The Breeding System and Genetic Diversity of the Enfield Grevillea (Proteaceae) Conservation

Unveiling the Reproductive Secrets of the Endangered Enfield Grevillea

Abstract

Endemic to a small region of southeastern Australia, the Enfield Grevillea ( Grevillea bedggoodiana ) faces heightened vulnerability to environmental changes and disturbances. This study delves into its reproductive biology and population genetics, crucial aspects for effective conservation. Through a controlled pollination experiment, we discovered that this species exhibits a strong preference for cross-pollination, showcasing its inherent need for genetic mixing. Interestingly, while facing potential threats from the decline of native pollinators, European honeybees appear to have effectively taken their place, ensuring continued seed production. Genetic analyses revealed surprisingly good news: the Enfield Grevillea boasts high levels of genetic diversity and healthy reproductive rates, even in populations impacted by historical disturbances. This suggests a resilient species capable of bouncing back from challenges and adapting to changing environments. Although moderate genetic differentiation exists between populations, it’s largely attributed to natural isolation by distance. Overall, our findings paint an optimistic picture for the future of G. bedggoodiana.

Keywords: Endangered species, Endemism, Genetic diversity, Microsatellites, Plant breeding system, Population structure

Introduction

Effective conservation strategies hinge on a deep understanding of a species’ biology, particularly for those teetering on the brink of extinction. Narrow endemics, like the Enfield Grevillea, face a double-edged sword: their limited range makes them susceptible to catastrophic events, and their specialization might hinder adaptation to new challenges. To fully grasp the resilience of G. bedggoodiana, we must unravel the secrets of its reproductive prowess, genetic diversity, and population structure.

This study addresses three critical questions:

1. What is the breeding system of the Enfield Grevillea? Understanding whether a plant prefers self-fertilization or relies on cross-pollination unlocks vital information about its potential for genetic diversity and adaptability.

2. How genetically diverse are its populations, and how is this diversity distributed across its range? Assessing genetic diversity provides insights into a species’ evolutionary potential and its ability to withstand environmental changes.

3. How efficiently are the different populations reproducing, and is their reproductive success linked to genetic diversity? Correlating reproductive output with genetic parameters unveils potential vulnerabilities and points towards targeted conservation efforts.

Unraveling the Enfield Grevillea’s Story

The Enfield Grevillea, an understorey shrub embellishing the Eucalyptus-dominated forests of Victoria, bears the scars of historical anthropogenic disturbances. Gold mining and logging have left their mark, potentially causing population bottlenecks and disrupting the delicate dance of pollination. This study steps into this complex ecological stage, seeking to illuminate the species’ current status and equip conservationists with the knowledge needed to safeguard its future.

Materials and methods

Our research unfolds in three distinct acts:

1. Unmasking the Breeding System: A controlled pollination experiment, meticulously designed to mimic natural settings, reveals the Enfield Grevillea’s preferred mode of reproduction.

2. Decoding Genetic Diversity: We journey into the intricate world of genetics, analyzing DNA samples from multiple populations to map genetic diversity and uncover hidden relationships between geographically separated groups.

3. Connecting Reproduction and Genes: By meticulously quantifying the reproductive output of different populations and linking it to their genetic diversity, we gain a deeper understanding of G. bedggoodiana’s resilience in the face of environmental change.

Site and study species

The Enfield Grevillea graces a small realm of approximately 150 km², primarily within the bounds of Enfield State Park (ESP) and Enfield State Forest (ESF) in Victoria, Australia (Fig. 1). Historically, these areas have endured the pressures of gold mining and logging, leaving a legacy of fragmented habitats. While the long-term impacts of these activities on the local flora remain largely unknown, we can surmise their contribution to the decline of native vertebrate pollinators and potential disruptions to plant population connectivity.

Within this nuanced landscape, G. bedggoodiana persists, favoring the gravelly clay soils of ridges and north-facing slopes. The species demonstrates a tendency to form distinct stands comprising hundreds of individuals, with solitary plants occasionally appearing, especially along roadsides. Our study focuses on these spatially discrete populations, mirroring the definition employed in the National Recovery Plan for Grevillea bedggoodiana where a ‘population’ represents a group exceeding 1000 individuals.

A closer look at the Enfield Grevillea

This low-growing shrub, often reaching heights of 0.5 m with trailing branches up to 1.9 m long, bursts into bloom during the Australian spring. Its toothbrush-like inflorescences, adorned with 10–20 pairs of flowers, present a remarkable spectacle as they sequentially unfurl over several days. Interestingly, the individual flowers exhibit protandry, a clever reproductive strategy where the male reproductive parts mature before the female ones, reducing the chances of self-fertilization.

