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.

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.

Influence of historical landscapes, drainage evolution and ecological traits on patterns of genetic diversity in Southeast Asian freshwater snakehead fishes

Snakehead fishes in the family Channidae are obligate freshwater fishes represented by two extant genera, the African Parachannna and the Asian Channa. These species prefer still or slow flowing water bodies, where they are top predators that exercise high levels of parental care, have the ability to breathe air, can tolerate poor water quality, and interestingly, can aestivate or traverse terrestrial habitat in response to seasonal changes in freshwater habitat availability. These attributes suggest that snakehead fishes may possess high dispersal potential, irrespective of the terrestrial barriers that would otherwise constrain the distribution of most freshwater fishes. A number of biogeographical hypotheses have been developed to account for the modern distributions of snakehead fishes across two continents, including ancient vicariance during Gondwanan break-up, or recent colonisation tracking the formation of suitable climatic conditions. Taxonomic uncertainty also surrounds some members of the Channa genus, as geographical distributions for some taxa across southern and Southeast (SE) Asia are very large, and in one case is highly disjunct. The current study adopted a molecular genetics approach to gain an understanding of the evolution of this group of fishes, and in particular how the phylogeography of two Asian species may have been influenced by contemporary versus historical levels of dispersal and vicariance. First, a molecular phylogeny was constructed based on multiple DNA loci and calibrated with fossil evidence to provide a dated chronology of divergence events among extant species, and also within species with widespread geographical distributions. The data provide strong evidence that trans-continental distribution of the Channidae arose as a result of dispersal out of Asia and into Africa in the mid–Eocene. Among Asian Channa, deep divergence among lineages indicates that the Oligocene-Miocene boundary was a time of significant species radiation, potentially associated with historical changes in climate and drainage geomorphology. Mid-Miocene divergence among lineages suggests that a taxonomic revision is warranted for two taxa. Deep intra-specific divergence (~8Mya) was also detected between C. striata lineages that occur sympatrically in the Mekong River Basin. The study then examined the phylogeography and population structure of two major taxa, Channa striata (the chevron snakehead) and the C. micropeltes (the giant snakehead), across SE Asia. Species specific microsatellite loci were developed and used in addition to a mitochondrial DNA marker (Cyt b) to screen neutral genetic variation within and among wild populations. C. striata individuals were sampled across SE Asia (n=988), with the major focus being the Mekong Basin, which is the largest drainage basin in the region. The distributions of two divergent lineages were identified and admixture analysis showed that where they co-occur they are interbreeding, indicating that after long periods of evolution in isolation, divergence has not resulted in reproductive isolation. One lineage is predominantly confined to upland areas of northern Lao PDR to the north of the Khorat Plateau, while the other, which is more closely related to individuals from southern India, has a widespread distribution across mainland SE Asian and Sumatra. The phylogeographical pattern recovered is associated with past river networks, and high diversity and divergence among all populations sampled reveal that contemporary dispersal is very low for this taxon, even where populations occur in contiguous freshwater habitats. C. micropeltes (n=280) were also sampled from across the Mekong River Basin, focusing on the lower basin where it constitutes an important wild fishery resource. In comparison with C. striata, allelic diversity and genetic divergence among populations were extremely low, suggesting very recent colonisation of the greater Mekong region. Populations were significantly structured into at least three discrete populations in the lower Mekong. Results of this study have implications for establishing effective conservation plans for managing both species, that represent economically important wild fishery resources for the region. For C. micropeltes, it is likely that a single fisheries stock in the Tonle Sap Great Lake is being exploited by multiple fisheries operations, and future management initiatives for this species in this region will need to account for this. For C. striata, conservation of natural levels of genetic variation will require management initiatives designed to promote population persistence at very localised spatial scales, as the high level of population structuring uncovered for this species indicates that significant unique diversity is present at this fine spatial scale.

