Microsatellite primers for Australian recreationally and commercially important estuarine fishes.

Eight primer sets that amplify microsatellite DNA loci were developed for the estuarine fishes, dusky flathead Platycephalus fuscus and sand whiting Sillago ciliata, which are targets of recreational and commercial fishers on the east coast of Australia. Surveys across these loci revealed remarkably similar levels of genetic diversity within samples of c. 22 P. fuscus and S. ciliata from each of the two estuaries. These loci will be useful in determining the genetic stock structure of these two species.

Is the post-disturbance composition of a plant population determined by selection for outcrossed seedlings or by the composition of the seedbank?

Seedbanks are expected to buffer populations against disturbances, such as fire, that could alter the genetic composition of smaller, ephemeral adult populations. However, seedling genotypes may be influenced by the spatially heterogeneous nature of both the seedbank and the disturbance (for example, germination may vary with local disturbance) and also by selection acting on germination and post-germination performance. We used microsatellite-DNA surveys of seedlings emerging from the soil-stored seedbanks of Grevillea macleayana after wildfire to compare diversity and spatial structure in seedlings and adults, and through resampling of the seedling data set, to determine whether the resultant adult population reflected the effects of selection or random seedling mortality. The large post-fire seedling cohorts captured the full allelic diversity of the pre-fire adult population. However, we found a mismatch in the genotypic structure of adults and seedlings. Seedlings displayed larger heterozygous deficits than adults; however, over the ensuing 11 years, seedling heterozygosity eventually matched values for the pre-fire adults. Increasing heterozygosity among adults has generally been attributed to heterosis and/or reduction in Wahlund effects via self-thinning. Resampling of early post-fire seedlings to generate samples of equivalent size to survivors at 11 years showed that increases in heterozygosity must be driven by selection favouring outcrossed seed. This finding is important in an evolutionary context but also has implications for the restoration of natural or managed populations where a seedbank is a viable source of recruits.

Patterns of hybridization and asymmetrical gene flow in hybrid zones of the rare Eucalyptus aggregata and common E. rubida.

The patterns of hybridization and asymmetrical gene flow among species are important for understanding the processes that maintain distinct species. We examined the potential for asymmetrical gene flow in sympatric populations of Eucalyptus aggregata and Eucalyptus rubida, both long-lived trees of southern Australia. A total of 421 adults from three hybrid zones were genotyped with six microsatellite markers. We used genealogical assignments, admixture analysis and analyses of spatial genetic structure and spatial distribution of individuals, to assess patterns of interspecific gene flow within populations. A high number of admixed individuals were detected (13.9-40% of individuals), with hybrid populations consisting of F(1) and F(2) hybrids and backcrosses in both parental directions. Across the three sites, admixture proportions were skewed towards the E. aggregata genetic cluster (x=0.56-0.65), indicating that backcrossing towards E. aggregata is more frequent. Estimates of long-term migration rates also indicate asymmetric gene flow, with higher migration rates from E. aggregata to hybrids compared with E. rubida. Taken together, these results indicate a greater genetic input from E. aggregata into the hybrid populations. This asymmetry probably reflects differences in style lengths (E. rubida: ~7 mm, E. aggregata: ~4 mm), which can prevent pollen tubes of smaller-flowered species from fertilizing larger-flowered species. However, analyses of fine-scale genetic structure suggest that localized seed dispersal (<40 m) and greater clustering between hybrid and E. aggregata individuals may also contribute to directional gene flow. Our study highlights that floral traits and the spatial distributions of individuals can be useful predictors of the directionality of interspecific gene flow in plant populations.

Gamete compatibility between marine and estuarine Acanthopagrus spp. (Sparidae) and their hybrids.

On Australia's south-east coast, hybridization between estuary-restricted black bream Acanthopagrus butcheri Munro and its migratory coastal congener yellowfin bream Acanthopagrus australis (Günther) has led to estuarine populations largely composed of hybrids that are most genetically similar to A. butcheri. The fertilization success achieved when ova of estuary-caught A. butcheri were fertilized with the cryogenically preserved sperm of either ocean-caught A. australis or estuary-caught A. butcheri-like was compared. The experimental crosses, which by chance included both pure parental and hybrid bream, revealed no evidence that gametic incompatibility provides a barrier to fertilization among both pure species and their hybrids.

Window of opportunity: an episode of recruitment in a Banksia hybrid zone demonstrates continuing hybridization and phenotypic plasticity.

