Ex situ germplasm collections of exceptional species are a vital part of the conservation of Australia's national plant treasures

Abstract

Seed banking is well established to contribute to the conservation of many seed-bearing plant species ex situ for future use in restoration, translocation, agriculture and horticulture. In Australia, over 67% of currently listed threatened plants are represented in conservation seed banks. However, there are challenges to conserving the full extent of plant diversity in seed banks, with growing recognition that we need to think beyond conventional seed banking methods to conserve ‘exceptional’ plant species that are difficult to collect, store and germinate. We examine how the framework for identification of such species can be applied to the Australian flora, using examples from the recently published guidelines for ‘Plant Germplasm Conservation in Australia’ and case studies and data arising from the Australian Academy of Science Fenner Conference on the Environment ‘Exceptional Times, Exceptional Plants’. We present a workflow to assist conservation decision-makers and practitioners in identifying exceptional species and overcoming barriers to storage of germplasm, enabling appropriate ex situ collection types to be established via seeds, living collections, tissue culture, cryopreservation or a combination of these. Australia's seed conservation sector continues to expand, with increasing expertise, facilities and networks established to conserve a diversity of plant species; however, resolving the challenges relating to each exceptionality factor requires significantly more time, labour and collaboration than current capacity allows. Understanding the barriers to conservation and production of healthy plants, via germination or other methods of propagation, is a critical component of conserving species long-term and ultimately returning plants to the landscape.

INTRODUCTION

Australia is home to a rich and diverse flora estimated to include more than 21,000 species, with many unique to the continent (Cassis et al., 2017; CHAH, 2021; Chapman, 2009). Biodiversity loss is increasing in Australia, with 1411 species (more than 6%) currently nationally listed as threatened (DCCEEW, 2023) increasing to 10% when state and territory data are included. Key threatening processes include past and ongoing habitat loss, inappropriate fire regimes, competition from invasive weeds and diseases caused by pathogens such as Phytophthora cinnamomi (Dieback) and Austropuccinia psidii (Myrtle Rust). Superimposed on these individual threatening processes, and of potentially greater impact, are the compounding effects of interactions between threats (Gallagher et al., 2022; Keith et al., 2022; Silcock & Fensham, 2018). For example, climate change, when combined with changed fire regimes, habitat fragmentation, and disease, is predicted to substantially increase the magnitude of adverse impacts on the Australian flora (Enright et al., 2015; Yates et al., 2021) and ecosystems (Keith et al., 2022). These combined threats are likely to have an irreversible impact on vegetation communities especially in alpine regions (Cotto et al., 2017; Nicotra et al., 2016; Theurillat & Guisan, 2001), rainforests (and cloud forests) (Costion et al., 2015; Sommerville et al., 2019; Sommerville, Errington, Newby, et al., 2021) and the eucalypt forests of southwest Western Australia (Enright et al., 2014; Matusick et al., 2013; Nolan et al., 2021; Yates et al., 2021). This range of threats and their interactions has resulted in increased in situ management and greatly increased use of ex situ conservation techniques for many threatened species. Ex situ conservation can enable in situ conservation goals via the maintenance of transition populations pending return to the wild (via augmentation, reintroduction, or introduction translocations), as well as permanent ex situ ‘insurance populations’ (Breman et al., 2021; Commander et al., 2018; White et al., 2023). Ex situ collections are particularly critical when in situ reproduction and survival is not adequate to meet conservation goals (Potter et al., 2017; Smith, 2006).

Seed banking is well established as the most efficient method of ex situ conservation for many seed-bearing plants as it provides for the collection and storage of genetically representative germplasm in a low maintenance environment for long periods (Breman et al., 2021; FAO, 2014; Martyn Yenson et al., 2021; Smith et al., 2003). Conservation seed banking principally involves the storage of seeds at low moisture content and temperature (i.e., −18°C and 15% RH; FAO, 2014). However, there are many cases for wild plant species in which conventional seed banking is challenging or requires further research (Hay & Probert, 2013). Challenges include low seed availability, poor seed quality, unorthodox storage behaviour and difficult germination (Pence, Bruns, et al., 2022; Pence, Meyer, et al., 2022; Sommerville, Errington, Newby, et al., 2021; Wyse et al., 2018). Such plants have been recently termed ‘Exceptional’ and categorised according to four ‘exceptionality factors’ (EF) based on whether the challenges relate to seed collection, drying, cold storage or utilisation—the main processes in seed banking (Pence, Bruns, et al., 2022, Pence, Meyer, et al., 2022). Species within Exceptionality Factor 1 (EF1) produce few, or no, viable seeds, or are difficult to access, meaning seeds are not readily available for banking. Species within EF2 are desiccation sensitive and cannot survive drying to the low water contents necessary for long-term storage (known as recalcitrant storage behaviour). Species within EF3 may be partially desiccation tolerant but cannot survive long-term under gene banking conditions at −18°C or −20°C, so they are either freeze-sensitive or short-lived in storage (e.g., defined as a half-life ≤20 years at −20°C and 15% RH by Pence, Meyer, et al., 2022). Species within EF4 produce seeds with complex dormancy that precludes reliable germination for viability monitoring, research or translocation. For exceptional species, other forms of germplasm conservation such as living collections (in ground and nursery collections) (Griffith et al., 2019; Westwood et al., 2020), tissue culture, or cryopreservation (Hay & Probert, 2013; Streczynski et al., 2019; Walters & Pence, 2020) may be required to supplement, or replace, conventional seed banking to achieve ex situ conservation goals. These exceptionality factors serve as a useful framework for prioritising the type(s) of germplasm to source and for identifying research priorities for effective ex situ conservation.

Despite continued advances in seed conservation of wild species (Hay & Probert, 2013), exceptional species frequently present challenges to those working with individual wild species. Conservation scientists, practitioners, and students recognise the need to identify the best means to conserve certain exceptional species, as shown by the level of engagement in the Australian Academy of Science Fenner Conference on the Environment (Martyn Yenson et al., 2022). The first day of the conference, a virtual event organised as part of the Australasian Seed Science Conference, attracted registrations from more than 300 people working in 29 countries. Polls of participants (n = 89, Table 1) indicated that close to one third were working on, or intending to work on, exceptional species; however, almost half found it difficult to identify which species were exceptional. Challenges also related to access to resources, issues with germination and dormancy, and, to a lesser extent, access to information. Many of these challenges, including a lack of information on target species, and lack of networking and partnerships supporting exceptional species conservation, were similarly identified as major challenges by institutions participating in exceptional species conservation within the United States (Philpott et al., 2022).

TABLE 1 Responses to online polls for participants in the Australian Academy of Science Fenner Conference on the Environment Day 1.

Poll question	Number of respondents (total n = 89)	% of respondents
1. What is your motivation for attending the Fenner conference?
Working on exceptional species	25	28
Intend to work on exceptional species	3	3
Interested in different methods of storing germplasm	17	19
Interest in plant conservation in general	44	50
2. What are the challenges in conserving exceptional species, for you?a
Identifying exceptional species	44	49
Access to resources	41	46
Issues with germination and propagation	42	47
Difficulty finding/accessing relevant information	21	24
Lack of capacity, decided not to pursue	13	15

^aMore than one response was possible for this question.

In this paper, we consider examples of plant species in Australia for which complementary methods of germplasm conservation have been developed and adopted, and test the application of the exceptionality factor framework of Pence, Meyer, et al. (2022) to the Australian flora. Using the framework to prioritise practical conservation action, we highlight gaps in knowledge of Australian plants, identify future research priorities, and consider the essential resources and networks required to support their successful conservation.

Ex situ germplasm conservation in Australia

Conservation seed banking of wild plant species in Australia is primarily undertaken by a network of seed banks in conservation agencies including botanic gardens, and organisations such as Greening Australia, the Australian Tree Seed Centre and the Australian Grains Genebank (Gibson-Roy, Hancock, et al., 2021; Van Moort et al., 2021; Lott & Read, 2021). Many of these organisations also participate in nationally collaborative programmes coordinated through the Australian Seed Bank Partnership (ASBP, Figure 1). Since 2000, seeds of over 10,000 species have been banked through the ASBP network (D. Wrigley, pers. comm.), including over 67% of species currently listed as threatened under Australia's Environment Protection and Biodiversity Conservation Act 1999 (EPBC Act) (Commonwealth of Australia, 2020). Seeds of most Australian species are desiccation tolerant and are expected to possess orthodox storage behaviour, that is, their longevity in storage increases as moisture content and storage temperature are reduced (within a certain range). Orthodox seeds are expected to survive for many decades when stored under conventional seed banking conditions (Merritt et al., 2021). Nevertheless, recalcitrant seeds, which are sensitive to desiccation and not suited to conventional storage, have been identified in 90 genera, across 36 plant families, that occur in Australia with most growing in tropical or subtropical rainforests (Sommerville et al., 2018, see also Figure 2 and case study for EF2). Intermediate seed storage behaviour includes a broad range of responses to desiccation and cold storage, for species classified as neither truly recalcitrant nor truly orthodox; it has been identified in 28 species across 18 plant families, with examples including both desiccation-intermediate and freezing-intermediate species (Sommerville, Errington, Funnekotter, & Newby, 2021). Examples of seeds that are desiccation tolerant, but short-lived in storage include those of Nymphaea spp. (Dalziell et al., 2019, see also Figure 2 and case study for EF3), many alpine endemics (Satyanti et al., 2018), and orchids (Hay et al., 2010; Merritt, Hay, et al., 2014). When conservation seed banking is not possible, other methods need to be utilised to conserve germplasm (Figure 3).

