Sequence Changes in Six Variants of Rice Tungro Bacilliform Virus and Their Phylogenetic Relationships

The DNA of three biological variants, G1, Ic and G2, which originated from the same greenhouse isolate of rice tungro bacilliform virus (RTBV) at the International Rice Research Institute (IRRI), was cloned and sequenced. Comparison of the sequences revealed small differences in genome sizes. The variants were between 95 and 99% identical at the nucleotide and amino acid levels. Alignment of the three genome sequences with those of three published RTBV sequences (Phi-1, Phi-2 and Phi-3) revealed numerous nucleotide substitutions and some insertions and deletions. The published RTBV sequences originated from the same greenhouse isolate at IRRI 20, 11 and 9 years ago. All open reading frames (ORFs) and known functional domains were conserved across the six variants. The cysteine-rich region of ORF3 showed the greatest variation. When the six DNA sequences from IRRI were compared with that of an isolate from Malaysia (Serdang), similar changes were observed in the cysteine-rich region in addition to other nucleotide substitutions and deletions across the genome. The aligned nucleotide sequences of the IRRI variants and Serdang were used to analyse phylogenetic relationships by the bootstrapped parsimony, distance and maximum-likelihood methods. The isolates clustered in three groups: Serdang alone; Ic and G1; and Phi-1, Phi-2, Phi-3 and G2. The distribution of phylogenetically informative residues in the IRRI sequences shared with the Serdang sequence and the differing tree topologies for segments of the genome suggested that recombination, as well as substitutions and insertions or deletions, has played a role in the evolution of RTBV variants. The significance and implications of these evolutionary forces are discussed in comparison with badnaviruses and caulimoviruses.

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.

Dating of divergences within the Rattus genus phylogeny using whole mitochondrial genomes

The timing and order of divergences within the genus Rattus have, to date, been quite speculative. In order to address these important issues we sequenced six new whole mitochondrial genomes from wild-caught specimens from four species, Rattus exulans, Rattus praetor, Rattus rattus and Rattus tanezumi. The only rat whole mitochondrial genomes available previously were all from Rattus norvegicus specimens. Our phylogenetic and dating analyses place the deepest divergence within Rattus at ∼3.5 million years ago (Mya). This divergence separates the New Guinean endemic R. praetor lineage from the Asian lineages. Within the Asian/Island Southeast Asian clade R. norvegicus diverged earliest at ∼2.9 Mya. R. exulans and the ancestor of the sister species R. rattus and R. tanezumi subsequently diverged at ∼2.2 Mya, with R. rattus and R. tanezumi separating as recently as ∼0.4 Mya. Our results give both a better resolved species divergence order and diversification dates within Rattus than previous studies.

Accounting for calibration uncertainty in phylogenetic estimation of evolutionary divergence times

The estimation of phylogenetic divergence times from sequence data is an important component of many molecular evolutionary studies. There is now a general appreciation that the procedure of divergence dating is considerably more complex than that initially described in the 1960s by Zuckerkandl and Pauling (1962, 1965). In particular, there has been much critical attention toward the assumption of a global molecular clock, resulting in the development of increasingly sophisticated techniques for inferring divergence times from sequence data. In response to the documentation of widespread departures from clocklike behavior, a variety of local- and relaxed-clock methods have been proposed and implemented. Local-clock methods permit different molecular clocks in different parts of the phylogenetic tree, thereby retaining the advantages of the classical molecular clock while casting off the restrictive assumption of a single, global rate of substitution (Rambaut and Bromham 1998; Yoder and Yang 2000).

The evolutionary history of cockatoos (Aves: Psittaciformes: Cacatuidae)

Cockatoos are the distinctive family Cacatuidae, a major lineage of the order of parrots (Psittaciformes) and distributed throughout the Australasian region of the world. However, the evolutionary history of cockatoos is not well understood. We investigated the phylogeny of cockatoos based on three mitochondrial and three nuclear DNA genes obtained from 16 of 21 species of Cacatuidae. In addition, five novel mitochondrial genomes were used to estimate time of divergence and our estimates indicate Cacatuidae diverged from Psittacidae approximately 40.7 million years ago (95% CI 51.6–30.3 Ma) during the Eocene. Our data shows Cacatuidae began to diversify approximately 27.9 Ma (95% CI 38.1–18.3 Ma) during the Oligocene. The early to middle Miocene (20–10 Ma) was a significant period in the evolution of modern Australian environments and vegetation, in which a transformation from mainly mesic to xeric habitats (e.g., fire-adapted sclerophyll vegetation and grasslands) occurred. We hypothesize that this environmental transformation was a driving force behind the diversification of cockatoos. A detailed multi-locus molecular phylogeny enabled us to resolve the phylogenetic placements of the Palm Cockatoo (Probosciger aterrimus), Galah (Eolophus roseicapillus), Gang-gang Cockatoo (Callocephalon fimbriatum) and Cockatiel (Nymphicus hollandicus), which have historically been difficult to place within Cacatuidae. When the molecular evidence is analysed in concert with morphology, it is clear that many of the cockatoo species’ diagnostic phenotypic traits such as plumage colour, body size, wing shape and bill morphology have evolved in parallel or convergently across lineages.

