Is Penguin Circovirus Circulating Only in the Antarctic Circle? Lack of Viral Detection in Namibia.

The known host range of circoviruses is continuously expanding because of more intensive diagnostic activities and advanced sequencing tools. Recently, a new circovirus (penguin circovirus (PenCV)) was identified in the guano and cloacal samples collected from Adélie penguins (Pygoscelis adeliae) and chinstrap penguins (Pygoscelis antarcticus) in Antarctica. Although the virus was detected in several asymptomatic subjects, a potential association with feather disease was speculated. To investigate the occurrence and implications of PenCV in other penguin species located outside of Antarctica, a broad survey was undertaken in African penguins (Spheniscus demersus) on two islands off the southern Namibian coast. For this purpose, specific molecular biology assays were developed and validated. None of the 151 blood samples tested positive for PenCV. Several reasons could explain the lack of PenCV positive samples. African penguins and Pygoscelis species are separated by approximately 6000 km, so there is almost no opportunity for transmission. Similarly, host susceptibility to PenCV might be penguin genus-specific. Overall, the present study found no evidence of PenCV in African penguin colonies in Namibia. Further dedicated studies are required to assess the relevance of PenCV among different penguin species.

Haemoproteus jenniae (Haemoproteidae, Haemosporida) infects gulls (Larus spp.) in South Africa, with redescription of Haemoproteus skuae.

Haemoproteus spp. are dipteran-borne protozoa that infect erythrocytes and reticulo-endothelial cells of birds. These parasites are not usually transmitted between birds belonging to different orders. The suborder Lari (order Charadriiformes) comprises ~170 avian species, the majority of which are aquatic, including gulls, terns, auklets, murres and skuas, among others. In spite of the diversity of this avian group, there is limited known diversity of haemosporidian parasites, with only 4 recorded Haemoproteus morphospecies thus far. We examined the blood smears of 21 kelp gulls (Larus dominicanus) captured at a breeding colony in South Africa, as well as Haemoproteus-positive archival blood smears of 15 kelp gulls and 1 Hartlaub's gull (Larus hartlaubii) sampled while under care at seabird rehabilitation facilities in South Africa. Haemoproteus sp. infection was detected in 19% of wild-caught kelp gulls. All parasites from the gulls were morphologically identified as Haemoproteus jenniae, a species previously recorded in Lari birds at the Galapagos Islands (Ecuador), Rocas Atoll (Brazil) and Poland. Gene sequencing uncovered a new cytochrome b lineage, LARDOM01, which was closely related to the previously reported H. jenniae lineage CREFUR01. Additionally, we evaluated a hapantotype blood smear of Haemoproteus skuae, which had been described infecting a brown skua (Catharacta antarctica) in South Africa. We provide a redescription of H. skuae and discuss the morphological characters distinguishing it from H. jenniae. Further research is necessary to improve our knowledge about the host and geographic distribution, health effects and phylogeny of H. jenniae and H. skuae.

Primary pollinator exclusion has divergent consequences for pollen dispersal and mating in different populations of a bird-pollinated tree.

Pollination by nectarivorous birds is predicted to result in different patterns of pollen dispersal and plant mating compared to pollination by insects. We tested the prediction that paternal genetic diversity, outcrossing rate and realized pollen dispersal will be reduced when the primary pollinator group is excluded from bird-pollinated plants. Pollinator exclusion experiments in conjunction with paternity analysis of progeny were applied to Eucalyptus caesia Benth. (Myrtaceae), a predominantly honeyeater-pollinated tree that is visited by native insects and the introduced Apis mellifera (Apidae). Microsatellite genotyping at 14 loci of all adult E. caesia at two populations (n = 580 and 315), followed by paternity analysis of 705 progeny, revealed contrasting results between populations. Honeyeater exclusion did not significantly impact pollen dispersal or plant mating at Mount Caroline. In contrast, at the Chiddarcooping site, the exclusion of honeyeaters led to lower outcrossing rates, a threefold reduction in the average number of sires per fruit, a decrease in intermediate-distance mating and an increase in near-neighbour mating. The results from Chiddarcooping suggest that bird pollination may increase paternal genetic diversity, potentially leading to higher fitness of progeny and favouring the evolution of this strategy. However, further experimentation involving additional trees and study sites is required to test this hypothesis. Alternatively, insects may be effective pollinators in some populations of bird-adapted plants, but ineffective in others.

