Genomic analyses and cloning of novel chicken natriuretic peptide genes reveal new insights into natriuretic peptide evolution

The natriuretic peptide (NP) family consists of multiple subtypes in teleosts, including atrial, B-type, ventricular, and C-type NPs (ANP, BNP, VNP, CNP-1–4, respectively), but only ANP, BNP, CNP-3, and CNP-4 have been identified in tetrapods. As part of understanding the molecular evolution of NPs in the tetrapod lineage, we identified NP genes in the chicken genome. Previously, only BNP and CNP-3 have been identified in birds, but we characterized two new chicken NP genes by cDNA cloning, synteny and phylogenetic analyses. One gene is an orthologue of CNP-1, which has only ever been reported in teleostei and bichir. The second gene could not be assigned to a particular NP subtype because of high sequence divergence and was named renal NP (RNP) due to its predominant expression in the kidney. CNP-1 mRNA was only detected in brain, while CNP-3 mRNA was expressed in kidney, heart, and brain. In the developing embryo, BNP and RNP transcripts were most abundant 24 h post-fertilization, while CNP mRNA increased in a stage-dependant manner. Synthetic chicken RNP stimulated an increase in cGMP production above basal level in chicken kidney membrane preparations and caused a potent dose-dependant vasodilation of pre-constricted dorsal aortic rings. From conserved chromosomal synteny, we propose that the CNP-4 and ANP genes have been lost in chicken, and that RNP may have evolved from a VNP-like gene. Furthermore, we have demonstrated for the first time that CNP-1 is retained in the tetrapod lineage.

Molecular phylogenetic relationships between Albugo candida collections on the Brassicaceae in Australia

Recent phylogenetic analyses of Albugo candida using the mitochondrial cytochrome c oxidase subunit II (cox2) gene, the nuclear ribosomal RNA large subunit (LSU) gene and the nuclear ribosomal RNA internal transcribed spacer (ITS) gene region have revealed significant genetic variation and led to the description of new species in the A. candida complex. This study examined the genetic diversity within Australian collections of A. candida from various Brassicaceae species in a range of geographic locations. Phylogenetic analysis of 31 Australian A. candida collections from 11 hosts using the rDNA ITS region, rDNA LSU region and cox2 mtDNA showed that the majority of Australian A. candida collections were the common form of A. candida. One collection from a common weed host, hairy bitter cress (Cardamine hirsuta), was found to belong to a previously reported but undescribed species, while three collections, also from C. hirsuta, were found to belong to a new undescribed species.

A molecular and morphological investigation of species boundaries and phylogenetic relationships in Australian free-tailed bats Mormopterus (Chiroptera:Molossidae)

The taxonomic uncertainty surrounding several prominent genera of Australian microbat has been a long-standing impediment to research and conservation efforts on these groups. The free-tail bat genus Mormopterus is perhaps the most significant example, with a long history of acknowledged species-level confusion. This study uses a combined molecular and morphological approach to conduct a comprehensive assessment of species and subgeneric boundaries, between-species phylogenetic affinities and within-species phylogeographic structure in Australian members of Mormopterus. Phylogenetic analyses based on 759 base pairs of the NADH Dehydrogenase subunit 2 mitochondrial gene were concordant with species boundaries delineated using an expanded allozyme dataset and by phallic morphology, and also revealed strong phylogeographic structure within two species. The levels of divergence evident in the molecular and morphological analyses led us to recognise three subgenera within Australia: Micronomus, Setirostris subgen. nov. and Ozimops subgen. nov. Within Ozimops we recognise seven Australian species, three of which are new, and none are conspecific with Indo-Papuan species. The family Molossidae now comprises eleven species across three subgenera in Australia, making it the continent's second most speciose family of bats. © CSIRO 2014.

Isolation and phylogenetic identification of halotolerant/halophilic bacteria from the salt mines of Karak, Pakistan

Extreme environments like salt mines are inhabited by a variety of bacteria that are well-adapted to such environments. The bacterial populations provide economic benefits in terms of enzymes synthesis. The salt mines of Karak region in Pakistan are extremely saline and the microbial communities found here have not yet been explored. In the present study, 57 halotolerant/halophilic bacterial strains were isolated from the salt mines of Karak. These strains were grown in media with 0-35% NaCl concentration. The morphological and physiological characteristics of the isolated strains were studied to optimize the growth conditions and to classify the isolated bacterial strains into slightly halotolerant/halophilic, moderately halophilic and extreme halophilic. The phylogenetic analyses inferred from 16S rRNA gene sequence of the isolated strains demonstrated that the major population were closely related to species belonging to Planococcus, Jeotgalicoccus, Staphylococcus, Halobacillus, Halomonas, Brevibacterium, Gracilibacillus, Kocuria, Salinivibrio, Salinicoccus, Oceanobacillus and Bacillus genera. Results showed that the salt mines of Karak region are rich in halotolerant/halophilic bacterial population with diverse bacterial communities, which may be utilized in various industrial applications after proper screening and identification.

