Reproductive biology of the Magenta Lilly Pilly



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Reproductive biology of the Magenta Lilly Pilly (Syzygium 

paniculatum) and its implications for conservation 

 

Katie A. G. Thurlby



1, 2

, William B. Sherwin

2

, Maurizio Rossetto



1

, and Peter G. 

Wilson



1



National Herbarium of NSW, Botanic Gardens Trust, Mrs Macquaries Rd Sydney, NSW 

2000 


2

School of Biotechnology and BioMolecular Sciences, University of New South Wales, 

NSW 2052 

 

 



 

TABLE OF CONTENTS

 

Abstract 

 

 

 

 

 

 

 

 

 



1. Introduction 

 

 

 

 

 

 

 

 



1.1.

 

Syzygium paniculatum: a brief description 

 

 

 



1.2.

 

Threatened species: population size and diversity  

 

 



1.3.

 

 Polyembryony, clonality and Syzygium paniculatum 

 

 



1.4.

 

 Fitness, clonality and Syzygium paniculatum 

 

 

 

11 

1.5.

 

Ploidy in Syzygium paniculatum 

 

 

 

 

 

11 

1.6.

 

Applicability of nuclear microsatellite 

markers 

   11 

1.7.

 

Aims   

 

 

 

 

 

 

 

 

12 

2. Materials and Methods   

 

 



 

 

 



 

12 

2.1.

 

Obtaining and analyzing diversity data   

12 

2.1.1.

 

Sample collection  

 

 

 

 

 

 

12 

2.1.2.

 

DNA 

preparation 

      12 

2.1.3.

 

Ploidy 

identification 

      14 

2.1.4.

 

Analysis 

of 

diversity 

data 

    

2.2.

 

Obtaining and analyzing germination and reproduction data   

16 

3. Results 

 

 

 

 

 

 

 

 

 

16 

3.1 

Ploidy 

         16 

3.2 

Diversity 

 

        18 

 

2



 

3.3 

Reproduction 

        21 

3.4 

Germination 

        21 

4. 

Discussion 

       

 

 

  

 

 

 

 

23 

 

4.1.

 

Ploidy 

 

 

 

 

 

 

        

 

23 

4.2.

 

Diversity 

 

 

 

 

 

 

        

 

23 

4.3.

 

Reproduction 

 

 

 

 

 

 

 

24 

4.4.

 

 Germination 

 

 

 

 

 

        

 

24 

4.5.

 

Conservation and horticultural implications 

 

 

 

26 

References 

       

 

 

 

 

 

 

 

  27 

Abstract 

The Magenta Lilly Pilly (Syzygium paniculatum), endemic to a narrow strip along the 

New South Wales coast, is currently listed as vulnerable at both state and national 

levels. At present management of the species focuses on minimizing currently known 

threats, such as weed invasion, while little is known about the reproductive biology of 

the species. S. paniculatum is the only recorded polyembryonic Australian species of 

Syzygium; polyembryony being the development of multiple (and often asexual) 

embryos in one seed. Nuclear microsatellite markers were used to investigate the 

genetic outcome of polyembryony on the reproductive and population biology of the 

species focusing particularly on the population located on The Entrance Peninsular. 

Low within-population diversity was found, with low heterozygosity levels and a low 

level diversity indices when compared to other rare or rainforest species. Multiple 

embryos from single seeds were found to be identical to the mother. Multiple embryos 

 

3



 

germinated and survived but one seedling was always significantly taller than all 

others in the seed but was not considered sexual. It was concluded that the rare S. 

paniculatum is an apomictic clonal species with extremely low genetic diversity.  

1

 

Introduction

 

1.1.

 

Syzygium paniculatum: a brief description 

Syzygium paniculatum Gaertn., commonly known as Magenta Lilly Pilly, is a small 

to medium tree in the family Myrtaceae. S. paniculatum is endemic to New South Wales, 

occurring along a narrow, linear, coastal strip in five separate geographical areas: Jervis Bay, 

Coalcliff, Botany Bay, Central Coast and Seal Rocks (Figure 1). At Jervis Bay and Coalcliff, 



S. paniculatum grows in remnant stands of littoral rainforest in grey soils over sandstone. At 

Botany Bay, the Central Coast, and Seal Rocks, the species can be found growing in 

remnant littoral rainforest patches and riverside gallery rainforests on sandy soils, stabilized 

dunes, gravels, silts or clays. Plants produce clusters of white flowers at the ends of 

branches, usually between November and February. The fruits are deep magenta, maturing 

from March to June, and containing a single seed, which is often polyembryonic (containing 

more than one embryo, see Figures 2, 3, 4 & 5).  