Conservation genetics of the water mouse, Xeromys myoides Thomas, 1889

The water mouse, Xeromys myoides, is currently recognised as a vulnerable species in Australia, inhabiting a small number of distinct and isolated coastal regions of Queensland and the Northern Territory. An examination of the evolutionary history and contemporary influences shaping the genetic structure of this species is required to make informed conservation management decisions. Here, we report the first analysis undertaken on the phylogeography and population genetics of the water mouse across its mainland Australian distribution. Genetic diversity was assessed at two mitochondrial DNA (Cytochrome b, 1000 bp; D-loop, 400 bp) and eight microsatellite DNA loci. Very low genetic diversity was found, indicating that water mice underwent a recent expansion throughout their Australian range and constitute a single evolutionarily significant unit. Microsatellite analyses revealed that the highest genetic diversity was found in the Mackay region of central Queensland; population substructure was also identified, suggesting that local populations may be isolated in this region. Conversely, genetic diversity in the Coomera region of south-east Queensland was very low and the population in this region has experienced a significant genetic bottleneck. These results have significant implications for future management, particularly in terms of augmenting populations through translocations or reintroducing water mice in areas where they have gone extinct.

Cryptic diversity and evolutionary relationships among Australian closed-forest Melomys (Rodentia: Muridae) and related Australo-Papuan mosaic-tailed rats

This thesis investigates patterns of evolution in a group of native Australo-Papuan rodents. Past climatic change and associated sea level fluctuations, and fragmentation of wet forests in eastern Australia has facilitated rapid radiation, diversification and speciation in this group. This study adds to our understanding of the evolution of Australia’s rainforest fauna and describes the evolutionary relationships of a new genus of Australian rodent.

The dominant 55kDa allergen of the subtropical Bahia grass (Paspalum notatum) pollen is a group 13 pollen allergen, Pas n 13

Bahia grass, Paspalum notatum, is an important pollen allergen source with a long season of pollination and wide distribution in subtropical and temperate regions. We aimed to characterize the 55. kDa allergen of Bahia grass pollen (BaGP) and ascertain its clinical importance. BaGP extract was separated by 2D-PAGE and immunoblotted with serum IgE of a grass pollen-allergic patient. The amino-terminal protein sequence of the predominant allergen isoform at 55. kDa had similarity with the group 13 allergens of Timothy grass and maize pollen, Phl p 13 and Zea m 13. Four sequences obtained by rapid amplification of the allergen cDNA ends represented multiple isoforms of Pas n 13. The predicted full length cDNA for Pas n 13 encoded a 423 amino acid glycoprotein including a signal peptide of 28 residues and with a predicted pI of 7.0. Tandem mass spectrometry of tryptic peptides of 2D gel spots identified peptides specific to the deduced amino acid sequence for each of the four Pas n 13 cDNA, representing 47% of the predicted mature protein sequence of Pas n 13. There was 80.6% and 72.6% amino acid identity with Zea m 13 and Phl p 13, respectively. Reactivity with a Phl p 13-specific monoclonal antibody AF6 supported designation of this allergen as Pas n 13. The allergen was purified from BaGP extract by ammonium sulphate precipitation, hydrophobic interaction and size exclusion chromatography. Purified Pas n 13 reacted with serum IgE of 34 of 71 (48%) grass pollen-allergic patients and specifically inhibited IgE reactivity with the 55. kDa band of BaGP for two grass pollen-allergic donors. Four isoforms of Pas n 13 from pI 6.3-7.8 had IgE-reactivity with grass pollen allergic sera. The allergenic activity of purified Pas n 13 was demonstrated by activation of basophils from whole blood of three grass pollen-allergic donors tested but not control donors. Pas n 13 is thus a clinically relevant pollen allergen of the subtropical Bahia grass likely to be important in eliciting seasonal allergic rhinitis and asthma in grass pollen-allergic patients.

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.

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.

Why is there no genetics in phylogenetics : misconceptions, clarification and solutions

Fundamental misconceptions regarding some basic phylogenetic terminology are presented in this opinion piece. An attempt is made to point out why these misconceptions exist and what may be causing the misapplication of terminology. Clarification is providing via basic definitions and simple explanations. Differences between the scientific fields of genetics and population genetics are discussed. The appropriate use of terminology is advocated and alternative terms are proposed to eliminate one potential source of confusion. It is suggested we use 'sequence data' instead of molecular data and 'non-sequence data' instead of morphological data in the field of phylogenetics and systematics.

