleles per locus (A); the number of which differed from the maternal alleles) was cal- alleles found in only one population [private al- culated for each family and compared with indi- leles, p(1)], observed heterozygosity (HO); unbi- vidual-tree estimates from MLTR. Associations ased expected heterozygosity (HE), and the between outcrossing rate, seed set and germination panmictic index or inbreeding coefficient (f) cal- rate were examined by linear regression. culated according to Weir and Cockerham (1984). For the two remnant populations, Camden and These parameters were calculated using the GDA Wallacia, paternal genotypes with the highest package (Lewis and Zaykin 2001). Allelic richness likelihood were determined using the package (AR) was calculated as the mean number of alleles CERVUS (Marshall et al. 1998). This allowed per locus, adjusted for differences in sample size estimation of the approximate number of pollen using the rarefaction option in the FSTAT pack- donors to each half-sib family. age (Goudet 2001). Genetic differentiation among all populations and pairs of population were compared using FST Results statistics, calculated according to Weir (1996) using the GDA package. Confidence intervals were Seed set and viability estimated by bootstrapping over loci 1000 times. Pairwise genotypic differentiation among popula- The mean weight of capsule contents, number of tions was also tested using exact tests for each seeds per capsule, seed weight and germination locus in the GENEPOP package (Version 3.3; rate for each of the natural populations are pre- Raymond and Rousset 1995a). Global p-values sented in Table 2. The mean weight of capsule were calculated across the 22 loci using Fisher’s contents was significantly higher in the remnant method (Raymond and Rousset 1995b). Patterns Camden, Bents Basin and Wallacia populations in allele frequencies were investigated by principal but a higher proportion of the contents was chaff component analysis (PCA) using the PCAGEN (82% compared with 72% in Kedumba). Fewer package (http://www2.unil.ch/izea/softwares/ seeds were produced per capsule in Camden, pcagen.html). Wallacia and Bents Basin, but the average weight of seeds was significantly higher than in the Ke- Mating system dumba population. Germination rates were sig- Single-locus (ts) and multilocus (tm) estimates of nificantly higher in Kedumba samples despite the the proportion of progeny arising from outcross- smaller average seed size. ing in each family and population were made using maximum likelihood procedures and the MLTR Genetic diversity parameters program (Ritland 2002). Maternal genotypes were known because leaf from the maternal trees was Comparison of genetic diversity measures (A, AR, genotyped to estimate the genetic diversity HO and HE) in the four populations, calculated parameters. The difference between the multilocus over 22 loci, showed no clear trend of lower estimate and the mean single-locus estimate diversity in the remnant populations (Table 3). (tm ) ts) was calculated to determine the lower Allelic richness and heterozygosity were lowest in bound for selfing due to biparental inbreeding the Wallacia population but highest in Camden. (Brown 1990). The correlation of paternal geno- There was evidence of significant departures from types among outcrossed sibs (rpm), the correlated random mating not only in the Camden popula- selfing rate (rs) which reflects variation in selfing tion but also in Kedumba. In total, 307 alleles were rates among families, and the correlation of selfing detected, 60% of which occurred at a frequency of among loci (rsm) which approximates the fraction less than 5%. A high proportion (45%) of alleles of selfing due to uniparental inbreeding, were also were detected in only one of the four populations, calculated using MLTR. To ensure estimates of most at frequencies of less than 5%. More private outcrossing were not biased by the assumption alleles were detected in the Camden population; an 218

Table 2. Mean weight of capsule contents, mean number of seeds per capsule, mean seed weight and percentage germination for four natural populations of Eucalyptus benthamii

Population Weight of capsule contents (mg) Number of seeds per capsule Seed weight (mg) Germination (%)

Wallacia 5.18 a 2.21 a 0.330 b 53.0 b Camden 5.22 a 2.23 a 0.439 a 71.8 a Bents Basin 4.88 a 2.84 ab 0.351 b 65.3 a Kedumba 3.15 b 4.09 b 0.235 c 83.9 c

Mean values sharing the same letter are not signiﬁcantly diﬀerent at P = 0.05 (ANOVA).