A preliminary study of 16S rRNA sequence variation in Australia Macrobrachium shrimps (Palaemonidae: Decapoda) reveals inconsistencies in their current classification

The systematic relationships among Australian palaemonid shrimps have been the subject of speculation for some time. A preliminary phylogenetic study was undertaken to clarify the relationships of five species, Macrobrachium intermedium (Stimpson), M. australiense (Holthuis), M. atactum (Riek), M. rosenbergii (de Man) and Palaemon serenus (Heller), using 16S rRNA mitochondrial gene sequences. Phylogenetic analyses indicated inconsistencies with the current classification in two respects. First, M. intermedium formed a very well-supported clade with P. serenus distinct from M. australiense, M. atactum and M. rosenbergii. Second, the two species from inland Australia, M. australiense and M. atactum, showed a high level of genetic similarity over a substantial geographic range, suggesting that they may represent conspecific populations. The taxonomic and biogeographic implications of these findings for Macrobrachium in Australia are discussed.

Molecular taxonomy and phylogenetics of some species of Australian palaemonid shrimps

The evolutionary history and classification of the palaemonid shrimps has been the subject of constant speculation and debate. At present, all major systematic treatments have been based on morphological characteristics. To help resolve the phylogenetic relationships, and thus enable the creation of a classification system that reflects evolutionary history, a region of the 16S mitochondrial rRNA gene was sequenced for a number of Australian Palaemonidae. The resulting phylogenetic analyses indicated the presence of major anomalies in the current classification of Australian Palaemonidae. Significantly, three species belonging to three separate genera, Macrobrachium intermedium, Palaemon serenus, and Palaemonetes australis, are closely related, with genetic differences more characteristic with that of congeneric species. The results also demonstrate non-monophyly in Australian palaemonids with respect to both Palaemonetes and Macrobrachium.

Utility of mitochondrial DNA sequences from four gene regions for systematic studies of Australian freshwater crayfish of the Genus Cherax (Decapoda: Parastacidae)

One of the most important requirements for systematic and phylogenetic studies is the identification of gene regions with the appropriate level of variation for the question of interest. Molecular phylogenetic and systematic studies of freshwater crayfish have made use of DNA sequences mainly from ribosomal genes, especially the 16S rRNA gene region. Thus, little information is available on other potentially useful mitochondrial gene regions for systematic studies in these animals. In this study, we look at nucleotide variation and phylogenetic relations within and between four species of freshwater crayfish of the genus Cherax from the southwest of Western Australia using four fragments amplified from the 16S rRNA, 12S rRNA, Cytochrome Oxidase I (COI), and Cytochrome b (Cyt b) gene regions. Samples of Engaeus strictifrons, Euastacus bispinosus, and Geocharax falcata were also sequenced for comparative purposes. The size of the fragments varied from 358 bp to 600 bp. Across all samples, the four fragments showed significant phylogenetic signal and showed similar proportions of variable sites (28.81–37.33%). Average divergence within species for the mitochondrial gene regions varied from 1.18% to 4.91%, with the 16S rRNA being the least variable and Cyt b the most variable. Average divergence between species ranged 7.63–15.53%, with 16S rRNA being the least variable and COI the most variable. At the generic level, average divergence ranged 17.21–23.82%. Phylogenetic analyses of the 16S rRNA, 12S rRNA, and COI regions generated four clades consistent with the presence of four species previously identified on the basis of allozyme and morphological studies. The relationships among samples were largely congruent across the data set, although some relationships remained unresolved. Not all samples could be amplified using the Cyt b primers, and some of those that were showed quite anomalous relationships, suggesting that one or more Cyt b pseudogenes were being amplified.

The systematics of freshwater crayfish of the genus Cherax Erichson (Decapoda : Parastacidae) in eastern Australia re-examined using nucleotide sequences from 12S rRNA and 16S rRNA genes

Nucleotide sequence data were used to re-examine systematic relationships and species boundaries within the genus Cherax from eastern Australia. Partial sequences were amplified from the 12S (~365 bp) and 16S (~545 bp) rRNA mitochondrial gene regions. Levels of intra- and inter-specific divergence for Cherax species were very similar between the two gene regions and similar to that reported for other freshwater crayfish for 16S rRNA. Phylogenetic analyses using the combined data provided strong support for a monophyletic group containing 11 eastern Australian species and comprising three well-defined species-groups: the 'C. destructor' group containing three species, the 'C. cairnsensis' group containing four species and the 'C. cuspidatus' group containing two species. Cherax dispar and C. robustus are distinct from all other species and each other. In addition, two northern Australian and a New Guinean species were placed in the 'Astaconephrops' group, which is the sister-group to the eastern Australian Cherax lineage. Several relationships were clarified, including: the status of northern and southern C. cuspidatus as separate species; a close relationship between C. cairnsensis and C. depressus; the validity of C. rotundus and C. setosus as separate species and their close affinities with C. destructor; and the distinctiveness of the northern forms of Cherax. The analysis of the 12S rRNA and 16S rRNA data is highly concordant with the results of previous allozyme studies.