FIGURE 1. Location of major ex situ conservation facilities for Australian flora, including Australian Seed Bank Partnership Partners, the Australian Tree Seed Centre, the Australian Grains Genebank and Australian Pastures Genebank (both storing crop wild relatives) and major forestry seed banks with conservation collections. (Source: Offord et al., 2021). The icon for the Victorian Conservation Seedbank (No. 8) has been updated to illustrate that cryostorage capability was incorporated in 2022 through the addition of ultra-low temperature freezers (Wrigley & Desmond, 2023). 1. George Brown Darwin Botanic Gardens conservation seed bank. 2. Alice Springs Desert Park. 3. Western Australian Seed Centre, Department of Biodiversity, Conservation and Attractions, Kensington, and Kings Park and Botanic Garden. 4. Forest Products Commission Seed Centre. 5. Australian Pastures Genebank, South Australian Research and Development Institute. 6. South Australian Seed Conservation Centre, Botanic Gardens and State Herbarium of South Australia (BGSH). 7. Australian Grains Genebank, Agriculture Victoria. 8. Victorian Conservation Seedbank, Royal Botanic Gardens Victoria. 9. Tasmanian Seed Centre, Sustainable Timber Tasmania. 10. Tasmanian Seed Conservation Centre, Royal Tasmanian Botanical Gardens. 11. National Seed Bank, Australian National Botanic Gardens. 12. Australian Tree Seed Centre, CSIRO. 13. Australian PlantBank, Botanic Gardens of Sydney. 14. Brisbane Botanic Gardens Conservation Seed Bank, Brisbane Botanic Gardens, Mt Coot-tha.

FIGURE 2. Species or genera presented in case studies as examples of each exceptionality factor. (a) Muehlenbeckia tuggeranong (Tuggeranong Lignum, Polygonaceae; Box 1), an endangered species listed under the Commonwealth Environment Protection and Biodiversity Conservation Act 1999, is not known to produce fruit or seed in situ (EF1). In recent decades an ex situ collection has been established in the Australian National Botanic Gardens nursery. Recently, plants in the ex situ collection produced fruit, leading to the collection of 53 viable seeds. The plant accession pictured here is presumed extinct in the wild. Credit: Zoe Knapp ANBG. (b) Syzygium hodgkinsonii (Smooth-barked Rose Apple, Myrtaceae; Box 2) has been severely impacted by Myrtle Rust in the wild and is listed as Vulnerable under the NSW Biodiversity Conservation Act 2016 and the Commonwealth Environment Protection and Biodiversity Conservation Act 1999. Like all Syzygium species for which storage behaviour data have been published, this species produces seeds that do not tolerate the drying necessary for seed banking (EF2). Credit: Karen Sommerville. (c) Nymphaea lukei (Nymphaeaceae; Box 3), like other Australian species of Nymphaea, appears to be desiccation tolerant and survives drying to 15% RH but germination declines markedly after 12 months storage at −20°C (EF3). Credit: Emma Dalziell. (d) Cross section of a Persoonia hirsuta (Hairy Geebung, Proteaceae; Box 4) drupe, a dispersal unit that contains an endocarp that can act as a water-permeable mechanical barrier to germination. The outcomes of >20 years of research on Persoonia seed biology have demonstrated the complex germination system of this genus (EF4), with recent advances showing that physiological dormancy in this genus may be relieved through wet-dry seasonal climate cycles or seed burial leading to warm and cold stratification. Credit: Nathan Emery.

FIGURE 3. Flow chart with guiding questions in blue to ensure species that need to be stored ex situ (1) can be collected (2), tolerate desiccation to 15% RH (3), store well under conventional seed banking conditions of −18° C and 15% RH (4), and can be reliably regenerated for utilisation (5). Species that meet these criteria are deemed non-exceptional but still require long term curation, including viability monitoring, to meet conservation goals. Negative responses to each guiding question relate to the exceptionality factors proposed by Pence, Meyer, et al. (2022) in green. Definition of exceptionality factors provides a useful framework for prioritising the type(s) of germplasm to source for ex situ conservation (germplasm icons) and for identifying research priorities (orange). Species within EF1a may be stored ex situ as living collections or via micropropagation, with due consideration for maintaining genetic diversity, and some may eventually produce seed that can be assessed for desiccation tolerance (dashed line). Species within EF2 can be stored in tissue culture using seed or vegetative material, as living collections, or as embryonic axes, spores, pollen or vegetative material in cryostorage. Species within EF3 may require alternative storage conditions, including cryostorage, with optimisation of protocols required for each species. Some collections in cryostorage need to transition through tissue culture prior to storage. As in EF2, vegetative material from tissue culture can also be cryopreserved. Species within EF4 can be banked under conventional seed banking conditions but require additional time and resources to overcome dormancy and develop reliable germination methods. Collection and propagation of vegetative material to establish a living collection for EF3 or EF4 provides a resource for research, refinement of horticultural protocols and education. Finally, sufficient seed must be available for utilisation, with the quantity and diversity depending on the conservation goal. If insufficient seed is available (EF1b), establishing complementary ex situ collections may supplement seed collections, with consideration for the cost effectiveness of this decision. A seed orchard approach for multiplication may also be required. Once issues with dormancy (EF4) or insufficient seed (EF1b) are addressed, species may be reclassified as non-exceptional.

Tissue culture, cryopreservation and living plant collections are becoming increasingly important for conserving species (Figures 1 and 3). Tissue culture supports ex situ conservation of species that are highly threatened (Sommerville et al., 2018, 2019; Sommerville, Bunn, Rollason, & Turner, 2021), do not produce viable seeds, and/or do not produce seeds suitable for banking (Bunn et al., 2007; Sommerville, Bunn, Rollason, & Turner, 2021). It is currently employed for 72 Australian native plant species across 21 families at the Australian PlantBank (NSW) and Botanic Gardens and Parks Authority (WA) (Sommerville, Bunn, Rollason, & Turner, 2021). Tissue culture facilities at these two organisations, and at Royal Botanic Gardens Victoria, Royal Tasmanian Botanical Gardens and the South Australian Seed Conservation Centre, are also used to symbiotically germinate seeds of threatened orchid species for translocation projects (Reiter, Dimon, Freestone, Davis, et al., 2021). Facilities at the University of Queensland are used to develop protocols for culturing species of horticultural importance, such as Macadamia, as well as threatened wild species (A. Hayward, pers. comm). Cryopreservation as a method of germplasm storage has been investigated in Australia since the early 1990's, with cryogenic storage facilities available at the Australian PlantBank (NSW), Botanic Gardens and Parks Authority (WA) and the Royal Botanic Gardens Victoria (Figure 1). Cryopreservation has been applied to seeds, seed axes, spores, protocorms, mycorrhizal fungi, callus tissues and shoot tips of various Australian plants (including many threatened species) across a wide range of taxonomic groups and life histories with varying levels of success (Funnekotter et al., 2021 and references therein). To date, 145 Australian plant species from 35 families have been successfully stored and revived via the cryopreservation process (Funnekotter et al., 2021) with some species subsequently utilised for successful translocation (Bunn & Turner, 2021; Funnekotter et al., 2013; Touchell et al., 1992).

Living plant collections (i.e., whole plants in potted collections, botanic gardens, arboreta, and plantations) as well as field genebanks (Said Saad & Ramanatha Rao, 2001) and seed production areas (Nevill et al., 2016) are another key germplasm type for plant species that require conservation action. Seed and/or vegetative material from these living collections may be used in restoration, translocation, research, education, horticultural development and display (Shade et al., 2021). Living plant collections are increasingly used to bridge the gap between ex situ conservation and recovery in situ (Volis & Blecher, 2010). They are especially useful when continuing access to in situ populations places a species under additional pressure or where so few individuals remain it may be the only practical conservation option available to capture genetic diversity (Shade et al., 2021). Living collections can be resource (largely labour) intensive, and it is challenging to maintain their genetic integrity over time (Ensslin & Godefroid, 2019; Fant et al., 2016), but this can usually be overcome for species where other ex situ options are not feasible. Collections of threatened species for display and education are maintained in many botanic gardens in Australia. Genetically diverse collections maintained for conservation purposes are less common but include potted collections of Wollemia nobilis (Wollemi Pine) (Offord & Zimmer, 2021), several species critically endangered by Myrtle Rust (Sommerville et al., 2019; Viler & Offord, 2020), as well as orchids (Reiter, Dimon, & Freestone, 2021).