Isolation and Lack of Potential Mates may Threaten an Endangered Arid-Zone Acacia.

Clonality may provide reproductive assurance for many threatened plants while limiting sexual reproductive success either through energetic tradeoffs or because clones are self-incompatible. Most stands of the Australian arid-zone plant Acacia carneorum, flower annually but low seed set and an absence of sexual recruitment now suggest that this species and other, important arid-zone ecosystem engineers may have low genotypic diversity. Indeed, our recent landscape-scale genetic study revealed that stands are typically monoclonal, with genets usually separated by kilometers. An inability to set sexually produced seed or a lack of genetically diverse mates may explain almost system-wide reproductive failure. Here, using microsatellite markers, we genotyped 100 seeds from a rare fruiting stand (Middle-Camp), together with all adult plants within it and its 4 neighboring stands (up to 5 km distant). As expected, all stands surveyed were monoclonal. However, the Middle-Camp seeds were generated sexually. Comparing seed genotypes with the single Middle-Camp genotype and those of genets from neighboring and other regional stands (n = 26), revealed that 73 seeds were sired by the Middle-Camp genet. Within these Middle-Camp seeds we detected 19 genotypes in proportions consistent with self-fertilization of that genet. For the remaining 27 seeds, comprising 8 different genotypes, paternity was assigned to the nearest neighboring stands Mallee and Mallee-West, approximately 1 km distant. Ironically, given this species' vast geographic range, a small number of stands with reproductively compatible near neighbors may provide the only sources of novel genotypes.

Conservation of old individual trees and small populations is integral to maintain species' genetic diversity of a historically fragmented woody perennial.

Historically fragmented and specialized habitats such as granite outcrops are understudied globally unique hot spots of plant evolution. In contrast to predictions based on mainstream population genetic theory, some granite outcrop plants appear to have persisted as very small populations despite prolonged geographic and genetic isolation. Eucalyptus caesia Benth. is a long-lived lignotuberous tree endemic with a naturally fragmented distribution on granite outcrops in south-western Australia. To quantify population to landscape-level genetic structure, we employed microsatellite genotyping at 14 loci of all plants in 18 stands of E. caesia. Sampled stands were characterized by low levels of genetic diversity, small absolute population sizes, localized clonality and strong fine-scale genetic subdivision. There was no significant relationship between population size and levels of heterozygosity. At the landscape scale, high levels of population genetic differentiation were most pronounced among representatives of the two subspecies in E. caesia as originally circumscribed. Past genetic interconnection was evident between some geographic neighbours separated by up to 20 km. Paradoxically, other pairs of neighbouring stands as little as 7 km apart were genetically distinct. There was no consistent pattern of isolation by distance across the 280 km range of E. caesia. Low levels of gene flow, together with strong drift within stands, provide some explanation of the patterns of genetic differentiation we observed. Individual genet longevity via the ability to repeatedly resprout and expand from a lignotuber may enhance the persistence of some woody perennial endemic plants despite small population size, minimal genetic interconnection and low heterozygosity.

Near-neighbour optimal outcrossing in the bird-pollinated Anigozanthos manglesii.