S. paniculatum is of conservation significance as it is listed as endangered under 

NSW Threatened Species Conservation Act 1995 (TSC Act) and vulnerable under Federal 

legislation. If appropriate conservation of S. paniculatum is to take place, to increase 

population numbers and protect the species from extinction, natural populations need to be 

studied to determine genetic diversity, breeding strategy and fitness potential of offspring. 

 

4



 

An understanding of the reproductive biology of this species is likely to be crucial because 

the polyembryonic seeds may have arisen through asexual apomixis.  

If polyembryony in Syzygium paniculatum is a product of apomixis, it is likely to 

have substantial implications for any recovery plan because apomixis may reduce genetic 

diversity within each population and increase genetic divergence between populations. 

Although uncommon in native vegetation communities, the species is widely 

cultivated and a number of cultivars are on sale commercially. Genetic distinctiveness and 

reproductive biology could also have important implications for potential interactions 

between cultivars and natural populations.  

 

 

5



 

 

Seal  



Rocks 

Botany Bay 

Coalcliff 

Central Coast 

Jervis Bay 

Figure 1. Distribution of S. paniculatum along the NSW coast and indicates the five 

geographically separated areas in which Syzygium paniculatum occurs; Seal Rocks 

(orange), Central Coast (blue), Botany Bay (red), Coalcliff (pink) and Jervis Bay 

(green).  

 

 

6



 

 

Figure 2. Botanical illustration of S. paniculatuma. fruiting branch – scale bar 3cm, 



b. leaf venation – scale bar 2.5cm, c. opening bud – scale bar 1cm, d. flower side-view 

 scale bar 1cm, e. transverse section of fruit showing seed containing multiple 

embryos – scale bar 1.5cm. (L. Elkan 2008 © Botanic Gardens Trust) 

 

1.2



 

Threatened species: population size and diversity 

The fragmentation and isolation of habitats, due to human disturbance or geological 

change, has resulted in the reduction in population size of many species of plants. Small 

 

7



 

population size is often a characteristic of threatened species (Frankham, 1996) and has been 

found to be positively correlated with genetic variation (Leimu et al., 2006). Reduced 

population size can lead to reduced evolutionary potential due to genetic drift and inbreeding 

(Frankham, 1999). Genetic drift and inbreeding may result in marked genetic effects for 

populations such as reduced genetic diversity through changes in allele frequencies and loss 

of heterozygosity (Allendorf & Luikart, 2007; Frankham, 1996; Frankham, 1999). Thus the 

potential for rapid erosion of genetic variation may make small populations poor candidates 

for conservation efforts (Lesica & Allendorf, 1992). However,

 

it is not always the case that 



rare species with reduced population sizes have low diversity (Gitzendanner & Soltis, 2000; 

Lesica & Allendorf, 1992; Lewis & Crawford, 1995; Ranker, 1994). Because genetic 

diversity is more likely to depend on the life history and evolutionary history of species, it 

cannot be assumed that all rare species will maintain low levels of genetic diversity 

(Gitzendanner & Soltis, 2000)

.

  



1.3

 

Polyembryony, clonality and Syzygium paniculatum 

There are a number of different processes by which multiple embryos (polyembryony) can 

arise in plant seeds (Stebbins, 1941; Naumova, 1992; Ozias-Askins, 2006; Richards, 2003). 

Polyembryony can occur through the division of a fertilized diploid zygotic embryo 

(cleavage polyembryony), resulting in the development of multiple yet identical sexually 

derived embryos. It can also arise through various forms of apomixis, where additional 

asexual embryos develop from diploid cells, usually alongside the fertilized diploid zygotic 

embryo. The latter may result in the development of both sexual and asexual embryos (if a 

zygote develops and survives), or only asexually derived embryos (if the zygote fails to 

develop or develops and subsequently degenerates or is out-competed). Apomixis can 

therefore, in some cases, result in the production of high levels of clonal individuals in a 

 

8



 

population, who have inherited their entire genome from their mother. This has the potential 

to drastically reduce the genetic diversity in the population or across the species if the clonal 

offspring of one mother happen to predominate. Also, if sexual reproduction is reduced (in 

facultative apomicts) or virtually non-existent (in obligate apomicts), the rate at which new 

combinations of alleles are made will also be reduced, in effect, hindering potential increase 

in genetic diversity. Apomicts can acquire genetic variability through means such as somatic 

mutation or somatic recombination; however most of the genetic variability found within 

apomictic species is a product of past sexuality (Richards, 2003). 

