Ancient fragmentation, speciation, and diversity within the Romnalda genus
All extant species in the genus Romnalda appear to be restricted to rainforest habitats. Rainforests (megathermal moist forests) are thought to have evolved in the Cretaceous 100 Mya, and to have connected Australia and New Guinea from at least the Oligocene (approximately 30 Mya) to the mid-Miocene (approximately 13 Mya) when Australia and New Guinea were connected by land bridges (Morley, 2000; Maslin et al., 2005). Bowman & Yeates (2006) identified that the late Miocene to early Pliocene (7–10 Mya) period was critical in establishing the phylogenetic composition of modern Australian flora, being a cooler, drier period sandwiched between warmer, more humid environments. The association of R. strobilacea with Trapezites butterflies suggests a long history of mutualism (Atkins, 2004).
The Pickett et al. (2004) reconstruction of the SEAPAC region during the last glacial maximum, based on pollen records, found that the climate was drier and cooler than at present and that WTRF and TRFO forest types (where Romnalda occurs) were more restricted in distribution than at present. However, this restriction was mostly a reduction in range at higher altitudes, as these areas were more sensitive to climate change, and lowland rainforest persisted more extensively than previously thought (Pickett et al., 2004). However, the Pickett et al. (2004) reconstruction also found that the mid-Holocene climate was slightly warmer and moister than today, with more extensive occurrence of WTRF vegetation than today. This was mainly the result of a higher tree line and lower sea levels between New Guinea and Australia. Thus, the potential Romnalda habitat was probably slightly more continuous than at present.
Ladiges et al. (2003) compared the phylogeny and biogeography of the Australian Eucalyptus and Melaleuca groups in the Myrtaceae with geological history. These are also members of a Gondwanan family whose distribution extends to New Guinea and contains some species with rainforest affinities and similar geographical distributions to Romnalda. Ladiges et al. (2003) concluded that these distributions were related to the geological history of South-east Asia and Australia, and dispersal and colonization. This was aided by rafting on microcontinental fragments by accretion of arc terranes onto New Guinea and land brought in to closer proximity during periods of low sea level from the late Miocene and Pliocene. Heads (2001) used similar patterns to explain the distributions of birds of paradise, and employed the distribution of R. papuana in New Guinea to support this. Van Welzen (1997) also found a strong correlation between species distributions and groups of terranes of 961 endemic New Guinean plants. The highly dynamic geological history plays a significant role in explaining species distributions in New Guinea and may be significant for the highly disjunct Romnalda distribution.
Romnalda species seem to be found in less disturbed vegetation communities that are very high in species diversity, endemic species, and species thought to have relictual distributions. For example, the forests at Cape Tribulation and Mt Lewis have very high levels of species endemism and diversity (Tracey, 1981; Crisp et al., 2001). The Romnalda populations at these locations were also the most genetically diverse within the genus. Although Johns (1986) found that New Guinean forests had high rates of natural disturbance caused by, for example, earthquakes, landslips, and tree falls, R. papuana is found in less disturbed sites (P. Katik, pers. observ.). The forests in a 1 ha plot at 900 m altitude in New Guinea were found by Wright et al. (1997) to have one of the highest species diversity records for such a plot in the world. They attributed this to the frequent natural disturbance, rich soil, and mix of ancestry of the flora with both Gondwanan and Laurasian components (Wright et al., 1997). The areas inhabited by Romnalda appear not to have been affected greatly by past climate change, being continuously occupied by rainforest types, which is thought to have led to higher species diversity (Wright et al., 1997).
Pye & Gadek (2004), in their study of Bunya pine, Araucaria bidwillii, the distribution of which extends across the range of the Australian Romnalda species, found evidence of historical fragmentation and major genetic differences between the northern and southern populations. They found that Mt Lewis populations represented a significant reservoir for genetic diversity in Bunya pine (Pye & Gadek, 2004). The levels of genetic diversity within Romnalda are lowest at the southern and northern extremes of the genus distribution and highest at the centre of the distribution (which includes populations at Mt Lewis; Fig. 1).
