Restriction Site-associated Sequencing Research Articles

Effects of inbreeding on reproductive success in endangered North Atlantic right whales.

Only approximately 356 North Atlantic right whales (Eubalaena glacialis) remain. With extremely low levels of genetic diversity, limited options for mates, and variation in reproductive success across females, there is concern regarding the potential for genetic limitations of population growth from inbreeding depression. In this study, we quantified reproductive success of female North Atlantic right whales with a modified de-lifing approach using reproductive history information collected over decades of field observations. We used double-digest restriction site-associated sequencing to sequence approximately 2% of the genome of 105 female North Atlantic right whales and combined genomic inbreeding estimates with individual fecundity values to assess evidence of inbreeding depression. Inbreeding depression could not explain the variance in reproductive success of females, however we present evidence that inbreeding depression may be affecting the viability of inbred fetuses-potentially lowering the reproductive success of the species as a whole. Combined, these results allay some concerns that genetic factors are impacting species survival as genetic diversity is being retained through selection against inbred fetuses. While still far fewer calves are being born each year than expected, the small role of genetics underlying variance in female fecundity suggests that variance may be explained by external factors that can potentially be mitigated through protection measures designed to reduce serious injury and mortality from human activities.

Genetic Diversity and Classification of Colletotrichum sublineola Pathotypes Using a Standard Set of Sorghum Differentials.

Anthracnose, incited by Colletotrichum sublineola, is the most destructive foliar disease of sorghum and, under severe conditions, yield losses can exceed 80% on susceptible cultivars. The hyper-variable nature of the pathogen makes its management challenging despite the occurrence of several resistant sources. In this study, the genetic variability and pathogenicity of 140 isolates of C. sublineola, which were sequenced using restriction site-associated sequencing (RAD-Seq), resulted in 1244 quality SNPs. The genetic relationship based on the SNP data showed low to high genetic diversity based on isolates' origin. Isolates from Georgia and North Carolina were grouped into multiple clusters with some level of genetic relationships to each other. Even though some isolates from Texas formed a cluster, others clustered with isolates from Puerto Rico. The isolates from Puerto Rico showed scattered distribution, indicating the diverse nature of these isolates. A population structure and cluster analysis revealed that the genetic variation was stratified into eight populations and one admixture group. The virulence pattern of 30 sequenced isolates on 18 sorghum differential lines revealed 27 new pathotypes. SC748-5, SC112-14, and Brandes were resistant to all the tested isolates, while BTx623 was susceptible to all. Line TAM428 was susceptible to all the pathotypes, except for pathotype 26. Future use of the 18 differentials employed in this study, which contains cultivars/lines which have been used in the Americas, Asia, and Africa, could allow for better characterization of C. sublineola pathotypes at a global level, thus accelerating the development of sorghum lines with stable resistance to the anthracnose pathogen.

Species-specific effects of production practices on genetic diversity in plant reintroduction programs.

Plant production practices can influence the genetic diversity of cultivated plant materials and, ultimately, their potential to adapt to a reintroduction site. A common step in the plant production process is the application of seed pretreatment to alleviate physiological seed dormancy and successfully germinate seeds. In production settings, the seeds that germinate more rapidly may be favored in order to fill plant quotas. In this study, we investigated how the application of cold-moist stratification treatments with different durations can lead to differences in the genetic diversity of the propagated plant materials. Specifically, we exposed seeds of three Viola species to two different cold stratification durations, and then we analyzed the genetic diversity of the resulting subpopulations through double-digestion restriction site-associated sequencing (ddRADseq). Our results show that, in two out of three species, utilizing a short stratification period will decrease the genetic diversity of neutral and expressed loci, likely due to the imposition of a genetic bottleneck and artificial selection. We conclude that, in some species, the use of minimal stratification practices in production may jeopardize the adaptive potential and long-term persistence of reintroduced populations and suggest that practitioners carefully consider the evolutionary implications of their production protocols. We highlight the need to consider the germination ecology of target species when selecting the length of dormancy-breaking pretreatments.

Breeding origins of a uniquely regular migrant songbird in the Galápagos Islands.

