There are three key findings from this study that are highly relevant not only to the domestication and breeding of Fontainea picrosperma for plantation production of EBC-46, but also to understanding the biogeographic history of the species. (1) The overall genetic diversity of F. picrosperma was relatively low but the seven populations sampled from across the natural range were genetically distinct. (2) The levels of inbreeding in the individual populations were negligible despite their current discontinuous distribution and fragmentation. (3) Within the context of the low levels of genetic diversity and weak genetic structure observed for this species, two putative long-term refugial areas were identified in the eastern (Boonjie) and western (Evelyn Highlands) parts of the natural distribution of the species, which align with the refugial rainforest areas of Bartle-Frere Uplands and western Atherton Uplands identified by Hilbert et al. [25].
Genetic diversity
This is the first study to utilise microsatellites to examine genetic structure in the genus Fontainea. We investigated the levels and partitioning of genetic variation across the known range of F. picrosperma and found that the seven populations surveyed were genetically distinct despite having uniformly low levels of genetic diversity. This finding was not unexpected as many Australian plant species are characterised by low levels of genetic diversity, often as an adaptation to harsh environmental conditions [29, 51, 55], but also as a result of belonging to ancient lineages [45]. For instance, contrary to the accepted anthropomorphic view that a high level of genetic diversity bestows optimal evolutionary capability under conditions of environmental stress, James [29] found low levels of diversity in many successful species of Australia’s southwestern flora due to the purging of recombinational impedimenta (genetic load), allowing them to operate in harsh conditions at a highly adapted level. This counter-intuitive finding may also explain low genetic diversity in many of the ancient lineages in the Australian rainforest flora [22], including the results of this study for F. picrosperma. In essence, these rainforest taxa are highly adapted over long time periods to specific niches provided by the rainforest environment. As a consequence of this specialisation and niche differentiation in an essentially stable local environment, they experience only modest selection pressure during periods of climatic stability and when environmental conditions change, they retreat into the remaining environmental habitat to which they are so well adapted.
Inbreeding
In general, the results indicate extremely low levels of inbreeding (F = −0.003), which despite local populations having been isolated through glacial events, and more recently by anthropogenic habitat fragmentation, would be expected in an obligate outcrossing, dioecious species like F. picrosperma. Even though proximate trees are likely to be siblings or half-sibs, due to the limited dispersal capabilities of F. picrosperma’s relatively large drupaceous fruit, this suggests that deleterious mutations may have been purged over time, as most of the diversity resolved was between individuals within populations, not among populations.
The slight excess of heterozygosity detected in some populations suggests that recent bottlenecks with subsequent founder effects due to the expansion/contraction dynamics of small populations located outside of the main refugia may be responsible for a minor degree of genetic drift causing the random fixation of alleles. However, several populations were found to exhibit an equally slight excess of homozygosity, either as a result of the lack of overall genetic variation in the species or because of consanguineous matings. Although allelic diversity was found to be low, the fact that only two individuals shared the same multilocus genotype indicates that ‘selfing’ among proximate sibs or half-sibs was of limited occurrence; in fact these two individuals may be clones. Numerous studies have found pollen travel in continuous rainforest vegetation may be within the order of several kilometres [3]; more detailed, parent-progeny research to investigate fine-scale patterns of gene flow within wild populations, aimed at maintaining optimal among production seed crops of F. picrosperma, is required.
Population structure and gene flow
Stands of F. picrosperma occur in the upland and highland rainforests of the Atherton Tableland within a 15–20 km radius of Malanda. As such, the seven populations selected for population genetic analysis in this study likely represent a considerable proportion of the available genetic diversity within the species. It is entirely plausible that the low levels of genetic diversity and weak population structure that we have observed within F. picrosperma could merely be reflective of a random distribution of the diversity between individuals and populations. However, we believe that our observations reflect the existence of three distinct races or forms, including two long-term refugial races where suitable habitat is known to have persisted during less favourable times [25].
The population genetic structure of F. picrosperma is likely heavily influenced by the species’ life-history attributes and the effects of a long history of rainforest attrition followed by successive cycles of glacial-induced expansion and contraction upon the distribution of remaining populations. The Quaternary glacial cycles of recent geological times are known to have played a significant role in the current distributions and genetic signatures of many species [24] and based on our results this would seem to apply to F. picrosperma. Episodes of range expansion and contraction can have considerable genetic consequences [42] and the dynamics of the Wet Tropics rainforests corresponding to the glacial cycles of the Plio-Pleistocene are well documented [23, 33, 57]. Hence, the present-day configuration of F. picrosperma’s population genetic structure is likely a direct product of re-colonisation of dry sclerophyllous vegetation by tropical rainforest from refugial pockets of suitable habitat, following amelioration of the cool, dry conditions associated with past glacial cycles [8, 15, 23, 25, 33, 34, 37, 38, 51, 54, 56, 57]. It is likely that during this period several of the central populations assessed here have undergone at least some degree of geographic and genetic isolation.