The data from this study, together with the gross morphological characteristics (Stevens, 1978; Henderson, 1986), indicate clearly that there are at least four species in the genus Romnalda. Further studies using molecular markers, such as chloroplast DNA, internal transcribed spacer (ITS) sequencing, or microsatellites, are required to confirm the order of species differentiation from the ancestral Romnalda species. However, these initial results and observations point to isolation within the genus as a result of the ancient fragmentation of the three major historical rainforest blocks of New Guinea, north Queensland, and southern Queensland, leading to allopatric speciation and the extant species R. papuana, R. grallata, and R. strobilacea. The gross morphological differences suggest isolation and evolution in significantly different environments. The patterns of geographical distribution, genetic differentiation, and diversity within the genus support this theory (Table 1; Fig. 2).
Although the similarity of gross morphology suggests that R. sp.‘Cooper Ck’ is most closely related to R. papuana, the results of this study indicate that these two species are significantly genetically distinct (Fig. 2; Table 2). Further sampling of the additional known R. papuana populations is needed to determine the genetic relationships between these species more convincingly. The data from this study indicate that, although R. grallata and R. sp.‘Cooper Ck’ are clearly distinctive in gross morphology, they are genetically more closely related than is R. sp.‘Cooper Ck’ to R. papuana (Fig. 2). There is evidence that, at locations in which these species come into immediate geographical contact, there is hybridization and some evidence of introgression (Figs 2, 3). This supports the theory that R. grallata and R. sp.‘Cooper Ck’ originally speciated in geographical isolation and have subsequently come back into contact (Levin, 2000). This is consistent with the possibility that R. papuana migrated south from PNG, possibly during later periods of contact, and subsequently became isolated and differentiated whilst in Australia. Having evolved allopatrically, R. papuana and R. grallata would not necessarily have strong reproductive isolating mechanisms, apart from reproductive timing and habitat preferences. However, occasionally, such as in the Mt Sorrow and Mt Peiter Botte sites, the two species came into contact and some hybridization occurred, followed by introgression, as seen in this study (RG7, RC7). This produced further genetic differentiation of the Australian and New Guinean populations as genes from R. grallata found their way into R. papuana, increasing the differences between the Australian and New Guinean populations and eventually leading to R. sp.‘Cooper Ck’. Romnalda sp.‘Cooper Ck’ thus resulted from a second more recent speciation process, explaining the gross morphological similarity with R. papuana as well as the genetic relationship with R. grallata (Fig. 2). The fact that R. grallata is apparently restricted to altitudes above 600 m, whereas R. sp.‘Cooper Ck’ is found at both higher and lower altitudes, is consistent with the R. papuana climatic profile. Alternatively, R. grallata and R. sp.‘Cooper Ck’ may be sister taxa, possibly evolving by reproductive isolation along an altitudinal gradient, but are incompletely reproductively isolated and have formed a zone of hybridization. Further investigation of the relationships amongst these taxa using a combination of molecular markers would help to clarify the evolutionary history of these species.
Reproduction, hybridization, and diversity
The higher levels of genetic diversity found in R. sp.‘Cooper Ck’ than would be expected on the basis of its small population size and limited distribution (Table 1) may be explained by hybridization and introgression with R. grallata. Hybridization has long been recognized as a major phenomenon promoting genetic diversity in plants (Riesberg, 1997). Ainouche et al. (2003) found that hybridization was important in speciation in the genus Spartina (Poaceae). They found examples of hybridization when geographically separate species came into contact, even in vegetatively spreading species (Ainouche et al., 2003). It has been found that polyploidy is not required for sympatric speciation following hybridization, which can result from backcrossing to one of the parent species combined with sib mating (Riesberg & Carney, 1998). Hardig et al. (2000) found morphological and molecular evidence for hybridization and historic introgression, suggesting that perhaps hybridization occurred in glacial refugia as well as more contemporary hybridization. As these species appear to be associated with rainforest refugia, such scenarios would be consistent with the results of this study. Founder events, in which two species colonize a new location and hybridize, can rapidly form new species, as there is a low likelihood of backcrossing to either parental species populations because of isolation (Riesberg & Carney, 1998). Smissen, Breitwieser & Ward (2007) found an important role for small population size and rarity in the formation of hybrid lineages in New Zealand everlasting daisies. Such scenarios may also explain the Romnalda results.
