Hedging in a Rare Yet Widespread Rainforest Tree, Syzygium

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Austral Ecology (2012) 37, 936–944

Reproductive bet-hedging in a rare yet widespread

rainforest tree, Syzygium paniculatum (Myrtaceae)aec_2353 936..944

KATIE A. G. THURLBY,1,2,3 PETER G. WILSON,1* WILLIAM B. SHERWIN,2 CAROLYN CONNELLY1 AND MAURIZIO ROSSETTO1 1National Herbarium of NSW,Royal Botanic Gardens and Domain Trust, Mrs Macquaries Road, Sydney, NSW 2000, Australia (Email: [email protected]), 2School of Biological, Earth and Environmental Sciences, and 3School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, New South Wales, Australia

Abstract The rare rainforest tree species, Syzygium paniculatum, is the only known Australian species of the genus to produce seeds that regularly have multiple embryos. Evidence from other species suggests that this is a case of adventitious polyembryony, with the embryos arising from maternal nucellar tissue. In the present study we use microsatellite data to determine whether sexual reproduction does occur and, if it does, to investigate the relative fitness of asexual versus sexual seedlings. Genotyping suggested that the species is a polyploid and our results found very little genetic diversity within and among populations (with a total of nine genotypic combinations across the entire species).The only significant variation was between the three northernmost populations and the other eight populations sampled. Analysis of individual embryos showed that sexually derived embryos did occur in some seeds but that these were not necessarily the fittest. In general, the seedling from the largest embryo is the first to emerge and maintains a competitive advantage over the other seedlings from the same seed. We discuss the ramifications of the low levels of genetic diversity and consider whether there is a direct relationship between polyembryony and the inferred polyploidy of the species. We consider the possible advantages of reproductive bet-hedging but also highlight the susceptibility of a species with low genetic diversity to extreme stochastic events. Syzygium paniculatum occurs in areas heavily impacted by human activity and these findings should contribute to improved management of this threatened species.

Key words: agamospermy, clonality, littoral rainforest, polyembryony, polyploidy, Syzygium.

INTRODUCTION tions unfavourable for sexual reproduction, such as strong pollen limitation in species-rich communities (Whitton et al. 2008); they could also increase the Sexual versus asexual reproduction survival of advantageous genotypes, enable a genotype to sequester space and monopolize resources The major benefit of sexual reproduction is generally (including establishment of new populations from a believed to be the ability of an organism to produce single individual), or provide the potential to accu- new and potentially advantageous combinations of mulate variability through extended lifespan (Cal- genes, an advantage that is only available to obligate laghan et al. 1992; Hörandl 2004; Houliston & asexual species through somatic mutation. However, Chapman 2004; Paun et al. 2006; Vallejo-Marín et al. sexual reproduction can also be disadvantageous. A 2010; van der Merwe et al. 2010). As a result, some sexual parent transmits only 50% of its genes to its asexual species are as genetically diverse and wide- offspring, which could potentially lead to the break- spread as sexual species (Gitzendanner & Soltis down of favourable genic combinations and the loss of 2000; Paun et al. 2006). Asexual reproduction also local adaptation (Otto 2009). poses possible disadvantages, particularly if it results Asexual reproduction may provide genetic and in loss of genetic diversity. Reduced genetic variation ecological advantages, particularly in the short term and drift (Harper 1978; Eckert et al. 1999; Paun (e.g. van Dijk & van Damme 2000). These advan- et al. 2006) can result in the loss of short-term fitness tages might include the ability to persist in condi- and of long-term adaptive potential (Richards 2003; Zhang & Zhang 2007). *Corresponding author. Interestingly, models show that the benefits Accepted for publication December 2011. of sex may be obtained through facultative sexual

© 2012 The Authors doi:10.1111/j.1442-9993.2011.02353.x Austral Ecology © 2012 Ecological Society of Australia REPRODUCTIVE BET-HEDGING IN SYZYGIUM PANICULATUM 937 reproduction without the cost (Green & Noakes 1995).This suggests that the combination of a range of reproductive mechanisms may provide the beneﬁts of both sexuality and asexuality while reducing overall costs (Green & Noakes 1995; Paun et al. 2006; Vallejo- Marín et al. 2010).

