Cascading questions for leafless Bossiaea

Working with stakeholders from different Australian states, geneticists have helped to clarify taxonomy and conservation planning for seven leafless Bossiaea species.

Often in science one question will lead to another. And another. When geneticists from the Research Centre for Ecosystem Resilience (ReCER) started working with the NSW Saving Our Species team to investigate the quirky group of peas known as the "leafless bossiaea", the project bounced in more than one unexpected direction. Genetic profiling of seven species enabled:

• identification of a possible new subspecies
• collapse of two species into one and
• ongoing research to ask, at what point do we intentionally mix isolated plant populations to reduce inbreeding, and could this mixing include hybridisation?

Australia is famous for its unique and sometimes unusual plant species, and among them is a fascinating group known as the ‘leafless Bossiaea.’ As the name suggests, these plants stand out for their flattened, photosynthetic stems and highly reduced leaves that have evolved into tiny scales, giving them the appearance of being entirely leafless.

There are 78 recognized species of Bossiaea, ranging from shrubs to small trees. While most species have traditional stems, leaves, and the characteristic pea flowers that rely on pollinators to transfer pollen (see the video below for more info) a subset of species along Australia’s east coast has evolved differently. These plants have dispensed with their leaves entirely (aphyllous) and replaced them with flattened, photosynthetic stems called cladodes, reinforced with woody, lignified tissue.

The evolution of aphyllous or leafless plants is theorised to be an adaptation to stressful environments. Instead of leaves, the stems of these plants have adapted to contain chlorophyll meaning they can photosynthesise. In dispensing with leaves these Bossiaea have less lignin meaning they can’t grow as tall (and are consequentially green not brown stemmed) but the trade-off is they can persist in more hostile environments. The absence of leaves reduces water loss and heat stress, through reducing the total surface area used for photosynthesis and respiration. Having less surface area is also theorised to reduce the nutrients required to grow and maintain leaves which means leafless species can grow in nutrient poor soils. Leaflessness occurs in many plant families across the globe, such as Cactacaeae (Cactus) and Euphorbiacaea (Euphorbias). These plant families are often associated with hot, arid, nutrient poor environments.

When taxonomists classify genera and species within them, one feature strongly relied on is floral structure. Bossiaea can be particularly tricky to classify because the floral characteristics normally used to differentiate between species are surprisingly similar.

This means that taxonomists must rely on leaf, indumentum (hair), stipule (small outgrowths enfolding the leaf stalk) and fruit traits, to classify species. The leafless Bossiaea group are, perhaps not surprisingly, an additional challenge because they are aphyllous, with compressed photosynthetic stems (cladodes), and lacking stipules. This group are also the most widely distributed, with the 12 known species ranging from far north Queensland to Victoria and Tasmania.

To classify leafless Bossiaea, taxonomists have relied on a variety of key traits, such as the number of scales on the stem, flower arrangement/quantity, and the size of the non-floral structures that surround the flowers (bracts and bracteoles) as well as growth form and geographic distribution. Differences in the morphological features, plus discrete location, support the assumption that an isolated population is a unique species. But is this supported by genetics?

Working with the NSW Saving Our Species program ReCER geneticists have be able to review the classification of seven leafless Bossiaea, five of which are threatened with extinction.

Eilish McMaster, a geneticist with the Conservation Genomics team at the Research Centre for Ecosystem Resilience (ReCER), analysed population-level Single Nucleotide Polymorphism (SNP) data to examine the relatedness among seven leafless Bossiaea species

SNPs represent natural variations in genome base pairs (alleles) that can influence specific traits within a species (for example, in radishes, a single SNP can determine whether flowers are purple or white) but often do not have any noticeable physical expression. These variations are more frequently shared among related individuals, allowing them to be used to assign individuals to specific populations and species.

Using SNPs, Eilish and the team found that for many leafless Bossiaea species, gene flow is limited beyond distances of one kilometre, indicating restricted movement of seeds and pollen. This suggests that numerous populations, located in discrete and isolated areas, are persisting through a combination of clonal reproduction and within family pollination, highlighting the potential benefits of genetic rescue or outcrossing.

Genetics also enabled the identification an entirely new population of the Bossiaea vombata, which was previously thought to occur only in the Wombat State Forest (Victoria). As well, the team suggested that two species should be collapsed into one.

The most perplexing result was very strong indication that two distinct species, with locally isolated populations 300 km apart, are probably a recent new lineage and could be classified as one species with two disjunct subspecies.

