Population structure, effective population size and adverse effects of stocking in the endangered Australian eastern freshwater cod Maccullochella ikei.

Microsatellite markers were used to examine spatio-temporal genetic variation in the endangered eastern freshwater cod Maccullochella ikei in the Clarence River system, eastern Australia. High levels of population structure were detected. A model-based clustering analysis of multilocus genotypes identified four populations that were highly differentiated by F-statistics (F(ST) = 0·09 - 0·49; P < 0·05), suggesting fragmentation and restricted dispersal particularly among upstream sites. Hatchery breeding programmes were used to re-establish locally extirpated populations and to supplement remnant populations. Bayesian and frequency-based analyses of hatchery fingerling samples provided evidence for population admixture in the hatchery, with the majority of parental stock sourced from distinct upstream sites. Comparison between historical and contemporary wild-caught samples showed a significant loss of heterozygosity (21%) and allelic richness (24%) in the Mann and Nymboida Rivers since the commencement of stocking. Fragmentation may have been a causative factor; however, temporal shifts in allele frequencies suggest swamping with hatchery-produced M. ikei has contributed to the genetic decline in the largest wild population. This study demonstrates the importance of using information on genetic variation and population structure in the management of breeding and stocking programmes, particularly for threatened species.

Genetic control of monoterpene composition in the essential oil of Melaleuca alternifolia (Cheel).

Previous studies into the population structure of Melaleuca alternifolia by both isozyme and microsatellite analysis revealed little evidence for genetic structuring within genetic provenances. In contrast, analysis of the oil composition within these same regions showed distinct clustering of chemotypes within the provenances suggesting either that chemotype was not under genetic control, or that there is strong environmental selection for plant chemotypes. To investigate the level of genetic control of monoterpene composition in the essential oil of M. alternifolia, individuals representing the three extreme chemotypes of high terpinen-4-ol, high 1,8-cineole and high terpinolene were crossed with an individual with the commercially desirable high terpinen-4-ol oil profile. The progeny resulting from these crosses displayed oil profiles that were intermediate to that of the parent. Further analysis of the survey of oil chemotypes within the natural population also suggests that these intermediate chemotypes may arise naturally between regions containing high proportions of the extreme chemotypes. These results imply that there is a level of genetic structure for chemotype determination within the genetic provenance that is undetected by isozyme and microsatellite analysis. This information could play a vital role in the selection of appropriate genetic material to be used in future essential oil selection and breeding programs.

The phylogenetic relationships of the bilby, Macrotis lagotis (Peramelimorphia: Thylacomyidae), to the bandicoots- DNA sequence evidence.

Partial sequencing of the 12S ribosomal RNA gene was used to test two competing hypotheses concerning the phylogenetic relationship of the bilby (Macrotis lagotis) to the Australian and New Guinean species of bandicoot. The first hypothesis proposes that the Australian and New Guinean bandicoots are in a monophyletic clade to the exclusion of the bilbies, whereas the second hypothesis proposes that the bilby is monophyletic with the Australian bandicoots to the exclusion of the New Guinean bandicoots. Phylogenies determined by both maximum-likelihood and neighbour-joining approaches supported the first hypothesis in which the bilby is excluded from the clade represented by the Australian and New Guinean bandicoots. Monophyly of the Australian and New Guinean bandicoots is consistent with the biogeographical scenario in which Australia and Papua New Guinea have undergone repeated connection and disconnection over the last 20 million years.

Phylogenetic relationships of Australian members of the family percichthyidae inferred from mitochondrial 12S rRNA sequence data.

Phylogenetic relationships among 20 Australian species of the family Percichthyidae were investigated from sequence data of two portions of the mitochondrial 12S rRNA gene. The molecular data indicate that Australian genera within this family cluster into three distinct clades. The first clade is composed of some species currently ascribed to the genus Macquaria, along with Nannatherina, Nannoperca, and Bostockia, the second of Maccullochella and two catadromous Macquaria species, and the third of Gadopsis. However, the positioning of Gadopsis within this family was unresolved. Monophyly within each genus was well supported, except for Macquaria, which is clearly polyphyletic. The molecular data were used to examine two hypotheses of Australian percichthyid evolution and favor a freshwater origin for the family.