Agamospermy

One particular case of asexual reproduction is agamospermy, the production of seed without sex (Talent 2009). It is a mechanism that affords offspring the advantages of seed production normally only available to sexually produced progeny: protection, disease resistance, dormancy and improved dispersal (Rich- ards 2003; Silvertown 2008). Sex can exist simultaneously with agamospermy because most forms of agamospermy require the initial development of a sexual embryo or, at the very least, fertilization and development of the endosperm (Ganeshaiah et al. 1991; Koltunow & Grossniklaus 2003; Whitton et al. 2008). So, in some polyembryonic seeds, sexual embryos may persist alongside the devel- oping agamospermic embryos (Ganeshaiah et al. 1991; Asker & Jerling 1992; Richards 2003; Whitton et al. 2008). However, stable coexistence of both sexual and asexual progeny may be difﬁcult to accom- plish within a single population (Silvertown 2008) because embryos must compete for the resources afforded by the endosperm. As a result, the percentage Fig. 1. Distribution and sampling sites of Syzygium paniculatum. The asterisks identify collection sites along the of fruits containing sexual embryos will vary depend- New South Wales coast. ing on the outcome of this competition (Roy 1953, 1961; Narayanaswami & Roy 1960; Naumova 1992; Whitton et al. 2008). Studies in Citrus indicate that the zygotic embryo develops rather slowly, and may be 1961). In adventitious polyembryony the embryos outcompeted by the more vigorous nucellar embryos are genetically identical and are derived from the and fail to survive in seeds where many of these occur integument or the nucellus (the parts of the ovule (Koltunow 1993). adjacent to the embryo sac). The embryos in S. paniculatum vary in size and often have markedly unequal storage cotyledons. Embryos with cotyledons Study system of this type are cryptocotylar and germination is often hypogeal. The rare Australian rainforest tree Syzygium panicu- Syzygium paniculatum is locally rare and represented latum Gaertn. (Myrtaceae) may prove to have a by small populations distributed across a geographical complex mating system, combining multiple repro- range of about 400 km (Fig. 1). Because of its mode of ductive mechanisms. Fruits contain a single seed reproduction, the species is likely to have low diversity, which is usually polyembryonic, a trait not seen in which may be detrimental to ﬁtness. Understanding any other Australian native Syzygium species. Poly- the relative contribution of sexual and asexual repro- embryony can arise by several means. One possible duction might help us to understand the causes of mechanism is cleavage polyembryony (the separation rarity in S. paniculatum, and this species may provide a of a zygote into two or more units; see Webber means for exploring the consequences of mating 1940), although this happens more often in gymno- mechanisms on the expansion or decline of rare sperms than angiosperms (Batygina & Vinogradova species. To better understand the impact of sexual 2007). It is most likely that polyembryony in S. pan- reproduction on rarity, current distribution and long- iculatum is due to agamospermy, as has been docu- term potential, this study aims to test the hypotheses mented in some Asian Syzygium species (Roy 1953, arising from the following points:

1. Despite its wide distribution range, the rare the PCR and genotyping conditions reported in Thurlby S. paniculatum has low genetic diversity. The level et al. (2011). Genotyping traces were analysed using Gen- and distribution of genetic diversity are important emapper v4.0 (Applied Biosystems). To test genotyping elements in the development of appropriate con- accuracy, PCRs were repeated for each primer across 20% servation planning for this species. of the individuals. Fewer than 5% of repeats identified errors needing confirmation with further PCR and 2. As suggested by other studies, there are asso- genotyping. ciations between polyembryony, apomixis and During the development of the SSR markers, it was polyploidy. noticed that multiple loci produced polyploid amplification 3. If both sexual and asexual reproduction mecha- patterns (Thurlby et al. 2011). In order to confirm the nisms are identified, it is expected that they polyploid nature of S. paniculatum and to verify the number could result in differential levels of fitness (as of alleles per locus, genotyping traces were obtained for measured by germination success and seedling three other Syzygium species: Syzygium corynanthum growth). (F.Muell.) L.A.S.Johnson, Syzygium francisii (F.M.Bailey) L.A.S.Johnson and Syzygium jambos (L.) Alston. Of the four Syzygium species tested, S. corynanthum and S. francisii showed one (homozygous) or two (heterozygous) MATERIALS AND METHODS alleles for all loci tested, as is expected for a diploid organism. The third species, S. jambos, a known tetraploid (cited as Eugenia jambos in Roy 1953, and Eugenia jambolana in Study species and sampling Singhal et al. 1985), displayed four alleles at locus SP33 and three alleles at locus SP43.1. Four alleles were also Syzygium paniculatum is a small to medium rainforest tree amplified at locus SP33 and three alleles at locus SP38 and that is restricted to littoral rainforest of New South Wales SP54 for S. paniculatum, suggesting that the study species is (NSW, Australia), occurring in disjunct populations along a also a polyploid. long, narrow, linear coastal strip (approx. 400 km N–S and <20 km inland) in five geographically separated areas (Fig. 1). Syzygium paniculatum is listed as endangered in the state Threatened Species Conservation (TSC) Act 1995 (NSW) Genetic diversity and vulnerable in the Federal Environment Protection and Biodiversity Conservation (EPBC) Act 1999, partly because Because of the unconventional nature of the genotyping data population sizes are small, rarely exceeding 20 individuals. It obtained for S. paniculatum, genetic variation within and has dark dense foliage and produces white flowers in summer between populations was measured using both an allelic and bright-pink to magenta fruit from late summer into approach (where alleles present at each locus were scored by autumn. As in most Myrtaceae, the floral morphology sug- size) and a binary approach (where all alleles were scored as gests a generalist pollination syndrome and this has been either present or absent). demonstrated in other Syzygium species by Hopper (1980) The program atetra (van Puyvelde et al. 2009) was used and Crome and Irvine (1986), who recorded a wide range of for the allelic approach. atetra performs Monte Carlo simu- floral visitors. However, little is known about fruit dispersal in lations to account for probable combinations of allele copy S. paniculatum; the fruits are palatable, but we saw no evi- number in polyploid datasets with partial heterozygotes. dence of birds or small mammals feeding on them during Genetic diversity statistics were calculated including Hardy- fieldwork for this study. Weinberg expected heterozygosity (He) and Shannon- Sampling was aimed at obtaining an extensive account of Weiner diversity index (H′) (Nei 1987), Nei’s measure of the genetic diversity across the geographical distribution of population differentiation (GST), the interpopulational gene the species. Eleven populations were sampled across the diversity in relation to intrapopulational gene diversity (RST), known range of S. paniculatum (Fig. 1). As populations were and Gene Diversity (Dm) (Nei 1973) as well as Nei’s generally small, samples from all accessible individuals iden- Genetic Distance (D) (Nei 1972, 1978). tified in this study and during previous surveys (Mills 1996; Using the binary approach (each allele for each locus was A.N. Rodd, pers. comm., 2008) at each of the listed sites scored as either present or absent across all individuals), were collected. In total, samples from 79 adult trees were principal coordinates analysis (PCoA; calculated at popula- obtained.The largest population consisted of 31 individuals, tion level because of the limited number of genets through- with all other populations comprising one to 12 individuals. out the sample) and analysis of molecular variance Leaf samples were freeze-dried or silica-gel-dried, and stored (AMOVA) were calculated using the program GenAlEx v6.3 at -20°C for later DNA extraction. Total genomic DNA was (Peakall & Smouse 2006). extracted using DNeasy 96 plant kits (Qiagen, Hilden, Germany). Polyembryony and relative fitness