Polyembryony has not been recorded for any other Australian species of Syzygium

but has been observed in a number of Asian species: S. malaccense (L.) Merr. & L.M.Perry

S. jambos (L.) Alstonand S. cumini (L.) Skeels. In S. jambos, Roy (1953) described the 

embryos as deriving from diploid cells in the nucellus, the maternal tissue surrounding the 

embryo sac. In Myrtaceae, formation of triploid endosperm (after pollination) is a source of 

nutrition to developing sexual embryos and this is also the case for nucellar embryos (Roy, 

1961). This pollination requirement suggests that polyembryony in Syzygium paniculatum is 

not brought on by male sterility or lack of pollination. Sahai & Roy (1962) report the 

formation of a zygote that degenerates soon after fertilization so that only asexually derived 

embryos remain. 

Understanding the immediate genetic outcome of polyembryony in S. paniculatum

will help in gaining an understanding of the effect that polyembryony has on population 

biology; both in S. paniculatum, as well as in other species. To determine which type of cells 

asexual embryos are derived from in S. paniculatum, would require a detailed embryological 

study and this was beyond the scope of this study. Regardless which type of maternal tissue 

gives rise to asexual embryos, they do not undergo meiosis and hence there is no 

 

9


 

recombination to facilitate chromosomal crossover and embryos will be genetically identical 

to the maternal plant, except in rare cases of somatic mutation or mitotic crossover. By using 

suitable molecular techniques to study the population genetics of the species and the 

genotypes of offspring, it may be possible to define the reproductive biology of S. 

paniculatum as either sexual or asexual, or both. If embryos or offspring show genetic 

evidence of recombination, reproduction in S. paniculatum may be defined as sexual. If all 

offspring are found to be identical to the mother, reproduction in the species may be defined 

as asexual and the species could be regarded as clonal. 

 

 

    1cm 



 

Figure 3. Fruit, seed and embryos of S. paniculatum. From left to right: the magenta 

coloured fruit; a single seed with seed coat left on; three embryos all dissected from a 

single seed.   

 

 

10



 

 

Figure 4. Five embryos of varying size, dissected from a single S. paniculatum seed, 

collected from Abrahams Bosom. Each square in the scale bar represent 1 cm. 

 

 



 

 

 



 

 

 



 

 

 



 

1cm 


 

Figure 5. Polyembryonic seed of S. paniculatum with seed coat removed (left) and the 

same seed dissected to reveal five embryos and an under-developed embryo (right).   

 

1.4

 

Clonality, fitness and Syzygium paniculatum 

The process of clonality can be seen as having a fitness trade-off between 

proliferation of provisionally fit clonal genotypes and the ability to generate potentially fitter 

 

11



 

sexually derived genotypes. Plants that maintain purely clonal reproductive habits have the 

potential for high genetic costs and disadvantages in changing environments (Callaghan et 

al., 1992). In some species, such as Syzygium jambos, polyembryony has been suggested to 

be so prolific that sexual reproduction is suppressed altogether (Sahai & Roy, 1962). If 

polyembryony in S. paniculatum is also found to be a completely clonal process, with all 

embryos identical to the parent plant, the number of potential new genotypic combinations 

will be limited. This could make the species even more vulnerable to future environmental 

change than originally thought. Even if some sexual reproduction is occurring alongside 

clonal reproduction, the consequence could still be a dramatic reduction in genetic diversity 

(Richards, 2003; Silvertown, 2008).  

1.5

 

Ploidy in Syzygium paniculatum 

Polyploidy is known to be linked to polyembryony and apomixis. It is thought that 

polyploidy may serve as a barrier for sexual reproduction (Andrew et al., 2003; Lo et al.

2009; Ozias-Askins, 2006; Van Der Hulst et al., 2003; Van Dijk & Van Damme, 2000) 

whilst duplicate genes may be expressed asynchronously causing multiple embryos to arise 

(Carman, 1997). Alternatively, it has also been suggested that the occurrence of apomixis 

can actually promote polyploidy (Richards, 2003).  

It is possible that polyembryonic S. paniculatum may also be a polyploid. In the 

Myrtaceae, diploid (2n=22) is the most common ploidy level but polyploidy has been 

documented, mainly in fleshy-fruited species (Da Costa & Forni-Martins, 2006 & 2007). 