BACKGROUND AND AIMS: In plants, the spatial and genetic distance between mates can influence reproductive success and offspring fitness. Negative fitness consequences associated with the extremes of inbreeding and outbreeding suggest that there will be an intermediate optimal outcrossing distance (OOD), the scale and drivers of which remain poorly understood. In the bird-pollinated Anigozanthos manglesii (Haemodoraceae) we tested (1) for the presence of within-population OOD, (2) over what scale it occurs, and (3) for OOD under biologically realistic scenarios of multi-donor deposition associated with pollination by nectar-feeding birds. METHODS: We measured the impact of mate distance (spatial and genetic) on seed set, fruit size, seed mass, seed viability and germination success following hand pollination from (1) single donors across 0 m (self), <1 m, 1-3 m, 7-15 m and 50 m, and (2) a mix of eight donors. Microsatellite loci were used to quantify spatial genetic structure and test for the presence of an OOD by paternity assignment after multi-donor deposition. KEY RESULTS: Inter-mate distance had a significant impact on single-donor reproductive success, with selfed and nearest-neighbour (<1 m) pollination resulting in only ~50 seeds per fruit, lower overall germination success and slower germination. Seed set was greatest for inter-mate distance of 1-3 m (148 seeds per fruit), thereafter plateauing at ~100 seeds per fruit. Lower seed set following nearest-neighbour mating was associated with significant spatial genetic autocorrelation at this scale. Paternal success following pollination with multiple sires showed a significantly negative association with increasing distance between mates. CONCLUSIONS: Collectively, single- and multi-donor pollinations indicated evidence for a near-neighbour OOD within A. manglesii. A survey of the literature suggests that within-population OOD may be more characteristic of plants pollinated by birds than those pollinated by insects.

Empirical Primaquine Treatment of Avian Babesiosis in Seabirds.

Babesia species are tickborne hemoprotozoans. Although experiments have shown that primaquine is highly effective in the treatment of Babesia species infections in mammals, this drug has not been widely used for the treatment of avian babesiosis. Consequently, the treatment of this disease for avian patients has traditionally relied on an empirically established imidocarb treatment. In this study, the authors examined the efficacy of primaquine as a treatment alternative for avian babesiosis (Babesia peircei and Babesia ugwidiensis) in seabirds. Retrospective analysis was performed on the medical records and blood smears of 446 B peircei-positive African penguins (Spheniscus demersus) and 41 B ugwidiensis-positive Cape cormorants (Phalacrocorax capensis) admitted for rehabilitation at the Southern African Foundation for the Conservation of Coastal Birds (SANCCOB, Cape Town, South Africa). Treatment with primaquine (1 mg/kg PO q24h for 10 days) was effective in rapidly and markedly decreasing the proportion of Babesia-positive blood smears in African penguins and Cape cormorants. No regurgitation, loss of appetite, or any other signs after administration of primaquine were observed during the study period. The use of primaquine can be a particularly advantageous treatment alternative for avian babesiosis in circumstances in which it is not possible to determine confidently whether the intraerythrocytic inclusions seen in blood smears correspond to Babesia or Plasmodium or in cases in which there is a coinfection by Babesia and Plasmodium.

Effectiveness of camera traps for quantifying daytime and nighttime visitation by vertebrate pollinators.

Identification of pollen vectors is a fundamental objective of pollination biology. The foraging and social behavior of these pollinators has profound effects on plant mating, making quantification of their behavior critical for understanding the ecological and evolutionary consequences of different pollinators for the plants they visit. However, accurate quantification of visitation may be problematic, especially for shy animals and/or when the temporal and spatial scale of observation desired is large. Sophisticated heat- and movement-triggered motion-sensor cameras ("camera trapping") provide new, underutilized tools to address these challenges. However, to date, there has been no rigorous evaluation of the sampling considerations needed for using camera trapping in pollination research.We measured the effectiveness of camera trapping for identifying vertebrate visitors and quantifying their visitation rates and foraging behavior on Banksia menziesii (Proteaceae). Multiple still cameras (Reconyx HC 500) and a video camera (Little Acorn LTL5210A) were deployed.From 2,753 recorded visits by vertebrates, we identified five species of nectarivorous honeyeater (Meliphagidae) and the honey possum (Tarsipedidae), with significant variation in the species composition of visitors among inflorescences. Species of floral visitor showed significant variation in their time of peak activity, duration of visits, and numbers of flowers probed per visit. Where multiple cameras were deployed on individual inflorescences, effectiveness of individual still cameras varied from 15% to 86% of all recorded visits. Methodological issues and solutions, and the future uses of camera traps in pollination biology, are discussed. Conclusions and wider implications: Motion-triggered cameras are promising tools for the quantification of vertebrate visitation and some aspects of behavior on flowers. However, researchers need to be mindful of the variation in effectiveness of individual camera traps in detecting animals. Pollinator studies using camera traps are in their infancy, and the full potential of this developing technology is yet to be realized.