Molecular analyses Experimental trials were conducted to investigate the origin of embryos, the viability of embryos and the relative ﬁtness of Nine nuclear simple sequence repeat (nSSR) markers spe- offspring. Albeit scarce, fruit was collected during the two ciﬁcally developed for S. paniculatum were used, based on collecting seasons over which the study was conducted doi:10.1111/j.1442-9993.2011.02353.x © 2012 The Authors Austral Ecology © 2012 Ecological Society of Australia REPRODUCTIVE BET-HEDGING IN SYZYGIUM PANICULATUM 939

(2008, 2009) from four populations, Abrahams Bosom The most common genotype (genotype 1) was Reserve (AB), Towra Point Nature Reserve (TP), The present in 56 (71.79%) individuals across six popu- Entrance Peninsular (TE) and Cams Wharf (CW) (Fig. 1). lations: CW, TE, WL, TP, CC and Conjola National Over both seasons, 129 seeds were dissected and embryos Park (CN) (Appendix S1). At four of these popula- counted, and the 68 collected in the second season were also tions – CW,TE, TP and CC – four unique secondary weighed. An unpaired t-test was performed to determine genotypes were also found (one genotype in each whether the difference in size between embryo 1 (largest by weight and/or first to germinate) and all other embryos in the population found in one individual). The secondary seed was statistically significant. genotype found at TP was the only genotype present Fifty-one whole seeds (13 with single embryos) and seven at AB; this genotype possessed only a single base pair seeds with embryos separated, with a total of 30 embryos, change in length (Appendix S1). Four populations were germinated in controlled conditions at the Australian possessed a single unique genotype that was not Botanic Garden, Mount Annan, NSW. Seeds were sown found in any other populations. These were the three fresh on agar plates (non-aseptic 8g L-1, non-nutrient) and northernmost populations, Green Point (GP), Salts germinated at 20°C on a cycle of 12-h light, 12-h dark. Bay (SB) and Sugarloaf Point (SP), as well as Germinated seedlings were measured for leaf number and Ourimbah Creek Valley (OC), which was the popu- height at regular intervals. Using an unpaired t-test, height, lation furthest from the coast. Genotypes were leaf number and branch number across weeks were com- distributed along a longitudinal gradient with north- pared between seedling 1 and all other seedlings in the seed. A linear regression analysis was then performed in Microsoft ernmost genotypes displaying the most allelic devia- Excel for embryo weight versus seedling height at 21 weeks. tions from genotype 1 (Appendix S1). The above statistical methods were repeated using only indi- All analyses supported significant differentiation viduals that had been genotyped, to look for correlations between a southern group of eight populations and the between height, seed size and sexuality. three northern populations. The within-north and

north versus south GST,RST, D and Dm values were higher than the within-south values, suggesting much higher genetic differentiation and diversity in the Sexuality and relative ﬁtness northern populations. The PCoA (Fig. 2) showed the southern popula- Leaf material was collected from the germinated seedlings tion group to be strongly differentiated from the from AB, TP, TE and CW (16 family groups – 10 samples from monoembryonic seeds and 21 samples from six poly- northern populations. Analysis of molecular variance embryonic seeds) in addition to collections of wild seedlings comparing northern and southern regions partitioned from Captain Cook Drive (CC) (three samples) and the variance with 4% (PhiRT 0.762; P < 0.01) within Wamberal Lagoon Nature Reserve (WL) (four samples). populations, 19% (PhiPT 0.956; P < 0.01) among Leaf material from maternal parents and seedlings was geno- populations and 77% (PhiPR 0.816; P < 0.01) typed to determine sexual or asexual origin. Once recombinant (‘sexual’) or asexual origin was determined, means were calculated for height of seedling 1 (sexual) versus height of embryo 1 (asexual) and a t-test performed to determine if the difference between sexual and asexual embryos was signiﬁcant.