Table 3. Estimated population size (N), number of trees sampled (n) and genetic diversity parameters for adult trees in four natural populations and a seed orchard estimated from 22 microsatellite loci

Population Nn AAR HE HO fp(1)

Wallacia 9 9 4.32 3.98 0.618 0.652 –0.058 8 Camden 30 16 8.14 5.81 0.747 0.622 0.173* 53 Bents Basin 300 17 6.41 4.85 0.712 0.714 –0.002 19 Kedumba 6550 32 7.77 4.98 0.708 0.639 0.099* 44 Mean 6.66 4.91 0.696 0.656 0.060* Seed orchard 20 9.00 6.13 0.784 0.627 0.206* 21

unexpected result given the relatively small size of The level of genetic diversity in the seed orch- the population and the number of individuals ard was higher than in the natural populations sampled. For comparison, allele frequencies were (Table 3). This is expected because different alleles estimated for the 456 progeny assayed to estimate in the source populations of Camden and Bents outcrossing rates. Despite the larger number of Basin are combined in the seed orchard. Differ- individuals analysed and fewer loci, 37% of alleles ences between the source populations would result at seven loci were detected in only one of the four in substructuring in the seed orchard, reflected in populations. Again, more private alleles were de- the significant inbreeding coefficient. The high tected in the Camden population (Table 4). inbreeding coefficient may also indicate that selfs

Table 4. Number of progeny sampled (np) from na mother trees and genetic diversity parameters for progeny from four natural populations estimated from seven microsatellite locia

Population np na AAR HE HO fp(1)

Wallacia 57 5 9.14 9.11 0.664 0.589 0.114 7 Camden 104 9 12.57 10.93 0.774 0.643 0.169* 17 Bents Basin 129 11 9.28 7.87 0.765 0.689 0.099* 10 Kedumba 178 16 10.71 8.97 0.714 0.599 0.061* 11 Mean 10.43 9.22 0.729 0.630 0.136*

A, mean number of alleles per locus; AR, allelic richness; HE, expected heterozygosity; HO, observed heterozygosity; f, inbreeding coefficient (*P<0.001 from exact tests); p(1), number of private alleles. aMicrosatellite loci used to measure outcrossing were the most polymorphic, therefore estimates of genetic diversity are not directly comparable with those in Table 3. 219 are more likely to survive in a cultivated stand of the variance respectively. The lack of gene flow than in the natural populations. was also evident among progeny from the Bents A significant proportion of variation in Basin and Camden populations that were estab- E. benthamii was due to differences among popu- lished in a seed orchard. The seed orchard trees lations (FST ¼ 0.105; confidence interval 0.087– clustered with their respective source populations 0.121). Pairwise FST values, based on 22 loci, dif- in the PCA (Figure 3) indicating that mating is not fered significantly from zero for all populations occurring between these remnant populations. (Table 5). Similarly, the six exact tests of differentiation, over all loci, revealed highly significant Mating system differences among all population pairs (P <10)5; df ¼ 44). This evidence, together with the high The average level of outcrossing in E. benthamii proportion of private alleles, indicates that there (tm ¼ 0.62) was similar to that found in other insect- has been little gene flow among populations. The pollinated, temperate eucalypts (see Potts and divergence among populations is evident in the Wiltshire 1997 for review). However, outcrossing plot of principal component scores (Figure 2). rates in the smaller remnant populations of Camden This shows the Kedumba and Wallacia popula- and Wallacia were significantly lower than in Bents tions separated on the pca-1 axis and Bents Basin Basin and Kedumba (Table 6). Outcrossing rates of and Camden separated on the pca-2 axis. The first individual trees were highly variable ranging from two principal components accounted for 9 and 6% complete inbreeding to complete outcrossing (Ta-

Table 5. Pairwise tests for diﬀerentiation among populations

Camden versus Camden versus Bents Basin Camden versus Wallacia versus Bents Basin Bents Basin Wallacia versus Wallacia Kedumba Kedumba versus Kedumba

FST 0.094 0.128 0.136 0.089 0.157 0.078 Upper 95% CI 0.113 0.176 0.167 0.109 0.190 0.099 Lower 95% CI 0.074 0.088 0.103 0.068 0.119 0.059

CI, conﬁdence interval.