Evolutionary relationships and divergence times among the native rats of Australia

Background The genus Rattus is highly speciose and has a complex taxonomy that is not fully resolved. As shown previously there are two major groups within the genus, an Asian and an Australo-Papuan group. This study focuses on the Australo-Papuan group and particularly on the Australian rats. There are uncertainties regarding the number of species within the group and the relationships among them. We analysed 16 mitochondrial genomes, including seven novel genomes from six species, to help elucidate the evolutionary history of the Australian rats. We also demonstrate, from a larger dataset, the usefulness of short regions of the mitochondrial genome in identifying these rats at the species level. Results Analyses of 16 mitochondrial genomes representing species sampled from Australo-Papuan and Asian clades of Rattus indicate divergence of these two groups ~2.7 million years ago (Mya). Subsequent diversification of at least 4 lineages within the Australo-Papuan clade was rapid and occurred over the period from ~ 0.9-1.7 Mya, a finding that explains the difficulty in resolving some relationships within this clade. Phylogenetic analyses of our 126 taxon, but shorter sequence (1952 nucleotides long), Rattus database generally give well supported species clades. Conclusions Our whole mitochondrial genome analyses are concordant with a taxonomic division that places the native Australian rats into the Rattus fuscipes species group. We suggest the following order of divergence of the Australian species. R. fuscipes is the oldest lineage among the Australian rats and is not part of a New Guinean radiation. R. lutreolus is also within this Australian clade and shallower than R. tunneyi while the R. sordidus group is the shallowest lineage in the clade. The divergences within the R. sordidus and R. leucopus lineages occurring about half a million years ago support the hypotheses of more recent interchanges of rats between Australia and New Guinea. While problematic for inference of deeper divergences, we report that the analysis of shorter mitochondrial sequences is very useful for species identification in rats.

Persistence of multiple genetic lineages within intra-host populations of Ross River virus

We examined the structure and extent of genetic diversity in intrahost populations of Ross River virus (RRV) in samples from six human patients, focusing on the nonstructural (nsP3) and structural (E2) protein genes. Strikingly, although the samples were collected from contrasting ecological settings 3,000 kilometers apart in Australia, we observed multiple viral lineages in four of the six individuals, which is indicative of widespread mixed infections. In addition, a comparison with previously published RRV sequences revealed that these distinct lineages have been in circulation for at least 5 years, and we were able to document their long-term persistence over extensive geographical distances

Mitochondrial genome deletions and minicircles are common in lice (Insecta: Phthiraptera)

Background The gene composition, gene order and structure of the mitochondrial genome are remarkably stable across bilaterian animals. Lice (Insecta: Phthiraptera) are a major exception to this genomic stability in that the canonical single chromosome with 37 genes found in almost all other bilaterians has been lost in multiple lineages in favour of multiple, minicircular chromosomes with less than 37 genes on each chromosome. Results Minicircular mt genomes are found in six of the ten louse species examined to date and three types of minicircles were identified: heteroplasmic minicircles which coexist with full sized mt genomes (type 1); multigene chromosomes with short, simple control regions, we infer that the genome consists of several such chromosomes (type 2); and multiple, single to three gene chromosomes with large, complex control regions (type 3). Mapping minicircle types onto a phylogenetic tree of lice fails to show a pattern of their occurrence consistent with an evolutionary series of minicircle types. Analysis of the nuclear-encoded, mitochondrially-targetted genes inferred from the body louse, Pediculus, suggests that the loss of mitochondrial single-stranded binding protein (mtSSB) may be responsible for the presence of minicircles in at least species with the most derived type 3 minicircles (Pediculus, Damalinia). Conclusions Minicircular mt genomes are common in lice and appear to have arisen multiple times within the group. Life history adaptive explanations which attribute minicircular mt genomes in lice to the adoption of blood-feeding in the Anoplura are not supported by this expanded data set as minicircles are found in multiple non-blood feeding louse groups but are not found in the blood-feeding genus Heterodoxus. In contrast, a mechanist explanation based on the loss of mtSSB suggests that minicircles may be selectively favoured due to the incapacity of the mt replisome to synthesize long replicative products without mtSSB and thus the loss of this gene lead to the formation of minicircles in lice.