The fruits of F. picrosperma disperse primarily by gravity with secondary long-distance dispersal facilitated either by hydrochory along drainage lines or zoochorous vectors [11, 14]. Populations therefore do not spread as a continuous wave of advance but rather are found as small and often isolated clumps or clusters, which may help to explain patterns in the geographical distribution of alleles. Nonetheless, the population genetic structure of F. picrosperma and the degree of historical gene flow between populations has been sufficient to maintain species’ integrity, suggesting populations were likely more continuously distributed in the past. The fact that the genus, originally described as containing a single taxon, F. pancheri, is composed of several highly similar taxa [21, 30], suggests vicariance due to habitat contraction occasioning genetic drift and the eventual loss of species cohesion may have been responsible for species divergences in the past.
The UPGMA cluster, principal coordinates and STRUCTURE analyses all provide a clear indication about the genetic distribution of this species. When combined with the genetic diversity analysis, the data show that the geographically distant (~28 km), peripheral populations of Boonjie and Evelyn Highlands, are genetically most diverse in comparison to the other populations whilst having elements of similarity, and form two genetically similar groups. Four of the remaining populations (Topaz, Towalla, Malanda and Gadgarra) form another genetic group, whereas the population at East Barron is genetically more divergent. We speculate that the populations at Evelyn Highlands and Boonjie represent two, genetically similar races or forms representing the two main refugial areas, where F. picrosperma persisted during times of sclerophyll expansion, before re-radiating out across the landscape under more favourable climatic conditions. In contrast, we suggest that the central populations of Topaz, Towalla, Malanda and Gadgarra represent a ‘plateau’ race or form that have likely expanded from small refugia during less severe climatic cycles, forming a genetically divergent race or form of F. picrosperma. East Barron appears to be derived from the elevated population at Evelyn Highlands (~1100 m asl), but is a genetically more divergent population, probably due to random founder effects. Gadgarra on the other hand, is genetically distinct, most likely as it contains no unique alleles and is somewhat inbred; essentially Gadgarra is a genetically depauperate variation of the plateau form. Despite the fact that the data suggests the presence of these three groups, it is important to highlight that the genetic diversity within F. picrosperma is low and the genetic structure between these three groups is proportionately low. In fact, the pairwise F
ST
values between Evelyn Highlands and Boonjie, Evelyn Highlands and the plateau group, and Boonjie and the plateau group range from only 0.039 to 0.060. However, each value was significantly different from zero (p <0.001) and within the context of the low levels of genetic variation within this species, this is suggestive of the presence of relevant genetic structure.
It is likely that the genetic relationship between the populations can be explained not so much by linear geographic distance but by their distribution within major river catchments radiating from the putative refugial sites of Evelyn Highlands and Boonjie. However, we also recognise the possibility that our analysis could merely be reflective of a random distribution of the observed genetic diversity. Therefore, future research to test our hypothesis that two refugial races or forms and a plateau race or form of F. picrosperma exist will necessarily involve chloroplast DNA analysis and the sampling of additional individuals sourced along potential gene flow corridors, such as major river systems originating from the putative refugia at Evelyn Highlands and Boonjie.
Selection, breeding, and plantation management
Knowledge of the genetic structure of source populations, mating system and patterns of gene flow are vital to the efficient establishment and management of seed orchard plantations and the production of improved open-pollinated seed [6, 7, 39]. Although the level of microsatellite variation detected in F. picrosperma was comparatively low, high exclusion probabilities (P
ID
) confirm that these markers will be useful in future paternity analyses and breeding programs; the former to determine patterns of gene flow in natural populations that will guide plantation design of this dioecious species, and the latter to ensure maximal genetic diversity is maintained during breeding of commercially significant agronomic traits, both of which are critical aspects of developing F. picrosperma as a niche tree crop for the supply of EBC-46.
Significant variation has been observed among F. picrosperma individuals with regard to several commercially significant agronomic traits such as growth, fruit production and EBC-46 content, suggesting that the species will be ideally suited for genetic improvement to optimise production. However, even in dioecious species, the genetic diversity of seed orchards can be eroded by a number of factors including a high proportion of ‘selfs’ arising from consanguineous matings between sibs or half-sibs, and departures from random mating due to unequal contributions of individuals to seed crops [39]. In fact, obligate outcrossing species such as F. picrosperma may be particularly vulnerable to losses in reproductive fitness stemming from elevated rates of inbreeding, leading to reductions in both vigour and yield [7, 35]. Therefore, to implement suitable plantation design and management options, it is necessary to have an intimate knowledge of a species’ mating system, reproductive biology, outcrossing rate and gene flow patterns in order to maximise breeding progress whilst preserving genetic diversity [5, 6, 39].
Traditionally, the most cost-effective manner of limiting inbreeding in ex situ populations was to position individuals in such a way that the possibility of close relatives mating would be small and hope for the best, however new techniques based on the minimisation of the global probability of consanguinity by considering the genetic relationships among trees within the entire planting have been developed [20]. Microsatellites are powerful tools for tracing pollen flow using parent/progeny arrays and work is continuing in both wild and artificial populations of F. picrosperma to establish which seed source and orchard variables are most likely to govern the efficiency of production plantations.