Bents Basin

B4 B1 B17B2 B9 W7 B5 B3 W6 W5 B13 K11 B14 B16B15 B8B11 W9 Wallacia K7 K10 B6 W1 K5 K16 K27 K9 W8 W3 K24 K21 B12 K8K2 K20K33 K30 K31 K23 B7 W2 W4 K18 K17 K1K25 C10K12 C11

PCA[2] score K4 K15 K32 K13 C12 K19K14 C2 K22 K26 C14 K29 C1 C15 K28 C13 Kedumba C3 C8 C16 C9 Camden C6 C5 C4 C7 -0.3 -0.1 0.2 0 0.1 0.2 0.3

-0.3 -0.2 -0.1 0 0.1 0.2 0.3 PCA[1] score

Figure 2. Plot of scores from principal component analysis of allele frequencies from 76 Eucalyptus benthamii trees in four populations: Camden (C), Bents Basin (B), Wallacia (W) and Kedumba (K). 220 3 W5 Bents Basin W7 W1 W6 W3 0.2 0.2 0.

W8 W9 B9 W4 B2B5 B15 B17 B10B8 C12 B16B11 B4 B13 C13 B1 B7 C15 B12 C7 B14 K12 C8 C14 PCA[2] score C9 K23 C10 C16 C4 K3 C2 C5 K27K30 C3 K6 K13 C6 K16K3 K15K31 K17 K11 K29 K20 K32 K5 K12 K26 K24K2K18 K10 K7 K19K28K22 K25 K18 K4 K1 Camden -0.3 -0.2 -0.1 0 0.1

-0.3 -0.2 -0.1 0 0.1 0.2 0.3 PCA[1] score

Figure 3. Plot of scores from principal component analysis of allele frequencies from 20 Eucalyptus benthamii seed orchard trees sourced from Bents Basin (ﬁlled triangles) and Camden (ﬁlled circles) and 76 trees from native populations; Camden (C), Bents Basin (B), Wallacia (W) and Kedumba (K).

ble 7). Two trees in the Camden population (Fig- fing among loci (rsm) were highly significant in all ure 4) and one tree in Wallacia produced no out- populations with about 80% of inbreeding due to crossed seed (Table 7). These trees were isolated selfing in the smallest population at Wallacia from other E. benthamii trees by distances of over (Table 6). In the large Kedumba population sig- 100 m. There was also evidence of hybridisation in nificantly more inbreeding was due to mating the Camden and Wallacia populations. More than a among neighbours (50%) than in the smaller third of the outcrossed progeny in Wallacia had the populations. green, lanceolate leaf morphology typical of E. Estimates of correlated selfing (rs) were not viminalis seedlings. These seedlings also had alleles statistically significant (Table 6) reflecting the fact not recorded in other sampled E. benthamii trees, that most trees produced both selfed and out- consistent with hybrid origins. crossed seed (Table 7). There was evidence of sig- Significant differences between single-locus and nificant correlated paternity among outcrossed multilocus estimates of outcrossing indicate sibs in the Bents Basin and Kedumba populations neighbourhood inbreeding in all populations (Table 6) where the outcrossing rates were higher. except Wallacia (Table 6). However, most This suggests a limited number of males were inbreeding appears to be from selfing rather than contributing to the pollen pool. The effective biparental inbreeding. Estimates of correlated sel- paternal mating pool was only two to three trees

Table 6. Mating system parameters for four natural populations of Eucalyptus benthamii estimated from seven microsatellite loci (standard deviations in parentheses). Populations with outcrossing rates (tm) followed by the same letter are not signiﬁcantly diﬀerent at the 5% probability level

Population ntm tm ) ts rs rsm rpm

Wallacia 5 0.54 (0.14) ab 0.10 (0.06) 0.37 (0.30) 0.81 (0.13) 0.47 (0.36) Camden 9 0.45 (0.09) a 0.13 (0.06) 0.24 (0.30) 0.70 (0.19) 0.39 (0.33) Bents Basin 11 0.70 (0.08) b 0.11 (0.02) 0.31 (0.25) 0.70 (0.12) 0.28 (0.11) Kedumba 16 0.68 (0.05) b 0.17 (0.02) 0.13 (0.11) 0.51 (0.06) 0.29 (0.11)

n, number of families; tm, multilocus outcrossing rate; ts, mean single locus outcrossing rate; rs, correlated selﬁng; rsm, the correlation of selﬁng among loci; rpm, multilocus correlation of paternity. 221

Table 7. Genetic diversity (A), number of progeny sampled per family (n) and mating system parameters for Eucalyptus benthamii families estimated from seven microsatellite loci