Microsatellite analysis of genetic structure in the mangrove species Avicennia marina (Forsk.) Vierh. (Avicenniaceae).

The level of genetic variation throughout the entire worldwide range of the mangrove species Avicennia marina (Forsk.) Vierh. was examined using microsatellite markers. Three microsatellite loci detected high levels of allelic diversity (70 alleles in total), essential for an accurate estimation of population genetic parameters. The informativeness of the microsatellite loci tended to increase with increasing average number of repeats. The levels of heterozygosity detected for each population, over all loci, ranged from 0.0 to 0.8, with an average of 0.407, indicating that some populations had little or no genetic variation, whereas others had a large amount. Populations at the extremes of the distribution range showed reduced levels of heterozygosity, and significant levels of inbreeding. This is not unexpected as these populations may be subject to founder effects and environmental constraints. The presence of genetic structure was tested in A. marina populations using three models: (i) a single panmictic model; (ii) the discrete subpopulation model; and (iii) the isolation by distance model. The discrete subpopulations model was supported by the overall measures of population differentiation based on the infinite alleles model (F-statistics), and the stepwise mutation model (R statistics). In addition, an analysis of molecular variance (AMOVA), using both theoretical models, found that most of the variation was between populations (41-71%), and within individuals in the total population (31-49%). There was little variation among individuals within populations (0-10%). There was no significant isolation by distance. The high levels of genetic differentiation observed among populations of A. marina may be due to environmental and ecological factors, particularly past sea level and climatic changes.

Analysis of grape ESTs: global gene expression patterns in leaf and berry.

Analysis of 2479 ESTs from Vitis vinifera berry tissue and 2438 from leaf revealed that 1% of the ESTs match to known Vitis proteins, 72% to plant proteins, 11% to non-plant, and 16% had no match (P[N]>0.5). The levels of redundancy were similar in the leaf and berry libraries. Only 12% of the genes matched by the ESTs were common to both libraries indicating marked differences in the genes expressed in the two tissues. The abundance of transcripts with predicted cellular roles in leaf and berry were estimated by classifying the primary BLAST matches to known proteins (score >80) into functional categories. Thirty-six percent of the leaf transcripts were involved in photosynthesis, compared to 3% in the berry. This is a much higher proportion of transcripts involved with a function limited to specialized cells, than was found when transcripts of 33 human tissues were compared using a similar approach, suggesting plant cells may involve their cellular machinery to a greater extent in specialized activities than animal cells. Relatively enhanced expression of specific transcription factors, and genes involved in defense, detoxification, stress response, proteolysis, trafficing, and signal transduction, suggests berry tissue is actively engaged in responding to environmental stimuli.

Evolutionary lineages of RT1.Ba in the Australian Rattus.

In this study, the evolutionary history of the variable second exon of RT1.Ba and its adjoining intron b are compared across a number of species and subspecies of the Australian RATTUS: Three lineages are identified in the second intron across a range of Rattus species. Two of these lineages, separated by the insertion of a probable rodent short interspersed nucleotide element and by point mutations outside the indel region, are both found in each of the major clades of the endemic Australian RATTUS: This pattern of ancestral polymorphism is reflected in the adjoining exon 2 sequences, although phylogenetic constraints confirm that the clustering is not identical to that of the associated intron sequences. In addition, the coding sequences show evidence of the retention of ancestral polymorphism, with identical exon sequences found in two divergent species, and some indication of gene conversion detected for the exon sequences.

Population genetics of Escherichia coli in a natural population of native Australian rats.