One of these species, Bossiaea fragrans, is only known from the Abercrombie region in the Central West of New South Wales. The other, Bossiaea milesiae, only occurs in the Brogo region of south-eastern New South Wales. In light of their genetic similarity, the ReCER team proposed that the two species could be collapsed into one species, with two subspecies (B. fragrans subsp. fragrans and B fragrans subsp. milesiae). These subspecies may be reproductively compatible, meaning they have the ability to mate.

Officers within the NSW Saving Our Species program have noted that both B. milesiae and B. fragrans appear to be experiencing unpredictable seed production and recruitment. The genetic similarity of the two species has opened to door to both new questions and unexpected solutions. One working theory, currently being investigated in collaboration with the University of New South Wales Sydney, is that the variable seed production is a result of inbreeding and geneflow restricted to local kin groups (siblings). Just like animals, many plants are fittest when they mate with unrelated other plants, but isolation can result in limited mate options and consequentially reproduction within family groups or between siblings.

“To prevent continuing inbreeding and ensure the survival of these species, we recommend introducing genetic diversity through controlled crossbreeding. For instance, crossbreeding between subspecies could enhance fertility and seed viability.” - Eilish McMaster Research Centre for Ecosystem Resilience

 

If inbreeding is identified as a contributing factor to the limited seed production, a novel and unexpected solution would be to test hybridisation between B. fragrans and B. milesiae. Given the genetic evidence suggesting these lineages are closely related, they should be able to mate successfully. This would increase the genetic diversity of both populations, potentially enhancing their resilience and protecting them from long-term extinction.

Genetic rescue can be a powerful conservation tool for plant or animal species suffering inbreeding, but sometimes populations are so small, there are no unrelated individuals to introduce. In this case, should hybridisation—the introduction of genetic diversity from a closely related species—be considered?

Genetic rescue through hybridisation is widely recommended by conservation genetics researchers but is seldom implemented as a conservation strategy in practice. In the direst circumstances, hybridisation may enable once sterile or highly inbred populations to again reproduce and may prevent the extinction of a species or population. A well-known and very successful example of genetic rescue through hybridisation occurred in the mid 1990’s to save the Florida Panther (Puma concolor coryi) via introduction of Texan individuals from the more common subspecies Puma concolor concolor. Although contentious at the time, the hybridisation between subspecies effectively saved the Florida Panther population and preserved the species role within the ecosystem. These decisions begin to untangle questions about why we want to conserve species from extinction, and the motivation behind preserving particular genotypes.

Genetic Rescue is the addition of new genotypes to a population of plants or animals which is small, isolated, and often suffering inbreeding. New genotypes can be introduced through adding unrelated individuals to a population, or in the case of plants, intentional mixing of pollen or seeds between populations.

Hybridisation provides a powerful potential pathway to conserve species with limited gene pools. However, within our current Australian environmental laws, threatened species conserved through adding new genetics from other closely related species, may not be protected because they are hybrids. Bossiaea fragrans subsp. fragrans for example, is currently listed as a critically endangered species (EPBC Act 1999), however if it received pollen from B. milesiae subsp. milesiae the offspring may not be awarded any environmental protection.

Once a species reaches the point of ‘functional extinction’—when it can no longer reproduce due to inbreeding—hybridisation may become the only means of preserving both biodiversity and the unique genes of the species. Fortunately, neither Bossiaea subspecies is at this critical juncture, and dedicated research is needed to identify the factors limiting seed production. Nonetheless, it is essential to recognize that identifying opportunities for long-term conservation, even in the worst-case scenarios, is a vital aspect of proactive decision-making that we must begin to prioritize to ensure the survival of these species for future generations.

Plant specimens for this research were collected from Gundungurra, Ngarigu, Dhurga, Djirrigany, Wiradjuri, Dhudhuroa, Woiwurrung and Palawa Country.

No effective project can occur in isolation. Investigation of leafless Bossiaea taxonomy, and breeding systems has relied on support from the NSW Saving Our Species program (NSW Department of Climate Change, Energy, the Environment and Water), the NSW National Parks and Wildlife team and the University of New South Wales Sydney. Generous time was given by staff from the Royal Tasmanian Botanical Gardens, La Trobe University, The Royal Botanic Gardens Victoria, and the Office of Nature Conservation (ACT Environment Planning and Sustainable Development Directorate).

Special thanks are also extended to the coauthors of the Bossiaea paper, including botanists Keith McDougall, Neville Walsh, and Elizabeth James, as well as field experts Nic Jario and Jessica Peterie.