Clonality disguises the vulnerability of a threatened arid zone Acacia.

Long-lived, widespread plant species are expected to be genetically diverse, reflecting the interaction between large population sizes, overlapping generations, and gene flow. Such species are thought to be resilient to disturbance, but may carry an extinction debt due to reproductive failure. Genetic studies of Australian arid zone plant species suggest an unusually high frequency of asexuality, polyploidy, or both. A preliminary AFLP genetic study implied that the naturally fragmented arid zone tree, Acacia carneorum, is almost entirely dependent on asexual reproduction through suckering, and stands may have lacked genetic diversity and interconnection even prior to the onset of European pastoralism. Here we surveyed microsatellite genetic variation in 20 stands to test for variation in life histories and further assessed the conservation status of the species by comparing genetic diversity within protected stands in National Parks and disturbed range lands. Using herbarium records, we estimate that 219 stands are extant, all of which occur in the arid zone, west of the Darling River in southeastern Australia. With two exceptions, all surveyed stands comprised only one multilocus genet and at least eight were putatively polyploid. Although some stands comprise thousands of stems, our findings imply that the species as a whole may represent ~240 distinct genetic individuals, many of which are polyploid, and most are separated by >10 km of unsuitable habitat. With only 34% of stands (and therefore genets) occurring within conservation reserves, A. carneorum may be at much greater risk of extinction than inferred from on-ground census data. Land managers should prioritize on-ground preservation of the genotypes within existing reserves, protecting both vegetative suckers and seedlings from herbivory. Importantly, three stands are known to set viable seed and should be used to generate genetically diverse germ-plasm for ex situ conservation, population augmentation, or translocation.

Characterization and transferability of microsatellites for the Kangaroo Paw, Anigozanthos manglesii (Haemodoraceae).

PREMISE OF THE STUDY: Microsatellites were developed for the future assessment of population genetic structure, mating system, and dispersal of the perennial kangaroo paw, Anigozanthos manglesii (Haemodoraceae), and related species. METHODS AND RESULTS: Using a Personal Genome Machine (PGM) semiconductor sequencer, ca. 4.03 million sequence reads were generated. QDD pipeline software was used to identify 190,000 microsatellite-containing regions and priming sites. From these, 90 were chosen and screened using PCR, and 15 polymorphic markers identified. These sites amplified di-, tri-, and pentanucleotide repeats with one to 20 alleles per locus. Primers were also amplified across congeners A. bicolor, A. flavidus, A. gabrielae, A. humilis, A. preissii, A. pulcherrimus, A. rufus, and A. viridis to assess cross-species transferability. CONCLUSIONS: These markers provide a resource for population genetic studies in A. manglesii and other species within the genus.

Novel Consequences of Bird Pollination for Plant Mating.

Pollinator behaviour has profound effects on plant mating. Pollinators are predicted to minimise energetic costs during foraging bouts by moving between nearby flowers. However, a review of plant mating system studies reveals a mismatch between behavioural predictions and pollen-mediated gene dispersal in bird-pollinated plants. Paternal diversity of these plants is twice that of plants pollinated solely by insects. Comparison with the behaviour of other pollinator groups suggests that birds promote pollen dispersal through a combination of high mobility, limited grooming, and intra- and interspecies aggression. Future opportunities to test these predictions include seed paternity assignment following pollinator exclusion experiments, single pollen grain genotyping, new tracking technologies for small pollinators, and motion-triggered cameras and ethological experimentation for quantifying pollinator behaviour.