Little is known about the causes and consequences of alternative pathways flown by long-distance migratory birds. Bobolinks (Dolichonyx oryzivorus) breed in grasslands across northern North America and migrate from their breeding grounds toward the eastern Atlantic Coast and then proceed through the Caribbean to South America. However, a small but regular number of Bobolinks have been recorded on the Galapagos Islands. We collected genetic samples from nine Galapagos Bobolinks and performed double-digest restriction site-associated sequencing. We compared them with samples from seven locations across their breeding distribution to determine their population of origin. Galapagos Bobolinks shared the genetic structure of a cluster in the eastern portion of the breeding range that includes New Brunswick and Ontario, Canada, and Vermont, United States. Genetic assignment tests largely corroborated this finding, although slightly different results were obtained for the two methods. All individuals were assigned to the Ontario breeding population using AssignPop, while Rubias assigned six of the migrants to Ontario and three to a Midwest breeding population. Low average relatedness among Galapagos individuals indicates that they are not more related to one another than to individuals within a breeding population and are therefore likely not from a single, small isolated population. Our results do not support the probability hypothesis-that Galapagos Bobolinks originated from the region that includes the greatest proportion of their breeding range (Great Plains)-or the vagrant hypothesis-that migrants are displaced onto Galapagos due to weather events. Instead, our findings support the proximity hypothesis, where migrants originate from the geographically closest-breeding populations.

Mapping the root systems of individual trees in a natural community using genotyping-by-sequencing.

The architecture of root systems is an important driver of plant fitness, competition and ecosystem processes. However, the methodological difficulty of mapping roots hampers the study of these processes. Existing approaches to match individual plants to belowground samples are low throughput and species specific. Here, we developed a scalable sequencing-based method to map the root systems of individual trees across multiple species. We successfully applied it to a tropical dry forest community in the Brazilian Caatinga containing 14 species. We sequenced all 42 individual shrubs and trees in a 14 × 14 m plot using double-digest restriction site-associated sequencing (ddRADseq). We identified species-specific markers and individual-specific haplotypes from the data. We matched these markers to the ddRADseq data from 100 mixed root samples from across the centre (10 × 10 m) of the plot at four different depths using a newly developed R package. We identified individual root samples for all species and all but one individual. There was a strong significant correlation between belowground and aboveground size measurements, and we also detected significant species-level root-depth preference for two species. The method is more scalable and less labour intensive than the current techniques and is broadly applicable to ecology, forestry and agricultural biology.

Seasonal variation of genotypes and reproductive plasticity in a facultative clonal freshwater invertebrate animal (Hydra oligactis) living in a temperate lake.

Facultative sexual organisms combine sexual and asexual reproduction within a single life cycle, often switching between reproductive modes depending on environmental conditions. These organisms frequently inhabit variable seasonal environments, where favorable periods alternate with unfavorable periods, generating temporally varying selection pressures that strongly influence life history decisions and hence population dynamics. Due to the rapidly accelerating changes in our global environment today, understanding the population dynamics and genetic changes in facultative sexual populations inhabiting seasonal environments is critical to assess and prepare for additional challenges that will affect such ecosystems. In this study, we aimed at obtaining insights into the seasonal population dynamics of the facultative sexual freshwater cnidarian Hydra oligactis through a combination of restriction site‐associated sequencing (RAD‐Seq) genotyping and the collection of phenotypic data on the reproductive strategy of field‐collected hydra strains in a standard laboratory environment. We reliably detected 42 MlGs from the 121 collected hydra strains. Most of MLGs (N = 35, 83.3%) were detected in only one season. Five MLGs (11.9%) were detected in two seasons, one (2.4%) in three seasons and one (2.4%) in all four seasons. We found no significant genetic change during the 2 years in the study population. Clone lines were detected between seasons and even years, suggesting that clonal lineages can persist for a long time in a natural population. We also found that distinct genotypes differ in sexual reproduction frequency, but these differences did not affect whether genotypes reappeared across samplings. Our study provides key insights into the biology of natural hydra populations, while also contributing to understanding the population biology of facultative sexual species inhabiting freshwater ecosystems.

Gene flow influences the genomic architecture of local adaptation in six riverine fish species.

