Periodic Reporting for period 1 - ADAPTATION (Predicting adaptive responses of protected species to environmental changes to optimise conservation management frameworks in Europe)
Période du rapport: 2016-11-14 au 2018-11-13
Sea lampreys are native to Europe where their populations are endangered. This project has been designed to specifically gather the necessary information (genetic and environmental) in order to inform conservation practices for the species. The genetic information will contribute to understanding the population connectivity of this endangered species across freshwater sites and in the even of needing to genetically enhance populations this work will also provide a map on which genetic information can be used to decided where to and from individuals could be moved. There were therefore two major objectives inherent in the project, reflecting the need to fill knowledge gap regarding the evolutionary mode and life history of this endangered species. The first was to identify reproductive units, or populations, of sea lampreys across European freshwater systems. The second, was to understand how the species utilizes the freshwater habitats. The accomplishment of both objectives will facilitate the monitoring of sea lamprey abundance both at European and at a river-by-river basis. We also aimed at developing a non-invasive method to detect the presence of sea lamprey juveniles in the river sediment. This task was complementary to habitat characterization activities, and was accomplished by developing an assay to detect molecules of sea lamprey DNA in environmental samples (eDNA).
Population genomics:
Sampling strategy was designed to screen a geographic scale as wide as possible within sea lamprey distribution range in Europe. This task was accomplished with several collaborations: Marine and Environmental Sciences Centre (MARE) from Lisbon, Portugal; Natural England (England); Environmental Agency (United Kingdom). A total of 192 specimens, from 8 river basins, were collected for DNA extraction.
Reads were aligned against the P. marinus available genome. All bioinformatic processing was performed at Bournemouth’s University High Processing Computing cluster that allowed parallel computations. Genome-wide diversity indices were computed. To understand population structure we performed AMOVAs; applied Bayesian clustering; performed discriminant analysis of principal components (DAPC). In order to explore the hypothesis that host populations play a role in the way that sea lamprey populations are organized, we compiled a list of potential suitable hosts: Atlantic mackerel, Atlantic cod, Haddock, hake, Atlantic salmon, shad and mullet (thicklip and grey). We retrieved information on the presence and abundance of each from FishStat J (FAO). We collected abiotic variables such as temperature, salinity, dissolved oxygen and chlorophyll a concentration for GPS coordinates corresponding to collection sites and performed environmental correlations.
Identification of suitable habitats, species validation and environmental DNA assay:
Larvae identification was done via: a) In situ and microscopic screen for pigmentation patterns and b) Molecular identification - barcoding via amplification with species specific of discriminant region of the mitochondrial DNA. Phylogenetic trees were built to investigate the clustering of individuals in each lamprey species.
Design of environmental DNA assay: species-specific primers and probe were designed on the mitochondrial DNA with in silico cross species amplification. Synthesis was outsourced to Applied Biosystems to produce a Taqman MGB probe labelled with the fluorescent dye FAM at the 5’-end and with a non-fluorescent quencher MGBNFQ at the 3’-end. Primer testing and validation was done in a) extracted sea lamprey DNA b) against off-target river species c) screening on environmental samples
Preliminary results and discussion
Diversity estimates revealed an overall similarity in terms of genome-wide diversity. The exception is that of the Severn population, which was revealed to be a less genetically diverse population also with a high level of inbreeding. Still regarding inbreeding coefficient, the Frome was shown to have the lowest value. The fact that all specimens from the Frome were all 1-year-old max. ammocoetes (larvae) suggests that either a) the spawners cohort that originate them was genetically diverse b) selection throughout life history reduces genome-wide diversity, in the sense that collections of individuals that migrate upstream to spawn will always be less genetically diverse than the offspring produced from their crosses.
Population structure analyses suggested a high connectivity among sea lamprey spawning areas at the centre of the distribution, and a certain degree of differentiation (significant) between the edges.
These preliminary results are in line with what was reported for the pacific lamprey, where limited dispersal capacity, inferred from geographic distances among sites, was suggested to responsible for any degree of reproductive isolation.
We found quite distinguishable pigmentation patterns in the tail of captured juveniles, whose differentiation proven to correspond to two different species with genetic markers. The amplification of target DNA region was successful both in extracted DNA from sea lamprey tissue and from sediment collected on the sites on which sea lamprey were caught. Samples where no sea lamprey were found resulted in negative PCR reactions, as well as those samples were DNA of off-target species was examined. Overall, the eDNA assay proved to be effective in detecting DNA concentrations as low as 10-5ng/ul in densities of 1larvae/3L of sediment.
Sampling strategy was designed to screen a geographic scale as wide as possible within sea lamprey distribution range in Europe. This task was accomplished with several collaborations: Marine and Environmental Sciences Centre (MARE) from Lisbon, Portugal; Natural England (England); Environmental Agency (United Kingdom). A total of 192 specimens, from 8 river basins, were collected for DNA extraction.
