A transcriptome resource for the koala (Phascolarctos cinereus): insights into koala retrovirus transcription and sequence diversity.

BACKGROUND: The koala, Phascolarctos cinereus, is a biologically unique and evolutionarily distinct Australian arboreal marsupial. The goal of this study was to sequence the transcriptome from several tissues of two geographically separate koalas, and to create the first comprehensive catalog of annotated transcripts for this species, enabling detailed analysis of the unique attributes of this threatened native marsupial, including infection by the koala retrovirus. RESULTS: RNA-Seq data was generated from a range of tissues from one male and one female koala and assembled de novo into transcripts using Velvet-Oases. Transcript abundance in each tissue was estimated. Transcripts were searched for likely protein-coding regions and a non-redundant set of 117,563 putative protein sequences was produced. In similarity searches there were 84,907 (72%) sequences that aligned to at least one sequence in the NCBI nr protein database. The best alignments were to sequences from other marsupials. After applying a reciprocal best hit requirement of koala sequences to those from tammar wallaby, Tasmanian devil and the gray short-tailed opossum, we estimate that our transcriptome dataset represents approximately 15,000 koala genes. The marsupial alignment information was used to look for potential gene duplications and we report evidence for copy number expansion of the alpha amylase gene, and of an aldehyde reductase gene.Koala retrovirus (KoRV) transcripts were detected in the transcriptomes. These were analysed in detail and the structure of the spliced envelope gene transcript was determined. There was appreciable sequence diversity within KoRV, with 233 sites in the KoRV genome showing small insertions/deletions or single nucleotide polymorphisms. Both koalas had sequences from the KoRV-A subtype, but the male koala transcriptome has, in addition, sequences more closely related to the KoRV-B subtype. This is the first report of a KoRV-B-like sequence in a wild population. CONCLUSIONS: This transcriptomic dataset is a useful resource for molecular genetic studies of the koala, for evolutionary genetic studies of marsupials, for validation and annotation of the koala genome sequence, and for investigation of koala retrovirus. Annotated transcripts can be browsed and queried at http://koalagenome.org.

Rare specimen identification in an un-integrated taxonomy: implications of DNA sequences from a Taiwanese Philine (Mollusca, Philinidae).

Many species of the gastropod genus Philine have been named from northeastern Asia but scanty descriptions based predominantly on shells make it difficult to determine which are valid. This, plus the sporadic anatomical and genetic information available for many of these species has led to what may be described as an un-integrated taxonomy. In this situation, it is generally preferable to postpone dissection of rare and unusual specimens until relevant diagnostic characters can be established in broader studies. Micro-CT scanning and DNA sequencing were used to examine such a specimen collected recently from deep waters off northeastern Taiwan. Micro-CT examination of the morphology of the internal shell and gizzard plates suggested that, among named species, the sequenced specimen is most similar to P.otukai. It cannot, however, be definitively referred to P.otukai as that species lacks adequate anatomical description or known DNA sequences. Phylogenetic analyses of newly collected DNA sequences show the specimen to be most closely related to, but distinct from the northern Atlantic Ocean and Mediterranean species, Philinequadripartita. The sequences also confirm genetically that five or more species of Philine occur in northeast Asia, including at least three subject to considerable taxonomic uncertainty.

Phylogenetic relationships among the black fly species (Diptera: Simuliidae) of Thailand based on multiple gene sequences.

