Characterisation and cross-amplification of 42 microsatellite markers in two Amphiprion species (Pomacentridae) and a natural hybrid anemonefish to inform genetic structure within a hybrid zone.

The hybrid anemonefish, Amphiprion leucokranos, is known to be the product of ongoing, introgressive hybridization between parent taxa Amphiprion sandaracinos and Amphiprion chrysopterus. Hybridization is an important evolutionary phenomenon contributing to biodiversity within marine systems, where hybrid zones provide ideal systems in which to study hybridization events. Here, a suite of 42 Amphiprion microsatellite markers (including development of 8 novel markers) were cross-amplified in individuals from parent taxa and hybrid populations to facilitate investigation into the relatedness of hybridizing species across the A. leucokranos hybrid zone. Analysis revealed 15, 20 and 24 highly polymorphic loci (PIC > 0.5) in the two parent species and hybrid, respectively, for use in population genetic and parentage studies, with 305 unique alleles found overall (ranging from 1 to 13 alleles per locus) and 7 alleles per locus on average. Observed and expected heterozygosities ranged from 0.000 to 1.000 and 0.000 to 0.978, respectively. Significant deviations from Hardy-Weinberg equilibrium were found in eight loci, possibly due to relatedness among samples or the presence of null alleles. Use of the suite of markers tested here will provide valuable insights into the contemporary population structure and introgression among species and hybrids within the Amphiprion leucokranos hybrid zone, as well as inform future ecological and evolutionary studies of anemonefishes more broadly.

Hierarchical behaviour, habitat use and species size differences shape evolutionary outcomes of hybridization in a coral reef fish.

Hybridization is an important evolutionary process, with ecological and behavioural factors influencing gene exchange between hybrids and parent species. Patterns of hybridization in anemonefishes may result from living in highly specialized habitats and breeding status regulated by size-based hierarchal social groups. Here, morphological, ecological and genetic analyses in Kimbe Bay, Papua New Guinea, examine the hybrid status of Amphiprion leucokranos, a nominal species and presumed hybrid between Amphiprion sandaracinos and Amphiprion chrysopterus. We test the hypothesis that habitat use and relative size differences of the parent species and hybrids determine the patterns of gene exchange. There is strong evidence that A. leucokranos is a hybrid of smaller A. sandaracinos and larger A. chrysopterus, where A. chrysopterus is exclusively the mother to each hybrid, based on mtDNA cytochrome b and multiple nDNA microsatellite loci. Overlap in habitat, depth and host anemone use was found, with hybrids intermediate to parents and cohabitation in over 25% of anemones sampled. Hybrids, intermediate in body size, colour and pattern, were classified 55% of the time as morphologically first-generation hybrids relative to parents, whereas 45% of hybrids were more A. sandaracinos-like, suggesting backcrossing. Unidirectional introgression of A. chrysopterus mtDNA into A. sandaracinos via hybrid backcrosses was found, with larger female hybrids and small male A. sandaracinos mating. Potential nDNA introgression was also evident through distinct intermediate hybrid genotypes penetrating both parent species. Findings support the hypothesis that anemonefish hierarchical behaviour, habitat use and species-specific size differences determine how hybrids form and the evolutionary consequences of hybridization.

New Zealand's breath and blood alcohol testing programs: further data analysis and forensic implications.

Paired blood and breath alcohol concentrations (BAC, in g/dL, and BrAC, in g/210 L), were determined for 11,837 drivers apprehended by the New Zealand Police. For each driver, duplicate BAC measurements were made using headspace gas chromatography and duplicate BrAC measurements were made with either Intoxilyzer 5000, Seres 679T or Seres 679ENZ Ethylometre infrared analysers. The variability of differences between duplicate results is described in detail, as well as the variability of differences between the paired BrAC and BAC results. The mean delay between breath and blood sampling was 0.73 h, ranging from 0.17 to 3.1 8h. BAC values at the time of breath testing were estimated by adjusting BAC results using an assumed blood alcohol clearance rate. The paired BrAC and time-adjusted BAC results were analysed with the aim of estimating the proportion of false-positive BrAC results, using the time-adjusted BAC results as references. When BAC results were not time-adjusted, the false-positive rate (BrAC>BAC) was 31.3% but after time-adjustment using 0.019 g/dL/h as the blood alcohol clearance rate, the false-positive rate was only 2.8%. However, harmful false-positives (defined as cases where BrAC>0.1 g/210L, while BAC< or =0.1g/dL) occurred at a rate of only 0.14%. When the lower of duplicate breath test results were used as the evidential results instead of the means, the harmful false-positive rate dropped to 0.04%.

Conclusive breath alcohol testing in New Zealand: results obtained using Intoxilyzer 5000 devices.

Intoxilyzer 5000 Conclusive Breath Testing Devices have been in wide operational use in New Zealand since June 1989. From the monitoring of these devices, large number of duplicate subject test results and blood alcohol/breath alcohol pairs have been obtained. Information on the agreement between duplicate subject breath samples, the blood/breath correlation, and the distribution of breath alcohol levels encountered at each testing location will be presented. Each device has been closely monitored in the field with respect to its calibration status and stability; the devices have proved to be very satisfactory in the field.