Understanding how gene flow influences adaptive divergence is important for predicting adaptive responses. Theoretical studies suggest that when gene flow is high, clustering of adaptive genes in fewer genomic regions would protect adaptive alleles from recombination and thus be selected for, but few studies have tested it with empirical data. Here, we used restriction site-associated sequencing to generate genomic data for six fish species with contrasting life histories from six reaches of the Upper Mississippi River System, USA. We used four differentiation-based outlier tests and three genotype-environment association analyses to define neutral single nucleotide polymorphisms (SNPs) and outlier SNPs that were putatively under selection. We then examined the distribution of outlier SNPs along the genome and investigated whether these SNPs were found in genomic islands of differentiation and inversions. We found that gene flow varied among species, and outlier SNPs were clustered more tightly in species with higher gene flow. The two species with the highest overall FST (0.0303-0.0720) and therefore lowest gene flow showed little evidence of clusters of outlier SNPs, with outlier SNPs in these species spreading uniformly across the genome. In contrast, nearly all outlier SNPs in the species with the lowest FST (0.0003) were found in a single large putative inversion. Two other species with intermediate gene flow (FST ~0.0025-0.0050) also showed clustered genomic architectures, with most islands of differentiation clustered on a few chromosomes. Our results provide important empirical evidence to support the hypothesis that increasingly clustered architecture of local adaptation is associated with high gene flow.

Evolutionary history of CAM photosynthesis in NeotropicalClusia: insights from genomics, anatomy, physiology and climate

AbstractClusia is a remarkable genus of Neotropical woody plants as its members engage in either C3 photosynthesis or employ, to varying degrees, crassulacean acid metabolism (CAM) photosynthesis. Information about the evolutionary history of CAM in Clusia is scarce. Restriction site-associated sequencing of 64 species (20% of the genus) provided strong support for most of the previously recognized nine lineages. Ancestral reconstruction using maximum parsimony or maximum likelihood under a one-rate model suggested that CAM evolved at least four times independently from a most recent common ancestor (MRCA) with C3, whereas a maximum likelihood two-rate model suggested that CAM was already present in the MRCA followed by reversions to C3 in several lineages. Phylogenetic generalized least square analysis assessed variation in seven leaf anatomical traits and CAM activity measured as δ 13C. Results indicate that CAM is highly correlated with palisade mesophyll layer thickness and cell size. In addition, correlation between 19 bioclimatic variables and δ 13C was evaluated. It was found that CAM is positively correlated with habitats with a more severe dry season and greater precipitation seasonality. Since CAM is weakly and/or only periodically expressed in many Clusia spp., and thus not readily reflected in δ 13C, future analysis of phylogenetically-informed CAM expression in Clusia must include physiological measurements such as CO2 exchange and/or diel changes in leaf acidity for each species under investigation.

Shedding Light on Specificity: Population Genomic Structure of a Symbiosis Between a Coral Reef Fish and Luminous Bacterium.

All organisms depend on symbiotic associations with bacteria for their success, yet how these interspecific interactions influence the population structure, ecology, and evolution of microbial symbionts is not well understood. Additionally, patterns of genetic variation in interacting species can reveal ecological traits that are important to gene flow and co-evolution. In this study, we define patterns of spatial and temporal genetic variation of a coral reef fish, Siphamia tubifer, and its luminous bacterial symbiont, Photobacterium mandapamensis in the Okinawa Islands, Japan. Using restriction site-associated sequencing (RAD-Seq) methods, we show that populations of the facultative light organ symbiont of S. tubifer exhibit genetic structure at fine spatial scales of tens of kilometers despite the absence of physical barriers to dispersal and in contrast to populations of the host fish. These results suggest that the host’s behavioral ecology and environmental interactions between host and symbiont help to structure symbiont populations in the region, consequently fostering the specificity of the association between host generations. Our approach also revealed several symbiont genes that were divergent between host populations, including hfq and a homolog of varS, both of which play a role in host association in Vibrio cholerae. Overall, this study highlights the important role that a host animal can play in structuring the distribution of its bacterial symbiont, particularly in highly connected marine environments, thereby promoting specificity of the symbiosis between host generations.

Population Genomics of Bettongia lesueur: Admixing Increases Genetic Diversity with no Evidence of Outbreeding Depression.

