Phylogeography of Echinocladius martini Cranston (Diptera: Chironomidae) in closed forest streams of eastern Australia

The eastern Australian rainforests have experienced several cycles of range contraction and expansion since the late Miocene that are closely correlated with global glaciation events. Together with ongoing aridification of the continent, this has resulted in current distributions of native closed forest that are highly fragmented along the east coast. Several closed forest endemic taxa exhibit patterns of population genetic structure that are congruent with historical isolation of populations in discrete refugia and reflect evolutionary histories dramatically affected by vicariance. Currently, limited data are available regarding the impact of these past climatic fluctuations on freshwater invertebrate taxa. The non-biting midge species Echinocladius martini Cranston is distributed along the east coast and inhabits predominantly montane streams in closed forest habitat. Phylogeographic structure in E. martini was resolved here at a continental scale by incorporating data from a previous pilot study and expanding the sampling design to encompass populations in the Wet Tropics of north-eastern Queensland, south-east Queensland, New South Wales and Victoria. Patterns of phylogeographic structure revealed several deeply divergent mitochondrial lineages from central and south-eastern Australia that were previously unrecognised and were geographically endemic to closed forest refugia. Estimated divergence times were congruent with late Miocene onset of rainforest contractions across the east coast of Australia. This suggested that dispersal and gene flow among E. martini populations isolated in refugia has been highly restricted historically. Moreover, these data imply, in contrast to existing preconceptions about freshwater invertebrates, that this taxon may be acutely susceptible to habitat fragmentation.

Evolutionary biology of Gondwanan non-biting midges (Diptera: Chironomidae)

The potential restriction to effective dispersal and gene flow caused by habitat fragmentation can apply to multiple levels of evolutionary scale; from the fragmentation of ancient supercontinents driving diversification and speciation on disjunct landmasses, to the isolation of proximate populations as a result of their inability to cross intervening unsuitable habitat. Investigating the role of habitat fragmentation in driving diversity within and among taxa can thus include inferences of phylogenetic relationships among taxa, assessments of intraspecific phylogeographic structure and analyses of gene flow among neighbouring populations. The proposed Gondwanan clade within the chironomid (non-biting midge) subfamily Orthocladiinae (Diptera: Chironomidae) represents a model system for investigating the role that population fragmentation and isolation has played at different evolutionary scales. A pilot study by Krosch et al (2009) indentified several highly divergent lineages restricted to ancient rainforest refugia and limited gene flow among proximate sites within a refuge for one member of this clade, Echinocladius martini Cranston. This study provided a framework for investigating the evolutionary history of this taxon and its relatives more thoroughly. Populations of E. martini were sampled in the Paluma bioregion of northeast Queensland to investigate patterns of fine-scale within- and among-stream dispersal and gene flow within a refuge more rigorously. Data was incorporated from Krosch et al (2009) and additional sites were sampled up- and downstream of the original sites. Analyses of genetic structure revealed strong natal site fidelity and high genetic structure among geographically proximate streams. Little evidence was found for regular headwater exchange among upstream sites, but there was distinct evidence for rare adult flight among sites on separate stream reaches. Overall, however, the distribution of shared haplotypes implied that both larval and adult dispersal was largely limited to the natal stream channel. Patterns of regional phylogeographic structure were examined in two related austral orthoclad taxa – Naonella forsythi Boothroyd from New Zealand and Ferringtonia patagonica Sæther and Andersen from southern South America – to provide a comparison with patterns revealed in their close relative E. martini. Both taxa inhabit tectonically active areas of the southern hemisphere that have also experienced several glaciation events throughout the Plio-Pleistocene that are thought to have affected population structure dramatically in many taxa. Four highly divergent lineages estimated to have diverged since the late Miocene were revealed in each taxon, mirroring patterns in E. martini; however, there was no evidence for local geographical endemism, implying substantial range expansion post-diversification. The differences in pattern evident among the three related taxa were suggested to have been influenced by variation in the responses of closed forest habitat to climatic fluctuations during interglacial periods across the three landmasses. Phylogeographic structure in E. martini was resolved at a continental scale by expanding upon the sampling design of Krosch et al (2009) to encompass populations in southeast Queensland, New South Wales and Victoria. Patterns of phylogeographic structure were consistent with expectations and several previously unrecognised lineages were revealed from central- and southern Australia that were geographically endemic to closed forest refugia. Estimated divergence times were congruent with the timing of Plio-Pleistocene rainforest contractions across the east coast of Australia. This suggested that dispersal and gene flow of E. martini among isolated refugia was highly restricted and that this taxon was susceptible to the impacts of habitat change. Broader phylogenetic relationships among taxa considered to be members of this Gondwanan orthoclad group were resolved in order to test expected patterns of evolutionary affinities across the austral continents. The inferred phylogeny and estimated divergence times did not accord with expected patterns based on the geological sequence of break-up of the Gondwanan supercontinent and implied instead several transoceanic dispersal events post-vicariance. Difficulties in appropriate taxonomic sampling and accurate calibration of molecular phylogenies notwithstanding, the sampling regime implemented in the current study has been the most intensive yet performed for austral members of the Orthocladiinae and unsurprisingly has revealed both novel taxa and phylogenetic relationships within and among described genera. Several novel associations between life stages are made here for both described and previously unknown taxa. Investigating evolutionary relationships within and among members of this clade of proposed Gondwanan orthoclad taxa has demonstrated that a complex interaction between historical population fragmentation and dispersal at several levels of evolutionary scale has been important in driving diversification in this group. While interruptions to migration, colonisation and gene flow driven by population fragmentation have clearly contributed to the development and maintenance of much of the diversity present in this group, long-distance dispersal has also played a role in influencing diversification of continental biotas and facilitating gene flow among disjunct populations.