RESULTS

Genetic diversity Fig. 2. Principal coordinates analysis plot for Syzygium A total of 39 alleles were found across nine nSSR paniculatum populations illustrating pairwise genetic distance between the 11 populations sampled. Principal coor- markers, with the number of alleles per locus ranging dinates analysis was produced from a binary distance from one (SP116) to six (SP33, SP54, SP85.1). Allele matrix and performed in GenAlEx. Southern populations size range was greatest in SP33 (28 bp), while a single (Abrahams Bosom Reserve (AB), Cams Wharf (CW), allele was ampliﬁed for SP116 across the entire species Captain Cook Drive (CC), Conjola National Park (CN), (Appendix S1). Average within-population diversity Towra Point Nature Reserve (TP), Wamberal Lagoon measures for S. paniculatum were He = 0.466 and Nature Reserve (WL), Ourimbah Creek Valley (OC) and The Entrance Peninsular (TE)) are grouped together H′ 0.740. The 79 adult individuals representing 11 = (shown in ﬁgure by dotted line), while the northernmost populations and the entire geographical range of populations (Green Point (GP), Salts Bay (SB) and Sugar- S. paniculatum produced only nine genotypic combi- loaf Point (SP)) remain distinctly separated from the south- nations from those 39 alleles. ern group.

between regions. Conjola National Park and GP were 80 a) excluded because AMOVA does not allow populations 70 with only one individual. 60 50 40 First Polyembryony and relative ﬁtness 30 Rest 20 Leaves (No.) Seed dissections conﬁrmed polyembryony in 10 S. paniculatum with embryo number ranging from one 0 to nine per seed (mean = 3.08; n = 129). Seeds that –10 contained only one embryo totalled 28.8% of all seeds dissected; however, this is likely to be an overestima- tion of monoembryony as many undifferentiated cell 7 b) structures (potential embryos) were observed at the 6 time of dissection and there was evidence of embryos 5 having been attacked by insect larvae. 4 The majority of polyembryonic seeds contained 3 one large embryo (embryo 1) and any subsequent 2

embryos were much smaller. An unpaired t-test on Branches (No.) the 58 second-year seeds for which embryos were 1 weighed revealed a signiﬁcant difference between the 0 weight of embryo 1 and embryo 2 (two-tail t = 10.06, –1 d.f. = 52, P < 0.001) and embryo 1 and all other embryos in the seed (two-tail t = 6.15, d.f. = 52, < 50 c) P 0.001). 45 All 58 seeds (51 whole and seven dissected) germi- 40 nated, with multiple seedlings arising from the polyem- 35 bryonic seeds. In 90.3% of seeds, embryo 1 became 30 seedling 1 (the tallest seedling) for the duration of the 25 measurement period (Fig. 3 summarizes the results for 20

Height (cm) 15 the three measurements). An unpaired t-test showed 10 that mean height for seedling 1 across the entire mea- 5 surement period was significantly different from that 0 of all other seedlings (two-tail t = 20.96, d.f. = 93, –5 6 weeks 12 weeks 21–22 weeks P < 0.001). A linear regression analysis performed on a smaller subset of weighted embryos showed a signifi- Fig. 3. Box plots summarizing measured values at cant association between embryo weight and seedling 6, 12 and 21/22 weeks for first seedling (grey shading) height (R2 = 0.573; P < 0.001). Leaf number and and the rest of the seedlings (no shading): (a) data for leaf number, (b) data for branch number, (c) data for branch number followed the same pattern as height height. throughout the period of measurement, with seedling 1 having the highest leaf number and branch number (Fig. 3). However, while differences in leaf number were significant (two-tail t = 15.02, d.f. = 93, to the parent plant. Considering that testing for P < 0.001), differences in branch length across the linkage disequilibrium in polyploid organisms is entire measurement period were not significant. problematic, similar patterns could be obtained in