Where seeds are not produced, or require symbionts to germinate, other forms of germplasm may be needed to support conservation goals. Species such as bryophytes and pteridophytes can be stored using germplasm types including spores (which may or may not be orthodox in their storage requirements), gametophytes, shoot tips and gemmae (Ballesteros & Pence, 2018; Nebot et al., 2021; North et al., 2021). In Australia, developing hubs of expertise dealing with these germplasm types are located at the National Seed Bank (ACT) and the Royal Botanic Gardens Victoria (Chong et al., 2021; Ohlsen & Miller, 2023). Storage of pollen is often utilised in conservation of crop species and wild relatives and can be important in the recovery of wild species separated by time or distance (Foster et al., 2022; North et al., 2021), including those established as ‘metacollections’ across botanic gardens worldwide (Griffith et al., 2019). Conservation of additional germplasm types, for example symbionts such as mycorrhizal fungi (for orchids) and rhizobia (for species in the family Fabaceae), may be required for successful utilisation (Reiter, Dimon, Freestone, Davis, et al., 2021).

APPLYING THE EXCEPTIONALITY FRAMEWORK TO AUSTRALIAN SPECIES

Our aim was to (a) test the applicability of the Pence, Meyer, et al. (2022) exceptionality factor framework to the Australian flora and (b) understand practical conservation actions that can be taken once an EF is assigned. To test applicability, we sought examples of exceptional species from the Australian flora from data collated in Martyn Yenson et al. (2021), a recently updated resource reviewing literature and compiling knowledge on the Australian flora from over 70 authors and including 50 case studies. We also relied on the knowledge of the expert working group, who have diverse roles in on-ground conservation and research, initially through discussions during Day 2 of the ‘Australian Academy of Science Fenner Conference on the Environment’, and thereafter from the authors' expertise and networks.

In applying the EF framework, the expert working group identified 37 species across 17 families with EF1 characteristics (Table 2a, Box 1). These examples represent the gamut of challenges identified by Pence, Meyer, et al. (2022) including low or no seed production, low seed viability, extremely limited quantities of seed and difficulties accessing seed (Table 2a). We extended the framework of Pence, Meyer, et al. (2022) to partition EF1 species into two subgroups: EF1a for species that do not produce seeds, or only produce non-viable seeds, and EF1b for species with seeds that are limited in either quantity or accessibility. A major insight revealed through this process was that the majority of these EF1 species are listed as threatened species under the Environment Protection and Biodiversity Conservation Act 1999.

TABLE 2a Examples of Australian species within exceptionality factor 1 (EF1, Pence, Meyer, et al., 2022). EF1 has been separated into EF1a where seeds are not produced or not viable, necessitating vegetative propagation, and EF1b for species with seeds limited in quantity or accessibility.

EF	Family	Species	Source	EPBC	Comment
1a	Atherospermataceae	Daphnandra johnsonii	NSW Office of Environment and Heritage, 2022	E	Low levels of seed production; non-viable seed.
1a	Boryaceae	Borya mirabilis	Reiter et al., 2015	E	Likely polyploid; vegetative reproduction.
1a	Fabaceae	Acacia anomala	Coates, 1988	V	Southern populations of species.
1a	Fabaceae	Daviesia speciosa	Schwarten, 1995	E
1a	Fabaceae	Indigofera efoliata	G. Errington, pers. comm.	E
1a	Fabaceae	Ptychosema pusillum	Thompson, 2011; A. Crawford, pers. comm.	V	Seed never seen.
1a	Myrtaceae	Eucalyptus recurva	Department of the Environment, 2015	CE	Very small number of capsules, average of 0.6 viable seeds per capsule. Seeds often hybrids.
1a	Myrtaceae	Verticordia apecta	Department of Environment and Conservation, 2012	CE	Lignotuberous species; seed not seen to date.
1a	Proteaceae	Banksia ionthocarpa subsp. chrysophoenix	Millar et al., 2010	E
1a	Proteaceae	Banksia prionophylla	A. Crawford, pers. comm.	-	Seed never seen.
1a	Proteaceae	Grevillea althoferorum subsp. althoferorum	Burne et al., 2003	E	G. althoferorum Eneabba population in paper
1a	Proteaceae	Grevillea pythara	Krauss & Zawko, 2004	E
1a	Proteaceae	Grevillea renwickiana	McAuliffe, 2020, James & McDougall, 2014	-	Flowers putatively sterile.
1a	Proteaceae	Hakea aenigma	Haegi & Barker, 1985	-	Fruits never forming.
1a	Proteaceae	Hakea pulvinifera	Threatened Species Scientific Committee, 2016	E	No sexual reproduction, extremely low pollen viability.
1a	Violaceae	Melicytus latifolius	M. Wilson, pers. comm.	CE	Very few seeds produced, low viability.
1b	Amaranthaceae	Ptilotus pyramidatus	A. Crawford, pers. comm.	CE	Low seed set.
1b	Apocynaceae	Parsonsia bartlensis	S. Worboys, pers. comm.	-	Seeds are not produced reliably or predictably.a
1b	Cunoniaceae	Ceratopetalum corymbosum	S. Worboys, pers. comm.	-	Seeds are not produced reliably or predictably.a
1b	Ericaceae	Dracophyllum oceanicum	PlantNET, 2023; Desmond et al., 2022	-	Seeds not observed, difficult to access oceanic cliff habitat. Seeds produced in cultivation.
1b	Escalloniaceae	Polyosma reducta	S. Worboys, pers. comm.	-	Seeds are not produced reliably or predictably.a
1b	Fabaceae	Acacia volubilis	Harris & Brown, 2003	E	Evidence of clonal regeneration. Low seed set.
1b	Fabaceae	Daviesia cunderdin	Subroy et al., 2021	E	Limited plants. Low seed set
1b	Goodeniaceae	Scaevola macrophylla	A. Crawford, pers. comm.	CE	Only two plants currently known.
1b	Haloragaceae	Myriophyllum lapidicola	A. Crawford, pers. comm.	E	Limited plants in a very specific habitat.
1b	Lamiaceae	Pityrodia scabra subsp. scabra	Subroy et al., 2021	E	A. Crawford reports no extant plants.
1b	Myrtaceae	Eucalyptus dolorosa	Stack et al., 2004	E	Low seed set.
1b	Myrtaceae	Eucalyptus imlayensis	McDougall et al., 2023	CE	Very few seeds.
1b	Myrtaceae	Gossia gonoclada	K. Sommerville, pers. comm., Austromyrtus gonoclada Recovery Team, 2001	E	Low fruit production, variable seed viability.
1b	Myrtaceae	Pilidiostigma sessile	S. Worboys, pers. comm.	-	Seeds are not produced reliably or predictably.a
1b	Myrtaceae	Rhodomyrtus psidioides	Sommerville et al. (2019); R.O. Makinson, pers. comm.	CE	Only producing seeds ex situ.
1b	Polygonaceae	Muehlenbeckia tuggeranong	Case study EF1 (Z. Knapp), G. Hoyle, unpublished.	E	Only producing seeds ex situ.
1b	Proteaceae	Banksia mimica	Millar & Byrne, 2020	E	Low seed set.
1b	Proteaceae	Synaphea quartzitica	Stack & English, 2003	E	Low seed set.
1b	Ranunculaceae	Clematis dubia	M. Wilson, pers. comm.	CE	Seeds limited in quantity and accessibility.
1b	Symplocaceae	Symplocos graniticola	S. Worboys, pers. comm.	-	Seeds are not produced reliably or predictably.a
1b	Symplocaceae	Symplocos oresbia	S. Worboys, pers. comm.	-	Seeds are not produced reliably or predictably.a

^aSeeds may be produced during the rainy season in December–March when collection sites are difficult to access.

1 BoxCase study EF1—Muehlenbeckia tuggeranong.

Muehlenbeckia tuggeranong Mallinson (Polygonaceae) is one of Australia's rarest plant species and is listed as Endangered under the Australian Environment Protection and Biodiversity Conservation Act 1999. The species occurs in a single population along the Murrumbidgee River, where fruit and seed production have not been observed (originally EF1a). The cause of reproductive failure is unknown, though may be related to its dioecy, the small number of plants, or the distance between individuals. In recent years, flooding and river morphology changes have left some plants under water and the potential loss of these individuals may further limit breeding opportunities.

Since the formal description of the species in 1997 (Makinson & Mallinson, 1997), the Australian National Botanic Gardens has worked in partnership with the ACT Government Environment, Planning and Sustainable Development Directorate to establish an ex situ living plant collection. Over time, as new plants have been discovered in the wild, additional plants have been propagated from cuttings, potentially increasing the genetic diversity held ex situ. Population genetic analysis (L. Broadhurst, pers. comm.) has informed management of the ex situ collection, including replication and maintenance of each known wild genotype, and has shown that some clones held in the ANBG ex situ collection may no longer be extant in the wild population. The ex situ collection also includes clones of the last remaining female plants in the wild.

