Integrated genetic and morphological data support eco-evolutionary divergence of Angolan and South African populations of Diplodus hottentotus.

The genus Diplodus presents multiple cases of taxonomic conjecture. Among these the D. cervinus complex was previously described as comprising three subspecies that are now regarded as separate species: Diplodus cervinus, Diplodus hottentotus and Diplodus omanensis. Diplodus hottentotus exhibits a clear break in its distribution around the Benguela Current system, prompting speculation that Angolan and South African populations flanking this area may be isolated and warrant formal taxonomic distinction. This study reports the first integrated genetic [mitochondrial (mt)DNA and nuclear microsatellite] and morphological (morphometric, meristic and colouration) study to assess patterns of divergence between populations in the two regions. High levels of cytonuclear divergence between the populations support a prolonged period of genetic isolation, with the sharing of only one mtDNA haplotype (12 haplotypes were fully sorted between regions) attributed to retention of ancestral polymorphism. Fish from the two regions were significantly differentiated at a number of morphometric (69·5%) and meristic (46%) characters. In addition, Angolan and South African fish exhibited reciprocally diagnostic colouration patterns that were more similar to Mediterranean and Indian Ocean congeners, respectively. Based on the congruent genetic and phenotypic diversity we suggest that the use of hottentotus, whether for full species or subspecies status, should be restricted to South African D. cervinus to reflect their status as a distinct species-like unit, while the relationship between Angolan and Atlantic-Mediterranean D. cervinus will require further demo-genetic analysis. This study highlights the utility of integrated genetic and morphological approaches to assess taxonomic diversity within the biogeographically dynamic Benguela Current region.

Isolation by distance and non-identical patterns of gene flow within two river populations of the freshwater fish Rutilus rutilus (L. 1758).

The spatial distribution of organisms is maintained by a combination of in situ reproduction and dispersal of conspecifics from elsewhere within its habitable range. The determination of dispersal origin and sub-population connectivity has a vital role to play in forming effective management policies. The common roach (Rutilus rutilus) is an important component of the economically and socially valuable recreational fishery and represents a well-studied member of the Cyprinidae. Microsatellite allele data were used to investigate hypothetically variant levels of microevolutionary structuring and isolation-by-distance (IBD) in in the Rivers Stour and Thames. A strong signal of IBD was found in the Stour, probably due to the limited capacity for unrestricted bidirectional dispersal in this river compared with the Thames. A weak inference of IBD in the Thames is likely erroneous and effected by a strong localised genetic signal from a recent stocking event. Whilst we found significantly genetically divergent upstream areas in the River Stour, a strong signal of IBD remained when the headwater sub-population was removed, suggesting that that the signal is not biased by non-equilibrium conditions in upstream reaches. We discuss these results with reference to the management of aquatic bioresources and emphasise the idiosyncrasy that aquatic biota and hydrological complexity may imprint upon patterns of biodiversity within any given system.

Historical and contemporary population genetic connectivity of the European short-snouted seahorse Hippocampus hippocampus and implications for management.

This first genetic study of Hippocampus hippocampus covers the species' entire geographic range and employs two mtDNA markers (control region and cytochrome b) to establish patterns of population structuring. A total of 255 specimens from 21 locations were used to obtain 89 concatenated haplotypes. The common haplotype was present in all but one population, however, most haplotypes were unique. The haplotype network had a star-like construction, suggesting expansion from a bottleneck event. F(ST) and AMOVA revealed population subdivision into three geographic regions (English Channel + Bay of Biscay, Mediterranean Sea + Atlantic Ocean Iberian coast + Macaronesian Islands, and West Africa) with barriers to gene flow indentified at Cape Finisterre and the Cape Verde frontal zone. Neutrality tests and nested clade analysis suggest a complex demographic history, with both historic events and contemporary processes shaping patterns of genetic differentiation. The genetic population subdivision detected in this study indicates that H. hippocampus should be managed as three separate units. This is especially pertinent as H. hippocampus populations within the West African region are the only ones known to be specifically targeted for exploitation.

First occurrence of the lined seahorse Hippocampus erectus in the eastern Atlantic Ocean.

A seahorse specimen from Banco Açores (Azores Archipelago) was identified using morphological and molecular genetic data as Hippocampus erectus. This specimen represents the first record of H. erectus in the eastern Atlantic Ocean, well outside its reported range, and may provide evidence of long-distance translocation in what are assumed to be relatively sedentary fish.

Genetic structuring of Patagonian toothfish populations in the Southwest Atlantic Ocean: the effect of the Antarctic Polar Front and deep-water troughs as barriers to genetic exchange.

