L'article 2 de la tesi enviat a la revista Conservation Biology, finalment va ser publicat a la revistat Conservation Genetics 3:385:400, 2002 / L'estudi de la diversitat i la diferenciació genètiques de les poblacions de truita comuna (Salmo trutta L.) a la Península Ibèrica ha confirmat l'elevada diferenciació observada en treballs previs i la divergència, ja descrita, entre les poblacions de la vessant atlàntica i la mediterrània. El resultats obtinguts, però, ens permeten observar patrons d'estructura poblacional tant en les poblacions atlàntiques com les mediterrànies. A l'Atlàntic s'observa un marcat patró hidrogràfic en la distribució de la diferenciació genètica, que contrasta fortament amb la distribució d'aquesta diferenciació en les poblacions mediterrànies, caracteritzades pels contactes secundaris entre llinatges durant les expansions pleniglacials i una forta divergència local conseqüència de la seva marginalitat i aïllament en els períodes interglacials. El manteniment d'aquesta diferenciació i individualitat descrites en les poblacions de truita de la Península, es veu seriosament compromès per les contínues repoblacions dels rius amb exemplars exògens d'origen nord europeu. La substitució dels genomes autòctons per la introducció de gens al.lòctons provoca una erosió dels patrimonis genètics natius i una homogeneïtzació de les poblacions, destruint els patrons de diferenciació existents. Al mateix temps, els nostres resultats indiquen que les conseqüències de les repoblacions no són sempre les mateixes. Concretament, es constata un fracàs de les repoblacions en rius intensament repoblats i sotmesos a pesca intensiva, que contrasta amb una enorme erosió de les poblacions quan les repoblacions s'efectuen sobre àrees protegides i sense cap mena de pressió pesquera. Això suggereix que múltiples factors com la gestió dels rius posterior a les repoblacions, l'estat de les poblacions o les condicions de l'hàbitat són determinants de la introducció efectiva dels exemplars alliberats; fet que dificulta la predicció sobre actuacions particulars. Malgrat aquesta introgressió de gens exògens que es detecta en moltes de les poblacions analitzades, els gens natius predominen en gairebé tots els rius de la Península. La conservació d'aquesta elevada riquesa genètica que encara resta en les poblacions de truita de la Península Ibèrica ha de ser l'objectiu final de qualsevol programa de gestió. Per això, defensem una gestió basada en el propi riu mitjançant una pesca sostinguda per la reproducció natural de les poblacions salvatges, acompanyada d'una millora i recuperació d'hàbitats adequats per la truita, i evitant, per sobre de tot, la introducció en els rius d'exemplars exògens, degut als efectes nocius i incontrolables que comporta aquest procés. / Our study on the genetic diversity and differentiation of brown trout (Salmo trutta L.) population of the Iberian Peninsula confirmed the high levels of differentiation and the divergence between Atlantic and Mediterranean populations previously described. However, additional substructuring among Atlantic rivers and among Mediterranean ones has been detected. In the Atlantic, genetic diversity is related with the river network, producing a marked hydrographical pattern in sharp contrast with a mosaic distribution of genetic variation among the Mediterranean collections. In this later area, genetic resources distributed as a result of distinct colonization episodes during pleniglacial expansions, secondary contacts, and local divergence due to its marginality and isolation in the interglacial periods. Continuous stocking with hatchery-reared fish of north European origin, seriously compromises the conservation of this differentiation and individuality described in brown trout populations of the Iberian Peninsula. Replacement of the autochthonous genome due to introduction of exogenous genes is leading to an erosion of the native gene pools, a homogenisation of populations, and a destruction of the ancestral patterns of differentiation. At the same time, our results indicated that successful stocking appears to be limited to wild populations subjected to occasional releases in protected or unfished areas. Extensive stocking in fished areas result in a more limited impact on the recipient native population. These results suggest that stocking success is unpredictable and controlled by different factors including habitat and population conditions at the time of stocking, as well as post-release management actuations. Although many of the analysed populations are introgressed with exogenous genes, native gene pools predominate in almost all the Iberian rivers. Conservation of this high genetic richness that still remains in brown trout Iberian populations must be the final goal of any management program. So, our management proposal is based on fisheries sustained by natural reproduction of the respective wild populations, coupled with an improvement and recovery of habitat suitable for brown trout; avoiding stocking with exogenous fish due to the harmful and incontrollable risks that this process involves for the genetic resources of the species.