Deeply divergent mitochondrial lineages reveal patterns of local endemism in chironomids of the Australian Wet Tropics

The Wet Tropics bioregion of north-eastern Australia has been subject to extensive fluctuations in climate throughout the late Pliocene and Pleistocene. Cycles of rainforest contraction and expansion of dry sclerophyll forest associated with such climatic fluctuations are postulated to have played a major role in driving geographical endemism in terrestrial rainforest taxa. Consequences for the distributions of aquatic organisms, however, are poorly understood.The Australian non-biting midge species Echinocladius martini Cranston (Diptera: Chironomidae), although restricted to cool, well-forested freshwater streams, has been considered to be able to disperse among populations located in isolated rainforest pockets during periods of sclerophyllous forest expansion, potentially limiting the effect of climatic fluctuations on patterns of endemism. In this study, mitochondrial COI and 16S data were analysed for E. martini collected from eight sites spanning theWet Tropics bioregion to assess the scale and extent of phylogeographic structure. Analyses of genetic structure showed several highly divergent cryptic lineages with restricted geographical distributions. Within one of the identified lineages, strong genetic structure implied that dispersal among proximate (<1 km apart) streams was extremely restricted. The results suggest that vicariant processes, most likely due to the systemic drying of the Australian continent during the Plio-Pleistocene, might have fragmented historical E. martini populations and, hence, promoted divergence in allopatry.

Cassowary dispersal of the invasive pond apple in a tropical rainforest: the contribution of subordinate dispersal modes in invasion.

Dispersal is a significant determinant of the pattern and process of invasions; however, weed dispersal distances are rarely described and descriptions of dispersal kernels are completely lacking for vertebrate-dispersed weeds. Here, we describe dispersal kernels generated by a native disperser, the endangered southern cassowary (Casuarius casuarius, L.) for an invasive, tropical rainforest plant, pond apple (Annona glabra, L.). Pond apple is primarily water-dispersed and is managed as such. We consider whether cassowary dispersal, as a numerically subordinate dispersal mode, provides an additional dispersal service that may modify the invasion process. In infested areas, pond apple seed was common in cassowary dung. Gut passage had no effect on the probability of single seed germination but deposition in clumps or as whole fruits reduced the probability of germination below that of single seeds. Gut passage times ranged from 65 to 1675 min. Combined with cassowary movement data, this resulted in estimated dispersal distances of 12.5-5212 m, with a median distance of 387 m (quartile range 112-787 m). Native frugivores can be effective dispersers of weeds in rainforest and even terrestrial dispersers can provide long-distance dispersal. Importantly, though pond apple might be expected to be almost entirely dispersed downstream and along the margins of aquatic and marine habitats, cassowaries provide dispersal upstream and between drainages, leading to novel dispersal outcomes. Even through the provision of small quantities of novel dispersal outcomes, subordinate dispersal modes can play a significant role in determining invasion pattern and influence the ultimate success of control programs by providing dispersal to locations unattainable via the primary mode.