In spring 2022, fruits were observed on ex situ female plants, and subsequently 53 viable seeds were collected (now EF1b). The pollination mechanism is unknown, but flies were observed visiting flowers. Seeds have since been successfully germinated in the ANBG Nursery, and the National Seed Bank is investigating seed germination requirements (G. Hoyle, unpublished data). This project demonstrates the significant value of ex situ living plant collections for exceptional species that do not readily produce seed in the wild, including opportunities for further research to understand threatened species' genetics, biology, and ecology.

Exceptional species with storage issues (EF2 and EF3) and dormancy issues (EF4) are collated in Table 2b. In total, we collated data for 65 species across 22 families with EF2 characteristics, with most growing in tropical or subtropical rainforests (see also Box 2). An additional 88 species across 44 families exhibit EF3 characteristics, including species identified as short-lived (using real time or rapid ageing data) or freezing sensitive (see also Box 3). Seeds were considered short-lived if their real-time half-life (p50) was ≤ 20 years (Pence, Bruns, et al., 2022) or artificial ageing p50 was ≤ 10 days (Mondoni et al., 2011). Data represent species showing a significant reduction in germination after a brief period in storage at −20°C (Dalziell et al., 2019; Sommerville, Errington, Newby, et al., 2021; Sommerville et al., 2023) or an artificial ageing p50 ≤ 10 days (Cross et al., 2016; Hay et al., 2010; Merritt, Martyn, et al., 2014; Satyanti et al., 2018; Tuckett et al., 2010). Indicative data from artificial ageing studies suggest that these collections should be prioritised for more frequent viability tests than species known to be long-lived under conventional storage conditions, as definitive data on seed longevity under gene banking conditions are not yet available for most Australian species (Merritt et al., 2021).

TABLE 2b Examples of Australian species within Exceptionality Factors 2–4 (EF2, EF3, EF4, Pence, Meyer, et al., 2022). Species within EF2 are desiccation sensitive (do not survive drying to equilibration with 15%RH). Species within EF3 are freezing sensitive or short lived in storage (Pence, Meyer, et al., 2022). Seeds are considered short-lived if real-time p50 ≤ 20 years (Pence, Bruns, et al., 2022) or artificial ageing p50 ≤ 10 days (Mondoni et al., 2011). Data represent species showing a significant reduction in germination after a brief period in storage at −20°C (Dalziell et al., 2019; Sommerville, Errington, Newby, et al., 2021; Sommerville et al., 2023) or p50 ≤ 10 days (Cross et al., 2016; Hay et al., 2010; Merritt, Martyn, et al., 2014; Satyanti et al., 2018; Tuckett et al., 2010). Species within EF4 have deeply dormant seeds (Pence, Meyer, et al., 2022). In this list, we highlight examples for which germination ex situ has not yet been successful despite research on the species.