BACKGROUND AND AIMS: In perennial plants (especially post-fire resprouters), extant populations may reflect recruitment events in the distant past. This is true of hybrid zones formed by two Banksia species of swamps and woodlands in south-eastern Australia, Banksia robur and B. oblongifolia. Both resprout after fire but recruitment is dependent on periodic fires. Although plants of intermediate morphology have also been identified as hybrids using allozyme markers, the extent of ongoing hybridization is unknown. This study investigates whether both microsatellite markers and morphological measurements can be used to distinguish between the two species and their hybrids. A recent recruitment event and microsatellite markers allow the frequency of ongoing hybridization to be estimated, and also the effects of environmental variation on the morphology of plants and seedlings to be tested. METHODS: Variation at seven microsatellite loci was scored and seven leaf characteristics within putatively pure stands and mixed stands of both species were measured, revealing that the two species were genetically and morphologically distinct and that mixed stands also contained genetically and sometimes morphologically distinct hybrids. An opportunity created by wildfires was used to analyse the genetics and morphometrics of adults and seedlings from two hybrid zones. KEY RESULTS: Approximately 9 % of adults and 21 % of seedlings were identified as genetic hybrids in both hybrid zones. Within these sites, the genotype of mature plants correlated well with morphology, except for some hybrid plants that had parental morphology. However, seedling morphology was highly variable and insufficient to describe the composition of the hybrid zone in this cohort. Greater phenotypic plasticity was evident among seedlings growing within the hybrid zones than seedlings growing in pots. CONCLUSIONS: The hybrid zones are complex and the range of genotypes detected in seedlings reveals both continuing hybridization and introgression.

Does life history predict past and current connectivity for rocky intertidal invertebrates across a marine biogeographic barrier?

The southeast Australian coast potentially includes a complex biogeographic barrier, largely lacking exposed rocky shore that may limit the dispersal of rocky intertidal taxa and contribute to the maintenance of two biogeographic regions. Surprisingly, within the 300-km barrier region, several species considered exposed rocky shore specialists occurred within sheltered sites. We analysed COI sequence variation for 10 rocky intertidal invertebrate species, with a range of life histories, to test the hypotheses that larval type and habitat specificity are strong predictors of gene flow between biogeographic regions. Our data revealed that the southeast corner of Australia includes a strong barrier to gene flow for six of eight species with planktonic larvae, and a coalescence analysis of sequence differentiation (IM model) suggests that a barrier has existed since the Pleistocene. In contrast, two direct developers were not affected by the barrier. Our comparative approach and data from earlier studies (reviewed here) do not support the hypothesis that larval type predicts gene flow across this barrier, instead we found that the ability to utilize sheltered habitat provides a clearer explanation of the phylogeographic break. Indeed, the species that displayed little or no evidence of a phylogeographic break across the barrier each displayed unexpectedly relaxed habitat specificity.

Evolutionary impacts of hybridization and interspecific gene flow on an obligately estuarine fish.

For free-spawning estuarine taxa, gene flow among estuaries may occur via hybridization with mobile congeners. This phenomenon has rarely been investigated, but is probably susceptible to anthropogenic disturbance. In eastern Australia, the estuarine Black Bream Acanthopagrus butcheri and marine Yellowfin Bream Acanthopagrus australis have overlapping distributions and the potential to hybridize. We used surveys of microsatellite and mtDNA variation in 565 adults from 25 estuaries spanning their distributional range to characterize the species and their putative hybrids. Hybrids were widespread (68% of estuaries) and hybrid frequencies varied greatly among estuaries (0-58%). Most (88%) were classed as advanced generation backcrosses with A. butcheri and displayed A. butcheri mtDNA haplotypes. We found most hybrids in the three estuaries within the zone of sympatry (57%). Our study highlights the underemphasized importance of estuaries as sites of hybridization and suggests that hybridization is driven both by opportunity for contact and human activity.

The role of sexual and asexual reproduction in structuring high latitude populations of the reef coral Pocillopora damicornis.