Population and family An td tm (SD) tm ) ts (SD)

Wallacia W1 4.0 12 0.33 0.32 (0.14) 0.02 (0.05) W2 5.6 10 0.80 0.82 (0.17) )0.03 (0.10) W4 4.0 12 0.75 0.74 (0.14) 0.11 (0.06) W8 1.7 11 0.00 0.00 (0.00) 0.00 (0.00) W9 3.4 12 0.83 0.85 (0.15) 0.34 (0.13) Camden C1 1.7 7 0 0.00 (0.00) 0.00 (0.00) C3 1.7 13 0 0.00 (0.00) 0.00 (0.00) C4 4.0 13 0.23 0.23 (0.12) )0.02 (0.01) C5 4.3 12 0.58 0.58 (0.14) 0.16 (0.05) C6 6.2 12 0.67 0.68 (0.14) )0.02 (0.03) C7 3.7 11 0.46 0.46 (0.14) 0.19 (0.07) C10 3.8 13 0.62 0.61 (0.15) 0.19 (0.06) C11 3.6 11 0.91 0.97 (0.16) 0.48 (0.14) C17 2.9 12 0.42 0.51 (0.15) 0.25 (0.09) Bents Basin BB1 4.0 12 1.00 1.20 (0.00) 0.34 (0.05) BB2 4.8 12 1.00 1.20 (0.00) 0.37 (0.11) BB3 4.6 13 1.00 1.20 (0.00) 0.41 (0.08) BB4 4.9 11 0.73 0.71 (0.17) 0.13 (0.06) BB5 4.4 13 0.54 0.54 (0.13) 0.04 (0.07) BB7 2.0 10 0.18 0.11 (0.09) 0.09 (0.08) BB8 4.2 12 0.42 0.39 (0.16) 0.09 (0.07) BB9 4.2 10 0.70 0.70 (0.16) 0.16 (0.14) BB10 4.4 12 0.92 0.96 (0.15) 0.15 (0.15) BB12 4.2 11 0.73 0.73 (0.14) 0.06 (0.06) BB14 5.3 11 0.46 0.47 (0.14) )0.07 (0.03) Kedumba K1 3.4 12 0.75 0.76 (0.13) 0.26 (0.08) K2 3.2 9 0.22 0.28 (0.16) 0.02 (0.10) K3 3.3 10 0.55 0.49 (0.15) 0.21 (0.10) K4 4.4 12 0.75 0.75 (0.17) 0.17 (0.09) K5 3.8 11 0.55 0.52 (0.15) 0.26 (0.08) K7 4.2 12 0.75 0.76 (0.16) 0.16 (0.09) K9 2.3 9 0.11 0.22 (0.16) 0.10 (0.08) K12 4.2 11 0.73 0.74 (0.16) 0.29 (0.10) K16 4.6 12 0.83 0.85 (0.15) 0.16 (0.08) K17 4.4 11 0.73 0.72 (0.14) 0.16 (0.07) K18 4.1 12 0.83 0.87 (0.15) 0.13 (0.09) K19 2.8 11 0.46 0.46 (0.17) 0.08 (0.06) K21 4.1 12 0.75 0.78 (0.15) 0.09 (0.08) K24 4.1 11 0.55 0.56 (0.15) 0.10 (0.05) K25 4.7 12 0.92 0.98 (0.17) 0.26 (0.12) K28 4.2 12 1.00 1.20 (0.00) 0.51 (0.06)

A, mean number of alleles per locus; td, proportion of progeny with an allele not detected in the mother i.e., detectable outcross; tm, multilocus outcrossing rate; ts, average single locus outcrossing rate. 222