Simulium is a very speciose genus of the black fly family Simuliidae that includes many important pests of humans and animals. Cytotaxonomic and morphological studies have made substantial progress in Simulium systematics. 16S rRNA and ITS-1 DNA sequence studies have assisted this progress. Intensive multi-gene molecular systematic investigations will, however, be required for a comprehensive understanding of the genus' taxonomy and evolution. Our research was conducted to investigate the relationships of Thai Simulium at the subgeneric, species group and species levels. We also examined the possibility of using mitochondrial DNA sequences to facilitate Simulium species identification. Data were collected from three mitochondrial genes (COI, ND4 and 16S rRNA) and two segments of the nuclear 28S ribosomal RNA (the D1 to D2 and the D4 expansion regions). The subgenera Simulium and Gomphostilbia were monophyletic in most analyses. Nevermannia included Montisimulium but was otherwise monophyletic in multigene analyses. In most analyses, Simulium and Nevermannia were more closely related to each other than to Gomphostilbia which was usually basal. Species groups were generally monophyletic. Within Gomphostilbia, however, the batoense species group was always paraphyletic to the other two species groups found in Thailand. Three species groups in Simulium were not monophyletic. The tendency to gill filament number reduction for some species groups in the subgenus Simulium was associated with a derived position in multigene analyses. Most species were monophyletic with two exceptions that probably represent species complexes and will present difficulties for rapid mitochondrial DNA identification.

Monsoon-influenced speciation patterns in a species flock of Eophreatoicus Nicholls (Isopoda; Crustacea).

A species flock of the freshwater isopod genus Eophreatoicus Nicholls lives in seeps, springs and perched aquifers at the base of the Arnhem Plateau and associated sandstone outliers in Australia's Northern Territory. These species have been found to have surprisingly high levels of genetic divergence and narrow range endemism, despite potential opportunities for dispersion during the summer monsoon season when streams flow continuously and have connectivity. Species of Eophreatoicus were identified morphologically as distinct taxa, sometimes with two or three species occurring at the same site. DNA sequence data from the mitochondrial 16S rRNA and cytochrome c oxidase subunit I genes corroborate our morphological concepts to a high level of resolution, with the exception of two distinct species that are identical genetically. The value of mtDNA data for identification of these species, therefore, is limited. These isopods disperse downstream from their home springs to a limited extent during the wet season, but the genetic data show that migration to non-natal springs, and reproduction there, may be rare. We argue that the multiplication of the narrow-range endemic species is the result of their homing behaviour combined with monsoonal alternation between aridity and flooding over recent and geological time scales since the Miocene period.

Arthropod Cladistics: Combined Analysis of Histone H3 and U2 snRNA Sequences and Morphology.

Morphological, developmental, ultrastructural, and gene order characters are catalogued for the same set of arthropod terminals as we have scored in a recent study of histone H3 and U2 snRNA sequences (D. J. Colgan et al., 1998, Aust. J. Zool. 46, 419-437). We examine the implications of separate and simultaneous analyses of sequence and non-sequence data for arthropod relationships. The most parsimonious trees based on 211 non-sequence characters (273 apomorphic states) support traditional higher taxa as clades, including Mandibulata, Crustacea, Atelocerata, Myriapoda, and Hexapoda. Combined analysis of morphology with histone H3 and U2 sequences with equal character weights differs from the morphological results alone in supporting Progoneata + Hexapoda (= Labiophora) in favor of a monophyletic Myriapoda, resolves the entognathous hexapods as a grade, and supports pycnogonids as sister group to Euchelicerata (rather than as basal euarthropods). Monophyly of Chelicerata (including pycnogonids), Mandibulata, Crustacea, Progoneata, Chilopoda, and Hexapoda is maintained under a range of transition/transversion and third codon weights, whereas Atelocerata and Myriapoda/Labiophora do not withstand all sensitivity analyses.

Phylogenetics of the brachyuran crabs (Crustacea: Decapoda): the status of Podotremata based on small subunit nuclear ribosomal RNA.