Small and isolated populations are subject to the loss of genetic variation as a consequence of inbreeding and genetic drift, which in turn, can affect the fitness and long-term viability of populations. Translocations can be used as an effective conservation tool to combat this loss of genetic diversity through establishing new populations of threatened species, and to increase total population size. Releasing animals from multiple genetically diverged sources is one method to optimize genetic diversity in translocated populations. However, admixture as a conservation tool is rarely utilized due to the risks of outbreeding depression. Using high-resolution genomic markers through double-digest restriction site-associated sequencing (ddRAD-seq) and life history data collected over nine years of monitoring, this study investigates the genetic and fitness consequences of admixing two genetically-distinct subspecies of Bettongia lesueur in a conservation translocation. Using single nucleotide polymorphisms (SNPs) identified from 215 individuals from multiple generations, we found an almost 2-fold increase in genetic diversity in the admixed translocation population compared to the founder populations, and this was maintained over time. Furthermore, hybrid class did not significantly impact on survivorship or the recruitment rate and therefore we found no indication of outbreeding depression. This study demonstrates the beneficial application of mixing multiple source populations in the conservation of threatened species for minimizing inbreeding and enhancing adaptive potential and overall fitness.

Attack of the PCR clones: Rates of clonality have little effect on RAD-seq genotype calls.

Interpretation of high-throughput sequence data requires an understanding of how decisions made during bioinformatic data processing can influence results. One source of bias that is often cited is PCR clones (or PCR duplicates). PCR clones are common in restriction site-associated sequencing (RAD-seq) data sets, which are increasingly being used for molecular ecology. To determine the influence PCR clones and the bioinformatic handling of clones have on genotyping, we evaluate four RAD-seq data sets. Data sets were compared before and after clones were removed to estimate the number of clones present in RAD-seq data, quantify how often the presence of clones in a data set causes genotype calls to change compared to when clones were removed, investigate the mechanisms that lead to genotype call changes and test whether clones bias heterozygosity estimates. Our RAD-seq data sets contained 30%-60% PCR clones, but 95% of RAD-tags had five or fewer clones. Relatively few genotypes changed once clones were removed (5%-10%), and the vast majority of these changes (98%) were associated with genotypes switching from a called to no-call state or vice versa. PCR clones had a larger influence on genotype calls in individuals with low read depth but appeared to influence genotype calls at all loci similarly. Removal of PCR clones reduced the number of called genotypes by 2% but had almost no influence on estimates of heterozygosity. As such, while steps should be taken to limit PCR clones during library preparation, PCR clones are likely not a substantial source of bias for most RAD-seq studies.

Iterative allogamy-autogamy transitions drive actual and incipient speciation during the ongoing evolutionary radiation within the orchid genus Epipactis (Orchidaceae).

The terrestrial orchid genus Epipactis has become a model system for the study of speciation via transitions from allogamy to autogamy, but close phylogenetic relationships have proven difficult to resolve through Sanger sequencing. We analysed with restriction site-associated sequencing (RAD-seq) 108 plants representing 29 named taxa that together span the genus, focusing on section Epipactis. Our filtered matrix of 12 543 single nucleotide polymorphisms was used to generate an unrooted network and a rooted, well-supported likelihood tree. We further inferred genetic structure through a co-ancestry heat map and admixture analysis, and estimated inbreeding coefficients per sample. The 27 named taxa of the ingroup were resolved as 11 genuine, geographically widespread species: four dominantly allogamous and seven dominantly autogamous. A single comparatively allogamous species, E. helleborine, is the direct ancestor of most of the remaining species, though one of the derived autogams has generated one further autogamous species. An assessment of shared ancestry suggested only sporadic hybridization between the re-circumscribed species. Taxa with the greatest inclination towards autogamy show less, if any, admixture, whereas the gene pools of more allogamous species contain a mixture alleles found in the autogams. This clade is presently undergoing an evolutionary radiation driven by a wide spectrum of genotypic, phenotypic and environmental factors. Epipactis helleborine has also frequently generated many local variants showing inclinations toward autogamy (and occasionally cleistogamy), best viewed as incipient speciation from within the genetic background provided by E. helleborine, which thus becomes an example of a convincingly paraphyletic species. Autogams are often as widespread and ecologically successful as allogams.

DepthFinder: a tool to determine the optimal read depth for reduced-representation sequencing.