The polyembryonic species, Syzygium jambos, has been found to be tetraploid (2n=44) 

(Oginuma et al., 1993; Roy, 1953; Singhal et al., 1985). However, diploid apomicts do exist 

(Bicknell, 1997) so polyploidy in S. paniculatum is speculative at best and needs to be 

investigated. 

 

12


 

1.6

 

Applicability of nuclear microsatellite markers  

The most suitable molecular markers for population studies are nuclear microsatellite 

markers (nSSRs) since they show co-dominance, simple Mendelian inheritance, high rate of 

polymorphism and mutation, abundance throughout the genome, reproducibility and relative 

ease of screening (Rossetto, 2001). They are also suitable

 

for studying parentage, when the 



reproductive biology of a species is essentially unknown, but clonality is considered a 

possibility (Jones et al., 2009). 



1.7

 

Project outline 

This research project will focus on the largest and better documented population 

(Payne 1991) located on the North Entrance Peninsula (c. 33º 18' 30″S, 151º 31' 10″E). The 

population will be sampled at three hierarchical levels in order to obtain information on the 

occurrence of apomixis, and on its potential effect on native populations as well as on the 

horticultural trade.  

We will test mature trees as a representation of genetic diversity across the 

population. We will test embryos to determine the likely origin (whether sexual, asexual 

reproduction or both, occur in S. paniculatum) We will also test embryos to assess 

germinability and fitness and the likely survival of apomictic vs. zygotic embryos within 

controlled conditions (i.e. information relevant to the horticultural trade) and within natural 

conditions (i.e. within the representative individuals sampled; information relevant to the 

conservation of this rare species).  

 

Highly informative molecular tools (microsatellite loci) will be used to obtain the 



relevant genetic information without the need for complex morphological and 

 

13



 

developmental investigations. Co-dominant and highly variable molecular tools such as 

microsatellites (SSR) are particularly suited to these investigations as changes in allelic 

frequencies and heterozygosity measures can differentiate between outcrossed, selfed and 

apomictic progenies.  

This is a unique approach, not been attempted before on a rare tree and likely to 

provide relevant information for conservation and management. For instance, cultivated 

material could include greater levels of apomixis than natural populations, and since S. 



paniculatum is a popular garden plant, dispersal of cultivated material onto native 

populations could potentially result in significant losses of natural diversity.  

This project will provide enhanced understanding of genetic diversity at the population 

level, indication as to whether apomictically derived embryos can be used in propagating 

individuals with a particular genotype rather than vegetative propagation, and results 

relevant for the development of conservation and management strategies for wild 

populations and cultivated plants  

 

14



 

 

2 Materials and Methods 



2.1

 

Obtaining and analyzing diversity data  

2.1.1

 

Sample collection  

Leaf sample collections were made at the main study site, The Entrance, with the 

aim of collecting over 30 individuals across the site.  

In addition to leaf collections from The Entrance, collections were also made for ten 

other populations of Syzygium paniculatum (Figure 6). At two populations, Captain Cook 

Drive and Wamberal Lagoon this included wild juveniles. Additional fruit samples were 

also collected at Abrahams Bosom, Towra Point and Cams Wharf, to supplement those 

collected at the Entrance (as fruit set was generally low in the two years the project was 

running and the proposed targets could not be achieved). Collections at additional 

populations was work extra to the AFF funded project, and was conducted as part of an 

Honours project (Katie Thurlby, UNSW) which is currently in preparation for publication. 

 

2.1.2



 

DNA preparation  

Total genomic DNA was extracted from dried leaf tissue and frozen embryo tissue 

using the Qiagen® DNeasy® 96 Plant Kit protocol. Nine nuclear microsatellite markers 

were developed for S. paniculatum and were used to determine genotypes of adults in wild 

populations as a measure of diversity and to study the parentage or breeding system through 

offspring genotypes. Preliminary genotyping results showed extremely low variation, so 

primers were also tested in eight diverse species of the tribe Syzygieae, including the 

polyploid, Syzygium jambos.  

 

15


 

 

 

Seal Rocks 



Jervis Bay 

Central Coast 

Botany Bay 

Towra Point

Captain Cook Drive 

Sugarloaf 

Point 

Wamberal Lagoon 



The Entrance 

Cams Wharf

Salts Bay 

Green Point

Ourimbah Creek

Abrahams Bosom

Conjola National Park 

Figure 6. Populations sampled are grouped according to the geographical area by 

coloured symbols: Seal Rocks (orange triangle), Central Coast (blue circle), Botany 

Bay (red square), Jervis Bay (green star).  