The true crabs, the Brachyura, are generally divided into two major groups: Eubrachyura or 'advanced' crabs, and Podotremata or 'primitive' crabs. The status of Podotremata is one of the most controversial issues in brachyuran systematics. The podotreme crabs, best recognised by the possession of gonopores on the coxae of the pereopods, have variously been regarded as mono-, para- or polyphyletic, or even as non-brachyuran. For the first time, the phylogenetic positions of the podotreme crabs were studied by cladistic analysis of small subunit nuclear ribosomal RNA sequences. Eight of 10 podotreme families were represented along with representatives of 17 eubrachyuran families. Under both maximum parsimony and Bayesian Inference, Podotremata was found to be significantly paraphyletic, comprising three major clades: Dromiacea, Raninoida, and Cyclodorippoida. The most 'basal' is Dromiacea, followed by Raninoida and Cylodorippoida. Notably, Cyclodorippoida was identified as the sister group of the Eubrachyura. Previous hypotheses that the dromiid crab, Hypoconcha, is an anomuran were unsupported, though Dromiidae as presently composed could be paraphyletic. Topologies constrained for podotreme monophyly were found to be significantly worse (P < 0.04) than unconstrained topologies under Templeton and S-H tests. The clear pattern of podotreme paraphyly and robustness of topologies recovered indicates that Podotremata as a formal concept is untenable. Relationships among the eubrachyurans were generally equivocal, though results indicate the majoids or dorippoids were the least derived of the Eubrachyura. A new high level classification of the Brachyura is proposed.

Molecular phylogeny and biogeography of spring-associated hydrobiid snails of the Great Artesian Basin, Australia.

The Great Artesian Basin (GAB) of Australia underlies some of the driest parts of South Australia and Queensland and feeds numerous freshwater springs. Prominent and endangered components of the GAB spring community are snails of the family Hydrobiidae. This paper examines the evolutionary relationships of the entire hydrobiid fauna associated with the GAB, and includes appropriate non-GAB species to place the GAB fauna in a broader phylogenetic context. The Queensland genus Jardinella is a focus of this paper, providing a fine scale examination of relationships between spring supergroups in the northeastern regions of the GAB. Maximum parsimony and Bayesian analyses performed on 16S, CO1, and combined sequence data from 40 hydrobiid taxa found four major clades of Australian taxa. The analysis revealed that at least three separate colonization events of the GAB spring fauna have occurred. Two of these are represented by considerable radiations, (1) Jardinella to the north and east and (2) Caldicochlea, Fonscochlea, and possibly Trochidrobia in South Australia. The phylogenetic position of the latter is uncertain so it may represent yet another invasion. The third definite invasion is represented by a single species of the speciose SE Australian genus Austropyrgus in the Dalhousie Springs in South Australia. Jardinella is found to be monophyletic, and with one exception, its members in each of the Queensland spring supergroups are found to be monophyletic.

Evolution of the major histocompatibility complex: Isolation of class II beta cDNAs from two monotremes, the platypus and the short-beaked echidna.

Extant mammals are composed of three lineages: the eutherians, the marsupials and the monotremes. The majority of the mammalian major histocompatibility complex (MHC) data is based on the eutherian mammals, which generally have three classical MHC class II beta chain gene clusters - DRB, DQB and DPB, as well as the non-classical DMB and DOB. Marsupial DMB, DAB and DBB have been characterised. Confusion still surrounds the relationship of the marsupial DAB and DBB genes with the classical eutherian class II clusters. Here we present the first monotreme MHC class II beta chain sequences. Four MHC class II beta chain sequences were isolated from a spleen cDNA library from the short-beaked echidna, and one from a spleen cDNA library from platypus using a brushtail possum DAB probe. Given the non-orthologous relationship of the monotreme sequences with marsupial and eutherian beta chain clusters, we recommend that the five new monotreme sequences be assigned the nomenclature 'DZB', signifying the description of a new mammalian beta chain cluster. Our analysis suggests that all mammalian beta chain sequences (except DMB) evolved from a common ancestor. Maximum likelihood analysis places the monotreme beta chain sequences at the base of the mammalian clade, indicating their ancestral status. However, within the mammalian clade, monophyletic clades are not robust, and elucidation of the order of gene duplication that gave rise to the present-day gene clusters is not yet possible.