Escherichia coli, a normal inhabitant of the intestinal tract of mammals and birds, is a diverse species. Most studies on E. coli populations involve organisms from humans or human-associated animals. In this study, we undertook a survey of E. coli from native Australian mammals, predominantly Rattus tunneyi, living in a relatively pristine environment in the Bundjalung National Park. The genetic diversity was assessed and compared by multilocus enzyme electrophoresis (MLEE), sequence analysis of the mdh (malate dehydrogenase) gene and biotyping using seven sugars. Ninety-nine electrophoretic types were identified from the 242 isolates analysed by MLEE and 15 sequences from the mdh genes sequenced from 21 representative strains. The Bundjalung isolates extend the diversity represented by the E. coli reference (ECOR) set, with new MLEE alleles found in six out of 10 loci. Many of the Bundjalung isolates fell into a discrete group in MLEE. Other Bundjalung strains fell into the recognized E. coli ECOR set groups, but tended to be at the base of both the MLEE and mdh gene trees, implying that these strains are derived independently from ancestral forms of the ECOR groups and that ECOR strains represent only a subset of E. coli adapted to humans and human-associated animals. Linkage disequilibrium analysis showed that the Bundjalung population has an 'epidemic' population structure. The Bundjalung isolates were able to utilize more sugars than the ECOR strains, suggesting that diet plays a prominent role in adaptation of E. coli.

Phylogeographic differentiation in the mitochondrial control region in the koala, Phascolarctos cinereus (Goldfuss 1817).

The koala, Phascolarctos cinereus, is a geographically widespread species endemic to Australia, with three currently recognized subspecies: P.c. adustus, P.c. cinereus, and P.c. victor. Intraspecific variation in the mitochondrial DNA (mtDNA) control region was examined in over 200 animals from 16 representative populations throughout the species' range. Eighteen different haplotypes were defined in the approximately 860 bp mtDNA control region, as determined by heteroduplex analysis/temperature gradient gel electrophoresis (HDA/TGGE). Any single population typically possessed only one or two haplotypes yielding an average within-population haplotypic diversity of 0.180 +/- 0.003, and nucleotide diversity of 0.16%. Overall, mtDNA control region sequence diversity between populations averaged 0.67%, and ranged from 0% to 1.56%. Nucleotide divergence between populations averaged 0.51%, and ranged from 0% to 1.53%. Neighbour-joining methods revealed limited phylogenetic distinction between geographically distant populations of koalas, and tentative support for a single evolutionarily significant unit (ESU). This is consistent with previous suggestions that the morphological differences formalized by subspecific taxonomy may be interpreted as clinal variation. Significant differentiation in mtDNA-haplotype frequencies between localities suggested that little gene flow currently exists among populations. When combined with microsatellite analysis, which has revealed substantial differentiation among koala populations, we conclude that the appropriate short-term management unit (MU) for koalas is the local population.

Microsatellite variation and assessment of genetic structure in tea tree (Melaleuca alternifolia-Myrtaceae).

Analysis of five microsatellite loci in 500 Melaleuca alternifolia individuals produced 98 alleles that were useful for population genetic studies. Considerable levels of observed heterozygosity were recorded (HO = 0.724), with approximately 90% of the variability being detected within populations. A low level of selfing (14%) was suggested to be the principal cause of excess homozygosity in a number of populations (overall FIS = 0.073). This study showed low levels of inbreeding in certain populations as well as a significant isolation-by-distance model. Only two groups of populations (Queensland and New South Wales) constituted different genetic provenances as a result of geographical isolation. The M. alternifolia data suggest that microsatellite loci did not always arise by a stepwise mutation process but that larger jumps in allele size may be involved in their evolution.

Variation on islands: major histocompatibility complex (Mhc) polymorphism in populations of the Australian bush rat.