EF		Family	Species	Source	EPBC
2		Araceae	Gymnostachys anceps	Sommerville, Errington, Newby, et al., 2021	-
2		Araceae	Pothos longipes	Sommerville, Errington, Newby, et al., 2021	-
2		Arecaceae	Archontophoenix alexandrae, A. cunninghamiana	Hamilton et al., 2013	-
2		Arecaceae	Linospadix monostachyos	Sommerville, Errington, Newby, et al., 2021	-
2		Cunoniaceae	Davidsonia pruriens	Sommerville, Errington, Newby, et al., 2021	-
2		Ebenaceae	Diospyros australis, D. mabacea	Sommerville, Errington, Newby, et al., 2021	E (D. mabacea)
2		Ebenaceae	Diospyros compacta	Thusitana et al., 2018	-
2		Elaeocarpaceae	Elaeocarpus obovatus	Sommerville, Errington, Newby, et al., 2021	-
2		Elaeocarpaceae	Sloanea australis	Hamilton et al., 2013, Sommerville, Errington, Newby, et al., 2021	-
2		Fabaceae	Castanospermum australe	Hamilton et al., 2013	-
2		Fabaceae	Archidendron hendersonii	Sommerville, Errington, Newby, et al., 2021	-
2		Flacourtiaceae	Scolopia braunii	Hamilton et al., 2013	-
2		Lauraceae	Cinnamomum oliveri	Sommerville, Errington, Newby, et al., 2021	-
2		Lauraceae	Cryptocarya laevigata	Hamilton et al., 2013, Sommerville, Errington, Newby, et al., 2021	-
2		Lauraceae	Cryptocarya rigida	Sommerville, Errington, Newby, et al., 2021	-
2		Lauraceae	Endiandra pubens	Hamilton et al., 2013	-
2		Lauraceae	Endiandra introrsa	Sommerville, Errington, Newby, et al., 2021	-
2		Lauraceae	Litsea reticulata	Sommerville, Errington, Newby, et al., 2021	-
2		Lauraceae	Neolitsea dealbata	Sommerville, Errington, Newby, et al., 2021	-
2		Malvaceae	Argyrodendron actinophyllum, A. trifoliolatum	Sommerville, Errington, Newby, et al., 2021	-
2		Malvaceae	Sterculia quadrifida	Sommerville, Errington, Newby, et al., 2021, Thusithana et al., 2018	-
2		Meliaceae	Dysoxylum mollissimum, D. rufum	Sommerville, Errington, Newby, et al., 2021	-
2		Meliaceae	Synoum glandulosum	Hamilton et al., 2013	-
2		Menispermaceae	Carronia multisepalea	Sommerville, Errington, Newby, et al., 2021	-
2		Monimiaceae	Wilkiea huegeliana	Sommerville, Errington, Newby, et al., 2021	-
2		Moraceae	Maclura cochinchinensis	Sommerville, Errington, Newby, et al., 2021	-
2		Myrtaceae	Acmena ingens, A. smithii	Hamilton et al., 2013	-
2		Myrtaceae	Pilidiostigma glabrum	Sommerville, Errington, Newby, et al., 2021	-
2		Myrtaceae	Syzygium australe, S. fullagarii, S. moorei, S. paniculatum, S. pseudofastigiatum, S. wilsonii	Hamilton et al., 2013	V (S. moorei)
2		Myrtaceae	Syzygium unipunctatum	Sommerville, Errington, Newby, et al., 2021	-
2		Opiliaceae	Opilia amentacea	Thusithana et al., 2018	-
2		Pennantiaceae	Pennantia cunninghamii	Sommerville, Errington, Newby, et al., 2021	-
2		Piperaceae	Piper hederaceum	Hamilton et al., 2013, Sommerville, Errington, Newby, et al., 2021	-
2		Proteaceae	Helicia ferruginea	Sommerville, Errington, Newby, et al., 2021	-
2		Proteaceae	Hicksbeachia pinnatifolia	Sommerville, Errington, Newby, et al., 2021	V
2		Rubiaceae	Atractocarpus chartaceus, A. fitzalanii, A. stipularis	Hamilton et al., 2013	-
2		Rutaceae	Clausena smyrelliana	Hamilton et al., 2013, Sommerville, Errington, Newby, et al., 2021	-
2		Rutaceae	Glycosmis trifoliata	Hamilton et al., 2013, Thusithana et al., 2018	-
2		Rutaceae	Micromelum minutum	Hamilton et al., 2013, Sommerville, Errington, Newby, et al., 2021, Thusithana et al., 2018	-
2		Sapindaceae	Castanospora alphandii	Sommerville, Errington, Newby, et al., 2021	-
2		Sapindaceae	Cupaniopsis flagelliformis	Sommerville, Errington, Newby, et al., 2021	-
2		Sapindaceae	Diploglottis australis	Sommerville, Errington, Newby, et al., 2021	-
2		Sapindaceae	Diploglottis campbellii	Hamilton et al., 2013	E
2		Sapindaceae	Guioa coriacea	Hamilton et al., 2013	-
2		Sapindaceae	Harpullia arborea, H. pendula, H. ramiflora	Hamilton et al., 2013	-
2		Sapindaceae	Lepiderema pulchella	Sommerville, Errington, Newby, et al., 2021	-
2		Sapindaceae	Mischarytera lautereriana	Sommerville, Errington, Newby, et al., 2021	-
2		Sapindaceae	Rhysotoechia robertsonii	Hamilton et al., 2013	-
2		Sapindaceae	Toechima dasyrrhache	Hamilton et al., 2013	-
2		Sapotaceae	Planchonella australis	Sommerville, Errington, Newby, et al., 2021	-
2		Zamiaceae	Macrozamia macdonnellii	Nano & Pavey, 2008	V
3		Apiaceae	Aciphylla glacialis	Satyanti et al., 2018	-
3		Apiaceae	Gingidia algens. G. harveyana	Satyanti et al., 2018	-
3		Apocynaceae	Melodinus australis	Sommerville, Errington, Newby, et al., 2021	-
3		Araliaceae	Hydrocotyle pedicellosa	Sommerville et al., 2023	-
3		Asteliaceae	Cordyline sp. Mt Banda Banda	Sommerville, Errington, Newby, et al., 2021	-
3		Asteraceae	Celmisia costiniana	Satyanti et al., 2018	-
3		Asteraceae	Craspedia leucantha	Satyanti et al., 2018	-
3		Asteraceae	Podolepis robusta	Merritt, Martyn, et al., 2014	-
3		Asteraceae	Senecio linearifolius	Merritt, Martyn, et al., 2014	-
3		Asteraceae	Vittadinia tenuissima	Sommerville, Errington, Newby, et al., 2021	-
3		Atherospermataceae	Doryphora sassafrass	Sommerville, Errington, Newby, et al., 2021	-
3		Bignoniaceae	Pandorea pandorana	Merritt, Martyn, et al., 2014	-
3		Boraginaceae	Ehretia acuminata	Sommerville, Errington, Newby, et al., 2021	-
3		Brassicaceae	Cardamine papillata	Merritt, Martyn, et al., 2014	-
3		Brassicaceae	Cardamine robusta	Satyanti et al., 2018	-
3		Campanulaceae	Wahlenbergia ceracea	Satyanti et al., 2018	-
3		Capparaceae	Apophyllum anomalum	Sommerville, Errington, Newby, et al., 2021	-
3		Celastraceae	Denhamia silvestris	Sommerville, Errington, Newby, et al., 2021	-
3		Cunoniaceae	Ackama paniculosa	Sommerville et al., 2023	-
3		Cunoniaceae	Ceratopetalum gummiferum	Sommerville et al., 2023	-
3		Cunoniaceae	Karrabina benthamiana	Sommerville et al., 2023	-
3		Cunoniaceae	Vesselowskya venusta	Sommerville et al., 2023	-
3		Cupressaceae	Callitris pyramidalis	Merritt, Martyn, et al., 2014	-
3		Cyperaceae	Carex cephalotes	Satyanti et al., 2018	-
3		Cyperaceae	Carpha nivicola	Satyanti et al., 2018	-
3		Droseraceae	Aldrovanda vesiculosa	Cross et al., 2016	-
3		Ericaceae	Richea continentis	Satyanti et al., 2018	-
3		Euphorbiaceae	Acalypha capillipes	Sommerville, Errington, Newby, et al., 2021	-
3		Euphorbiaceae	Baloghia inophylla	Sommerville, Errington, Newby, et al., 2021	-
3		Gesneriaceae	Fieldia australis	Sommerville et al., 2023	-
3		Haloragaceae	Myriophyllum balladoniense, M. petreanum	Tuckett et al., 2010	-
3		Lamiaceae	Callicarpa pedunculata	Sommerville, Errington, Newby, et al., 2021	-
3		Lamiaceae	Gmelina leichhardtii	Sommerville, Errington, Newby, et al., 2021	-
3		Lamiaceae	Plectranthus nitidus	Sommerville et al., 2023	-
3		Liliaceae	Astelia alpina var. novae-hollandiae	Satyanti et al., 2018	-
3		Malvaceae	Corchorus cunninghamii	Sommerville et al., 2023	-
3		Melastomataceae	Melastoma affine	Sommerville, Errington, Newby, et al., 2021	-
3		Menispermaceae	Stephania japonica var. discolor	Sommerville, Errington, Newby, et al., 2021	-
3		Menyanthaceae	Ornduffia albiflora	Merritt, Martyn, et al., 2014	-
3		Myrtaceae	Archirhodomyrtus beckleri	Sommerville, Errington, Newby, et al., 2021	-
3		Myrtaceae	Austromyrtus dulcis	Sommerville, Errington, Newby, et al., 2021	-
3		Myrtaceae	Lenwebbia prominens	Sommerville et al., 2023	-
3		Myrtaceae	Melaleuca styphelioides	Sommerville et al., 2023	-
3		Myrtaceae	Rhodamnia maideniana	Sommerville, Errington, Newby, et al., 2021	CE
3		Myrtaceae	Rhodomyrtus psidioides	Sommerville, Errington, Newby, et al., 2021	CE
3		Myrtaceae	Uromyrtus australis	Sommerville, Errington, Newby, et al., 2021	E
3		Nymphaeaceae	Nymphaea immutabilis, N. lukei, N. macrosperma, N. violacea	Dalziell et al., 2019	-
3		Onagraceae	Ludwigia octovalvis	Sommerville et al., 2023	-
3		Orchidaceae	Caladenia flava, C. huegelii	Hay et al., 2010	E (C. huegelii)
3		Orchidaceae	Diuris fragrantissima	Hay et al., 2010	E
3		Orchidaceae	Pterostylis recurva, P. sanguinea	Hay et al., 2010	-
3		Orchidaceae	Thelymitra crinita	Hay et al., 2010	-
3		Paracryphiaceae	Quintinia verdonii	Sommerville et al., 2023	-
3		Pittosporaceae	Pittosporum multiflorum	Sommerville, Errington, Newby, et al., 2021	-
3		Plantaginaceae	Plantago drummondii	Merritt, Martyn, et al., 2014	-
3		Plantaginaceae	Plantago glacialis	Satyanti et al., 2018	-
3		Poaceae	Australopyrum velutinum	Satyanti et al., 2018	-
3		Poaceae	Entolasia marginata	Sommerville et al., 2023	-
3		Poaceae	Panicum bisulcatum	Sommerville, Errington, Newby, et al., 2021	-
3		Poaceae	Poa costiniana, P. hiemata	Satyanti et al., 2018	-
3		Proteaceae	Lomatia arborescens	Sommerville et al., 2023	-
3		Proteaceae	Lomatia myricoides	Merritt, Martyn, et al., 2014	-
3		Proteaceae	Telopea speciosissima	Merritt, Martyn, et al., 2014	-
3		Ranunculaceae	Clematis linearifolia	Merritt, Martyn, et al., 2014	-
3		Ranunculaceae	Psychrophila introloba	Satyanti et al., 2018	-
3		Ranunculaceae	Ranunculus acrophilus, R. clivicola, R. lappaceus	Satyanti et al., 2018	-
3		Rhamnaceae	Alphitonia oblata	Sommerville, Errington, Newby, et al., 2021	-
3		Rousseaceae	Abrophyllum ornans	Sommerville et al., 2023	-
3		Rousseaceae	Cuttsia viburnea	Sommerville et al., 2023	-
3		Rubiaceae	Gynochthodes jasminoides	Sommerville, Errington, Newby, et al., 2021	-
3		Rubiaceae	Psychotria loniceroides	Sommerville et al., 2023	-
3		Rutaceae	Acradenia euodiiformis	Sommerville, Errington, Newby, et al., 2021	-
3		Rutaceae	Flindersia schottiana	Sommerville, Errington, Newby, et al., 2021	-
3		Scrophulariaceae	Glossostigma drummondii	Tuckett et al., 2010	-
3		Simaroubaceae	Quassia sp. Mount Nardi	Sommerville, Errington, Newby, et al., 2021	-
3		Solanaceae	Nicotiana forsteri	Sommerville et al., 2023	-
3		Solanaceae	Solanum vicinum	Sommerville et al., 2023	-
3		Urticaceae	Boehmeria macrophylla	Sommerville et al., 2023	-
4		Apiaceae	Eryngium ovinum	Hodges et al., 2019	-
4		Asparagaceae	Arthropodium strictum	Hodges et al., 2019	-
4		Asparagaceae	Sowerbaea laxiflora	Tieu et al., 2001	-
4		Celastraceae	Stackhousia monogyna	Vening et al., 2017	-
4		Cyperaceae	Gahnia sieberiana	G. Liyanage, pers.comm.	-
4		Cyperaceae	Lepidosperma concavum, L. laterale, L. longitudinale	Kodym et al., 2010	-
4		Cyperaceae	Lepidosperma scabrum	Turner, 2013	-
4		Dilleniaceae	Hibbertia commutata, H. huegelii, H. hypericoides, H. racemosa	Schatral, 1996; Hidayati et al., 2012	-
4		Ericaceae	Astroloma xerophyllum	Turner et al., 2009	-
4		Ericaceae	Leucopogon esquamatus, L. exolasius, L. setiger	Ooi et al., 2006	V (L. exolasius)
4		Goodeniaceae	Goodenia scaevolina	Merritt et al. 2007	-
4		Goodeniaceae	Goodenia cycloptera, G. fascicularis	Hoyle et al., 2008	-
4		Goodeniaceae	Velleia glabrata	Hoyle et al., 2008	-
4		Gyrostemonaceae	Tersonia cyathiflora	Baker et al., 2005; Merritt et al. 2007	-
4		Gyrostemonaceae	Codonocarpus cotinifolius	Baker et al., 2005	-
4		Gyrostemonaceae	Gyrostemon racemiger	Baker et al., 2005	-
4		Hemerocallidaceae	Dianella longifolia, D. revoluta	Vening et al., 2017; Hodges et al., 2019	-
4		Musaceae	Musa banksii	G. Liyanage, pers.comm.	-
4		Nymphaeaceae	Nymphaea pubescens	Dalziell et al., 2018	-
4		Poaceae	Entolasia stricta, Eragrostis leptostachya, Panicum effusum, P. simile, Paspaladium distans	Clarke & French, 2005	-
4		Poaceae	Triodia angusta, T. basedowii	Erickson et al., 2016	-
4		Pittosporaceae	Cheiranthera cyanea	Hodges et al., 2019	-
4		Pittosporaceae	Marianthus bicolor	Merritt et al., 2007	-
4		Proteaceae	Grevillea juniperina, G. sericea, G. speciosa	Morris, 2000	-
4		Proteaceae	Persoonia acerosa, P. levis, P. linearis, P. longifolia, P. nutans, P. pauciflora, P. pinifolia	Chia et al., 2016, Emery & Offord, 2019a, Catelotti et al., 2020	V (P. acerosa), E (P. nutans), CE (P. pauciflora)
4		Restionaceae	Loxocarya striatus, Schoenus unspiculatus	Tieu et al., 2001	-
4		Rutaceae	Asterolasia buxifolia	Collette & Ooi, 2017	Under assessment
4		Rutaceae	Boronia anemonifolia ssp. anemonifolia, B. floribunda, B. fraseri, B. ledifolia, B. pinnata, B. serrulata, B. thujona	Mackenzie et al., 2016	-
4		Rutaceae	Diplolaena dampieri, Rhadinothamnus anceps	Just et al., 2023	-
4		Rutaceae	Zieria alata	G. Liyanage, pers.comm.	-
4		Scrophulariaceae	Eremophila oldfieldii	Commander et al., 2009	-