E. benthamii. The outcrossing rate in Bents Basin, which has about 300 mature trees, was not significantly different from Kedumba with about 6500 trees. However, there appears to be a size or density threshold below which populations may be more susceptible to increased inbreeding and inter- species gene flow. In a survey of outcrossing rates in the rare mallee, E. argutifolia, Kennington and James (1997) also failed to find a relationship with population size and suggested increased inbreeding in small populations may be compensated by increased abortion of competitively inferior selfed seed. However, the lack of correlation between outcrossing rate and seed set indicates this is not the case in E. benthamii. While population size Figure 4. Distribution of sampled trees in the Camden population and outcrossing rates of trees with seed crops. alone was not correlated with outcrossing rate, the level of inbreeding was affected by fragmentation of populations and isolation of individual trees. for Wallacia and Camden and three to four trees The smaller remnant populations of Camden and for Bents Basin and Kedumba. This ‘pool’ was Wallacia had significantly higher rates of calculated as the inverse of rpm (Ritland 1989) and inbreeding than Kedumba and Bents Basin, pri- assumes all males have equal mating probabilities. marily due to the fact that isolated trees in the The estimate of two to three effective pollen do- Camden and Wallacia remnants produced only nors for trees in the Wallacia and Camden popu- self-pollinated seed. More inbreeding was due to lations was consistent with the absolute number of selfing (70–80%) in the remnant populations than pollen donors identified using likelihood-based in Kedumba where about 50% of inbreeding was paternity inference (95% probability). In Wallacia attributed to mating among relatives. The pro- 45% of progeny were selfs, 25% were sired from a portion of outcrossed seedlings with correlated neighbouring tree (generally 15–30 m distant) and paternity was also higher in Camden (39%) and for 30% the pollen parent was not sampled; these Wallacia (47%), compared with 28% in Bents progeny had morphological attributes consistent Basin and Kedumba. This suggests that once a with hybrids involving E. viminalis. In Camden population declines beyond a certain threshold 54% of progeny were selfs; 17% sired from (between 30 and 300 trees in E. benthamii), the neighbouring trees and for 29% the pollen donor limited number or low density of flowering trees was not sampled. Among the sampled trees in may adversely affect the abundance and behaviour Camden the identified pollen donors were all of pollinators. This is supported by the lack of adjacent trees with no evidence of pollen flow be- evidence of long-distance pollen flow in Camden tween clusters. Similarly, in Wallacia most identi- and Wallacia – most intra-species pollen flow was fied pollen donors were the neighbouring tree with with neighbouring trees. In a study of native spe- only two cases where pollen was transported more cies in mallee woodland, Cunningham (2000) than 100 m. No significant correlations were found habitat fragmentation can lead to a decline found between individual tree outcrossing rates in pollination and subsequent seed set, particularly and seed set, seed mass or seed viability. where the understorey, which provides alternative habitat for pollinators, has been reduced or removed. In the Camden and Wallacia populations Discussion the understorey has been cleared and is now dominated by introduced grasses and woody weed Seed production and mating system species. In addition, the density of mature trees in Camden and Wallacia is lower than in Bents Basin Population size alone does not appear to have a and Kedumba. While pollinator activity was not significant impact on outcrossing rates in directly monitored in E. benthamii, the fact that 223 only selfed seeds were detected on isolated trees between clones of E. grandis (Hodgson 1976) and indicates an absence of outcross pollen. Selfed between E. globulus individuals (Pound et al. embryos may be more likely to persist and develop 2002). Reductions in seed set would be expected into seed when there is no competition for re- only if trees were self-incompatible. In E. benth- sources with outcrossed embryos (James and amii, trees that produced only self-seed must be Kennington 1993). self-compatible. The range in outcrossing rates in The high level of correlated mating together the Kedumba populations (from 22 to 100%) may with evidence of neighbourhood inbreeding in also reflect variation in self-compatibility among E. benthamii, indicates a tendency to mate with trees. A similar range in outcrossing rates (0– near neighbours, even in the largest population. 