The genotypic composition of populations of the asexually viviparous coral Pocillopora damicornis varies in a manner that challenges classical models of the roles of sexual and asexual reproduction. On the geographically isolated Hawaiian reefs and high latitude reefs in Western Australia, P. damicornis populations are highly clonal although it has been argued that sexual reproduction via broadcast spawning generates widely dispersed colonists. In contrast, on eastern Australia's tropical Great Barrier Reef populations show little evidence of clonality. Here, we compare the genotypic diversity of adult and juvenile colonies of P. damicornis at seven sites on eastern Australia's high latitude Lord Howe Island reefs to determine if levels of clonality vary with habitat heterogeneity and age of colonies (as predicted by theory) or alternatively if clonality is again always high as for other isolated reef systems. We found 55-100% of the genotypic diversity expected for random mating at all seven sites and little evidence of asexual recruitment irrespective of habitat heterogeneity (sheltered versus wave exposed) or colony age. We found reduced levels of genetic diversity compared with tropical reefs (2.75 versus 4 alleles/locus), which supports earlier findings that Lord Howe Island is an isolated reef system. Furthermore, heterozygote deficits coupled with significant genetic subdivision among sites (FST=0.102+/-0.03) is typical of populations that have limited larval connections and are inbred. We conclude that the genetic structure of P. damicornis at Lord Howe Island reflects populations that are maintained through localised recruitment of sexually produced larvae.

Evidence for ancient genetic subdivision among recently fragmented populations of the endangered shrub Grevillea caleyi (Proteaceae).

The genetic effects of population fragmentation cannot be interpreted without understanding the underlying pattern of genetic variation resulting from historic population processes. We used AFLP markers to determine genetic structure and distribution of genetic diversity among populations of an endangered Australian shrub Grevillea caleyi (Proteaceae). Populations that occurred historically on four ridges have new been fragmented to varying degrees, producing some large, relatively pristine populations and very small populations consisting of fewer than 10 adult plants. We found marked population genetic structure (65.9% of genetic variation was among populations) and a significant relationship between genetic and geographic distance (rm=0.564, P=0.004). However, only 14% of overall genetic differentiation was attributable to variation among ridges, compared with 52% among populations within ridges. Moreover, genetic diversity within samples of plants did not vary with either population size or degree of isolation. Thus, the present genetic structure of populations is probably almost entirely the product of historical events. Fine-scale structuring within populations prior to fragmentation may have been caused by limited seed and pollen dispersal, despite a complex suite of (mostly avian) pollinators, and a mixed mating system that allows a large amount of selfing. The combined effects of adult longevity and a soil-stored seed bank may have buffered the recently fragmented populations against the effects of dramatic reductions in numbers of adult plants.

Effects of seed bank disturbance on the fine-scale genetic structure of populations of the rare shrub Grevillea macleayana.

Dispersal in most plants is mediated by the movement of seeds and pollen, which move genes across the landscape differently. Grevillea macleayana is a rare, fire-dependent Australian shrub with large seeds lacking adaptations for dispersal; yet it produces inflorescences adapted to pollination by highly mobile vertebrates (eg birds). Interpreting fine-scale genetic structure in the light of these two processes is confounded by the recent imposition of anthropogenic disturbances with potentially contrasting genetic consequences: (1) the unusual foraging behaviour of exotic honeybees and 2. widespread disturbance of the soil-stored seedbank by road building and quarrying. To test for evidence of fine-scale genetic structure within G. macleayana populations and to test the prediction that such structure might be masked by disturbance of the seed bank, we sampled two sites in undisturbed habitat and compared their genetic structure with two sites that had been strongly affected by road building using a test for spatial autocorrelation of genotypes. High selfing levels inferred from genotypes at all four sites implies that pollen dispersal is limited. Consistent with this, we observed substantial spatial clustering of genes at 10 m or less in the two undisturbed populations and argue that this reflects the predicted effects of both high selfing levels and limited seed dispersal. In contrast, at the two sites disturbed by road building, spatial autocorrelation was weak. This suggests there has been mixing of the seed bank, counteracting the naturally low dispersal and elevated selfing due to honeybees. Pollination between near neighbours with reduced relatedness potentially has fitness consequences for G. macleayana in disturbed sites.

Genotypic diversity and gene flow in brooding and spawning corals along the Great Barrier Reef, Australia.