Seed dispersal and predation in the endemic Atlantic rainforest palm Astrocaryum aculeatissimum across a gradient of seed disperser abundance

Tropical forests have been subject to intense hunting of medium and large frugivores that are important in dispersing large-seeded species. It has been hypothesized that in areas with extinction or low abundance of medium and large-bodied animals the density of small rodents may increase. Therefore, this increment in the density of small rodents may compensate for the absence or low abundance of medium and large frugivores on seed removal and seed dispersal. Here, we fill up this gap in the literature by determining if seed removal, seed dispersal, and seed predation by small rodents (spiny rats, Trinomys inheringi and squirrels, Sciurus ingrami) are maintained in defaunated areas. We accessed seed removal, seed dispersal, seed predation, and seedling recruitment of an endemic Atlantic rainforest palm, Astrocaryum aculeatissimum, in a gradient of abundance of agoutis. We found that seed removal, scatter hoarding, and seed predation increase with the abundance of agoutis. In contrast, the proportion of dispersed but non-cached seeds decreased with the abundance of agoutis. We did not find any effect of the abundance of agoutis on seed dispersal distance, but we did find a positive trend on the density of seedlings. We concluded that small rodents do not compensate the low abundance of agoutis on seed removal, scatter hoarding, and seed predation of this palm tree. Moreover, areas in which agoutis are already extinct did not present any seed removal or scatter hoarding, not even by small rodents. This study emphasizes both the importance of agoutis in dispersing seeds of A. aculeatissimum and the collapse in seed dispersal of this palm in areas where agoutis are already extinct.

Seed dispersal and predation in the endemic Atlantic rainforest palm Astrocaryum aculeatissimum across a gradient of seed disperser abundance

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

A new rain-operated seed dispersal mechanism in Bertolonia mosenii (Melastomataceae), a Neotropical rainforest herb

Although widespread among fungi, lichens, liverworts, and mosses, seed dispersal mechanisms operated by rain are unusual among flowering plants. Generally speaking, two mechanisms are involved in seed dispersal by rains: the splash-cup and the springboard. Here we describe a new seed dispersal mechanism operated by rain in a Neotropical rainforest herb Bertolonia mosenii Cogniaux (Melastomataceae). The study was carried out at the lowland Atlantic rainforest, southeastern Brazil. We experimentally demonstrate that rain is necessary to release the seeds from the capsules through what we call squirt-corner seed dispersal mechanism: when a raindrop strikes the mature fruit, the water droplet forces the seeds outward to the angles (corners) of the triangular capsule and the seeds are released. As far as we know squirt-corner represents a new rain-operated seed dispersal mechanism, and a novel seed dispersal mode both for Melastomataceae and for flowering plants from Neotropical forests.

Ancient Dispersal of the Human Fungal Pathogen Cryptococcus gattii from the Amazon Rainforest

Over the past two decades, several fungal outbreaks have occurred, including the high-profile 'Vancouver Island' and 'Pacific Northwest' outbreaks, caused by Cryptococcus gattii, which has affected hundreds of otherwise healthy humans and animals. Over the same time period, C. gattii was the cause of several additional case clusters at localities outside of the tropical and subtropical climate zones where the species normally occurs. In every case, the causative agent belongs to a previously rare genotype of C. gattii called AFLP6/VGII, but the origin of the outbreak clades remains enigmatic. Here we used phylogenetic and recombination analyses, based on AFLP and multiple MLST datasets, and coalescence gene genealogy to demonstrate that these outbreaks have arisen from a highly-recombining C. gattii population in the native rainforest of Northern Brazil. Thus the modern virulent C. gattii AFLP6/VGII outbreak lineages derived from mating events in South America and then dispersed to temperate regions where they cause serious infections in humans and animals.