 

 



16

 

2.1.3

 

Ploidy identification 

Genotyping traces were analysed in Genemapper 4.0 (Applied Biosystems) to look 

for similarities between species in microsatellite peak patterns, which may serve as an 

indicator for ploidy level. Genotyping traces of the known tetraploid Syzygium jambos and 

assumed diploid species, S. corynanthum and S. francisii, were compared with genotyping 

traces of S. paniculatum. Chromosome counts were also attempted, however time and 

resources did not allow for optimization of the necessary techniques and as such, 

chromosome counts are yet to be completed.  



2.1.4

 

Analysis of diversity data 

The preliminary results for ploidy identification indicated that the species might be 

tetraploid; having four copies of the genome rather than the usual two, making a co-

dominant analysis approach difficult. Standard analysis packages such as GenAlEx (Peakall 

& Smouse, 2005) require determination of microsatellite allele copy number. In diploid 

species, determining allele copy number is simple because a heterozygote will yield two 

peaks (two different microsatellite lengths) in a genotyping trace, whilst a homozygote will 

yield one peak (two copies of the one microsatellite length). For tetraploid species, with two 

copies of a microsatellite from each parent, determining allele copy number is not always 

possible. For this reason, diversity statistics were calculated using the program ATETRA 

(Van Puyvelde et al., 2009) which is designed specifically for analysing tetraploid data. Use 

of ATETRA required an assumption of ploidy level. After preliminary testing, tetraploidy 

was inferred for Syzygium paniculatum however, ploidy should ideally be confirmed by 

chromosome counts.  

 

17


 

Also, following other studies of clonal polyploid species (Andrew et al., 2003; 

Samadi et al., 1999) and to confirm results obtained through ATETRA, a dominant binary 

approach was also taken. This approach uses all the alleles present in the population to 

construct a genetic barcode for each individual by scoring alleles across all microsatellite 

loci as present or absent. Data could then be used in the program GenAlEx (Peakall & 

Smouse, 2005) to perform various data analyses.  

2.2

 

Obtaining and analyzing germination and reproduction data 

Fruit samples were collected from some individuals at The Entrance and were 

dissected and used for both the reproduction and germination trials. Fruit samples were also 

collected from Abrahams Bosom, Towra Point and Cams Wharf, for use in the Honours 

project (Katie Thurlby, UNSW) to supplement the data obtained at The Entrance. 

The germination and reproduction trials were designed to investigate the fitness and 

parentage of embryos. Both seeds with single and multiple embryos were germinated. The 

first embryo to germinate from each polyembryonic seed was recorded and measurements of 

seedling leaf number and height were taken every three to six weeks. Statistical analysis was 

then performed. 

Leaf samples were then taken from germinated seedlings, DNA extracted and 

genotyping conducted. Genotypes of each individual seedling were compared to other 

individual seedlings originally belonging to the same seed (family groups) to look for allelic 

differences which may suggest the occurrence of either sexual reproduction or 

recombination. Embryo material was also collected and DNA extracted however, the DNA 

obtained from embryos was not of satisfactory quality for use in genotyping and as such, 

only leaf material from the germinated embryos could be successfully genotyped. 

 

 



18

 

3 Results 



3.1.

 

Ploidy  

The genotyping data for Syzygium paniculatum showed four allele peaks at the SP33 

microsatellite locus and three allele peaks at the SP38, SP54 and SP86 loci, suggesting 

polyploidy (possibly tetraploid). Syzygium jambos (known tetraploid) also showed four 

alleles at marker SP33. The two other Syzygium species, S. corynanthum and S. francisii

showed normal diploid patterns of one (homozygous) and two (heterozygous) peaks 

respectively (Figure 7). 

 

 

19



 

 

Syzygium 



paniculatum 

 

 

 

Syzygium  

francisii 

 

 

 

Syzygium 

 jambos 

 

 

 

Syzygium 

corynanthum 

 

Figure 7. Genotyping traces for one microsatellite locus (SP33). Syzygium 



paniculatum (complete homozygous tetraploid= 4 peaks), Syzygium francisii (assumed 

heterozygous diploid =2 peaks), Syzygium jambos (complete heterozygous tetraploid= 

4 peaks) and Syzygium corynanthum (assumed homozygous diploid=1 peak). 

 

20



 

 

21



 

3.2.

 

Genetic Diversity 

Both low heterozygoisty and extremely low genetic diversity were found at The 

Entrance. Of the 31 individuals genotyped across nine microsatellite loci, 30 possessed an 

identical genotype with the single remaining individual possessing a genotype that showed 

allelic differences at four of the nine loci. These differences were three instances of 

homozygosity (in loci that were heterozygous in all other samples) and one instance of a 2bp 

change. These allelic differences may be attributable to a rare sexual or selfing event, a 

remnant of past sexual reproduction or a genotyping error. 

