Understanding population structure and genetic diversity is important for designing effective conservation strategies. As a critically endangered shrub, the six remaining extant populations of spiny daisy (Acanthocladium dockeri) are restricted to country roadsides in the mid-north of South Australia, where the species faces many ongoing abiotic and biotic threats to survival. Currently the spiny daisy is managed by selecting individuals from the extant populations and translocating them to establish insurance populations. However, there is little information available on the genetic differentiation between populations and diversity within source populations, which are essential components of planning translocations. To help fill this knowledge gap, we analysed population structure within and among all six of its known wild populations using 7,742 SNPs generated by a genotyping-by-sequencing approach. Results indicated that each population was strongly differentiated, had low levels of genetic diversity, and there was no evidence of inter-population gene flow. Individuals within each population were generally closely related, however, the Melrose population consisted entirely of clones. Our results suggest genetic rescue should be applied to wild spiny daisy populations to increase genetic diversity that will subsequently lead to greater intra-population fitness and adaptability. As a starting point, we suggest focussing on improving seed viability via inter-population crosses such as through hand pollination experiments to experimentally assess their sexual compatibility with the hope of increasing spiny daisy sexual reproduction and long-term reproductive fitness.
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Previous work has assessed the genetic diversity among four populations known at the time (Thornlea, Yangya, Hart and Rusty Cab) using allozymic markers (Jusaitis and Adams 2005). The authors found that each population represented a single, distinct genotype suggesting all individuals within each population were clones and there was no inter-population gene flow (Jusaitis and Adams 2005). Efforts to conserve the spiny daisy included regular site monitoring and maintenance (i.e. pest control and weeding), in addition to the establishment of at least one translocation site for each population (Sharp et al. 2010; Clarke et al. 2013). Subsequent conservation activities further led to the discovery of another two populations (Telowie and Melrose) (Clarke et al. 2013). Although informative, the analysis of allozyme markers only provides a limited representation of the allelic variation in a genome, in comparison to the use of more modern genome-wide techniques (Gaudeul et al. 2004; Narum et al. 2013). It is therefore necessary that population studies use high-resolution genomic methodologies to better inform management and conservation goals.
The aim of our study was to determine levels of genetic diversity within and among six extant populations of the spiny daisy using a genome-wide SNP dataset derived by GBS. We hope to use this information to help conservation management of this species. Based on previous findings, we predicted that each population would be genetically differentiated and contain low levels of genetic diversity. We addressed the following questions: (i) How strong is the genetic differentiation among populations? (ii) How variable is intra-populations genetic diversity? (iii) Is evidence of gene flow present among the extant populations?
All spiny daisy populations occur along roadsides within close proximity to country townships in the mid-north of South Australia (Fig. 1) (Jusaitis and Adams 2005; Clarke et al. 2013). Some of these sites are surrounded by several species of native vegetation identified in the Spiny Daisy Recovery Guide (Clarke et al. 2013). Thornlea, Yangya and Rusty Cab sites are situated East of Laura and are the closest geographically (4 km) to each other. The Hart site occurs within the Clare Valley, approximately 65 km south of the township Laura, between a main sealed road and an old railway reserve. The sites are located in semi-arid grasslands, adjacent to and sometimes partially on private property. The Telowie site is located approximately 20 km north of Port Pirie and its closest population, Thornlea is 30 km to the south-east of Telowie. Melrose represents the most northerly site, occurring 31 km north-east of Telowie. Plant density of these populations have been monitored over time and was recorded in 2007 by the Department of Environment, Water and Natural Resources (DEWNR) (Appendix Table S1) (Clarke et al. 2013).
This study confirms that strong genetic structure is present among all currently known extant populations of spiny daisy with little to no evidence of inter-population gene flow. This indicates that each site represents a unique subset of genetic variation of this species. The inclusion of Telowie and Melrose populations has increased the amount of known genetic diversity that exists within the species by approximately 33%, however the lack in connectivity among these isolated populations reinforces the extinction risk faced by this species (Ellstrand and Elam 1993; Young et al. 1996; Spielman et al. 2004; Frankham and Wilcken 2006). Although relatedness within each population was high, only Melrose represented a single genet. All other populations contained a small number of clones. Our findings therefore differ to that of previous genetic studies that described each population to consist entirely of clones (Jusaitis and Adams 2005). Levels of observed heterozygosity were greater than expected heterozygosity, likely as a result of high levels of clonal reproduction and/or mechanisms preventing sexual reproduction. Our study helps improve the knowledge of the distribution of limited genetic diversity within this critically endangered species and demonstrates that the spiny daisy has likely experienced strong genetic bottlenecks (Rodger et al. 2021), probably due to large-scale habitat disturbance (Jusaitis and Adams 2005; Clarke et al. 2013; Brown and Hodgkin 2015). The varying levels of genetic diversity detected among populations and subtle differences in population structure provide a deeper insight towards prioritization strategies for conservation management (Gardiner et al. 2017).