100%) was recorded in E. cladocalyx (McDonald The propensity of E. benthamii to mate with near et al. 2003), a species for which variation among neighbours has meant that fragmentation not only individuals in self-compatibility had previously increases the probability of inbreeding but also been demonstrated (Ellis and Sedgley 1992). increases the probability of hybridisation if com- Controlled pollination experiments are required to patible species with overlapping flowering times establish whether self-compatibility varies among are present. Seedling leaf morphology and allele maternal genotypes in E. benthamii. profiles suggest about 30% of outcrossed progeny While seed set was higher in the Kedumba in Wallacia and 10% of outcrossed progeny from population, the average seed size was smaller. The Camden are hybrids with E. viminalis. Eucalyptus trend to produce fewer larger seeds versus more, viminalis is in the same taxonomic section (Maid- smaller seeds may be explained by a simple trade- enaria)asE. benthamii and is in the upper decile of off of resources at the individual capsule level. In species ranked for the number of different hybrid contrast to reports for other eucalypts (Burrows species combinations recorded (Griffin et al. 1988). 2000), larger seeds did not have higher germina- The high incidence of hybrid and selfed progeny tion rates. means that only 25% of the seed collected from trees in the Wallacia population and 35% of the Patterns of genetic diversity seed from Camden would be suitable for revegetation. Comparison of the levels of diversity in the four Seed set and germination were significantly populations of E. benthamii suggests that frag- lower in the Camden and Wallacia populations mentation has not led to a decline in genetic than in Kedumba. Reduced seed set and viability diversity. Reductions in allelic richness in the were also reported in isolated E. melliodora trees remnant populations would be expected, due when compared with woodland trees and attrib- simply to reductions in the number of individuals, uted to increased self-fertilization in isolated trees particularly when individuals possess rare alleles. (Burrows 2000). Inbreeding can reduce seed set if Reductions in heterozygosity are more likely to self-pollen tubes fail to penetrate the ovule or if occur in succeeding generations as a result of self-fertilised ovules degenerate. Seed constituted, inbreeding and genetic drift (Crow and Kimura on average, only 18% of capsule contents for trees 1970). While there is evidence of a decline in in the remnant populations, while for the Ke- allelic richness and heterozygosity in the smallest dumba population 30% of the capsule contents remnant population at Wallacia, both parameters was seed. The lower proportion of seed to chaff in are surprisingly high in Camden. The high het- capsules from the remnant populations indicates erozygosity in the Camden population may reflect either lower rates of fertilisation or a higher rate of the fact that at least half the sampled trees pre- aborted seed. Seed set was not correlated with the date clearing and the impact of fragmentation outcrossing rates of individual trees, suggesting may not be evident in these trees. However, de- pollinator activity is more likely to be limiting spite higher inbreeding in the Camden popula- fertilisation. Attempts to find a relationship be- tion, heterozygosity in the progeny from these tween seed set and inbreeding in eucalypts are trees was also higher than in the larger Kedumba complicated by the fact that individual trees may population. differ in the level of self-compatibility. For exam- Attempts to find correlations between genetic ple, the degree of self-compatiblility varied widely diversity and population size in eucalypts have 224 had limited success (Moran and Hopper 1987; explanation is that past hybridisation in the Prober et al. 1990; Kennington and James 1997), Camden populations has contributed to diver- the exceptions being E. caesia (Moran and Hop- gence of this population and limited gene flow per 1983), E. crucis (Sampson et al. 1988) and among all populations has allowed local genetic E. albens (Prober and Brown 1994). These studies differentiation through drift. have been conducted using seedlings rather than The clustering in the PCA of trees that were in adult trees, and Prober and Brown (1994) suggest closest geographical proximity (Figure 2) suggests that other factors that differentially influence ge- that genetic differentiation among populations has netic diversity in the seed crop in different pop- resulted from neighbourhood formation. That is, ulations, such as variation in the number of trees trees are most likely to mate with nearest neigh- flowering in the year of seed collection, tree bours, thereby producing neighbourhoods of density and pollinator abundance, may make it similar genotypes. This is supported by evidence of difficult to detect a relationship between genetic significant correlated paternity in the Bents Basin diversity and population size. In E. benthamii the and Kedumba populations. The paternity analysis sampling of adult trees removed these sources of in Wallacia and Camden also revealed a propen- variation, yet similar trends were evident amongst sity to mate with nearest neighbours. adults and progeny. The low diversity in the Comparison of single- and multi-locus esti- Wallacia population suggests that fragmentation mates of outcrossing indicated the presence of may cause a decline in genetic diversity once biparental inbreeding, supporting the hypothesis population size drops below a critical threshold. of differentiation due to formation of localised Prober and Brown (1994) suggested such a neighbourhoods of related trees. To compare evi- threshold (500 trees) for E. albens. While the dence of departures from random mating among Camden population with only 30 trees may be generations, inbreeding coefficients