The environmental and/or life history factors affecting genetic exchange in marine species with potential for high dispersal are of great interest, not only from an evolutionary standpoint but also with regard to effective management. Previous genetic studies have demonstrated substantial differentiation among populations of the Patagonian toothfish around the Southern Ocean, indicating breakdown of gene flow across large distances between inhabited shelf areas. The present study examined genetic structuring through analysis of microsatellite loci and restriction fragment length polymorphism (RFLP) of the mitochondrial ND2 gene and control region of the toothfish population in the SW Atlantic, allowing examination of the relative effects of the Antarctic Polar Front (APF), deep-water troughs and distance between sites. Mitochondrial DNA (mtDNA) data indicated a sharp genetic division between the Patagonian Shelf/North Scotia Ridge and the Shag Rocks/South Georgia samples, whereas microsatellite data showed much less distinct structuring and an intermediate position of the North Scotia Ridge samples. We suggest these data indicate that the APF, as a barrier to larval dispersal, is the major inhibitor of genetic exchange between toothfish populations, with deep-water troughs and distance between sites contributing to genetic differentiation by inhibiting migration of relatively sedentary adults. We also suggest that differences between mtDNA and nuclear DNA population patterns may reflect either genome population size effects or (putative) male-biased dispersal.

Inbreeding depression and genetic load of sexually selected traits: how the guppy lost its spots.

To date, few studies have investigated the effects of inbreeding on sexually selected traits, although inbreeding depression on such traits can play an important role in the evolution and ecology of wild populations. Sexually selected traits such as ornamentation and courtship behaviour may not be primary fitness characters, but selection and dominance coefficients of their mutations will resemble those of traits under natural selection. Strong directional selection, for instance, through female mate-choice, purges all but the most recessive deleterious mutations, and the remaining dominance variation will result in inbreeding depression once populations undergo bottlenecks. We analysed the effects of inbreeding on sexually selected traits (colour pattern and courtship behaviour) in the male guppy, Poecilia reticulata, from Trinidad, and found a significant decline in the frequency of mating behaviour and colour spots. Such effects occurred although the genetic basis of these traits, many of which are Y-linked and hemizygous, would be expected to leave relatively little scope for inbreeding depression. Findings suggest that these sexually selected traits could reflect the genetic condition or health of males, and thus may be informative mate-cue characters for female choice as suggested by the 'good genes' model.

Extensive population subdivision of the cuttlefish Sepia officinalis (Mollusca: Cephalopoda) around the Iberian Peninsula indicated by microsatellite DNA variation.

The Atlantic Ocean-Mediterranean Sea junction has been proposed as an important phylogeographical area on the basis of concordance in genetic patterns observed at allozyme, mtDNA and microsatellite DNA markers in several marine species. This study presents microsatellite DNA data for a mobile invertebrate species in this area, the cuttlefish Sepia officinalis, allowing comparison of this relatively new class of DNA marker with previous allozyme results, and examination of the relative effects on gene flow of the Strait of Gibraltar and the Almería-Oran oceanographic front. Genetic variation at seven microsatellite loci screened in six samples from NE Atlantic and Mediterranean coasts of the Iberian Peninsula was high (mean Na = 9.6, mean H(e) = 0.725). Microsatellites detected highly significant subpopulation structuring (F(ST)= 0.061; R(ST) = 0.104), consistent with an isolation-by-distance model of low levels of gene flow. Distinct and significant clinal changes in allele frequencies between Atlantic and Mediterranean samples found at five out of seven loci, however indicate these results might be also consistent with an alternative model of secondary contact and introgression between previously isolated and divergent populations, as previously proposed for other marine species from the Atlantic-Mediterranean area. A pronounced 'step' change between SW Mediterranean samples associated with the Almería-Oran front suggests this oceanographic feature may represent a contemporary barrier to gene flow.

Genetic population structure indicates sympatric speciation of Lake Malawi pelagic cichlids.

Allopatric processes of speciation have routinely been presented to explain the extraordinary radiation of the East African Great Lakes cichlid fish species flocks. The 21 or more species of pelagic cichlids within the Lake Malawi flock appear to have lake-wide distributions that challenge such a concept. Data from six microsatellite DNA loci indicate single, panmictic populations across the lake of three Diplotaxodon species. Levels of variability at these loci suggest that populations have been large and stable. Mitochondrial DNA sequence data (872 bp of control region + 981 bp of the NADH-2) from 90 species, representing all major clades within the Lake Malawi flock, indicate reciprocal monophyly of the pelagic clade. We suggest that these data support a hypothesis that speciation in sympatry is more plausible (and widespread) within the cichlid species flocks than previously thought.

Microsatellite DNA analysis of population structure in Atlantic herring (Clupea harengus), with direct comparison to allozyme and mtDNA RFLP analyses.