Identification of DNA sequence variations such as single nucleotide polymorphisms (SNPs) is a fundamental step toward genetic studies. Reduced-representation sequencing methods have been developed as alternatives to whole genome sequencing to reduce costs and enable the analysis of many more individual. Amongst these methods, restriction site associated sequencing (RSAS) methodologies have been widely used for rapid and cost-effective discovery of SNPs and for high-throughput genotyping in a wide range of species. Despite the extensive improvements of the RSAS methods in the last decade, the estimation of the number of reads (i.e. read depth) required per sample for an efficient and effective genotyping remains mostly based on trial and error. Herein we describe a bioinformatics tool, DepthFinder, designed to estimate the required read counts for RSAS methods. To illustrate its performance, we estimated required read counts in six different species (human, cattle, spruce budworm, salmon, barley and soybean) that cover a range of different biological (genome size, level of genome complexity, level of DNA methylation and ploidy) and technical (library preparation protocol and sequencing platform) factors. To assess the prediction accuracy of DepthFinder, we compared DepthFinder-derived results with independent datasets obtained from an RSAS experiment. This analysis yielded estimated accuracies of nearly 94%. Moreover, we present DepthFinder as a powerful tool to predict the most effective size selection interval in RSAS work. We conclude that DepthFinder constitutes an efficient, reliable and useful tool for a broad array of users in different research communities. https://bitbucket.org/jerlar73/DepthFinder. Supplementary data are available at Bioinformatics online.

The identification of multiple SNP markers for scald resistance in spring barley through restriction-site associated sequencing

Leaf scald caused by Rhynchosporium commune (formerly R. secalis) is an important fungal disease in barley (Hordeum vulgare) in western Canada. Pyramiding multiple scald resistance qualitative trait loci (QTL) is considered an effective strategy for breeding durable scald resistance into barley. The objective of this study was to map QTLs with restriction site-associated (RAD) markers and identify single nucleotide polymorphism (SNP) markers for scald resistance. Two recombinant inbred line (RIL) populations (crosses Harrington/Seebe and Seebe/Shyri) with good resistance to local scald isolates were genotyped by RAD sequencing (RADseq). A total of 4203 SNPs were obtained from the RADseq analysis of 162 F7 RILs. Inclusive composite interval mapping identified multiple major QTLs for scald resistance on chromosomes 3H, 4H, and 5H in Shyri, and 2H and 6H in Seebe. SNP markers from Shyri were located in the same region as the previously reported 3H QTL Rrs1. Several SNP markers from Seebe clustered on chromosome 6H near a major scald resistance QTL. The 6H QTL appeared effective for both adult plant resistance and seedling resistance, and explained up to 70.9% of the phenotypic variation among the sequenced lines. A tightly linked SNP for the major 6H QTL was converted into an allele specific PCR marker. The QTLs and their genetic markers found in this study will be useful in barley breeding for the selection of resistance to barley leaf scald.

Genome-wide divergence patterns support fine-scaled genetic structuring associated with migration tendency in brown trout

Brown trout (Salmo trutta) exhibit highly diverse life histories varying from resident, slow-growing, and early maturing to migratory, fast-growing, and late maturing, even within single watersheds. We sampled 11 locations within the transboundary Finnish–Russian River Koutajoki watershed to evaluate genomic differences among mainstem and headwater sites, of which some are isolated by migration barriers. Restriction site associated sequencing (RADSeq) revealed that the most headwater localities supported unique, isolated populations with generally lower heterozygosity compared with the mainstem populations. The sampled migratory adults in the three main stems showed signals of admixture despite small but statistically significant genetic divergence, while the headwater populations, except for two, showed a high level of divergence and a lack of admixture. These results suggest that most of the headwater populations consist of resident brown trout and that the population genetic structuring is often maintained even in the absence of migration barriers. Our results have clear implications for fisheries management and conservation; each brown trout subpopulation represents an evolutionarily important unit with unique genetic makeup and life history variation.

Adaptation to reef habitats through selection on the coral animal and its associated microbiome.