the unlikely event of all loci being linked (e.g. Pcgen (Sydes & Peakall 1998) suggests that the likelihood Sexuality and relative fitness of genotype 1 to occur so many times by chance through recombination among the adult individuals All seedlings tested were from the southern group of is P = 2.4 ¥ 10-7). The term ‘sexual’ is used to repre- eight S. paniculatum populations where little or no sent offspring that differed from the parent plant diversity was found within or between populations. In and were likely to be recombinant. Seedlings from general, a higher proportion of sexuality was found in both monoembryonic and polyembryonic seeds offspring from populations at the northernmost end of showed both sexuality and asexuality in almost all the southern genetic group of populations (CW and combinations (Appendix S2). Seven out of nine seed- TE; Appendix S2). The term ‘asexual’ is used here lings from monoembryonic seeds were sexual, and five with caution to represent offspring that were identical of seven polyembryonic seeds produced one sexual doi:10.1111/j.1442-9993.2011.02353.x © 2012 The Authors Austral Ecology © 2012 Ecological Society of Australia REPRODUCTIVE BET-HEDGING IN SYZYGIUM PANICULATUM 941 seedling. Out of 38 offspring genotyped, 26 (72.7%) Loss of genetic diversity and increased genetic were asexual and the remaining 12 were sexual divergence along the latitudinal gradient could be the (Appendix S2). result of vicariance and drift caused by the combined Of the 12 sexual seedlings, one seedling possessed effect of the expansion/contraction cycles of the Qua- an allele that was not found elsewhere across the ternary and more recent anthropogenic pressures samples taken of the species. An additional five pos- (Floyd 1990; Payne 1991). Additionally, the lower sessed alleles that were not found in samples taken divergence among the southern populations (includ- from the population the seedling originated from, but ing multiple populations containing many ramets of were found elsewhere across the species. Two of the one genet) suggests that the southern distribution these, one from AB (from a monoembryonic seed) could be the result of a small number of founder and one from CC, contained only a single base-pair events (as previously described in Erythroxylum pusil- allelic change in a single locus (potentially a somatic lum for example; van der Merwe et al. 2010). The mutation rather than sexual recombination; van der production of dispersible asexually produced seed Merwe et al. 2010; Gross et al. 2012). The remain- could have facilitated the fast spread of genetically ing six showed changes in heterozygosity (parent identical individuals across the southern distribution was heterozygous or partially heterozygous and off- of this tree. Thus, in these genetically depleted popu- spring was partially heterozygous or homozygous; lations new diversity is most likely to arise via migra- Appendix S2). tion from the north, by occasional sexual events, or Sexual seedlings were not always tallest and sexual through somatic mutation (van der Merwe et al. seedling 1 was not significantly taller than asexual 2010; Gross et al. 2012). seedling 1. In polyembryonic seeds that contained one sexual and one asexual embryo, the tallest germinated seedling was sexual. However, in polyembryonic seeds Polyploidy and polyembryony that contained more than two embryos (one being sexual), the tallest germinated seedling was asexual. The usual ploidy level in the Myrtaceae is diploid An unpaired t-test for mean height of sexual seedling 1 (Rye 1979; Vijayakumar & Subramanian 1985; (tallest sexual seedling from a polyembryonic seed) Rye & James 1992), but polyploidy is a frequent occur- and mean height of asexual seedling 1 (tallest asexual rence in some genera, and in Syzygium it has been seedling from a polyembryonic seed) was not signifi- recorded in S. jambos and Syzygium cumini (L.) Skeels cant, suggesting that sexuality does not directly influ- (NicLughadha & Proenca 1996). Our genotypic data ence seedling height. indicate that S. paniculatum is a polyploid. An increase in ploidy levels has been frequently associated with apomixis (Rye 1979; Nogler 1984; Bierzychudek 1987; Mogie 1992; Roche et al. 2001; Richards 2003; Bicknell & Koltunow 2004). Carman (1997) sug- DISCUSSION gested a positive correlation between polyploidy and apomixis, and proposed the hypothesis that polyem- Distribution of diversity bryony and other reproductive anomalies ‘result from asynchronously-expressed duplicate genes in polyp- Syzygium paniculatum is an atypical rare species, loids, mesopolyploids, or paleopolyploids’. On the comprising a few small populations that cover a geo- other hand, Asker and Jerling (1992) indicated that, graphical range of about 400 km. In comparison to a in general, adventitious embryony is much more related species of similar geographical range within common in diploids than in polyploids.The hypothesis Australia, Syzygium nervosum DC. (Shapcott 1998), mentioned by Whitton et al. (2008), that pollen limi- S. paniculatum has extremely low genetic diversity tation might selectively favour adventitious embryony, with a large number of individuals having identical does not seem applicable here; the temperate littoral genotypes. Syzygium paniculatum also displays distinct rainforest communities where this species occurs are partitioning of genetic diversity. In particular, the three not sufficiently species-rich to significantly reduce northern populations (GP, SB and SP) each displayed pollinator visitation. distinctive genotypes, suggesting the presence of a Whitton et al. (2008) also suggested that both strong genetic and geographical divide between the apomixis and polyploidy may be selectively favoured northern populations and the larger southern group in allopolyploid hybrids as a mechanism to avoid of populations. The genetic diversity evident in hybrid breakdown. Soltis and Soltis (2000) reported S. paniculatum populations does not appear to relate to that most polyploid plant species that have been population size, with both the largest and smallest examined using molecular markers ‘have been populations existing within the southern, less geneti- shown to be polyphyletic, having arisen multiple cally diverse region. times from the same diploid species’, but we found