While observations initially suggested bird pollination, our concurrent camera trapping study revealed a more complex pollination web. Although the Yellow-faced Honeyeater (Lichenostomus chrysops) graced the flowers with its presence, along with occasional visits from small nocturnal mammals, the most frequent visitor proved to be the introduced European honeybee ( Apis mellifera ). This unexpected finding suggests a potential shift in the pollination dynamics, with honeybees potentially filling the void left by declining native pollinator populations.

Breeding System: A Tale of Outcrossing

To decipher the Enfield Grevillea’s breeding system, we conducted a controlled pollination experiment at one of its central populations (MCS, Fig. 1). Our experiment mimicked different pollination scenarios:

Autogamy: Self-fertilization within the same flower.
Geitonogamy: Pollen transfer between flowers on the same plant.
Xenogamy: Cross-pollination with pollen from a different plant.
Open pollination: Natural pollination without any manipulation.

We carefully manipulated flower access, hand-pollinated select blossoms, and tracked seed production. The results were striking:

Autogamy yielded no seeds at all, confirming the Enfield Grevillea’s aversion to self-fertilization.
Geitonogamy produced a meager number of seeds, significantly less than both xenogamy and open pollination.
Xenogamy, the champion of genetic mixing, produced similar seed quantities to open pollination.

These findings strongly suggest that the Enfield Grevillea thrives on cross-pollination, a strategy that fosters genetic diversity and enhances its resilience. Even with the potential decline of native pollinators, the presence of honeybees seemingly ensures adequate pollen transfer and successful seed set.

Microsatellite Markers: Unveiling Genetic Secrets

We delved into the Enfield Grevillea’s genetic makeup using microsatellite markers, powerful tools for assessing genetic diversity and uncovering relationships between populations. DNA samples were collected from 30 individuals across eight representative populations, encompassing both ESP and ESF.

After careful analysis, we focused on nine specific microsatellite loci, which revealed fascinating insights:

Genetic Diversity Thrives: The populations exhibited high levels of allelic richness, observed heterozygosity, and gene diversity, exceeding those of some closely related but less widespread grevillea species.
Inbreeding Occurs, but…: Despite high outcrossing rates, inbreeding coefficients were significant in several populations. This suggests that some level of self-fertilization might still occur, particularly in smaller populations.
No Evidence of Recent Bottlenecks: Except for one population showing potential signs of a historical bottleneck, the data suggests that most populations haven’t experienced severe population declines in recent times.

Population Structure: Moderate Differentiation, Largely due to Distance

Our genetic analyses unveiled a moderate level of differentiation between populations, with 12% of the genetic variation attributable to differences between them. This differentiation is largely explained by isolation by distance: populations farther apart geographically tend to be more genetically distinct. Further analysis using specialized software (Structure and DAPC) indicated the presence of at least three distinct genetic clusters, aligning with the geographic distribution of populations. Importantly, private alleles, unique genetic variations, were detected in every population, highlighting the importance of conserving each distinct group.

Reproductive Rates: No Correlation with Genetic Parameters

To assess reproductive success, we meticulously counted the number of juvenile plants within each population. While the number of seedlings varied, there was no correlation between reproductive rates and either gene diversity or inbreeding coefficients. This suggests that even populations with slightly lower genetic diversity or some level of inbreeding aren’t experiencing compromised reproductive output.

Interestingly, the population exhibiting the highest ratio of juveniles to adults was one that had experienced historical disturbance, potentially through gold mining. This finding points towards a species capable of vigorous reproduction, perhaps even spurred on by soil disruption.

Discussion: Resilience in the Face of Change

Our findings paint a surprisingly optimistic picture for the Enfield Grevillea:

Master of Cross-pollination: The species’ strong preference for outcrossing promotes genetic diversity and adaptability.
Honeybees Fill the Gap: Despite the potential decline of native pollinators, honeybees appear to have stepped in to ensure efficient pollination and seed production.
Thriving Genetic Diversity: The populations harbor high levels of genetic diversity, exceeding that of some related but more widespread grevillea species.
No Evidence of Widespread Inbreeding Depression: Even populations exhibiting some level of inbreeding maintain healthy reproductive rates.
Resilience to Disturbance: The species shows a remarkable ability to recover from past disturbances, suggesting an inherent toughness.

Pop	Nest	Na	Ar	A ■priv	Ho	He	Fi	(Fi)	1-s
OT	1400	5.3	4.5(1.1)	1	0.45(0.15)	0.60(0.16)	0.259	0.260	1.00(0.15)
WT	4800	7.1	5.9(1.4)	2	0.58(0.08)	0.73(0.09)	0.220	0.186	0.94(0.32)
HR	2100	6.9	5.6(1.4)	2	0.650.11)	0.69(0.10)	0.074	0.044	1.00(0.00)
MCSa	3000	7.9	6.3(1.0)	4	0.63(0.18)	0.75(0.07)	0.187	0.036	0.95(0.24)
MCN	1800	7.6	6.3(0.9)	2	0.60(0.19)	0.750.07)	0.225	0.035	1.00(0.19)
LGR	1300	6.7	5.5(0.9)	6	0.59(0.11)	0.70 (0.10)	0.177	0.101	0.90(0.13)
IGR	400	4.9	4.1(0.6)	3	0.52(0.16)	0.66(0.05)	0.233	0.101	0.96(0.39)
VR	300	4.8	3.8(0.5)	2	0.64(0.15)	0.60(0.11)	– 0.061	– 0.149b	0.99(0.17)
Mean	–	6.40	5.25	2.75	0.58	0.69	0.164	0.077	0.97
SE	–	0.41	0.33	0.52	0.02	0.02	0.035	0.040	0.01