Previous attempts to test for small-scale stock structuring within Atlantic herring (Clupea harengus L.) with molecular markers have been hampered by uninformative levels of genetic variation. Here we report the first application of microsatellite DNA markers to investigate population subdivision in Atlantic herring from Norwegian waters and the Barents Sea, and also examine microsatellite differentiation between C. harengus and Pacific herring (C. pallasi). Results from four microsatellite loci indicate high, and informative, variation compared to molecular markers used previously: number of alleles per locus=18-41; mean expected heterozygosity within samples=0.90-0.93. Significant genetic differences were detected between almost all samples representing postulated Icelandic summer-spawner, Norwegian spring-spawner and Norwegian fjord stocks, using Fisher's exact test, FST and RST values. Levels of allele frequency differentiation between Atlantic and Pacific herring overlapped the range seen among Atlantic herring samples, indicating that microsatellites are poor indicators of the degree of species differentiation. Comparison with allozyme and mitochondrial DNA restriction fragment length polymorphism (RFLP) datasets from the same samples suggests that microsatellites may detect structuring at a finer scale, but are less informative at larger scales of divergence.

United States v. Levin: entrapment by estoppel doctrine applied in Medicare reimbursement context.

The decision in Levin reaffirms that health care providers should be entitled to rely in good faith on official interpretations and representations by authorized government officials as to the propriety of their conduct under the Medicare program. Although the doctrine of entrapment by estoppel was developed as a due process defense to a criminal prosecution, the fundamental notions of fairness underlying the doctrine should be applicable as well as in a civil action. Thus, the defense of entrapment by estoppel should be available, for example, in the context of civil health care reimbursement recoupment audits, where a provider has acted in reliance on the interpretation of a statute or regulation by the appropriate administrative agency charged with its enforcement.

Vitrification of mouse oocytes using short cryoprotectant exposure: effects of varying exposure times on survival.

The effects on oocyte viability of varying the duration of exposure to cryoprotectants before rapid cooling to -196 degrees C were examined, using the vitrification protocol of Nakagata. A very short exposure (15 sec) was found to be optimal, resulting in an overall rate of development from vitrified oocytes to hatching blastocysts of 31.8%. Very high rates of survival (77-89%) of oocytes exposed to the cryoprotectant media, but without the vitrification, together with extreme variability in results between straws in the vitrified groups, suggest that losses in viability during vitrification may result from ice damage during devitrification of the medium.

Impact of a pharmacist on the educational value of pharmaceutical industry film showings.

A controlled trial was performed to assess the impact of drug information provided by a pharmacist on the educational value to physicians of pharmaceutical manufacturers' film showings. The trial consisted of two teams of physicians who attended pharmaceutical manufacturers' films and who afterward answered multiple choice questions on the drug being promoted. In one group, the liaison pharmacist, who had no knowledge of the content of the questionnaire, presented information on the drug being featured prior to the film showing while the control group did not have a pharmacist presentation. Out of a perfect score of five, there was a higher test score in the group of physicians who attended the pharmacist presentation/film showing (n = 75) than in the group which only attended the film (n = 65) (3.3 +/- 1.1 versus 2.8 +/- 1.2, respectively (p = 0.017)). While there was no difference in the scores obtained by the clerks, interns and residents (3.2 +/- 1.1, 3.3 +/- 0.9, 3.4 +/- 1.2 respectively) when a pharmacist was present, in his or her absence the scores for clerks, interns and residents were 2.5 +/- 1.3, 2.8 +/- 1.0, 3.6 +/- 1.2 respectively with residents scoring higher than clerks (p = 0.047). A pharmacist can enhance the educational value of a pharmaceutical manufacturer's film showing.

Vitrification of mouse oocytes: improved rates of survival, fertilization, and development to blastocysts.

Rall and Fahy's (1985) vitrification procedure for the cryopreservation of 8-cell embryos was applied to unfertilized mouse oocytes. Unchanged, this method resulted in a mean of 24.1% of vitrified oocytes fertilizing and developing to blastocysts in vitro. Exposure of oocytes to the cryoprotectant media, but without the vitrification, resulted in 30.8% developing to blastocysts. Modifications to the durations of and media used in the dilution and equilibration steps of the procedure produced a final protocol giving a mean of 55.4% of vitrified oocytes and 72.4% of nonvitrified VS1-exposed oocytes developing to blastocysts; 85.7% of control oocytes develop to blastocysts. Osmotically induced damage was found to be the most important cause of loss of viability in these methods. Cooling of oocytes to 5-8 degrees C during the procedure had no significant effect on their viability. No parthenogenetic activation of oocytes occurred as a result of exposure to the procedure.

Use of statistical sampling and extrapolation in overpayment audits of physicians.

The successful defense of claims for physician overpayments by insurers requires the health care attorney to be familiar with the statistical sampling procedures employed during these types of audits. In this article, the author describes when audits are initiated, the factors that are taken into account in determining if statistical sampling is appropriate, how the statistical sampling study is conducted, and what defenses the health care attorney can assert when confronted with the results of a statistical sampling audit.