© 2012 The Authors doi:10.1111/j.1442-9993.2011.02353.x Austral Ecology © 2012 Ecological Society of Australia 942 K. A. G. THURLBY ET AL. no evidence that this was the case with S. panicula- lations, S. paniculatum is currently persisting locally tum. Preliminary sequencing of other taxa could as well as producing viable, dispersible sexual and not identify likely parental sources for an allo- asexual seed. polyploid origin scenario for this species (data not Although sexual individuals do not always survive presented). in preference to asexual ones, this rainforest tree could be hedging its bets with the potential to receive the benefits of both reproductive mechanisms (Kol- tunow 1993). In the long term, sexual events might Sexual versus asexual reproduction and fitness enhance the adaptive potential of S. paniculatum, while asexual events give it the capacity to persist This study has shown that S. paniculatum seedlings locally and capitalize on expansion opportunities can be of sexual or asexual origin, but that successful even if sexual reproduction fails. Additionally, the recombinant events are relatively uncommon. Most polyploid nature of S. paniculatum in effect creates an forms of apomixis require fertilization to trigger the added buffering system whereby the potential rate production of single or multiple asexual embryos and at which fit alleles are lost through sexual recombi- sexual embryos can persist in the seed alongside devel- nation is reduced. Finally, polyembryony could oping adventive embryos (Ganeshaiah et al. 1991; also minimize the impact of insect seed predators. Asker & Jerling 1992; Koltunow & Grossniklaus 2003; In a recent study, Juniper and Britton (2010) Richards 2003; Whitton et al. 2008). However, detected a minimum of three seed predators in fruits because sexual and asexual embryos must compete for of this species: one weevil (Sigastus sp.), one wasp the available endosperm resources, the sexual embryo (Anselmella miltoni) and the Guava moth (Coscinopty- is sometimes outcompeted (Roy 1953; Naraya- cha improbana). Interestingly, these authors noted naswami & Roy 1960; Whitton et al. 2008). We found that despite fruits and seeds being severely damaged that when seeds in S. paniculatum contained two at times, some seedlings still emerged. embryos, the largest was the sexual embryo, but in Regardless, low population numbers and low diver- seeds containing more than two embryos the sexual sity still make S. paniculatum susceptible to extreme embryo was never the largest. This suggests that the stochastic events (such as current threats like myrtle sexual embryo might be outcompeted by proliferation rust; see Carnegie & Cooper 2011), especially with of asexual embryos. limited evidence for sexual individuals surviving in the Embryo weight is correlated with seedling size, but wild. While the species is currently able to persist with no evidence of genetic influence on seedling size. locally and colonize new habitat, suitable new habitat The largest embryo always produces a significantly may no longer exist because of extensive clearing of larger seedling, but the largest embryo is not always littoral rainforests. In the future, artificial cross- the sexual one. Large seeds (or embryos) provide seed- fertilization and fitness experiments will enable us to lings with an energy source that is important for test the relative fitness of specific genotype com- increased shade tolerance for seedlings germinating binations and further support decision making in a under dense canopies and for increasing height translocation scenario. where light gradients exist (Leishman & Westoby 1994; Myers & Kitajima 2007). Considering that S. paniculatum embryos have storage cotyledons, it is ACKNOWLEDGEMENTS likely that the largest seedling is produced by the embryo with the largest energy store, rather than by We acknowledge the Australian Flora Foundation for the one with the sexual origin. funding, Chris Allen, Sven Delaney, Louise Lutz- Mann, Margaret Heslewood, Robert Kooyman, Hannah McPherson, Rohan Mellick, Amanda Rolla- son, Tony Rodd, Paul Rymer and Marlien van der Bet-hedging: survival strategies for Merwe for technical support and/or advice, and Syzygium paniculatum reviewers and editors for their useful comments.

A number of studies (Wollemia nobilis Peakall et al. 2003; Elaeocarpus williamsianus Rossetto et al. 2004; REFERENCES Eidothea hardeniana Rossetto & Kooyman 2005) have shown that rare rainforest trees can successfully Asker S. E. & Jerling L. (1992) Apomixis in Plants. CRC Press, persist through historical environmental changes with Boca Raton. low diversity because of persistence mechanisms Batygina T. B. & Vinogradova G. Y. (2007) Phenomenon of such as asexual reproduction. Despite low popula- polyembryony. Genetic heterogeneity of seeds. Russ. J. Dev. tion numbers and low genetic diversity in wild popu- Biol. 38, 126–51. doi:10.1111/j.1442-9993.2011.02353.x © 2012 The Authors Austral Ecology © 2012 Ecological Society of Australia REPRODUCTIVE BET-HEDGING IN SYZYGIUM PANICULATUM 943