2 BoxCase study EF2—Syzygium.

Syzygium is a genus of trees and shrubs in the Myrtaceae family native to tropical and subtropical regions of Africa, Asia, Australia, and the Pacific islands (PlantNET, 2023). Around 70 species occur naturally in Australia where they chiefly grow in rainforest habitats (Harden et al., 2017; Zich et al., 2020). Nine Syzygium species are currently listed as threatened under State or Federal legislation. Diseases such as Myrtle Rust and Phytophthora have the potential to put further pressure on these already threatened species (Brown, 1999; Makinson et al., 2020). Populations of the vulnerable Syzygium hodgkinsoniae, for example, have been severely impacted by Myrtle Rust in the wild and this species has now been listed as a high priority for ex situ conservation (Makinson et al., 2020).

All Syzygium species for which storage behaviour data have been published produce seeds that do not tolerate the drying necessary for seed banking (Sommerville et al., 2018; Sommerville, Errington, Newby, et al., 2021). Ex situ conservation for species in this genus must therefore be based on living collections, tissue culture or cryopreservation of shoots or embryonic axes. Potted collections for two threatened species (S. hodgkinsoniae and Syzygium paniculatum), and the Lord Howe Island endemic Syzygium fullagarii, have been established at the Australian PlantBank from wild-sourced seed and cuttings. These collections have been crucial to providing the healthy shoots necessary to initiate the species into tissue culture, a sterile environment in which they can be maintained free from the impact of Myrtle Rust.

As tissue culture collections can be labour intensive to maintain, work to develop cryopreservation protocols for tissue-cultured shoot tips has now commenced in a collaboration with Curtin University, University of Queensland, Botanic Gardens and Parks Authority (WA) and several other partner organisations. Once protocols have been developed and optimised, it will be possible to store genetically representative tissue for each species at ultra-low temperatures (e.g., −192°C). This, in turn, will allow long-term preservation with minimal on-going costs.

3 BoxCase study EF3—Nymphaea.

Waterlilies are iconic wetland plants found across the world, of which 18 native species are found throughout the wet-dry tropics of northern Australia (Jacobs & Hellquist, 2011; Löhne et al., 2009). While most of these species are relatively common and are distributed across the Northern Territory, Queensland and Western Australia, several species are more restricted in their range and are of potential conservation concern (Western Australian Herbarium, 1998). The increasing impact of climate change via associated sea-level rise and saltwater intrusion of low-lying coastal freshwater wetlands is of significant concern for many species, given that even slight increases in salinity result in reduced germination and stunted seedling development (Dalziell et al., 2020). As a result, ex situ conservation is likely to be required to ensure the effective conservation of impacted species.

Nymphaea in Australia may be considered exceptional for several reasons. Freshwater wetlands in northern Australia are often remote and difficult to access, requiring the use of four-wheel drive vehicles, boats, or helicopters. Seed collection is difficult, as once pollinated, flowers sink below the water line and the developing fruit is recoiled towards the underlying substrate. In addition, the distribution of Nymphaea through the wetlands of northern Australia also overlaps with the distribution of fresh and saltwater crocodiles making seed collecting an extremely dangerous task in some areas.

Once seeds have been collected, overcoming dormancy and stimulating germination can be problematic for some species, making assessments of seed viability difficult (Dalziell et al., 2018, 2019). In terms of their seed storage behaviour, most Australian species of Nymphaea appear to be desiccation tolerant and survive drying to 15% RH (~5% internal seed moisture content; Dalziell et al., 2019). However, when exposed to a range of experimental storage temperatures (25°C, 5°C, −20°C and −196°C) at different seed moisture contents (15, 30, 70, 95% RH) individual species and collections responded differently in terms of viability and longevity (Dalziell et al., 2019). In several instances colder storage temperatures (i.e., at −20 and −196°C) did not translate to an increase in longevity in comparison with seeds stored above 0°C. For seeds of Nymphaea immutabilis, Nymphaea lukei, Nymphaea macrosperma, and two of four accessions of Nymphaea violacea, seed germination and viability declined markedly over the 12-month storage duration, even under conventional long-term storage conditions, that is, 15% RH and −20°C. If ex situ conservation for all species of Australian Nymphaea is to be successful, alternative storage conditions should be trialled to determine whether different storage temperatures or moisture contents improve viability. Alternatively, the storage of other forms of germplasm including bulbs, rhizomes and tubers may be beneficial.

Challenging seed dormancy syndromes (EF4) have been identified in Australian species in several plant families including Cyperaceae, Dasypogonaceae, Dilleniaceae, Ericaceae, Restionaceae and Rutaceae, in addition to some Persoonia species in the Proteaceae family (Hirst et al., 2021; Table 2b; Box 4). In total, we identified 61 species from 17 families as exemplars of EF4, with a focus on species for which germination ex situ has not yet been successful despite significant research (Table 2b). Rainforest species in plant families such as Celastraceae, Elaeocarpaceae and Podocarpaceae have also been noted with germination times of more than 1 year (Zich et al., 2020).

4 BoxCase study—EF4 Persoonia.

Persoonia (Proteaceae) is a genus of long-lived small trees and spreading or prostrate shrubs that are endemic to Australia. Most species are narrow range endemics occurring on acidic sandy or sandstone-derived soils with a low nutrient content in south-eastern and south-western Australia. A total of 43 species are listed as threatened and a further two species are extinct under the IUCN Red List (IUCN, 2023). The dispersal unit of Persoonia is a fleshy drupaceous fruit that contains an endocarp covering one or two seeds. Germination after fire is commonly known in the genus (Auld et al., 2007). Poor recruitment success or limited recruitment has been documented for several Persoonia species (Persoonia elliptica, Persoonia hirsuta and Persoonia pauciflora).

Early research into the germination of Persoonia seeds identified that removal of at least half of the endocarp was necessary for germination to occur (Ketelhohn et al., 1996; Nancarrow, 2001). Typically, entire endocarps are now removed before sowing seeds in the laboratory or nursery. More recently, imbibition tests and burial trials determined that the endocarp is not an absolute physical barrier to water uptake (i.e., seeds are not physically dormant), but a mechanical mechanism that is water-permeable and weakens in the soil seedbank from wet-dry seasonal climate cycling for up to 24 months or more (Chia et al., 2016; Emery & Offord, 2019a).

Despite the revelation of the endocarp as a mechanical barrier, germination success of Persoonia remained highly variable and standardised germination protocols among species were still not possible. The highest germination success of several eastern Australia Persoonia species was reported to occur following mild temperatures reflecting typical autumn averages (Catelotti et al., 2020). However, the proportion of germinated seeds was very low among the species tested. The hypothesis of a physiologically dormant embryo was recently supported after seed burial trials showed that germination significantly increased following cycles of warm and cold stratification (Chia et al., 2016; Emery & Offord, 2019a). In these studies, germination significantly increased when seeds were retrieved in autumn, suggesting that summer conditions relaxed physiological dormancy.

The outcomes of >20 years of research on Persoonia seed biology have demonstrated the complex germination system of this genus. The seasonal environmental conditions are critical for weakening the endocarp and alleviating physiological dormancy. Monitoring these conditions and the interaction with other factors that weaken the endocarp, including fire and consumption by vertebrate fauna, can provide insight into the timing of recruitment events in situ and the development of ex situ germination protocols (Emery & Offord, 2018). Significant progress in understanding the perplexities of Persoonia seeds, over many years of research, has culminated in achieving positive restoration outcomes for several threatened species through trial translocation programmes (Andres et al., 2022; Emery et al., 2018; Emery & Offord, 2019b).