Between population data, obtained as part of the honours project (Katie Thurlby et 

al., unpubl), showed that genotypes were distributed along a geographic gradient. It also 

showed that the main genotype found at The Entrance is the most common genotype across 

the entire species, being present in 70.89% of individuals. The majority of divergences from 

the most common genotype occurred in northern populations. All statistical tests showed the 

southern populations (most of which contain only the most common genotype) grouped 

together with northern populations as outliers. Almost all variation between populations is 

explained by this southern grouping. Variation was not found to be related to current 

population size, as the largest populations (The Entrance and Wamberal Lagoon) exhibited 

proportionally less variation than smaller populations.



 

3.3.

 

Reproduction 

Embryo dissection confirmed polyembryony, with embryo number ranging from 

one to nine per seed. In most seeds there is one large embryo, with each subsequent 

embryo diminishing in size.  

Leaves were taken from seedlings germinated ex-situ and family groups were 

genotyped. Embryos proved extremely difficult to genotype, probably due to the high 

carbohydrate levels in seeds. As fruit set was low, data was supplemented by including 

individuals from other populations collected as part of the aforementioned honours 

project

.

 One seedling (single embryo in a seed) from The Entrance showed evidence of 



sexual origin (two loci heterozygous in the parent were homozygous in the offspring 

and two new alleles were present). Four seedlings germinated from two seeds (two 

embryos per seed) showed evidence for the sexual origin of one embryo in the seed 

(multiple new instances of homozygosity) and the asexual origin of the other (identical 

genotype to parent). This suggests that both sexual reproduction and asexual 

reproduction is occurring at The Entrance. In other populations tested there was also 

evidence for the sexual origin of embryos (single embryos or one embryo from a 

polyembryonic seed) as well as extensive evidence for asexual origin of embryos 

(including polyembryonic seeds showing no evidence of sexual origin). 

Additionally, juveniles from two populations were sampled and genotyped. Of 

the eight wild juvenile leaf samples only one had a different genotype to the adults 

sampled at the same location. This individual had a 1bp change at one allele in one 

microsatellite locus. This single allele change most likely provides evidence of somatic 

mutation (rather than reproduction) as no changes in heterozygosity were found in the 

 

22


 

genotype as would be expected if recombination had occurred. It is possible that the 

parent plant was not sampled, and as such, that the juvenile is also a single surviving 

germinant from an apomictic seed. 

 

3.4.

 

Germination Trial 

From polyembryonic seeds, multiple embryos germinated and multiple seedlings 

survived (Figure 8 & 9). Embryos germinated successfully whether they were kept 

within the bounds of the seed coat or whether embryos were separated. One seedling 

was always taller than other seedlings arising from the same seed suggesting a possible 

fitness advantage and this was found to be statistically significant. Seedling height was 

highly correlated to embryo weight suggesting that embryo size is a determining factor 

in embryo height. When genotype was taken into account and all sexual embryos 

compared to all asexual embryos, it was found that sexual seedlings were significantly 

taller than asexual seedlings; however the test was no longer significant when the 

smaller embryos from polyembryonic seeds were removed from the test (leaving the 

largest embryo from a purely asexual seed and the sexual embryo). Seedling height was 

not correlated to embryo weight in sexual embryos but was found to be highly 

significant in polyembryonic asexual seedlings. Leaf number and branch number were 

also recorded but no statistically significant differences were found.  

 

 



23

 

 

24



 

Seedling 1 

Seedling 2 

Seedling 3 

Seedling 4 

Seedling 6 

Seedling 5 

Figure 8. Multiple seedlings arising from a single seed.  

 

 



 

 

 



 

 

 

 

 

Figure 9. Survival of the multiple seedlings (Figure 8) over time. Left to right: 

Weeks 12, 18, 21 and 27, (photographs not to same scale).


 

 

4 Discussion 



4.1.

 

Ploidy 

The results suggest that because Syzygium paniculatum displays similar genotypic 

patterns to the tetraploid S. jambosS. paniculatum is likely to be tetraploid; however, this will 

need confirmation through chromosome counts. As the multiple alleles were found at four loci, it 

is unlikely that the multiple alleles are caused by localized gene duplication and more likely that 

it is a result of whole genome duplication. Although our results do not exclude other higher 

ploidy levels, tetraploidy should be assumed to be most likely as it would require the least 

number of events to arise. It is not yet known whether S. paniculatum is an allopolyploid, or an 

autopolyploid. 