Riesberg & Carney (1998) found that reproductive isolation is often asymmetrical. For example, some species are more often pollen donors than the maternal parent of hybrids because, if a species that is self-incompatible crosses with a species that is self-compatible, usually only the self-compatible parent can produce viable hybrid offspring. Our study found evidence of allelic spread between R. grallata and R. sp.‘Cooper Ck’ where they co-occurred, but these preliminary results suggest that hybridization is asymmetric with offspring more closely aligned with R. sp.‘Cooper Ck’ than with R. grallata. This is consistent with the findings of Smissen et al. (2007), who also found backcrossing mostly in one direction following hybridization. Our results also indicated that either mating amongst sibs, possibly via selfing, or backcrossing to R. sp.‘Cooper Ck’ had resulted in individuals that were homozygous for R. grallata alleles, thereby introducing new alleles into R. sp.‘Cooper Ck’ (Table 1; Fig. 3). Cogolludo-Agustin, Agundez & Gil (2000) also showed that hybridization led to greater genetic diversity in populations of elm.
Some endangered species have been identified as being under threat as a result of hybridization (Levin, Francisco-Ortega & Jansen, 1996). For example, Schnabel & Krutovskii (2004) have reported that an endangered tree (Gleditsia caspica) in Azerbaijan is threatened because of genetic introgression through hybridization with a related species that is widely cultivated; they found that the populations in one reserve consisted of hybrids. Similarly, Cogolludo-Agustin et al. (2000) found that the native Iberian elm (Ulmus minor) was under threat as a result of hybridization with exotic Siberian elms. Siberian elms and their hybrids are resistant to Dutch elm disease, which has led to their increased abundance in the landscape, but threatens the genetic integrity of native U. minor. They found asymmetric hybridization, in which the hybrids were nearer to the exotic species than to the local native U. minor, and that backcrossing occurred more frequently to the Siberian elm than to the Iberian elm. Given the small number of known individuals of R. sp.‘Cooper Ck’, there is a possibility that continued hybridization could lead to a loss of genetic identity, at least for one population in the future. Further studies utilizing maternally inherited markers, such as chloroplast DNA, would be useful to confirm the direction of hybridization between these species.
Reproductive isolation as a result of the timing of phenology between co-occurring congenetic species has been found to be quite common (Lamont et al., 2003). However, Lamont et al. (2003) found that disturbance altered the phenology of two co-occurring Banksia species, and this led to hybridization. They found that, in undisturbed sites, there was a phenological barrier to gene flow, whereas, in disturbed sites, B. hookeriana flowering was earlier and flowering in B. prionotes was prolonged, breaking the phenological barrier between the co-occurring species and resulting in hybrid swarms (Lamont et al., 2003). The results of the present study indicate that the timing of flowering within Romnalda follows a climatic gradient, with R. sp.‘Cooper Ck’ populations apparently responding to an altitudinal gradient. It appears that differences in flowering time reduce the potential for hybridization between R. grallata and R. sp.‘Cooper Ck’ at mid-altitude sites, but, at the higher altitude site (RC7, RG7), the phenological barrier is removed.
Although phenology is under strong genetic control, new models for the effects of climate change are predicting changes in the onset of flowering in some species (Chuine, Cambon & Comtois, 2000). Osborne et al. (2000) found that olive phenology was likely to change with increased climate warming, specifically leading to both earlier flowering times and greater spatial variation in timing of flowering amongst populations. Menzel et al. (2006) also found evidence for an earlier onset of spring phenological events and greater spatial variability in the timing of plant phenological stages. They predicted that differential changes in the timing of flowering may affect interactions amongst populations. Pickett et al. (2004) found that higher altitude sites were more greatly affected than lower altitude sites by climate change historically. Climate change may thus have a long-term impact on the potential for hybridization in R. sp.‘Cooper Ck’.
Preliminary observations have shown that reproductive synchronicity amongst populations within R. sp.‘Cooper Ck’ could be affected by climate (altitude). Thus, altitude together with distance could lead to genetic isolation of Romnalda populations, hence making small populations more prone to a loss of genetic diversity as a result of drift, and increasing the importance of intermediate altitude populations for gene flow amongst R. sp.‘Cooper Ck’ populations. Gomes et al. (2004) found a weak relationship between genetic variability and altitude in a rare member of the Asteraceae in Brazil. Most variation was found within populations, but 17% of variation was amongst populations from different altitudes. In R. sp.‘Cooper Ck’, there was some evidence of genetic differentiation along an altitudinal gradient. Thus, climate change has the potential to impact on both gene flow within the species and hybridization frequency, and may lead to either increased or decreased phenological synchronicity amongst populations or between species along an altitudinal gradient. Further studies to confirm the timing of flowering of these species along the altitudinal and climate gradients in different years would provide insights to clarify this.