Overall, this preliminary assessment identified exceptional species in 73 families, with some exhibiting more than one exceptionality factor (Table 2a,b). Exceptional species have been identified among plant families in which conventional seed storage and utilisation is often applicable, including Fabaceae (8 species across EF 1 and 2) and Myrtaceae (24 species across EF 1, 2 and 3). Assessing species for exceptionality assists with the identification of knowledge gaps to be addressed and decisions around which conservation techniques should be applied to adequately conserve a species. Resolving which factor is relevant for each species, or whether multiple factors are hampering conservation efforts, may assist practitioners in developing suitable germplasm collections and deciding on research priorities. For example, in the family Rutaceae (16 species across EF 2, 3 and 4), challenges with germination may be the result of seed drying (EF2), time in storage (EF3), dormancy (EF4) or a combination of these.

In addition to the examples in Tables 2a and 2b, the expert working group documented case studies for each Exceptionality Factor to exemplify application to conservation of Australian flora, across diverse genera (Figure 2, Boxes 1–4).

Considerations for applying the EF framework

The EF framework provides a means to identify when conservation of a species may require, for example, successive seed collections over many years or multiple types of germplasm and storage techniques. The framework also facilitates the prioritisation of research needed to overcome barriers to the storage and use of germplasm, so that healthy plants may be generated and utilised in situ as required. We have developed a flow chart (Figure 3) to visualise this workflow.

Applying the EF framework led the working group to propose two changes to optimise its application to conservation practice in Australia. We propose that EF1 be further divided into two sub-categories reflecting the different conservation techniques that can be applied. For species for which seed is either not produced or is not viable (hereafter referred to as EF1a), then vegetative propagation methods provide the only means by which plants can be produced. To preserve an ex situ conservation collection of such species, a decision will need to be made as to whether to establish living collections, tissue culture or both. Whichever method is used, sufficient maternal lines should be conserved and maintained to adequately represent the genetic diversity of the source population. When translocation is considered, plants would be sourced from these living or tissue culture collections.

Some species, while producing seeds, do so in such small quantities that capturing sufficient seeds to bank or to facilitate on ground recovery, such as translocation, is difficult in the short term (<10 years) (Gibson-Roy, Breed, et al., 2021; Subroy et al., 2021) (hereafter referred to as EF1b). For these species, a decision must be made to determine the most appropriate method of conserving the species ex situ. For many species, if adequate seed quantities can be secured over time or through production, seed conservation is still the most cost-effective means of conserving a genetically representative sample of the species, particularly when the number of plants in the wild is still relatively high. In some instances, ex situ vegetative conservation measures as per EF1a species may be considered instead of, or in conjunction with, seed conservation. For long-term conservation and/or utilisation, a seed multiplication stage that maintains genetic diversity may be considered (Hay & Probert, 2013). For example, when translocation is required, EF1b species would require a multiplication (seed orchard) stage to obtain sufficient seed to produce adequate numbers of seedlings (Subroy et al., 2021; Figure 3). However, methods for multiplying material may be species specific, and losses can occur throughout the establishment process (Hay & Probert, 2013).

The categorisation of a species to EF may be temporal in nature, including EF1a that produce seed ex situ but not in situ, EF1b that may be classified as ‘non-exceptional’ if seed production can be resolved, and EF4 that may be classified as ‘non-exceptional’ if research can alleviate dormancy (Figure 3). Some EF1b species may be ephemeral, or rarely seen for most of the time, but be present in high numbers following certain environmental events such as fire (e.g., Bell, 2021) or heavy, episodic rainfall events (Silcock et al., 2011). Fecundity can be affected by population size, with small populations often producing less seed than larger ones (e.g., Lamont et al., 1993), thus taking advantage of a population when it is in a ‘boom’ phase may overcome the limitations to seed production brought about by small population size. Likewise, if seed production areas create a larger population size than present in the wild, seed availability may increase. Issues that limit seed collection from inaccessible species in EF1b may also be addressed by new technology, for example, the use of drones to collect some tropical or cliff top species that are otherwise inaccessible to collectors (La Vigne et al., 2022).

Where genetic data are available, categorisation of taxa into Exceptionality Factors might be better done at the level of the Conservation Unit (CU) (Coates et al., 2018) rather than the species, especially where CUs signify different reproductive strategies i.e., sexual v clonal within the same species. Conservation Units have been variously defined and interpretations and criteria differ, but the primary aim is to recognise major elements of intraspecific diversity for conservation actions noting that CUs usually do not correspond to taxonomically recognised infraspecific entities (see Coates et al., 2018). For example, Acacia anomala, a threatened species from Western Australia, occurs as two CUs 30 km apart, with the northern CU found to reproduce sexually, while the southern CU reproduces vegetatively with each population consisting of one or a few clones (Coates, 1988). As the aim of ex situ collections is to sample species diversity both within, and between populations, different strategies may be required for each of the CU's. In other species, after closer taxonomic scrutiny, CUs have been assigned to different taxonomic entities (e.g., Grevillea althoferorum: Burne et al., 2003, Olde & Marriott, 2008; Banksia ionthocarpa: George, 2005, Millar et al., 2010).

It is important to note that the procedures required to assign exceptionality are those intrinsic to management, curation and utilisation of germplasm, rather than a separate suite of tools. It is particularly helpful to identify which impediments to ex situ conservation exist for each species, to prioritise future research and focus on possible interim storage solutions (Figure 3). Storing germplasm of vegetative tissue, for example, is research and labour intensive (Wyse et al., 2018), with species-specific protocols required for the initiation of tissue culture systems and the development of cryostorage techniques, and such protocols have only been developed for a small number of Australian species (Funnekotter et al., 2021; Sommerville, Errington, Funnekotter, & Newby, 2021). Ideally the characteristics of exceptionality only apply for a period of time, as with sufficient resources for research (personnel, facilities, technology and curation) many species may be able to be progressed through the EF framework to develop appropriate collection, storage or germination protocols and thus reclassified as ‘non-exceptional’ (Figure 3).

Applying resources and research effort to understanding seed biology and storage behaviour pay dividends in maximising longevity in storage, and facilitating dormancy break and utilisation, for both non-exceptional and exceptional species. Seed of species with EF1b and EF4 can still be collected with confidence, though additional resources will be required to bulk up seed collections (for example, see Gibson-Roy, Breed, et al., 2021; Subroy et al., 2021) or understand quality, germination and dormancy limitations (Hirst et al., 2021; Merritt et al., 2021). Understanding the barriers to production of healthy plants, via germination or other methods of propagation, is critical to resolving exceptionality and returning plants into the landscape—the ‘exit plan’ that needs to be defined for all stored germplasm.

Understanding exceptionality is essential to holistic ex situ conservation

Identifying exceptional species within Australia's plant biodiversity is important to the development of ex situ conservation strategies and associated research that respond to the growing number of threatened plant species and ecological communities and address contemporary threats. The Australian Threatened Species Action Plan 2022–2032 (Commonwealth of Australia, 2022) and the Kunming-Montreal Global Biodiversity Framework (CBD, 2022) both highlight that in situ and ex situ conservation of a greater proportion of threatened plant species is a priority in order to halt extinction. This will require significantly greater effort and resources as we seek to conserve and use germplasm that is more difficult to collect and propagate. Within Australia, the number of orthodox and non-orthodox species held ex situ continues to expand, and the associated maintenance requirements of these collections will continue to increase. A lack of resources and investment in understanding exceptionality, alongside ongoing work on orthodox species, risks exclusion of a wide range of species from being secured ex situ and available for future translocation and restoration programmes. Addressing the difficulties and time frames associated with entry and exit from ex situ storage, for both exceptional and orthodox species, is essential to meeting conservation goals.

Prioritising assessment of exceptionality factors

Now that we have demonstrated that the EF framework can be applied to the Australian flora, a systematic assessment of exceptionality factors should be a high priority for Australia's current >1300 (EPBC Act) threatened plant species, which are more likely to exhibit at least one of these factors (Wyse et al., 2018). Assessment is particularly urgent for species identified as requiring translocation, as their exit from germplasm storage is imminent and both sufficient propagules and knowledge of propagation requirements are required for translocation success (Commander et al., 2018). Conserving species while they are healthy, productive, and genetically diverse means it is possible to make high quality collections that do not suffer the same limitations as encountered for threatened species. Having a flexible process of ex situ conservation prioritisation that can incorporate new and emerging threats, such as disease-causing pathogens, is critical to ensure the best use of limited resources. An understanding of exceptionality factors can also aid in the utilisation of more common species such as those required for restoration, species that restore landscape structure and function (ecosystem engineers), and species within threatened ecological communities. For example, for commercial operations, the recommendation for species exhibiting EF2 behaviour could be to use the seed immediately in restoration projects.