There is a strongly documented link between polyploidy and apomixis (Bicknell & 

Koltunow, 2004; Roche et al., 2001) and it is thought that polyembryony might be caused by 

duplicate genes being expressed asynchronously (Carman, 1997). It is possible that the 

polyembryony found in S. paniculatum is caused by the polyploid nature of the species; however 

this would need further investigation. 

 

4.2 Diversity 

The present study has shown that S. paniculatum has extremely low overall genetic 

diversity. Only nine genotypes were found among eleven populations across the entire 

geographic range of the species (Katie Thurlby unpubl. obs., 2011). S. paniculatum also showed 

rather low heterozygosity and genetic diversity when compared to other rare species (Katie 

Thurlby, unpubl. obs., 2011). 

 

25


 

There are a number of explanations as to why The Entrance population possesses such 

extremely low diversity and shares an identical genotype with almost all southern populations of 

S. paniculatum. It is possible that The Entrance population represents one part of a large clonal 

stand of S. paniculatum existing in the southern distribution of the species. This clonal stand is 

likely to have been broken into smaller populations due to the occupation of S. paniculatum in 

specific costal habitats of littoral rainforest which has been largely cleared or modified for coastal 

development, agriculture and sand mining (Floyd, 1990). This clearing has reduced population 

numbers substantially and reduced habitat to small, highly vulnerable fragments (Payne, 1991).  

Conversely, low levels of heterozygosity, extremely low within population diversity and 

almost no diversity, may be an indication of a founder effect. Clonal reproduction, through 

dispersible apomictic seed, would facilitate the fast spread of genetically identical individuals to 

colonize and expand populations with any new genetic diversity arising via somatic mutation. It 

is likely that The Entrance population, along with other southern populations of S. paniculatum

was colonized in such a way. 

It is possible that The Entrance population of S. paniculatum arose as one of multiple 

hybridization events. Variability across populations of an apomictic species can be described by 

the multiple origins of asexual polyploids by hybridization (Soltis & Soltis, 2000). S. 

paniculatum may have arisen in such a manner at a number of different sites where the two 

parental species co-occur (or did co-occur). This would result in the pattern of unique genotypes 

which we see across the range of S. paniculatum (Katie Thurlby, pers. obs. 2011) and could also 

explain the existence of polyembryony and polyploidy in the species. However, none of the 

results obtained from this study are able to directly substantiate this idea and further studies are 

required. 

 

26


 

4.3 Reproduction and Fitness 

Reproduction in Syzygium paniculatum appears to be asexual or, clonal. This study has 

shown that embryos produced in seeds of S. paniculatum are of both sexual and asexual origin. 

This supports the view that S. paniculatum is a facultative apomictic species i.e. able to reproduce 

both sexually and asexually, which is more common than obligate apomixis (Asker & Jerling, 

1992; Bicknell & Koltunow, 2004). Sexual embryos occurred both in monoembryonic seeds and 

polyembryonic seeds however most embryos from polyembryonic seeds showed no evidence of 

sexual origin. It is possible that a sexual embryo develops first in all seeds but is sometimes out-

competed by the proliferation of multiple asexual embryos. If sexual reproduction occurs and the 

sexual embryo is not out-competed, a sexual individual can survive either along in a seed or 

alongside asexual embryos. This study found limited evidence for recent recombination in adult 

populations, suggesting that if there is a sexual embryo it never (or hardly ever) survives in-situ. 

Also, as a sexual event may occur in as little as 2% of progeny of apomictic species (Bicknell & 

Koltunow, 2004) the small samples sizes in this study may have resulted in a gross overestimate 

of the sexual reproduction in the species as a whole. 

Apomixis can be influenced by environmental factors such as climate, nutritional supply 

or competition (Asker & Jerling, 1992) which suggest that environmental influences during 

particular seasons may result in a higher rate of sexual reproduction than others. Apomicts are 

often found in highly disturbed areas or where individuals are widely dispersed (Asker & Jerling, 

1992), so it is possible that habitat disruption and habitat fragmentation may be an influencing 

factor for sexuality in S. paniculatum.  