The levels of genetic diversity in all species in the genus Romnalda were quite high compared with other endemic rainforest or herbaceous species (Hamrick & Godt, 1989; Shapcott, 2000; Honnay et al., 2005). Rossetto & Kooyman (2005) suggested that vegetative regeneration in rainforest plants may be a significant factor in slowing the rate of loss of diversity caused by drift as it enables the persistence of genotypes within the population. It has been observed that, under low light conditions, forest herbs may exhibit prolonged clonal growth (Honnay et al., 2005). Richards et al. (2004) found that, even in clonally reproducing species, sexual reproduction is often underestimated, but can be responsible for high diversity within patches assumed to be largely clonal. Sexual reproduction in partially clonal species is important for dispersal between populations (Honnay et al., 2005). The ability of plants to maintain themselves via vegetative means may have been significant in Romnalda to reduce the loss of diversity caused by drift in its small populations.
Populations that are small and isolated are expected to become more inbred, which may lead to a decline in reproductive output and hence population growth (Ellstrand & Elam, 1993). These features leave them more susceptible to demographic chance events. Despite high genetic diversity, all Romnalda species in this study were found to be highly inbred with generally low levels of synchronous reproductive activity (Tables 3, 4). Low levels of synchronous flowering are, however, not uncommon in understorey rainforest species (De Steven et al., 1987; Shapcott, 2000). Culley & Grub (2003) found that increased homozygosity as a result of a decrease in the population size of pollinators led to increased selfing in Viola. The results of the present study, together with anecdotal evidence, suggest that all species are self-compatible to some extent, although it is unknown whether viable seed can be produced by autogamy or whether the species are reliant on pollinators. In Romnalda, the high allelic fixation in the genus appears to be a combined effect of self-compatibility, weak spatial aggregations, and vegetative spread, but not associated with low diversity, as would be expected after founder or bottleneck events (Litrico et al., 2005).
Romnalda sp.‘Cooper Ck’ is currently undescribed. Its distribution is highly restricted and, at present, only three populations are known (Fig. 1); there are probably fewer than 500 plants. Thus, it deserves the highest conservation status of ‘endangered’. Given the inconspicuous nature of the species, it is possible that it has been missed from surveys and more populations are yet to be found. Currently, the RC7 population is potentially under long-term threat of introgression and loss of identity as a result of hybridization with R. grallata. However, given the size of the R. sp.‘Cooper Ck’ populations and their high levels of genetic diversity, these populations appear to be worthy of conservation and are likely to remain viable for some time.
Romnalda papuana has been recorded from only four confirmed sites in PNG and there is one very old record in Irian Jaya (RBG KEW). Two of the PNG sites are remote and inaccessible mountain tops and are likely to have remained reasonably undisturbed since the species collections in the 1970s (P. Katik, pers. observ.). Since this study was completed, an additional population in PNG has been identified, indicating that the species is likely to be more abundant than its current distribution record shows. However, expert opinion (P. Katik, pers. observ.) suggests that the species is not common and should be classified as ‘rare’ until further studies indicate a different conservation status. Given its geographical distribution within PNG, it seems likely that the populations from the disjunct regions are genetically distinct from each other. It also seems likely that the species occurs in clusters of small subpopulations and that these are likely to be associated with relictual areas of low disturbance. Conserving blocks of habitat containing R. papuana populations is likely to be the simplest and most effective means to ensure long-term survival of the species. Romnalda grallata populations contain the highest diversity in the genus and, at present, are known from populations in two disjunct protected areas. In both areas, the species co-occurs with many highly restricted endemic species. Romnalda strobilacea is classified as ‘vulnerable’ and, although more populations are known to exist of this species than of the others, they contain significantly less genetic diversity than the other Australian species (Table 1). The preliminary evidence suggests that the presence of Romnalda species is likely to indicate relictual rainforest which may contain other significant species. However, further studies are needed to confirm this hypothesis. Given that Romnalda species are much easier to observe and identify than most canopy tree species, the usual focus for conservation assessments, this genus may be a potentially useful indicator species of high conservation status rainforest.