Comparison of intraspecific genetic structure among related chironomids (Diptera) from New Zealand and Patagonia : disparity between potential and realized dispersal

Population genetic studies of freshwater invertebrate taxa in New Zealand and South America are currently few despite the geologically and climatically dynamic histories of these regions. The focus of our study was a comparison of the influence on realized dispersal of 2 closely related nonbiting midges (Chironomidae) of population fragmentation on these separated austral land masses. We used a 734-base pair (bp) fragment of cytochrome c oxidase subunit I (COI) to investigate intraspecific genetic structure in Naonella forsythi Boothroyd in New Zealand and Ferringtonia patagonica Edwards in Patagonia. We proposed hypotheses about their potential dispersal and, hence, expected patterns of genetic structure in these 2 species based on published patterns for the closely related Australian taxon Echinocladius martini Cranston. Genetic structure revealed for both N. forsythi and F. patagonica was characterized by several highly divergent (2.0–10.5%) lineages of late Miocene–Pliocene age within each taxon that were not geographically localized. Many were distributed widely. This pattern differed greatly from population structure in E. martini, which was typified by much greater endemicity of divergent genetic lineages. Nevertheless, diversification of lineages in all 3 taxa appeared to be temporally congruent with the onset of late Miocene glaciations in the southern hemisphere that may have driven fragmentation of suitable habitat, promoting isolation of populations and divergence in allopatry. We argue that differences in realized dispersal post-isolation may be the result of differing availability of suitable habitat in interglacial periods.

The genographic project: Traditional knowledge and population genetics

This article considers the debate over patent law, informed consent, and benefit-sharing in the context of biomedical research in respect of Indigenous communities. In particular, it focuses upon three key controversies over large-scale biology projects, involving Indigenous populations. These case studies are representative of the tensions between research organisations, Indigenous communities, and funding agencies. Section two considers the aims and origins of the Human Genome Diversity Project, and criticisms levelled against the venture by Indigenous peak bodies and anti-biotechnology groups, such as the Rural Advancement Foundation International. It examines the ways in which the United Nations Educational, Scientific, and Cultural Organization (UNESCO) grappled with questions of patent law, informed consent, and benefit sharing in relation to population genetics. Section three focuses upon the ongoing litigation in Tilousi v. Arizona State University, and the Havasupai Tribe v. Arizona State University. In this matter, the Havasupai tribe from the Grand Canyon in the United States brought legal action against the Arizona State University and its researchers for using genetic data for unauthorised purposes - namely, genetic research into schizophrenia, migration, and inbreeding. The litigation raises questions about informed consent, negligence, and larger matters of human rights. Section four explores the legal and ethical issues raised by the Genographic Project. It considers the aims and objectives of the venture, and the criticisms levelled against it by Indigenous communities, and anti-biotechnology groups. It examines the response of the United Nations Permanent Forum on Indigenous Issues to the Genographic Project. It charts the debate over the protection of traditional knowledge in various international fora. The conclusion recommends a number of measures to better regulate large-scale biology projects involving the participation of Indigenous communities.

Spatial population genetic structure reveals strong natal site fidelity in Echinocladius martini (Diptera: Chironomidae) in northeast Queensland, Australia

1. A diverse array of patterns has been reported regarding the spatial extent of population genetic structure and effective dispersal in freshwater macroinvertebrates. In river systems, the movements of many taxa can be restricted to varying degrees by the natural stream channel hierarchy. 2. In this study, we sampled populations of the non-biting freshwater midge Echinocladius martini in the Paluma bioregion of tropical northeast Queensland to investigate fine scale patterns of within- and among-stream dispersal and gene flow within a purported historical refuge. We amplified a 639 bp fragment of mitochondrial COI and analysed genetic structure using pairwise ΦST, hierarchical AMOVA, Mantel tests and a parsimony network. Genetic variation was partitioned among stream sections using Streamtree to investigate the effect of potential instream dispersal barriers. 3. The data revealed strong natal site fidelity and significant differentiation among neighbouring, geographically proximate streams. We found evidence for only episodic adult flight among sites on separate stream reaches. Overall, however, our data suggested that both larval and adult dispersal was largely limited to within a stream channel. 4. This may arise from a combination of the high density of riparian vegetation physically restricting dispersal and from the joint effects of habitat stability and large population sizes. Together these may mitigate the requirement for movement among streams to avoid inbreeding and local extinction due to habitat change and may thus enable persistence of upstream populations in the absence of regular compensatory upstream flight. Taken together, these data suggest that dispersal of E. martini is highly restricted, to the scale of only a few kilometres, and hence occurs predominantly within the natal stream.