Marine organisms exhibit great variation in reproductive modes, larval types, and other life-history traits that may have major evolutionary consequences. We measured local and regional patterns of genetic variation in corals along Australia's Great Barrier Reef to determine the relative contributions of sexual and asexual reproduction to recruitment and to infer levels of gene flow both locally (among adjacent sites, < 5 km apart) and regionally (among reefs separated by 500-1,200 km). We selected five common brooding species (Acropora cuneata, A. palifera, Pocillopora damicornis, Seriatopora hystrix, and Stylophora pistillata) and four broadcast spawners (Acropora hyacinthus, A. cytherea, A. millepora, and A. valida), which encompassed a wide range of larval types and potential dispersal capabilities. We found substantial genotypic diversity at local scales in six of the nine species (four brooders, two spawners). For these six, each local population displayed approximately the levels of multilocus genotypic diversity (Go) expected for outcrossed sexual reproduction (mean values of Go:Ge ranged from 0.85 to 1.02), although consistent single-locus heterozygous deficits indicate that inbreeding occurs at the scale of whole reefs. The remaining three species, the brooder S. hystrix and the spawners A. valida and A. millepora displayed significantly less multilocus genotypic diversity (Go) than was expected for outcrossed sexual reproduction (Ge) within each of several sites. Acropora valida and A. millepora showed evidence of extensive localized asexual replication: (1) a small number of multilocus (clonal) genotypes were numerically dominant within some sites (Go:Ge values were as low as 0.17 and 0.20): (2) single-locus genotype frequencies were characterized by both excesses and deficits of heterozygotes (cf. Hardy-Weinberg expectations), and (3) significant linkage disequilibria occurred. For the brooding S. hystrix Go:Ge values were also low within each of four sites (x = 0.48). However, this result most likely reflects the highly restricted dispersal of gametes or larvae, because levels of genetic variation among sites within reefs were extremely high (FSR = 0.28). For all species, we detected considerable genetic subdivision among sites within each reef (high FSR-values), and we infer that larval dispersal is surprisingly limited (i.e., Nem among sites ranging from 0.6 to 3.3 migrants per generation), even in species that have relatively long planktonic durations. Nevertheless, our estimates of allelic variation among reefs (FRT) also imply that for all four broadcast spawning species and three of the brooders, larval dispersal is sufficient to maintain moderate to high levels of gene flow along the entire Great Barrier Reef (i.e., Nem among reefs ranged from 5 to 31). In contrast, widespread populations of S. hystrix and S. pistilata (the two remaining brooders) are relatively weakly connected (Nem among reefs was 1.4 and 2.5, respectively). We conclude that most recruitment by corals is very local, particularly in brooders, but that enough propagules are widely dispersed to ensure that both broadcast spawning and brooding species form vast effectively panmictic populations on the Great Barrier Reef.

Genetic evidence of widespread dispersal in a parthenogenetic freshwater ostracod.

We used an hierarchical analysis of allozyme variation to investigate for the freshwater ostracod Candonocypris novaezelandiae the relative contributions of sexual and asexual reproduction to recruitment into 42 local populations and to infer patterns of gene flow within and among four geographical regions (watersheds) in south-eastern Australia. Allele frequency variation among local populations was marked (mean F(ST)=0.228) but showed no regional differentiation. The allele frequency differences among local populations probably reflect the effects of stochastic processes, such as founder events, as well as variation in the success, and hence abundance, of particular clonal genotypes within water-bodies. Indeed, local populations were highly clonal, containing only females and displaying relatively low levels of genotypic diversity. Nevertheless, the distribution of genotypes within and among regions was surprising. The bulk of sampled individuals (88 per cent) were represented by just six common genotypes that were shared extensively among local populations and were geographically widespread. Individual samples contained a mean of 4.05, and up to 10, distinct four-locus genotypes and overall we detected a total of 26 electrophoretically distinct genotypes. In combination, our results suggest that either the south-eastern Australian populations of C. novaezelandiae arose through a recent colonization event (perhaps associated with an expansion of agricultural practices) or there is sufficient continuing gene flow between regions to prevent differentiation. However, the exact contributions of sexual and asexual reproduction to dispersal in this ostracod remain unclear.

Factors controlling fruit set in hermaphroditic plants: Studies with the Australian proteaceae.

The number of fruits produced by many hermaphroditic plants is usually far fewer than the number of flowers available for fertilization. There are various possible explanations for the low fruit:flower ratio, some proximate and others ultimate. Recent studies, especially in northern hemisphere systems, have used field experiments to test some of them, but there are potential difficulties with the methodology of some experiments and with the testing of ultimate hypotheses. It is important to examine the possible explanations for low fruit: flower ratios with a range of different systems. This article reviews studies on Australian species of woody, perennial shrubs in the family Proteaceae; this evolutionarily distinct group of plants and pollinators has several unusual and interesting characteristics, and provides a valuable addition to the better-known northern hemisphere studies.