Bicknell R. A. & Koltunow A. M. (2004) Understanding apo- Mills K. (1996) Illawarra Vegetation Studies: Littoral Rainforest in mixis: recent advances and remaining conundrums. Plant Southern NSW, Inventory, Characteristics and Management. Cell 16, S228–45. Illawarra Vegetation Studies (1). Coachwood Publishing, Bierzychudek P. (1987) Patterns in plant parthenogenesis. Expe- Jamberoo. rientia 41, 1255–64. Mogie M. (1992) The Evolution of Asexual Reproduction in Plants. Callaghan T.V., Carlsson B. A., Jonsdottir I. S., Svensson B. M. Chapman & Hall, London. & Jonasson S. (1992) Clonal plants and environ- Myers J. A. & Kitajima K. (2007) Carbohydrate storage mental change: introduction to the Proceedings and enhances seedling shade and stress tolerance in a Neotropi- summary. Oikos 63, 341–7. cal forest. J. Ecol. 95, 383–95. Carman J. G. (1997) Asynchronous expression of duplicate Narayanaswami S. & Roy S. K. (1960) Embryo sac development genes in angiosperms may cause apomixis, bispory, tet- and polyembryony in Syzygium cumini (Linn.) Skeels. Bot. raspory, and polyembryony. Biol. J. Linn. Soc. Lond. 61, Not. 113, 273–84. 51–94. Naumova T. (1992) Apomixis in Angiosperms: Nucellar and Integu- Carnegie A. J. & Cooper K. (2011) Emergency response to the mentary Embryony. CRC Press, Boca Raton. incursion of an exotic myrtaceous rust in Australia. Nei M. (1972) Genetic distance between populations. Am. Nat. Australas. Plant Pathol. 40, 346–59. 106, 283–92. Crome F. H. J. & Irvine A. K. (1986) ‘Two Bob Each Way’: the Nei M. (1973) Analysis of gene diversity in subdi- pollination and breeding system of the Australian rain forest vided populations. Proc. Natl Acad. Sci. USA 70, 3321– tree Syzygium cormiflorum (Myrtaceae). Biotropica 18, 115– 3. 25. Nei M. (1978) Estimation of average heterozygosity and genetic Eckert C. G., Dorken M. E. & Mitchell S. A. (1999) Loss of sex distance from a small number of individuals. Genetics 89, in clonal populations of a flowering plant, Decodon verticil- 583–90. latus (Lythraceae). Evol. Ecol. 53, 1079–92. Nei M. (1987) Molecular Evolutionary Genetics. Columbia Uni- Floyd A. G. (1990) Australian Rainforests in New South Wales. versity Press, New York. Surrey Beatty & Sons, Chipping Norton. NicLughadha E. & Proenca C. (1996) A survey of the reproduc- Ganeshaiah K. N., Shaanker R. U. & Joshi N. V. (1991) Evolu- tive biology of the Myrtoideae (Myrtaceae). Ann. Mo. Bot. tion of polyembryony: consequences to the fitness of mother Gard. 83, 480–503. and offspring. J. Genet. 70, 103–27. Nogler G. A. (1984) Gametophytic apomixis. In: Embryology of Gitzendanner M. & Soltis P. (2000) Patterns of genetic variation Angiosperms (ed. B. M. Johri) pp. 475–518. Springer-Verlag, in rare and widespread plant congeners. Am. J. Bot. 87, Berlin. 783–92. Otto S. P. (2009) The evolutionary enigma of sex. Am. Nat. 174, Green R. F. & Noakes D. L. G. (1995) Is a little bit of sex as good 1–14. asalot?J.Theor. Biol. 174, 87–96. Paun O., Greilhuber J., Temsch E. M. & Hörandl E. (2006) Gross C. L., Nelson P. A., Haddadchi A. & Fatemi M. (2012) Patterns, sources and ecological implications of clonal Somatic mutations contribute to genotypic diversity in diversity in apomictic Ranunculus carpaticola (Ranunculus sterile and fertile populations of the threatened shrub, Gre- auricomus complex, Ranunculaceae). Mol. Ecol. 15, 897– villea rhizomatosa (Proteaceae). Ann. Bot. doi: 10.1093/aob/ 910. mcr283. Payne R. (1991) New findings of the rare tree Syzygium panicu- Harper J. L. (1978) The demography of plants with clonal latum (Myrtaceae) in the Wyong area, New South Wales. growth. In: Structure and Functioning of Plant Populations Cunninghamia 2, 495–8. (eds A. J.W. Freysen & J.W. Woldendorp) pp. 27–48. North- Peakall R., Ebert D., Leon J. S., Meagher P. F. & Offord C. A. Holland Publishing, Amsterdam. (2003) Comparative genetic study confirms exceptionally Hopper S. D. (1980) Pollination of the rainforest tree Syzy- low genetic variation in the ancient and endangered relictual gium tierneyanum (Myrtaceae) at Kuranda, Northern conifer, Wollemia nobilis (Araucariaceae). Mol. Ecol. 12, Queensland. Aust. J. Bot. 28, 223–37. 2331–43. Hörandl E. (2004) Comparative analysis of genetic divergence Peakall R. & Smouse P. E. (2006) GENALEX 6: genetic analysis among sexual ancestors of apomictic complexes using in Excel. Population genetic software for teaching and isozyme data. Int. J. Plant Sci. 165, 615–22. research. Mol. Ecol. Notes 6, 288–95. Houliston G. J. & Chapman H. M. (2004) Reproductive Richards A. J. (2003) Apomixis in flowering plants: an over- strategy and population variability in the facultative view. Philos. Trans. R. Soc. Lond., B, Biol. Sci. 358, 1085– apomict Hieracium pilosella (Asteraceae). Am. J. Bot. 91, 93. 37–44. Roche D., Hanna W. W. & Ozias-Akins P. (2001) Is Juniper P. A. & Britton D. R. (2010) Insects associated with the supernumerary chromatin involved in gametophytic fruit of Syzygium paniculatum (Magenta Lillypilly) and apomixis of polyploid plants? Sex. Plant Reprod. 13, 343– Syzygium australe (Brush Cherry). Aust. J. Entomol. 49, 296– 9. 303. Rossetto M., Gross C. L., Jones R. & Hunter J. (2004) The Koltunow A. M. (1993) Apomixis: embryo sacs and embryos impact of clonality on an endangered tree (Elaeocarpus wil- formed without meiosis or fertilization in ovules. Plant Cell liamsianus) in a fragmented rainforest. Biol. Conserv. 117, 5, 1425–37. 33–9. Koltunow A. M. & Grossniklaus U. (2003) Apomixis: a devel- Rossetto M. & Kooyman R. M. (2005) The tension between opmental perspective. Annu. Rev. Plant Biol. 54, 547– dispersal and persistence regulates the current distribution 74. of rare palaeo-endemic rain forest flora: a case study. J. Ecol. Leishman M. R. & Westoby M. (1994) The role of large seed size 93, 906–17. in shaded conditions: experimental evidence. Funct. Ecol. 8, Roy S. K. (1953) Embryology of Eugenia jambos L. Curr. Sci. 8, 205–14. 249–50.