Ground beetle (Carabidae) abundance data from north-eastern Australian rainforest, between June 2008 - January 2010, using pitfall traps

Understanding how the environment influences patterns of diversity is vital for effective conservation management, especially in a changing global climate. While assemblage structure and species richness patterns are often correlated with current environmental factors, historical influences may also be considerable, especially for taxa with poor dispersal abilities. Mountain-top regions throughout tropical rainforests can act as important refugia for taxa characterised by low dispersal capacities such as flightless ground beetles (Carabidae), an ecologically significant predatory group. We surveyed flightless ground beetles along elevational gradients in five different subregions within the Australian Wet Tropics World Heritage Area to investigate (1) whether the diversity and composition of flightless ground beetles are elevationally stratified, and, if so, (2) what environmental factors (other than elevation per se) are associated with these patterns. Generalised linear models and model averaging techniques were used to relate patterns of diversity to environmental factors. Unlike most taxonomic groups, flightless ground beetles increased in species richness and abundance with elevation. Additionally, each subregion consisted of distinct assemblages containing a high level of regional endemic species. Species richness was most strongly positively associated with the historical climatic conditions and negatively associated with severity of recent disturbance (treefalls) and current climatic conditions. Assemblage composition was associated with latitude and current and historical climatic conditions. Our results suggest that distributional patterns of flightless ground beetles are not only likely to be associated with factors that change with elevation (current climatic conditions), but also factors that are independent of elevation (recent disturbance and historical climatic conditions). Variation in historical vegetation stability explained both species richness and assemblage composition patterns, probably reflecting the significance of upland refugia at a geographic time scale. These findings are important for conservation management as upland habitats are under threat from climate change.

Epiphytic orchid diversity found on trees and shrubs in coffee cultivated Afromontane rainforest, SW Ethiopia

The moist evergreen Afromontane forest of SW Ethiopia has become extremely fragmented and most remnants are intensively managed for cultivation of coffee (Coffea arabica). We investigated the distributions of epiphytic orchids in shade trees and their understory in forests with contrasting management intensity to determine biodiversity losses associated with coffee cultivation and to determine the capacity of coffee shrubs to act as refugia for orchid species. We studied epiphytic orchids in managed forests and natural forests and recorded orchid diversity and abundance in different tree zones of 339 trees and in the understory. Coffee management was associated with a downward shift of orchid species as orchid species were occurring in significantly lower tree zones in managed forest. The number of shrubs in the understory of managed forest was not higher than in natural forests, yet orchid abundance was higher in the understory of managed forests. Local extinctions of epiphytic orchids and species losses in the outer tree zones (a contraction of habitat) in managed forests are most likely driven by losses of large, complex-structured climax trees, and changes in microclimate, respectively. Coffee shrubs and their shade trees in managed forests are shown here to be a suitable habitat for only a limited set of orchid species. As farmers continue to convert natural forest into managed forest for coffee cultivation, further losses of habitat quality and collateral declines in regional epiphytic orchid diversity can be expected. Therefore, the conservation of epiphytic orchid diversity, as well as other components of diversity of the coffee forests, must primarily rely on avoiding coffee management intensification in the remaining natural forest. Convincing farmers to keep forest-climax trees in their coffee forest and to tolerate orchids on their coffee shrubs may also contribute to a more favorable conservation status of orchids in Ethiopian coffee agroecosystems.

A test of alternative models of diversification in tropical rainforests: Ecological gradients vs. rainforest refugia

Comparison of mitochondrial and morphological divergence in eight populations of a widespread leaf-litter skink is used to determine the relative importance of geographic isolation and natural selection in generating phenotypic diversity in the Wet Tropics Rainforest region of Australia. The populations occur in two geographically isolated regions, and within each region, in two different habitats (closed rainforest and tall open forest) that span a well characterized ecological gradient. Morphological differences among ancient geographic isolates (separated for several million years, judging by their mitochondrial DNA sequence divergence) were slight, but morphological and life history differences among habitats were large and occurred despite moderate to high levels of mitochondrial gene flow. A field experiment identified avian predation as one potential agent of natural selection. These results indicate that natural selection operating across ecological gradients can be more important than geographic isolation in similar habitats in generating phenotypic diversity. In addition, our results indicate that selection is sufficiently strong to overcome the homogenizing effects of gene flow, a necessary first step toward speciation in continuously distributed populations. Because ecological gradients may be a source of evolutionary novelty, and perhaps new species, their conservation warrants greater attention. This is particularly true in tropical regions, where most reserves do not include ecological gradients and transitional habitats.