Bold font indicates Fi values significantly greater than 0 (999 permutations, p < 0.05) ^aThis population was used in the pollination experiment ^bValue significantly lower than 0 (999 permutations, p < 0.001)

Table 3 Matrix of pairwise FST (AMOVA, 999 permutations,		OT	WT	HR	MCS	MCN	LGR	IGR	VR
all p-values < 0.01) (below	OT	–	1.2	3.2	2.7	4.5	3.8	8.5	11.3
diagonal), and pairwise geographic (Euclidean)	WT	0.095	–	2.0	2.6	4.3	4.0	8.9	11.5
distances (km) between	HR	0.114	0.012	–	3.1	4.1	4.7	9.3	11.5
the sampled populations of	MCS	0.117	0.036	0.047	–	1.8	1.7	6.4	8.9
Grevillea bedggoodiana (above	MCN	0.115	0.045	0.041	0.021	–	1.8	5.3	7.4
diagonal)	LGR	0.196	0.109	0.127	0.072	0.073	–	4.8	3.1
	IGR	0.202	0.112	0.122	0.112	0.088	0.134	–	3.1
	VR	0.286	0.194	0.203	0.186	0.155	0.166	0.211	–

Fig. 4 Isolation by distance plot of pairwise F_ST vs geographic (Euclidean) distances among eight populations of Grevillea bedg- goodiana

Fig. 5 a Structure Harvester’s L(K) plot and Delta K plot indicat­ing K = 3 and K = 5 models explained the data nearly equally well (Evanno method); b Structure bar plots illustrating patterns of genetic admixture in the sampled Grevillea bedggoodiana populations under

the two models. Each vertical line represents one individual, different colours indicate proportion of an individual’s genotype deriving from one of the K ancestral groups; c Structure tree plots showing the rela­tive genetic distances between the clusters

Fig. 6 Discriminant analysis of the principal components (DAPC) scatter plots: a includ­ing all populations sampled; b excluding the two outlying populations from ESF for better resolution of the structure of the ESP populations. In both analyses retained were the first 30 principal components (dimensions of variation in the genotypic data) and all discrimi­nant functions (synthetic vari­ables optimizing the clustering of groups)

Table 4 Estimated population area, estimated population size (N_est), estimated number of juveniles per 100 adults (Nj) and time since last fire at the sampled populations of Grevillea bedggoodiana

Pop	Area (× 1000 m2)	Nest	Nj	Time since last fire (years)
OT	11	1400	8	10, 9a
WT	22	4800	16	23, 14a
HR	17	2100	5	7, 6a
MCS	18	3000	6	23
MCN	16	1800	5	23
LGR	6.0	1300	23	23
IGR	5.5	400	93	23
VR	2.5	300	25	23

^a These populations were control-burnt in two stages (using a road as a fire break) or only a portion of the population was burnt since the 1995 wildfire. In each case, the lower of the two values was used in the regression analysis

Implications for Conservation: Protecting a Resilient Legacy

To effectively manage the Enfield Grevillea and its unique genetic legacy, conservation efforts must utilize the insights gleaned from this study:

Safeguarding Peripheral Populations: While the large populations within ESP are currently protected, attention must shift to the smaller, genetically distinct groups in ESF. These populations harbor unique genetic adaptations and require tailored conservation strategies to ensure their long-term survival.

Embracing a Balanced Fire Regime: Controlled burns, a vital tool in managing fire-prone landscapes, should be carefully planned to allow sufficient time for populations to replenish their seedbanks and for genetic exchange between burnt and unburnt areas. This approach fosters generational overlap and mitigates the risk of genetic loss through drift, particularly crucial for small, isolated populations susceptible to random fluctuations.

Restoring Native Pollinator Networks: While honeybees offer a reassuring safety net, relying solely on a single, introduced pollinator poses risks. Conservation efforts should prioritize the restoration of native pollinator communities, particularly nectarivorous birds and mammals, to ensure the long-term resilience of G. bedggoodiana. This involves habitat restoration and management strategies that promote the abundance and diversity of these vital ecosystem engineers.

By understanding the subtle complexities of the Enfield Grevillea’s reproductive biology and genetic makeup, we can tailor targeted conservation actions, maximizing our chances of preserving this remarkable species within the intricate tapestry of Australian biodiversity.