The presence of sporophytic self-incompatibility (SI) is present in many Asteraceae species and serves as an evolutionary mechanism to promote outcrossing within populations and restrict the build-up of deleterious alleles which is often associated with inbreeding in small, isolated populations (Zagorski et al. 1983; Les et al. 1991; Ferrer and Good-Avila 2007). Given the absence of excess homozygosity and sexual reproduction, high relatedness within the spiny daisy populations may reflect increased clonality, whereby the majority of the population represents descendants from a single lineage (Young and Brown 1999). Compatibility of the pollen grain is typically determined by the diploid genotype at the S locus of the paternal individual, inhibiting fertilization between individuals who contain the same S-alleles. The mating system within these spiny daisy populations may be attributed to this mating system (Frankel and Galun 1977; DeMauro 1993). Species which exhibit self-incompatibility often demonstrate limited reproductive performance, particularly via inhibition of the stigma, pollen tube growth and reduced seed set (Lloyd 1968; Oloumi and Rezanejhad 2009).
Although there was no evidence of inter-population gene flow, Thornlea, Rusty Cab and Yangya sites contained lower levels of pairwise genetic differentiation, higher levels of genetic diversity and fewer numbers of clones than Melrose and Telowie. A possible explanation for this is that these populations have been isolated for fewer generations and/or have experienced occasional sexual reproduction in the past, which would reduce genetic drift (Young et al. 2002; Milton et al. 2004). As these three sites occur proximal to each other, and face exposure to similar environmental conditions, increased genetic diversity may reflect historic opportunistic gene flow followed by genetic drift upon being isolated (Milton et al. 2004). Increased spatial separation among populations may have led to increased rates of clonality within populations as a result of limited mate availability. Changes in reproductive strategy from a sexual to vegetative mode in response to patchy geographic distribution has been observed in other Asteraceae species in Australia (Young et al. 2002; Blyth et al. 2021). For example, populations of Rutidosis leiolepis that occur in higher altitudes have been reported to contain greater levels of clonality and population genetic differentiation than those at lower altitudes. Reductions in pollinator activity within these areas, altered length of flowering season, as well as increased disturbance events were considered possible reasons towards increased clonality (Young et al. 2002). Variation in the level of clonality observed among spiny daisy populations may therefore have been influenced by changes in historic connectivity. High intra-population relatedness with strong differentiation, particularly within Melrose and Telowie, indicate effects of genetic drift are likely due to higher rates of cloning (Campbell and Husband 2005).
Our study helps improve the knowledge of genetic structure within the critically endangered spiny daisy, demonstrating that it is likely experiencing strong genetic drift probably due to processes that limit sexual reproduction (Sharp et al. 2010; Clarke et al. 2013; Bickerton et al. 2018; Rodger et al. 2021). However, as the evolutionary resilience of a species relies upon adequate levels of genetic diversity which can only be achieved through successful interbreeding, and our study therefore raises concerns about its adaptability and we call for updated conservation management interventions (Bijlsma et al. 2000; Brook et al. 2002; Young et al. 2002; Blambert et al. 2016). We urge the implementation of strategies which enhance reproductive fitness to facilitate sexual reproduction and encourage seed set as the initial step towards species recovery. Although preliminary hand pollination experiments between Hart and Thornlea have been trialled, limited seed viability has been a notable hurdle towards successful germination (Jusaitis and Adams 2005; Sharp et al. 2010; Clarke et al. 2013; Bickerton et al. 2018). Updated crosses among individuals from each population (propagated from vegetative cuttings) would help determine the severity of this problem and explore reproductive compatibility among populations. 2ff7e9595c
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