Loss of genetic variation in small, isolated populations is commonly observed at neutral or nearly neutral loci. In this study, the loss of genetic variation was assessed in island populations for a locus of major histocompatibility complex (Mhc), a locus shown to be under the influence of balancing selection. A total of 36 alleles was found at the second exon of RT1.Ba in 14 island and two mainland populations of Rattus fuscipes greyii. Despite this high overall diversity, a substantial lack of variation was observed in the small island populations, with 13 islands supporting only one to two alleles. Two populations, Waldegrave and Williams Islands, showed moderately high levels of heterozygosity (52-56%) which were greater than expected under neutrality, suggesting the action of balancing selection. However, congruence between the level of variation at this Mhc locus and in previous allozyme electrophoresis and mitochondrial DNA studies highlights the dominant influence of genetic drift and population factors, such as bottlenecks and structuring in the founding population, in the loss of genetic variation in these small, isolated populations.

Consequences of a catadromous life-strategy for levels of mitochondrial DNA differentiation among populations of the Australian bass, Macquaria novemaculeata.

The influence of a catadromous life-strategy on levels of spatial genetic structuring in fish is poorly understood. In an effort to gain a better appreciation of how this specialized life-strategy determines population genetic structuring, we assessed variation in the mitochondrial DNA (mtDNA) control region in a catadromous perciform, the Australian bass Macquaria novemaculeata. Nineteen putative haplotypes were resolved using temperature gradient gel electrophoresis from 10 geographically distinct populations. Significant heterogeneity was revealed in haplotype frequencies and their spatial distributions among many locales. Gene partitioning statistics (AMOVA) for both raw haplotype frequency data and frequency data with sequence divergences were concordant, indicating that M. novemaculeata populations were moderately genetically structured (phi ST = 0.05, 0.06; P < 0.001, respectively). Isolation by distance seems to be a strong structuring force in M. novemaculeata, culminating in no detectable phylogeographic structuring among haplotypes. Low sequence divergences were observed among many haplotypes and it is suggested that these are the result of pruning of maternal lineages by cyclical variations in female reproductive success. This study highlights the importance of life-history patterns and, in particular, spawning locality, in determining spatial structuring of mtDNA variation in catadromous species.

Biogeographic origins of goannas (Varanidae): a molecular perspective.

This project aims to clarify the phylogenetic relationships among the extant species of Varanus in order to elucidate the origins of Varanidae, using DNA sequences. Results obtained for a minimum of 662 nucleotides of 12S rRNA sequence data from each of 21 extant species of Varanus indicate that the Australian varanids form a single monophyletic clade and also suggest that within the Australian varanids, members of the subgenus Odatria (pygmy monitors) may from a clade separate from those in the subgenus Varanus (large monitors). The Asian species appear to be sister taxa to the Australian species, while the two African species investigated were most divergent, suggesting that the Varanidae are not Gondwanic in origin. Hypothesis testing analyses were performed and involved constraining the 12S sequence data according to previously described topologies and testing the difference using parametric and nonparametric statistics. The phylogeny generated using 12S sequence data was statistically different from previously described morphological trees, while there was some support for topologies based on chomosomal and immunological datasets. Overall, our results suggest that the Australian species may be derived from an Asian source and are, therefore, in agreement with the hypothesis based on the fossil record suggesting that Varanidae may be Asian in origin.

Myoglobin intron variation in the Gouldian Finch Erythrura gouldiae assessed by temperature gradient gel electrophoresis.

In this study we show how the use of exon-primed, intron-crossing (EPIC) polymerase chain reaction (PCR) of a diploid intronic region, in conjunction with temperature gradient gel electrophoresis (TGGE), can be used to detect and rapidly assess allelic variation at the nucleotide level. We developed passerine-specific primers to amplify and sequence a 762 bp region including the second intron of the myoglobin gene in the Gouldian Finch, Erythrura gouldiae. A POLAND plot based on this sequence indicated that TGGE in combination with heteroduplex analysis (TGGE/HA) should reveal nucleotide variation in the 160 bp low-melting domain. Sequencing of the entire fragment from 19 Er. gouldiae revealed five nucleotide substitution differences within the low-melt domain, all of which could be detected and differentiated by TGGE/HA, and an additional substitution in a section of the high-melt domain which characterised another allele. A total of 181 individuals from four populations were screened for these six alleles.