Microsatellite primers for vulnerable and thriving Acacia (Fabaceae) species from Australia's arid zone.

PREMISE OF THE STUDY: Microsatellite markers were developed for the common arid Australian shrub Acacia ligulata (Fabaceae) and the threatened overstory trees A. melvillei and A. pendula. METHODS AND RESULTS: DNA sequence data generated by 454 sequencing were used to identify microsatellite nucleotide repeat motifs. Including previously developed primer sets, we report on the development of 10 polymorphic microsatellite loci for each species. Six of these were novel for A. melvillei and A. ligulata, and five were novel for A. pendula, while five more each were transferred from primers developed for related species (A. carneorum and A. loderi). We found three to 17 alleles per locus for each species, with high multilocus genotypic diversity within each of two A. ligulata and A. pendula stands, and one A. melvillei population. A second A. melvillei stand appeared to be monoclonal. CONCLUSIONS: These markers will allow assessment of population genetics, mating systems, and connectedness of populations of these and possibly other arid-zone acacias.

Marine genetic swamping: hybrids replace an obligately estuarine fish.

Populations of obligately estuarine taxa are potentially small and isolated and may lack genetic variation and display regional differentiation as a result of drift and inbreeding. Hybridization with a wide-ranging marine congener should introduce genetic variation and reduce the effects of inbreeding depression and genetic drift. However, high levels of hybridization can cause demographic and genetic swamping. In southeastern Australia hybridization occurs between obligately estuarine Black bream (Acanthopagrus butcheri) and migratory marine Yellowfin bream (Acanthopagrus australis). Here, we surveyed genetic variation at eight microsatellite loci and the mitochondrial control region of juvenile fish from five coastal lagoons (including temporal replication in two lagoons) (total n = 970) to determine the frequency and persistence of hybridization, and its likely consequence for the estuarine restricted A. butcheri. Of 688 juvenile fish genotyped 95% were either A. australis (347) or hybrids (309); only 5% (32) were A. butcheri. Most hybrids were later generation hybrids or A. butcheri backcrosses, which are likely multi-generational residents within lagoons. Far greater proportions of hybrid juveniles were found within two lagoons that are generally closed to the ocean (>90% hybrid fish within generally closed lagoons vs. 12-27% in permanently or intermittently open lagoons). In both lagoons, this was consistent across multiple cohorts of fish [79-97% hybrid fish (n = 282)]. Hybridization and introgression represent a major threat to the persistence of A. butcheri and have yet to be investigated for large numbers of estuarine taxa.

Urban plants as genetic reservoirs or threats to the integrity of bushland plant populations.

Remnant plants in urban fringes and native plants in gardens have the potential to contribute to the conservation of threatened plants by increasing genetic diversity, effective size of populations, and levels of genetic connectedness. But they also pose a threat through the disruption of locally adapted gene pools. At Hyams Beach, New South Wales, Australia, four bushland stands of the rare shrub, Grevillea macleayana McGillivray, surround an urban area containing remnant and cultivated specimens of this species. Numbers of inflorescences per plant, fruits per plant, and visits by pollinators were similar for plants in urban gardens and bushland. Urban plants represented a substantial but complex genetic resource, displaying more genetic diversity than bushland plants judged byH(e), numbers of alleles per locus, and number of private alleles. Of 27 private alleles in urban plants, 17 occurred in a set of 19 exotic plants. Excluding the exotic plants, all five stands displayed a moderate differentiation (F(ST)= 0.14 +/- 0.02), although the urban remnants clustered with two of the bushland stands. These patterns may be explained by high levels of selfing and inbreeding in this species and by long-distance dispersal (several seeds in the urban stand were fathered by plants in other stands). Genetic leakage (gene flow) from exotic plants to 321 seeds on surrounding remnant or bushland plants has not occurred. Our results demonstrate the conservation value of this group of urban plants, which are viable, productive, genetically diverse, and interconnected with bushland plants. Gene flow has apparently not yet led to genetic contamination of bushland populations, but high levels of inbreeding would make this a rare event and difficult to detect.