Molecular Genetic Evidence for a New Species of Bream of the Genus Acanthopagrus Peters (Perciformes: Sparidae)

Fishes of the genus Acanthopagrus are found throughout the coastal waters of Asia and Australia with several species being of commercial significance. In this study, genetic comparisons are made between widely disjunct populations of Acanthopagrus australis (Günther) from Australian and Taiwanese waters and among samples of A. butcheri (Munro), A. berda, (Forskal), A schlegeli (Day) and A. latus (Houttuyn) using mitochondrial DNA sequences obtained from the control region. The mean interspecific pairwise sequence divergence for all species is 17%, while the divergence between A. australis from Australia and that of Taiwan is slightly larger at 18%. These values are considerably higher than those found for intraspecific control region comparisons in some fish species. Phylogenetic analyses indicate that A. australis from Australia is more closely related to the Australian species A. butcheri than to A. australis from Taiwan. These findings suggest that the northern and southern hemisphere forms of A. australis are not monophyletic, with the former possibly representing a new undescribed species of Acanthopagrus.

Virulence determinants of infectious bursal disease virus

The very virulent (vv) pathotype of infectious bursal disease virus (IBDV) has spread rapidly throughout Europe, Asia, and the Middle East. Although Australia is currently unaffected, there remains the potential for incursion of an exotic isolate. The aim of this study was to identify putative virulence determinants of IBDV to facilitate the development of improved diagnostic assays for detection and characterisation of vvIBDV isolates. Sequencing of Indonesian vvIBDV Tasik94 revealed a unique substitution [ A�¨S222] in the hypervariable region (HVR) of viral protein (VP) VP2, which did not appear to impinge on virulence or antigenicity. Phylogenetic analyses indicated that Tasik94 was closely related to Asian and European vvIBDV strains. Extensive alignment of deduced protein sequences across the HVR of VP2 identified residuesI242 I256 and I294 as putative markers of the vv phcnotype. Comparison of the pathology induced by mildly-virulent Australian IBDV 002/73 and Indonesian vvIBDV Tasik94, revealed that histological lesions in the spleen, thymus and bone marrow were restricted to Tasik94-infected birds, suggesting the enhanced pathogenicity of vvIBDV might be attributed to replication in non-bursal lymphoid organs. The biological significance of the VP2 HVR in virulence was assessed using recombinant viruses generated by reverse genetics. Both genomic segments of Australian IBDV 002/73, and recombinant segment A constructs in which the HVR of 002/73 was replaced with the corresponding region of either tissue culture-adapted virus or vvIBDV (Tasik94), were cloned behind T7 RNA polymerase promoter sequences. In vitro transcription/translation of each construct resulted in expression of viral proteins. Co-transfection of synthetic RNA transcripts initiated replication of both tissue culture-adapted parental and recombinant viruses, however attempts to rescue non-adapted viruses in specific-pathogen-free (SPF) chickens were unsuccessful. Nucleotide sequence variation in the HVR of VP2 was exploited for the development of a new diagnostic assay to rapidly detect exotic IBDV isolates, including vvIBDV, using reverse transcription polymerase chain reaction (RT-PCR) amplification and Bmrl restriction enzyme digestion. The assay was capable of differentiating between endemic and exotic IBDV in 96% of 105 isolates sequenced to date.

Plasmodium falciparum possesses two GRASP proteins that are differentially targeted to the Golgi complex via a higher- and lower-eukaryote-like mechanism

Plasmodium falciparum, the causative agent of malaria, relies on a complex protein-secretion system for protein targeting into numerous subcellular destinations. Recently, a homologue of the Golgi re-assembly stacking protein (GRASP) was identified and used to characterise the Golgi organisation in this parasite. Here, we report on the presence of a splice variant that leads to the expression of a GRASP isoform. Although the first GRASP protein (GRASP1) relies on a well-conserved myristoylation motif, the variant (GRASP2) displays a different N-terminus, similar to GRASPs found in fungi. Phylogenetic analyses between GRASP proteins of numerous taxa point to an independent evolution of the unusual N-terminus that could reflect unique requirements for Golgi-dependent protein sorting and organelle biogenesis in P. falciparum. Golgi association of GRASP2 depends on the hydrophobic N-terminus that resembles a signal anchor, leading to a unique mode of Golgi targeting and membrane attachment.