Spatially adjacent habitats on coral reefs can represent highly distinct environments, often harbouring different coral communities. Yet, certain coral species thrive across divergent environments. It is unknown whether the forces of selection are sufficiently strong to overcome the counteracting effects of the typically high gene flow over short distances, and for local adaptation to occur. We screened the coral genome (using restriction site-associated sequencing) and characterized both the dinoflagellate photosymbiont- and tissue-associated prokaryote microbiomes (using metabarcoding) of a reef flat and slope population of the reef-building coral, Pocillopora damicornis, at two locations on Heron Island in the southern Great Barrier Reef. Reef flat and slope populations were separated by <100m horizontally and ~5m vertically, and the two study locations were separated by ~1km. For the coral host, genetic divergence between habitats was much greater than between locations, suggesting limited gene flow between the flat and slope populations. Consistent with environmental selection, outlier loci primarily belonged to the conserved, minimal cellular stress response, likely reflecting adaptation to the different temperature and irradiance regimes on the reef flat and slope. The prokaryote community differed across both habitat and, to a lesser extent, location, whereas the dinoflagellate photosymbionts differed by habitat but not location. The observed intraspecific diversity associated with divergent habitats supports that environmental adaptation involves multiple members of the coral holobiont. Adaptive alleles or microbial associations present in coral populations from the environmentally variable reef flat may provide a source of adaptive variation for assisted evolution approaches, through assisted gene flow, artificial cross-breeding or probiotic inoculations, with the aim to increase climate resilience in the slope populations.

Integrating restriction site-associated DNA sequencing (RAD-seq) with morphological cladistic analysis clarifies evolutionary relationships among major species groups of bee orchids.

Background and AimsBee orchids (Ophrys) have become the most popular model system for studying reproduction via insect-mediated pseudo-copulation and for exploring the consequent, putatively adaptive, evolutionary radiations. However, despite intensive past research, both the phylogenetic structure and species diversity within the genus remain highly contentious. Here, we integrate next-generation sequencing and morphological cladistic techniques to clarify the phylogeny of the genus.MethodsAt least two accessions of each of the ten species groups previously circumscribed from large-scale cloned nuclear ribosomal internal transcibed spacer (nrITS) sequencing were subjected to restriction site-associated sequencing (RAD-seq). The resulting matrix of 4159 single nucleotide polymorphisms (SNPs) for 34 accessions was used to construct an unrooted network and a rooted maximum likelihood phylogeny. A parallel morphological cladistic matrix of 43 characters generated both polymorphic and non-polymorphic sets of parsimony trees before being mapped across the RAD-seq topology.Key ResultsRAD-seq data strongly support the monophyly of nine out of ten groups previously circumscribed using nrITS and resolve three major clades; in contrast, supposed microspecies are barely distinguishable. Strong incongruence separated the RAD-seq trees from both the morphological trees and traditional classifications; mapping of the morphological characters across the RAD-seq topology rendered them far more homoplastic.ConclusionsThe comparatively high level of morphological homoplasy reflects extensive convergence, whereas the derived placement of the fusca group is attributed to paedomorphic simplification. The phenotype of the most recent common ancestor of the extant lineages is inferred, but it post-dates the majority of the character-state changes that typify the genus. RAD-seq may represent the high-water mark of the contribution of molecular phylogenetics to understanding evolution within Ophrys; further progress will require large-scale population-level studies that integrate phenotypic and genotypic data in a cogent conceptual framework.

Genomic analysis of a cardinalfish with larval homing potential reveals genetic admixture in the Okinawa Islands

Discrepancies between potential and observed dispersal distances of reef fish indicate the need for a better understanding of the influence of larval behaviour on recruitment and dispersal. Population genetic studies can provide insight on the degree to which populations are connected, and the development of restriction site-associated sequencing (RAD-Seq) methods has made such studies of nonmodel organisms more accessible. We applied double-digest RAD-Seq methods to test for population differentiation in the coral reef-dwelling cardinalfish, Siphamia tubifer, which based on behavioural studies, have the potential to use navigational cues to return to natal reefs. Analysis of 11,836 SNPs from fish collected at coral reefs in Okinawa, Japan, from eleven locations over 3years reveals little genetic differentiation between groups of S.tubifer at spatial scales from 2 to 140km and between years at one location: pairwise FST values were between 0.0116 and 0.0214. These results suggest that the Kuroshio Current largely influences larval dispersal in the region, and in contrast to expectations based on studies of other cardinalfishes, there is no evidence of population structure for S.tubifer at the spatial scales examined. However, analyses of outlier loci putatively under selection reveal patterns of temporal differentiation that indicate high population turnover and variable larval supply from divergent source populations between years. These findings highlight the need for more studies of fishes across various geographic regions that also examine temporal patterns of genetic differentiation to better understand the potential connections between early life-history traits and connectivity of reef fish populations.