When vicars meet: A narrow contact zone between morphologically cryptic phylogeographic lineages of the rainforest skink, Carlia rubrigularis

Phylogeographic analyses of the fauna of the Australian wet tropics rainforest have provided strong evidence for long-term isolation of populations among allopatric refugia, yet typically there is no corresponding divergence in morphology. This system provides an opportunity to examine the consequences of geographic isolation, independent of morphological divergence, and thus to assess the broader significance of historical subdivisions revealed through mitochondrial DNA phylogeography. We have located and characterized a zone of secondary contact between two long isolated (mtDNA divergence > 15%) lineages of the skink Carlia rubrigularis using one mitochondrial and eight nuclear (two intron, six microsatellite) markers. This revealed a remarkably narrow (width < 3 km) hybrid zone with substantial linkage disequilibrium and strong deficits of heterozygotes at two of three nuclear loci with diagnostic alleles. Cline centers were coincident across loci. Using a novel form of likelihood analysis, we were unable to distinguish between sigmoidal and stepped cline shapes except at one nuclear locus for which the latter was inferred. Given estimated dispersal rates of 90-133 m x gen(-1/2) and assuming equilibrium, the observed cline widths suggest effective selection against heterozygotes of at least 22-49% and possibly as high as 70%. These observations reveal substantial postmating isolation, although the absence of consistent deviations from Hardy-Weinberg equilibrium at diagnostic loci suggests that there is little accompanying premating isolation. The tight geographic correspondence between transitions in mtDNA and those for nuclear genes and corresponding evidence for selection against hybrids indicates that these morphologically cryptic phylogroups could be considered as incipient species. Nonetheless, we caution against the use of mtDNA phylogeography as a sole criterion for defining species boundaries.

Can rodents enhance germination rates in rainforest seeds?

The decline of large coevolved frugivorous species within fragmented habitats can have an effect on ecological processes, for example, seed dispersal and germination. It is therefore necessary for more resilient species to ensure essential processes are maintained within the system. This study investigates the influence of two rodent species, Melomys cervinipes (Fawn-footed Melomys) and Rattus fuscipes (Bush Rat), on the germination process of rainforest fruits. Both species are endemic to north Queensland rainforest and commonly found in fragmented habitats in high densities. We found in 85% of fruit species tested, rodent feeding increased seed germination rate by a factor of 3.5. Our results suggest that rodents can play a significant role in enhancing germination rates of fruits in the tropical rainforest of far north Queensland.

Anthropogenic landscape change promotes asymmetric dispersal and limits regional patch occupancy in a spatially structured bird population

1. Local extinctions in habitat patches and asymmetric dispersal between patches are key processes structuring animal populations in heterogeneous environments. Effective landscape conservation requires an understanding of how habitat loss and fragmentation influence demographic processes within populations and movement between populations. 2. We used patch occupancy surveys and molecular data for a rainforest bird, the logrunner (Orthonyx temminckii), to determine (i) the effects of landscape change and patch structure on local extinction; (ii) the asymmetry of emigration and immigration rates; (iii) the relative influence of local and between-population landscapes on asymmetric emigration and immigration; and (iv) the relative contributions of habitat loss and habitat fragmentation to asymmetric emigration and immigration. 3. Whether or not a patch was occupied by logrunners was primarily determined by the isolation of that patch. After controlling for patch isolation, patch occupancy declined in landscapes experiencing high levels of rainforest loss over the last 100 years. Habitat loss and fragmentation over the last century was more important than the current pattern of patch isolation alone, which suggested that immigration from neighbouring patches was unable to prevent local extinction in highly modified landscapes. 4. We discovered that dispersal between logrunner populations is highly asymmetric. Emigration rates were 39% lower when local landscapes were fragmented, but emigration was not limited by the structure of the between-population landscapes. In contrast, immigration was 37% greater when local landscapes were fragmented and was lower when the between-population landscapes were fragmented. Rainforest fragmentation influenced asymmetric dispersal to a greater extent than did rainforest loss, and a 60% reduction in mean patch area was capable of switching a population from being a net exporter to a net importer of dispersing logrunners. 5. The synergistic effects of landscape change on species occurrence and asymmetric dispersal have important implications for conservation. Conservation measures that maintain large patch sizes in the landscape may promote asymmetric dispersal from intact to fragmented landscapes and allow rainforest bird populations to persist in fragmented and degraded landscapes. These sink populations could form the kernel of source populations given sufficient habitat restoration. However, the success of this rescue effect will depend on the quality of the between-population landscapes.