Roy S. K. (1961) Embryology of Eugenia fruticosa L. Proc. Natl van der Merwe M., Spain C. S. & Rossetto M. (2010) Enhanc- Acad. Sci. India Section B 31, 80–7. ing the survival and expansion potential of a founder Rye B. L. (1979) Chromosome number variation in the Myrta- population through clonality. New Phytol. 188, 868– ceae and its taxonomic implications. Aust. J. Bot. 27, 547– 78. 73. van Dijk P. & van Damme J. (2000) Apomixis technology and the Rye B. L. & James S. H. (1992) The relationship between dys- paradox of sex. Trends Plant Sci. 5, 81–4. ploidy and reproductive capacity in Myrtaceae. Aust. J. Bot. van Puyvelde K., van Geert A. & Triest L. (2009) ATETRA, a 40, 829–848. new software program to analyse tetraploid microsatellite Shapcott A. (1998) Vagile but inbred: patterns of inbreeding and data: comparison with TETRA and TETRASAT. Mol. Ecol. the genetic structure within populations of the monsoon Resour. 10, 331–4. rain forest tree Syzygium nervosum (Myrtaceae) in Northern Vijayakumar N. & Subramanian D. (1985) Cytotaxono- Australia. J.Trop. Ecol. 14, 595–614. mical studies in South Indian Myrtaceae. Cytologia 50, Silvertown J. (2008) The evolutionary maintenance of sexual 513–20. reproduction: evidence from the ecological distribution of Webber J. M. (1940) Polyembryony. Bot. Rev. 6, 575– asexual reproduction in clonal plants. Int. J. Plant Sci. 169, 98. 157–68. Whitton J., Sears C. J., Baack E. J. & Otto S. P. (2008) The Singhal V. K., Gill B. S. & Bir S. S. (1985) Cytology of woody dynamic nature of apomixis in the Angiosperms. Int. J. Plant species. Proc. Indiana Acad. Sci. 94, 607–17. Sci. 169, 169–82. Soltis P.S. & Soltis D. E. (2000) The role of genetic and genomic Zhang Y. & Zhang D. (2007) Asexual and sexual reproduc- attributes in the success of polyploids. Proc. Natl Acad. Sci. tive strategies in clonal plants. Front. Biol. China 2, 256– USA 97, 7051–7. 62. Sydes M. & Peakall R. (1998) Extensive clonality in the endangered shrub Haloragodendron lucasii (Haloragaceae) revealed by allozymes and RAPDs. Mol. Ecol. 7, 87– 93. SUPPORTING INFORMATION Talent N. (2009) Evolution of gametophytic apomixis in ﬂower- ing plants: an alternative model from Maloid Rosaceae. Theory Biosci. 128, 121–38. Additional Supporting Information may be found in Thurlby K. A. G., Connelly C., Wilson P. G. & Rossetto M. the online version of this article: (2011) Development of microsatellite loci for Syzygium paniculatum (Myrtaceae), a rare polyembryonic rainforest tree. Appendix S1. Summary of adult genotypic data. Conserv. Genet. Resour. 3, 205–8. Appendix S2. Summary of genotypic data from Vallejo-Marín M., Dorken M. E. & Barrett S. C. H. (2010) parents and offspring. Ecological and evolutionary consequences of clonality for plant mating. Annu. Rev. Ecol. Evol. Syst. 41, 193–213.