Phylogeographic structure of brown trout Salmo trutta in Britain and Ireland: glacial refugia, postglacial colonization and origins of sympatric populations.

The phylogeographical structure of brown trout Salmo trutta in Britain and Ireland was studied using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of four mitochondrial DNA segments (16S/ND1, ND5/6, COXIII/ND5 and ND5/12S). Analysis of 3636 individuals from 83 sites-morphotypes revealed a total of 25 haplotypes. These haplotypes were nested in seven two-step clades. Although there was a clear geographical patterning to the occurrence of derived clades, admixture among ancestral clades was extensive throughout the studied area. A relevant feature of the data was that some populations contained mixtures of highly divergent clades. This type II phylogeographic pattern is uncommon in nature. Clade intermixing is likely to have taken place during earlier interglacials as well as since the Last Glacial Maximum. The anadromous life history of many S. trutta populations has probably also contributed to clade mixing. Based on the data presented here and published data, postglacial colonization of Britain and Ireland most likely involved S. trutta from at least five potential glacial refuges. Probable locations for such refugia were: south of England-western France, east of the Baltic Sea, western Ireland, Celtic Sea and North Sea. Ferox S. trutta, as defined by their longevity, late maturation and piscivory, exhibited a strong association with a particular clade indicating that they share a common ancestor. Current evidence indicates that the Lough Melvin gillaroo S. trutta and sonaghen S. trutta sympatric types diverged prior to colonization of Lough Melvin and, although limited gene flow has occurred since secondary contact, they have remained largely reproductively isolated due to inlet and outlet river spawning segregation. Gillaroo S. trutta may reflect descendents of a previously more widespread lineage that has declined due to habitat alterations particularly affecting outlet rivers. The mosaic-like distribution of mtDNA lineages means that conservation prioritization in Britain and Ireland should be based on the biological characteristics of local populations rather than solely on evolutionary lineages.

Mitochondrial DNA polymorphism: its role in longevity of the Irish population.

The mtDNA genome has been implicated as playing a pivotal role in determining the longevity and success of the human lifespan. A PCR-RFLP methodology was used to identify polymorphic restriction enzyme sites within a 2643 bp region of the mtDNA genome and a table of genetic haplotypes for a healthy aged and a younger control cohort of patients was constructed. Forty-six different mtDNA haplotypes and 11 groups of related haplotypes were identified across the two age groups but statistical analysis failed to show any significant associations. The European J haplogroup, previously reported to be associated with longevity, was not found at an increased frequency within the Irish aged population (P=0.36). However, the haplotypes comprising the J haplogroup could be differentiated into two distinct branches by the presence or absence of the two polymorphic restriction sites, 16,389g and 16,000g. The branch of haplotypes defined by 16,389g displayed a significant increased frequency in the aged samples (8%) compared to the controls (1%), P=0.015. Inversely, the branch of haplotypes defined by 16,000g displayed a significant decreased frequency in the aged samples (4%) compared to the controls (13%), P=0.011. The polymorphism (mt5178A) associated with longevity in the Japanese was not found in the Irish population, while the polymorphism (mt9055A) associated with successful ageing in the French centenarians was found at an increased frequency in the Irish aged population (9%) compared to the younger control group (5%), but failed to reach a level of statistical significance, P=0.164.

Multi-centre testing and validation of current protocols for the identification of Gyrodactylus salaris (Monogenea).

Despite routine screening requirements for the notifiable fish pathogen Gyrodactylus salaris, no standard operating procedure exists for its rapid identification and discrimination from other species of Gyrodactylus. This study assessed screening and identification efficiencies under real-world conditions for the most commonly employed identification methodologies: visual, morphometric and molecular analyses. Obtained data were used to design a best-practice processing and decision-making protocol allowing rapid specimen throughput and maximal classification accuracy. True specimen identities were established using a consensus from all three identification methods, coupled with the use of host and location information. The most experienced salmonid gyrodactylid expert correctly identified 95.1% of G. salaris specimens. Statistical methods of classification identified 66.7% of the G. salaris, demonstrating the need for much wider training. Molecular techniques (internal transcribed spacer region-restriction fragment length polymorphism (ITS-RFLP)/cytochrome c oxidase I (COI) sequencing) conducted in the diagnostic laboratory most experienced in the analysis of gyrodactylid material, identified 100% of the true G. salaris specimens. Taking into account causes of potential specimen loss, the probabilities of a specimen being accurately identified were 95%, 87% and 92% for visual, morphometric and molecular techniques, respectively, and the probabilities of correctly identifying a specimen of G. salaris by each method were 81%, 58% and 92%. Inter-analyst agreement for 189 gyrodactylids assessed by all three methods using Fleiss' Kappa suggested substantial agreement in identification between the methods. During routine surveillance periods when low numbers of specimens are analysed, we recommend that specimens be analysed using the ITS-RFLP approach followed by sequencing of specimens with a "G. salaris-like" (i.e. G. salaris, Gyrodactylus thymalli) banding pattern. During periods of suspected outbreaks, where a high volume of specimens is expected, we recommended that specimens be identified using visual identification, as the fastest processing method, to select "G. salaris-like" specimens, which are subsequently identified by molecular-based techniques.