Conserving and utilising germplasm relies on a wide range of ex situ facilities

To successfully conserve a wide range of species ex situ, including exceptional species, consideration for the sourcing of multiple forms of germplasm is required (Wyse et al., 2018), and different conservation goals require the transitioning of germplasm through different collection types. For example, the use of banked seeds for ecological restoration or translocation may require transitioning seeds from the seedbank, to seedlings via the nursery, and into the landscape (Dillon et al., 2018; Turner et al., 2021). Seed collections identified as decreasing in viability may also need regeneration in the nursery to allow for replacement collections to be banked—replenishing seed stocks is a key consideration for crop genebanks and may become increasingly important for wild species that can no longer be collected (Gibson-Roy, Breed, et al., 2021); albeit regeneration for wild species can be fraught due to adult plant size, or long growing periods to reach reproductive maturity. Using seeds to initiate nursery living collections as seed production areas can also be necessary to support plant translocations (Subroy et al., 2021; Gibson-Roy, Breed, et al., 2021) or research to resolve issues with germination, dormancy and storage. Complementary storage methods such as cryopreservation often require transition through different germplasm types, such as tissue culture, prior to and following removal from storage at low temperatures.

Resourcing ex situ plant conservation must consider exceptional species

Inclusion of exceptional species in ex situ conservation programmes strengthens the potential for creating insurance populations of the widest possible range of species, as long as it is adequately resourced alongside in situ management to protect populations and resources to maintain existing ex situ collections. Conservation of most exceptional species is technically possible, and Australia is fortunate to have a network of personnel, expertise and facilities suited to the task (Figure 1) at current scales of activity, but the need is expected to grow rapidly in the near future. Botanic gardens and other conservation agencies act as knowledge hubs with the appropriate botanical expertise and specialist horticulture facilities required to identify, collect, research and conserve exceptional species (Westwood et al., 2020), with universities also playing a key role in research. Some restoration facilities have also utilised tissue culture to provide an appropriate suite of species for restoration (Koch, 2007; Willyams, 2005). With an increasing number of species recognised as exceptional, it is timely to consider the need to scale up existing resources to increase our capacity to understand and manage complex seed behaviour and curate more extensive living collections. Living plant collections are currently the only available option for many exceptional species, at least in the short term, until alternative methods are refined for each species (Fant et al., 2016).

Labour is the main cost for conserving exceptional species in vitro (in tissue culture) and via cryopreservation in the USA (Philpott et al., 2022) and the situation is similar in Australia. The cost to grow plants for living collections, seed orchards or translocation is also high, with the establishment of tested and reliable protocols for successful germination alone costed at between $3168 AUD and $125,630 for a subset of Endangered and Critically Endangered species in the Western Australian Wheatbelt (Subroy et al., 2021 and supplementary data therein). It is often the same people who work on orthodox and exceptional species, so there is a need to train additional personnel and expand the network of skilled practitioners who can identify exceptional species and work through the necessary steps to secure germplasm and make it available in the future (Philpott et al., 2022). Collaboration between larger well-established labs and smaller ones beginning work on exceptional species, supported with targeted investment, provides one model for scaling up this work (Philpott et al., 2022). Ex situ conservation is a long-term proposition, as species may be required in the landscape years or decades into the future. So appropriate resources and expertise must be maintained in organisations through robust succession planning, collection curation and data management, including transfer of information and processes over long time frames, and in concert with growing investment profiles that match the exponential increase in collection size and associated maintenance requirements (Fu, 2017).

Network approach to the conservation of exceptional species

Australia has benefited from investment in seed collection and germplasm storage facilities, bolstered by the Millennium Seed Bank Partnership, which led to the coordination of seedbanks and associated research facilities through the Australian Seed Bank Partnership (Wrigley & North, 2021; CHABG (Council of Heads of Australian Botanic Gardens), 2022). Further plant conservation support and expertise contributed through the Australian Network for Plant Conservation for knowledge sharing and communication, and Botanic Gardens Australia and New Zealand and Botanic Gardens Conservation International for horticulture in botanic gardens, are critical to comprehensive conservation outcomes. These collaborative partnerships and sophisticated management tools for plant collections are necessary to identify and store exceptional species (Martyn Yenson et al., 2022; Pence, Meyer, et al., 2022; Philpott et al., 2022). Within the Australian network, one project underway to preserve plants endemic to tropical montane cloud forests threatened by climate change has already identified exceptional species in all four categories (G. Hoyle, unpublished data; K. Sommerville, pers. comm.). Shared projects such as this offer a model for utilising and expanding on existing networks, examining existing data to understand which species are exceptional, applying and sharing expertise relating to these species, and securing a wider range of species in ex situ storage.

Plant conservation networks in Australia have reached a level of technical maturity that would lend itself to extending support for plant conservation across the Indo-Pacific region, in collaboration with other active global networks. Recently published Guidelines (Martyn Yenson et al., 2021), while tailored to Australian species, draw on the international plant conservation literature and present decision support tools that can provide a template for other countries aiming to conserve plant germplasm. In addition to formal guidelines, peer-reviewed journal papers and databases, practitioners rely on personal and regional or local contacts to access information on exceptional species and adapt them to suit their local context (A. Martyn Yenson, unpublished). This contact is facilitated through the global Exceptional Plant Conservation Network, as well as the Millennium Seed Bank Partnership and the Australian Seed Bank Partnership. This could be supplemented by a regional network, such as the Australasian Seed Science Network, for better support, coordination and awareness raising - a topic under active discussion at recent Australasian and global conferences (D. Wrigley, pers. comm.).

Practitioners and scientists throughout the region are urged to consolidate local, regional and personal contacts to support their work in this ‘critical window of opportunity’ before extinction may occur (Sommerville et al., 2018). Australian conservation leaders must be supported to share their expertise with others in less well-resourced countries to stem global biodiversity losses, particularly as global efforts focus on delivering the agreed priorities of the Kunming-Montreal Global Biodiversity Framework (CBD, 2022).

CONCLUSION

The methods to assess exceptionality are now available and the framework provides a workflow to identify and overcome barriers to storage of germplasm, so that healthy plants may be generated and utilised in situ as required (Figure 3). The framework supports decision-making relating to ex situ conservation, including more realistic appraisal of the challenges and resources required to create insurance collections of exceptional species. The framework helps prioritise research and development of appropriate storage methods for each EF identified, so that with sufficient time and resources some species can be reclassified as ‘non-exceptional’. The case studies presented are indicative of the detailed research required to address exceptionality factors and establish ex situ collections of exceptional species. The assessment of exceptionality should be considered alongside the existing suite of tools and research for ex situ germplasm conservation, and in addition to management of threats and care for plant diversity in situ. This preliminary assessment suggests that a proportion of Australian species may have one or more EF, which need to be resolved to successfully conserve the species ex situ and make them available for future utilisation. To address these issues with the urgency required in the current environmental crisis, long term funding and cohesive partnerships across disciplines must be maintained and expanded. A comprehensive review of exceptionality in the Australian flora would be a useful next step in identifying and managing these challenging species. Natural resource managers, threatened species experts, government agencies managing biodiversity and non-government organisations in this space must consider the need to significantly expand germplasm collections of all types including exceptional species, and provide resources for associated labour costs, to successfully conserve and restore Australia's national plant treasures.

AUTHOR CONTRIBUTIONS

Amelia J. Martyn Yenson, Karen D. Sommerville, Lydia K. Guja, Lucy Commander, Robert O. Makinson, Tony D. Auld, Damian Wrigley and Catherine A. Offord contributed to planning the Fenner Conference; Amelia J. Martyn Yenson, Karen D. Sommerville, Lydia K. Guja, David J. Merritt, Emma L. Dalziell, Leonie Monks, Damian Wrigley, Linda Broadhurst, Zoe Knapp, David J. Coates, Bryn Funnekotter, Robert O. Makinson and Catherine A. Offord wrote and edited the manuscript; Zoe Knapp, Karen D. Sommerville, Emma L. Dalziell and Nathan J. Emery contributed case studies; Karen D. Sommerville, Lydia K. Guja, Andrew D. Crawford, David J. Merritt, Emma L. Dalziell, Leonie Monks, Robert O. Makinson and Bryn Funnekotter contributed data (Table 2); Amelia J. Martyn Yenson, Bryn Funnekotter, Karen D. Sommerville, David J. Merritt, Emma L. Dalziell, Andrew D. Crawford, Lydia K. Guja, and Damian Wrigley contributed to the workflow (Figure 3).

ACKNOWLEDGEMENTS

The authors acknowledge The Ian Potter Foundation for funding the revision of ‘Plant Germplasm Conservation in Australia’, including salary for AJMY and an associated expert workshop; this was supplemented with funds from the Australian Academy of Science to hold the Australian Academy of Science Fenner Conference on the Environment 2021/22 with the theme ‘Exceptional Times, Exceptional Plants’. The authors acknowledge Australian Network for Plant Conservation staff Jo Lynch and Christine Fernance for their support of the Fenner Conference, the Australian Seed Bank Partnership for incorporating the Fenner Conference into the broader Australasian Seed Science Conference (2021) programme, and the Fenner Conference presenters and participants. Thanks to Craig Miskell (CAM Graphics) for layout of Figures 1 and 3, and to Gemma Hoyle for comments on an earlier version of the manuscript. ELD and DJM acknowledge financial support from the Australian Research Council (Project LP200200680).

CONFLICT OF INTEREST STATEMENT

All authors declare that they have no conflicts of interest.

DATA AVAILABILITY STATEMENT

The datasets used during the current work are available from the corresponding author upon request.