The effects of sexual and asexual reproduction on geographic variation in the sea anemone Actinia tenebrosa.

Allelic and genotypic frequencies were determined for samples from 35 widely distributed Australasian colonies of Actinia tenebrosa and 2 South African colonies of A. equina. These data provided no evidence of gene flow between Australisian and South African Actinia colonies and indicated that there may be some restriction of gene flow between widely separated Australasian colonies.Both species are viviparous, and brooded A. tenebrosa are known to be produced asexually. The present data indicate that, within both species, almost all genotypic diversity is generated by sexual reproduction with recombination. Sexually produced juveniles appear to be widely dispersed and panmixis may occur over thousands of kilometres. However, successful sexual recruitment must be episodic or rare. Colonies on stable shores displayed relatively low levels of genotypic diversity, as compared with expectations for sexually reproducing populations, indicating strong local effects of asexual recruitment. Clonal genotypes may be spread over hundreds of metres of shore, but are typically restricted to discrete colonies. Asexual recruitment is highly localised and asexual dispersal appears to be limited by lengths of shore (≤500 m) which are unsuitable for colonization. Colonies on unstable shores are significantly more diverse genotypically and show little evidence of clonal proliferation.

The effects of asexual reproduction and inter-genotypic aggression on the genotypic structure of populations of the sea anemone Actinia tenebrosa.

Genotype frequencies of adult and juvenile Actinia tenebrosa from 2 populations showed that settlement and recruitment predominantly involved the localised asexual (ameiotic) proliferation of established genotypes. However, there is strong indirect evidence that the genotypic variation was generated by sexual reproduction. Genotypic structuring of these populations was detected at 2 levels. First, coarse clumping of genotypically identical adults and juveniles occurred on a scale consistent with the pattern of asexual dispersal. Second, fine-scale clustering of genotypically identical adults and juveniles occurred on a scale consistent with predicted effects of inter-genotypic aggression. Inter-genotypic aggression seems certain to play an important role in inter-genotypic competition for recruitment space and should reduce the input of genotypic variation into established populations. The applicability of the Strawberry-Coral Model to the life history of this species is discussed.

A survey of pathological changes associated with long-term high tar tobacco smoke exposure in a murine model.

Balb/c mice were exposed to the fresh smoke of a daily equivalent of thirty high tar filtered cigarettes for periods of up to 95 weeks. Detailed surveys of the gross and histopathological data are presented which indicates that there is the induction or production of significant numbers of malignant tumours of several types in the animals exposed to tobacco smoke. There are also significant histopathological changes which consist mainly of interstitial pneumonia and focal low grade emphysema. These features contribute significantly to the reduction in life span of the test animals in which the main causes of death are malignancies and inflammatory diseases of the lungs.

Effects of chronic tobacco smoke exposure on immune responses in aged mice.

Balb/c mice were exposed to fresh smoke, equivalent to 30 high-tar filtered cigarettes/day, for 83 wk or less. After groups of mice were subjected to 56, 64, 72, and 80 wk of tobacco smoke (TS) exposure, serum antibody responses to both T-dependent and T-independent antigens, numbers of plaque-forming cells, spleen cell and lymph node PHA responses, and systemic clearance of normal and opsonised antigen were investigated. Aged TS-exposed mice showed some deterioration of immune responsiveness with age, but results varied; in extreme old age the immunological responsiveness of TS-exposed mice was similar to that of age-matched controls. In addition, groups of mice inoculated with fibrosarcoma cells after 78 wk TS-exposure did not show significantly greater susceptibility to the early development of pulmonary metastases.

Effects of tobacco smoke exposure on splenic architecture and weight, during the primary immune response of BALB/c mice.

Mice exposed to tobacco smoke (TS) for 3 days or 18 or 28 weeks, prior to SRBC inoculation, subsequently displayed less pronounced and/or "shorter-lived" splenomegaly than are matched controls. In addition mice exposed to TS for three days or 18 weeks displayed a reduction in both the magnitude and duration of the primary immune response as evidenced by the pattern of expansion of splenic white pulp and "RNA-rich" white pulp volumes. In contrast mice exposed to TS for 28 weeks, prior to inoculation, displayed white pulp] and "RNA-rich" white pulp volumes similar to those of control mice.