Freshwater sport fisheries contribute substantially to the economies of England and Wales. However, many trout fisheries rely partly or entirely on stocking farmed trout to maintain catches within freshwater fisheries. Farmed trout often differ genetically from their wild counterparts and wild trout could be at risk of reduced fitness due to interbreeding or competition with farmed fish. Therefore, to protect remaining wild brown trout (Salmo trutta L) populations and as a conservation measure, stocking policy has changed. Legislation introduced by the Environment Agency (EA, 2009) will now only give consent to stocking of rivers and some stillwaters with sterile, all-female triploid brown trout. There are reliable triploidy induction protocols for some other commercially important salmonid species however; there is limited knowledge on triploid induction in brown trout. Previously, triploid brown trout have been produced by heat shocks although reduced survivals were obtained suggesting that an optimised heat shock had not been identified, or that heat shock gives less consistent success than hydrostatic pressure shock (HP), which is now recognised as a more reliable technique to produce triploid fish. Thus the overall aim of this thesis was to conduct novel research to support the aquaculture and freshwater fisheries sector within the United Kingdom by optimising the production and furthering the knowledge of triploid brown trout. Firstly, this PhD project investigated an optimised triploidy induction protocol using hydrostatic pressure (Chapter 2). In order to produce an optimised hydrostatic pressure induction protocol three experiments were conducted to (1) determine the optimal timing of HP shock application post-fertilisation, (2) define optimal pressure intensity and duration of the HP shock and (3) study the effect of temperature (6-12 °C) on triploid yields. Results indicated high survival to yolk sac absorption stage (69.2 - 93.6 %) and high triploid yields (82.5 - 100 %) from the range of treatments applied. Furthermore, no significant differences in triploid rates were shown when shock timings and durations were adjusted according to the temperature used. In all treatments deformity prevalence remained low during incubation (<1.8 %) up to yolk sac absorption (~550 degree days post hatch). Overall, this study indicated that the optimised pressure shock for the induction of triploidy in brown trout delivering high survival and 100 % triploid rate (a prerequisite to brown trout restocking) is a shock with a magnitude of 689 Bar applied at 300 Centigrade Temperature Minutes (CTM) for 50 CTM duration. Regarding the assessment of triploid status, the second experimental chapter tested the accuracy and efficacy of three ploidy verification techniques (Chapter 3). Techniques studied were erythrocyte nuclei measurements (Image analysis), flow cytometry (Becton Dickinson Facscalibur flow cytometer) and DNA profiling (22 polymorphic microsatellite loci) to assess the effectiveness of triploidy induction in brown trout. Results indicated the validity of using erythrocyte indices major nuclear axis measurements, flow cytometric DNA distributions expressed as relative fluorescence (FL2-Area), and polymorphic microsatellite loci (Ssa410UOS, SSa197, Str2 and SsaD48) for assessing ploidy status in brown trout. Accuracy of each technique was assessed and indicated that all techniques correctly identified ploidy level indicating 100 % triploid rate for that commercial batch of brown trout. These techniques may be utilised within aquaculture and freshwater fisheries to ensure compliance with the legislation introduced by the EA. As a result of the legislation introduced by the Environment Agency triploid brown trout will freely interact with diploid trout therefore there is a need to assess feeding response and behavioural differences between diploid and triploid trout prior to release. Therefore, in the third experimental chapter (Chapter 4) diploid and triploid brown trout were acclimated for six weeks on two feeding regimes (floating/sinking pellet). Thereafter, aggression and surface feeding response was compared between pairs of all diploid, diploid and triploid and all triploid brown trout in a semi natural stream (flume). In each pairwise matching, fish of similar size were placed in allopatry and rank determined by the total number of aggressive interactions initiated. Dominant individuals initiated more aggression than subordinates, spent more time defending a territory and positioned themselves closer to the food source (Gammarus pulex) whereas subordinates occupied the peripheries. When ploidy was considered, diploid trout were more aggressive than triploid, and dominated their siblings when placed in pairwise matchings. However, surface feeding did not differ statistically between ploidy irrespective of feeding regime. Triploids adopted a sneak feeding strategy while diploids expended more time defending a territory. In addition, an assessment of whether triploids exhibited a similar social dominance to diploids when placed in allopatry was conducted. Although aggression was lower in triploid pairs than in the diploid/triploid pairs, a dominance hierarchy was observed between individuals of the same ploidy. Dominant triploid fish were more aggressive and consumed more feed items than subordinate individuals. Subordinate fish displayed a darker colour index than dominant fish suggesting increased stress levels. However, dominant triploid fish seemed more tolerant of subordinate individuals and did not display the same degree of invasive aggression as observed in the diploid/diploid or diploid/triploid matchings. These novel findings suggest that sterile triploid brown trout feed similarly but are less aggressive than diploid trout and therefore may provide freshwater fishery managers an alternative to stocking diploid brown trout. In addition to research at the applied level in triploid brown trout, this thesis also examined the fundamental physiological effects of ploidy in response to temperature regime. Triploid salmonids have been shown to differ in their tolerance to environmental temperature. Therefore the fourth experimental chapter (Chapter 5) investigated whether temperature tolerance affected feed intake and exercise recovery. Diploid and triploid brown trout were exposed to an incremental temperature challenge (10 and 19 °C) and subsequent survival and feed intake rates were monitored. Triploids took longer to acclimate to the increase in temperature however feed intake were significantly greater in triploids at high temperature. In a follow on study, we investigated post-exercise recovery processes under each temperature regime (10 and 19 °C). Exhaustion was induced by 10 minutes of forced swimming, with subsequent haematological responses measured to determine the magnitude of recovery from exercise. Plasma parameters (alkaline phosphatase, aspartate aminotransferase, calcium, cholesterol, triglycerides, phosphorous, total protein, lactate, glucose, pH, magnesium, osmolality, potassium, sodium, chloride, lactate dehydrogenase) were measured for each ploidy. Basal samples were taken prior to exercise and then at: 1; 4, and 24 hours post-exercise. Contrary to previous studies, there was no triploid mortality during or after the exercise at either temperature. Although diploid and triploid brown trout responded metabolically to the exercise, the magnitude of the response was affected by ploidy and temperature. In particular, triploids had higher levels of plasma lactate, osmolality, and lower pH than diploids at 1 hour post exhaustive exercise. By 4 hours post-exercise plasma parameters analysed had returned to near basal levels. It was evident that the magnitude of the physiological disturbance post-exercise was greater in triploids than diploids at 19 °C. This may have implications where catch and release is practiced on freshwater fisheries. Overall, this work aimed to develop and/or refine current industry induction and assessment protocols while better understand the behaviour and physiology of diploid and triploid brown trout. The knowledge gained from this work provides aquaculture and freshwater fisheries with an optimised protocol, which delivers 100 % triploid rates and profitability without compromising farmed trout welfare, thus ultimately leading towards a more sustainable brown trout industry within the United Kingdom.