The results of this study did not find a significant fitness advantage for sexual individuals 

in terms of height; rather, they suggest that embryo size is a significant contributor to seedling 

height while embryo origin is not. Sexual reproduction does involve some advantages however, 

 

27


 

because through sexual reproduction, species are equipped for relatively fast genetic changes 

should they be required by a changing environment conversely, the genotype of one sexual seed 

has one chance to survive whereas the identical genotype of multiple asexual seeds has multiple 

chances to survive. So it is possible that there underlying are trade-offs between the advantages 

and disadvantages of different reproductive modes. 

 

4.4 Conservation Implications 

The results of this study imply that Syzygium paniculatum may have some difficulty 

adapting to future environmental change. Until now, the species has survived with moderately 

low overall genetic diversity and extremely low (if any) variation between individuals. This 

survival is likely to be due to the ability of the species to reproduce prolifically by way of 

polyembryony and the theoretical fitness of the persistent genotypes in the current environment. 

It should however be noted, that whilst there is low between-individual variation in the species, 

the nature of apparent tetraploidy in S. paniculatum means that there is high within-individual 

variation. Nevertheless, the lack of variation between individuals and in the species as a whole, 

and the apparent asexual reproduction, means that the development of new variation is restricted 

to the slow process of somatic mutation.  

The detection of some sexual reproduction within the species is positive as it means there 

is potential within the species to adapt genetically to future environmental change however the 

distinct lack of variation particularly at The Entrance suggests that such sexual individuals rarely 

survive in-situ. 

It is unlikely that the reduced population sizes and low genetic variation found in S. 



paniculatum are completely due to human interference. However, human activity is likely to have 

 

28



 

caused significant reduction in the range and population sizes of the species. From a conservation 

perspective, there is a need to improve environmental conditions and habitats where practicable, 

not only to preserve current genetic diversity but to give the species the best possible chance for 

the accumulation of variation in the future, be that by somatic mutation or sexual reproduction. 

This particularly applies to the northern populations over southern populations, where the 

majority of known genetic diversity has been found (Katie Thurlby, pers. comm. 2011). 

Appropriate activities would include habitat protection and management, weed management, and 

fire management plus on-going monitoring and surveying as well as more extensive research.  

In addition to preserving habitats, in the interest of preserving allelic variation, it may be 

advisable to introduce each of the various different genotypes found across the species into 

cultivation to make sure that the variation found within the species is not diluted further by the 

cultivation of only the most common genotype.  

Propagation of S. paniculatum via seed may be a viable mode of cultivating genetically 

identical plants as multiple embryos in each seed are easily germinated more reliably than 

through methods of vegetative cultivation and a high proportion of individuals survive beyond 

the seedling and sapling stages. Further studies would need to determine the rate of possible 

sexual events and the practicality of cultivating via seed for large scale production. 

 

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Document Outline

  • 1.2. Threatened species: population size and diversity    5
  • 2. Materials and Methods        12
  • The Magenta Lilly Pilly (Syzygium paniculatum), endemic to a narrow strip along the New South Wales coast, is currently listed as vulnerable at both state and national levels. At present management of the species focuses on minimizing currently known threats, such as weed invasion, while little is known about the reproductive biology of the species. S. paniculatum is the only recorded polyembryonic Australian species of Syzygium; polyembryony being the development of multiple (and often asexual) embryos in one seed. Nuclear microsatellite markers were used to investigate the genetic outcome of polyembryony on the reproductive and population biology of the species focusing particularly on the population located on The Entrance Peninsular. Low within-population diversity was found, with low heterozygosity levels and a low level diversity indices when compared to other rare or rainforest species. Multiple embryos from single seeds were found to be identical to the mother. Multiple embryos germinated and survived but one seedling was always significantly taller than all others in the seed but was not considered sexual. It was concluded that the rare S. paniculatum is an apomictic clonal species with extremely low genetic diversity. 
  • 1 Introduction
    • 1.1. Syzygium paniculatum: a brief description
    • 1.2 Threatened species: population size and diversity
    • 1.3 Polyembryony, clonality and Syzygium paniculatum
    • 1.4 Clonality, fitness and Syzygium paniculatum
    • 1.5 Ploidy in Syzygium paniculatum
    • 1.6 Applicability of nuclear microsatellite markers 
    • 1.7 Project outline
    • 2.1 Obtaining and analyzing diversity data 
    • 2.1.3 Ploidy identification
    • 2.2 Obtaining and analyzing germination and reproduction data
    • 3.1. Ploidy 
    • 3.2. Genetic Diversity
    • 3.3. Reproduction
    • 3.4. Germination Trial
  • 4 Discussion
    • 4.1. Ploidy
    • 4.3 Reproduction and Fitness
    • 4.4 Conservation Implications
  • References


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