Insights into the evolution of a cryptic radiation of bats: dispersal and ecological radiation of Malagasy Miniopterus (Chiroptera: Miniopteridae)

The past decade has seen a proliferation of new species of Miniopterus bats (family Miniopteridae) recognized from Madagascar and the neighboring Comoros archipelago. The interspecific relationships of these taxa, their colonization history, and the evolution of this presumed adaptive radiation have not been sufficiently explored. Using the mitochondrial cytochrome-b gene, we present a phylogeny of the Malagasy members of this widespread Old World genus, based on 218 sequences, of which 82 are new and 136 derived from previous studies. Phylogenetic analyses recovered 18 clades, which divide into five primary lineages: (1) M. griveaudi; (2) M. mahafaliensis, M. sororculus and X3; (3) M. majori, M. gleni and M. griffithsi; (4) M. brachytragos; M. aelleniA, and M. aelleniB; and (5) M. manavi and M. petersoni recovered as sister species, which were in turn linked to a group comprising M. egeri and five genetically distinct populations referred to herein as P3, P4, P5, P6 and P7. Beast analysis indicated that the initial divergence within the Malagasy Miniopterus radiation took place 4.5 Myr; most species diverged between 4 and 2.5 Myr, and a secondary period was between 1.25 and 1 Myr. DNA K2P-distances between recognized taxa ranged from 12.9% to 2.5% and intraspecific variation was less than 1.8%. Of the 18 identified clades, Latin binomials are only associated with 11, which indicates much greater differentiation than currently recognized for Malagasy Miniopterus. These data are placed in a context of the dispersal history of this genus on the island and patterns of ecological diversity.

Evolutionary history and leaf succulence as explanations for medicinal use in aloes and the global popularity of Aloe vera

BACKGROUND: Aloe vera supports a substantial global trade yet its wild origins, and explanations for its popularity over 500 related Aloe species in one of the world's largest succulent groups, have remained uncertain. We developed an explicit phylogenetic framework to explore links between the rich traditions of medicinal use and leaf succulence in aloes. RESULTS: The phylogenetic hypothesis clarifies the origins of Aloe vera to the Arabian Peninsula at the northernmost limits of the range for aloes. The genus Aloe originated in southern Africa ~16 million years ago and underwent two major radiations driven by different speciation processes, giving rise to the extraordinary diversity known today. Large, succulent leaves typical of medicinal aloes arose during the most recent diversification ~10 million years ago and are strongly correlated to the phylogeny and to the likelihood of a species being used for medicine. A significant, albeit weak, phylogenetic signal is evident in the medicinal uses of aloes, suggesting that the properties for which they are valued do not occur randomly across the branches of the phylogenetic tree. CONCLUSIONS: Phylogenetic investigation of plant use and leaf succulence among aloes has yielded new explanations for the extraordinary market dominance of Aloe vera. The industry preference for Aloe vera appears to be due to its proximity to important historic trade routes, and early introduction to trade and cultivation. Well-developed succulent leaf mesophyll tissue, an adaptive feature that likely contributed to the ecological success of the genus Aloe, is the main predictor for medicinal use among Aloe species, whereas evolutionary loss of succulence tends to be associated with losses of medicinal use. Phylogenetic analyses of plant use offer potential to understand patterns in the value of global plant diversity.

Characterization of antibody V segment diversity in the Tasmanian devil (Sarcophilus harrisii)

The Tasmanian devil (Sarcophilus harrisii) immune system has recently been under scrutiny because of the emergence of a contagious cancer, which has decimated devil numbers. Here we provide a comprehensive description of the Tasmanian devil immunoglobulin variable regions. We show that heavy chain variable (VH) and light chain variable (VL) repertoires are similar to those described in other marsupial taxa: VL diversity is high, but VH diversity is restricted and belongs only to clan III. As in other mammals, one VH and one Vλ germline family and multiple incomplete Vκ germline sequences were identified in the genome. High Vκ variation was observed in transcripts and we predict that it may have arisen by gene conversion and/or somatic mutations, as it does not appear to have originated from germline variation. Phylogenetic analyses revealed that devil VL gene segments are highly complex and ancient, with some lineages predating the separation of marsupials and eutherians. These results indicate that although the evolutionary history of immune genes lead to the expansions and contractions of immune gene families between different mammalian lineages, some of the ancestral immune gene variants are still maintained in extant species. A high degree of similarity was found between devil and other marsupial VH segments, demonstrating that they originated from a common clade of closely related sequences. The VL families had a higher variation than VH both between and within species. We suggest that, similar to other studied marsupial species, the complex VL segment repertoire compensates for the limited VH diversity in Tasmanian devils.