The rate of mitochondrial 12S rRNA gene evolution is similar in freshwater turtles and marsupials.

Assertions that the "conventional" rate of mitochondrial DNA (mtDNA) evolution is reduced in poikilotherms in general and turtles in particular were tested for side-necked turtles (Pleurodira: Chelidae). Homologous data sets of mitochondrial 12S rRNA gene sequences were used to compare the average divergence between the Australian and South American species for two Gondwanan groups: the chelid turtles and the marsupials. The mean nucleotide divergences between continental groups for both the turtles and the marsupials are remarkably similar. These data suggest that the rate of evolution of mitochondrial 12S rRNA gene is not substantially slower in turtles than in the homeothermic marsupials.

Phylogenetic relationships of chelid turtles (Pleurodira: Chelidae) based on mitochondrial 12S rRNA gene sequence variation.

Conflicting phylogenies have been proposed for the Chelidae (Testudines: Pleurodira), a family of side-necked turtles found only in Australasia and South America. Sequence data from the mitochondrial 12S rRNA gene were used to test these phylogenies. In total, 411 nucleotides were sequenced for each of 16 chelid species, including all 11 recognized chelid genera and, as outgroups, 5 genera of Pelomedusidae (Testudines: Pleurodira). Analyses using parsimony and neighbor joining algorithms strongly support the division of Australian Chelidae into the three monophyletic groups initially suggested by Burbidge et al. (1974; Copeia 2: 392-409): Chelodina (bootstrap value 99%), the Emydura group (87%), and Pseudemydura. The analyses suggest that the Australian chelids are a monophyletic lineage (64%), with the Australian long-necked turtles, Chelodina, more closely related to the Australian short-necked chelids than to the long-necked South American species. These relationships are in contrast to those of Gaffney (1977; Am. Mus. Novitates 2620: 1-28). The species of Australian long-necked chelids consistently form a monophyletic clade, with Chelodina longicollis and Chelodina oblonga as sister taxa. The data failed to resolve relationships among the Australian short-necked taxa: Emydura, the Elseya latisternum group, the Elseya dentata group, Rheodytes, and Elusor. Unlike Gaffney (1977), we find some weak support (58%) for Pseudemydura as the closest relative of the other Australian short-necked taxa. With the exception of Hydromedusa, the South American taxa are monophyletic and the subgenera of Phrynops are paraphyletic.

Outgroup heteroduplex analysis using temperature gradient gel electrophoresis: high resolution, large scale, screening of DNA variation in the mitochondrial control region.

The ability of DNA screening techniques such as Temperature Gradient Gel Electrophoresis (TGGE) and Heteroduplex Analysis to provide resolution approaching that provided by DNA sequencing for a fraction of the time, effort and expense point to them as the logical successor to allozyme electrophoresis for population genetics. Here we present a novel alternative to the standard TGGE/Heteroduplex Analysis protocol - Outgroup Heteroduplex Analysis (OHA). We assess this technique's sensitivity in comparison to previous screening approaches using a known hierarchy of sequence differences. Our data show that Outgroup Heteroduplex Analysis has greatly increased sensitivity for screening DNA variants from that of TGGE used alone and is easily applicable to large numbers of samples. Using this technique we can consistently detect differences of as small as one base change in a 433-base-pair fragment of Control Region mitochondrial DNA from Melomys cervinipes (an Australian rodent). The approach should easily be extendable to nuclear loci and is not necessarily dependent on the use of a denaturing gradient. When combined with a targeted sequencing effort, OHA provides a sensitive and simple means of obtaining allele/haplotype frequencies and their phylogenies for population and phylogeographic studies in molecular ecology.