were estimated below such a threshold, the high numbers of from the seven microsatellite loci that were assayed private alleles and evidence of hybrid progeny in both the parent trees and the progeny. The suggest that alleles gained through hybridisation, inbreeding coefficients were lower for mature trees past as well as present, may be masking any loss in the natural populations (f ¼ 0.01; 95% of alleles due to fragmentation. Camden is the CI ¼ )0.04–0.08)) than for the progeny (f ¼ 0.14; southern limit of the species distribution and trees 95% CI ¼ 0.11–0.18) suggesting that there has there may have been more susceptible to hy- been selection against inbred individuals between bridisation. Private alleles were equally numerous the seedling and adult stage. This would limit any in trees pre-dating clearing as those derived from decline in genetic diversity in the smaller popula- post-fragmentation matings suggesting that hy- tions associated with inbreeding. bridisation was impacting on this population prior to land clearing. Implications for conservation The high level of genetic differentiation (FST ¼ 11%) and significant divergence among all The highly significant differences in allele frequen- pairs of populations was unexpected given the cies among populations of E. benthamii indicate short geographical distances between stands. The that to adequately conserve the species genetic high proportion of alleles that were restricted to a resources all remaining populations should be pro- single population, together with the age structure tected and managed as independent units. The of these populations (35–200 years), indicates that remnant Camden and Wallacia populations not genetic divergence among populations occurred only have similar levels of genetic diversity to the prior to land clearing. If gene dispersal is limited, larger Kedumba and Bents Basin populations, but local genetic differentiation may occur due to ei- they also contain a high proportion (29%) of alleles ther spatially variable selection or as a result of not detected in the larger populations. These pop- localised genetic drift. Differential selection ulations therefore play an important role in pressure in the Camden, Wallacia and Bents Basin maintaining the species genetic diversity. Conser- populations is unlikely given their close geo- vation efforts should focus on the remnant popu- graphical proximity and the absence of any geo- lations where natural regeneration is limited by graphical barriers to gene flow. The most likely competition with exotic species. Many of the mature 225 trees in Wallacia, Camden and Bents Basin popu- lite markers in Eucalyptus grandis and E. urophylla. Theor. lations are in poor health, which limits natural seed Appl. Genet., 97, 816–827. Brooker MIH, Kleinig DA (1999) Field Guide to Eucalypts production. Increased inbreeding and reduced seed Volume 1. South Eastern Australia (revised edn). Bloomings set and seed viability in the Camden and Wallacia Books, Melbourne. populations, together with evidence of increased Brown AHD (1990) Genetic characterisation of plant mating inter-species gene flow in the remnant populations, systems. Ch. 9 In: Plant Population Genetics, Breeding and Genetic Resources (eds. Brown AHD, Clegg MT, Kahler AL, reduce the value of seed for regeneration or plan- Weir BS), pp. 145–162. Sinauer Associates. Inc., Sunderland, tation establishment. 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The microsatellite markers used in this study loci in the genus Eucalyptus. Aust. J. Bot., 44, 331–341. Crow JF, Kimura M (1970) An Introduction to Population provide a reliable means of validating these crosses Genetics Theory. Harper and Row, New York. and determining the source population of seedlings. Cunningham SA (2000) Depressed pollination in habitat frag- An alternative approach to providing seedlings for ments causes low fruit set. Proc. R. Soc. Lond. B, 267, 1149– revegetation would be to germinate seeds from the 1152. Ellis MF, Sedgley M (1992) Floral morphology and breeding remaining trees in Wallacia and Camden, eliminate system of three species of Eucalyptus, section Bisectaria hybrids based on seedling morphology and use mi- (Myrtaceae). Aust. J. Bot., 40, 249–262. crosatellites to identify outcrossed progeny. These Frankel OH, Brown AHD, Burdon JJ (1995) The Conservation markers could also be used to identify hybrids if of Plant Biodiversity. Cambridge University Press, Cam- bridge. alleles can be found in E. viminalis trees in the Glaubitz JC, Emebiri LC, Moran GF (2001) Dinucleotide mi- Camden–Wallacia region that were not detected in crosatellites from Eucalyptus sieberi: inheritance, diversity, E. benthamii. and improved scoring of single-base differences. Genome, 44, 1041–1045. Goudet J (2001) FSTAT, a program to estimate and test gene Acknowledgements diversities and fixation indices (version 2.9.3). Available from http://www.unil.ch/izea/softwares/fstat.html. Griffin AR, Cotterill PP (1988) Genetic variation in growth of This project was supported by the New South outcrossed, selfed and open-pollinated progenies of Euca- Wales Government through its Environmental lyptus regnans and some implications for breeding strategy. Trust. Permission to sample on lands managed by Silv. Genet., 37, 124–131. 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