Reads were aligned against the P. marinus available genome. All bioinformatic processing was performed at Bournemouth’s University High Processing Computing cluster that allowed parallel computations. Genome-wide diversity indices were computed. To understand population structure we performed AMOVAs; applied Bayesian clustering; performed discriminant analysis of principal components (DAPC). In order to explore the hypothesis that host populations play a role in the way that sea lamprey populations are organized, we compiled a list of potential suitable hosts: Atlantic mackerel, Atlantic cod, Haddock, hake, Atlantic salmon, shad and mullet (thicklip and grey). We retrieved information on the presence and abundance of each from FishStat J (FAO). We collected abiotic variables such as temperature, salinity, dissolved oxygen and chlorophyll a concentration for GPS coordinates corresponding to collection sites and performed environmental correlations.
Identification of suitable habitats, species validation and environmental DNA assay:
Larvae identification was done via: a) In situ and microscopic screen for pigmentation patterns and b) Molecular identification - barcoding via amplification with species specific of discriminant region of the mitochondrial DNA. Phylogenetic trees were built to investigate the clustering of individuals in each lamprey species.
Design of environmental DNA assay: species-specific primers and probe were designed on the mitochondrial DNA with in silico cross species amplification. Synthesis was outsourced to Applied Biosystems to produce a Taqman MGB probe labelled with the fluorescent dye FAM at the 5’-end and with a non-fluorescent quencher MGBNFQ at the 3’-end. Primer testing and validation was done in a) extracted sea lamprey DNA b) against off-target river species c) screening on environmental samples
Preliminary results and discussion
Diversity estimates revealed an overall similarity in terms of genome-wide diversity. The exception is that of the Severn population, which was revealed to be a less genetically diverse population also with a high level of inbreeding. Still regarding inbreeding coefficient, the Frome was shown to have the lowest value. The fact that all specimens from the Frome were all 1-year-old max. ammocoetes (larvae) suggests that either a) the spawners cohort that originate them was genetically diverse b) selection throughout life history reduces genome-wide diversity, in the sense that collections of individuals that migrate upstream to spawn will always be less genetically diverse than the offspring produced from their crosses.
Population structure analyses suggested a high connectivity among sea lamprey spawning areas at the centre of the distribution, and a certain degree of differentiation (significant) between the edges.
These preliminary results are in line with what was reported for the pacific lamprey, where limited dispersal capacity, inferred from geographic distances among sites, was suggested to responsible for any degree of reproductive isolation.
We found quite distinguishable pigmentation patterns in the tail of captured juveniles, whose differentiation proven to correspond to two different species with genetic markers. The amplification of target DNA region was successful both in extracted DNA from sea lamprey tissue and from sediment collected on the sites on which sea lamprey were caught. Samples where no sea lamprey were found resulted in negative PCR reactions, as well as those samples were DNA of off-target species was examined. Overall, the eDNA assay proved to be effective in detecting DNA concentrations as low as 10-5ng/ul in densities of 1larvae/3L of sediment.
Progresses beyond the state of the art includes the sequencing technology and evolutionary information applied to the endangered species. This information contributes, in a first phase, to understand the population connectivity of this endangered species across freshwater sites. It provides a monitoring framework on which authorities, at national and European level, will be able to utilize as a non-invasive activity to follow sea lamprey abundances. Ultimately, in the event of stocking be deemed necessary to protect the species abundance in critically depleted sites, our work provides a map on which genetic information can be used to decided where to and from individuals could be moved. Expected results contribute to a better understanding of sea lamprey life cycle, habitat utilization and connectivity among freshwater sites and eventually the role of abiotic variables and fish hosts might have on the dispersal of sea lamprey individuals.
Because natural populations of sea lamprey were never investigated from an evolutionary perspective with this detail, this project provides a significant improvement to the ecological-only existing knowledge.
Impacts will be determined upon project termination and publication of the results. It is possible that directives for freshwater management and, specifically, sea lamprey monitoring at least in the United Kingdom, to be improved based on the knowledge here given.
From a socio-economic perspective, this project will have a large impact at southern european latitudes. Indeed, sea lamprey in UK is mostly fished for recreation,and not necessarily part of tradition or culture. Therefore, despite that the tools to preserve UK freshwater biodiversity will be greatly augmented with the outcomes of the project, most socio-economic impacts will occur in countries like Portugal (with whom we collaborate) Spain or France that actually target the species for fishing.
Because natural populations of sea lamprey were never investigated from an evolutionary perspective with this detail, this project provides a significant improvement to the ecological-only existing knowledge.
Impacts will be determined upon project termination and publication of the results. It is possible that directives for freshwater management and, specifically, sea lamprey monitoring at least in the United Kingdom, to be improved based on the knowledge here given.
From a socio-economic perspective, this project will have a large impact at southern european latitudes. Indeed, sea lamprey in UK is mostly fished for recreation,and not necessarily part of tradition or culture. Therefore, despite that the tools to preserve UK freshwater biodiversity will be greatly augmented with the outcomes of the project, most socio-economic impacts will occur in countries like Portugal (with whom we collaborate) Spain or France that actually target the species for fishing.