Low-frequency somatic mutations are heritable in tropical trees Dicorynia guianensis and Sextonia rubra.

Somatic mutations potentially play a role in plant evolution, but common expectations pertaining to plant somatic mutations remain insufficiently tested. Unlike in most animals, the plant germline is assumed to be set aside late in development, leading to the expectation that plants accumulate somatic mutations along growth. Therefore, several predictions were made on the fate of somatic mutations: mutations have generally low frequency in plant tissues; mutations at high frequency have a higher chance of intergenerational transmission; branching topology of the tree dictates mutation distribution; and exposure to UV (ultraviolet) radiation increases mutagenesis. To provide insights into mutation accumulation and transmission in plants, we produced two high-quality reference genomes and a unique dataset of 60 high-coverage whole-genome sequences of two tropical tree species, Dicorynia guianensis (Fabaceae) and Sextonia rubra (Lauraceae). We identified 15,066 de novo somatic mutations in D. guianensis and 3,208 in S. rubra, surprisingly almost all found at low frequency. We demonstrate that 1) low-frequency mutations can be transmitted to the next generation; 2) mutation phylogenies deviate from the branching topology of the tree; and 3) mutation rates and mutation spectra are not demonstrably affected by differences in UV exposure. Altogether, our results suggest far more complex links between plant growth, aging, UV exposure, and mutation rates than commonly thought.

Sea trout (Salmo trutta) straying rate decreases as distance from river mouth increases.

For both conservation and management purposes, it is important to identify the natal origin of migratory individuals entering a river, particularly in genetically spatially structured species like brown trout (Salmo trutta) where the migrant ecotype (called sea trout) can originate from different populations. Nonetheless, little attention has been paid to the spatial distribution of non-local fish at the level of an entire river catchment. The objective of the study was to quantify the proportion of non-local sea trout entering a river catchment (i.e., straying rate) and estimate the spatial extent of their upstream migration. Here, the authors considered dispersal in three distinct rivers, taking advantage of 10 sampling sites. Sea trout, either trapped or rod-caught, were genotyped and genetically assigned to their source populations using appropriate baselines. Based on 1437 sea trout fish classified as local or non-local, the authors empirically demonstrate that straying rate declines in each river as distance from the coast increases in a non-linear fashion. Straying rate exceeds 50% near the mouth, and then decreases gradually to reach <10% 40-50 km inland. A similar spatial pattern is found in the three rivers investigated suggesting an underlying common behaviour of non-local sea trout. The data and results presented here suggest that straying in is far more constrained spatially than first expected. The majority of non-local sea trout were found within the first 25 km of the estuary in the three rivers investigated.

Joint analysis of microsatellites and flanking sequences enlightens complex demographic history of interspecific gene flow and vicariance in rear-edge oak populations.

Inference of recent population divergence requires fast evolving markers and necessitates to differentiate shared genetic variation caused by ancestral polymorphism and gene flow. Theoretical research shows that the use of compound marker systems integrating linked polymorphisms with different mutational dynamics, such as a microsatellite and its flanking sequences, can improve estimation of population structure and inference of demographic history, especially in the case of complex population dynamics. However, empirical application in natural populations has so far been limited by lack of suitable methods for data collection. A solution comes from the development of sequence-based microsatellite genotyping which we used to study molecular variation at 36 sequenced nuclear microsatellites in seven Quercus canariensis and four Q. faginea rear-edge populations across Algeria. We aim to decipher their taxonomic relationship, past evolutionary history and recent demographic trajectory. First, we compare the estimation of population genetics parameters and simulation-based inference of demographic history from microsatellite sequence alone, flanking sequence alone or the combination of linked microsatellite and flanking sequence variation. Second, we apply random forest approximate Bayesian computation to identify which of these sequence types is most informative. Whereas analysing microsatellite variation alone indicates recent interspecific gene flow, additional information gained by integrating nucleotide variation in flanking sequences, by reducing homoplasy, suggests ancient interspecific gene flow followed by drift in isolation instead. The weight of each polymorphism in the inference also demonstrates the value of linked variations with contrasted mutation dynamic to improve estimation of both demographic and mutational parameters.

Iguanainsularis (Iguanidae) from the southern Lesser Antilles: An endemic lineage endangered by hybridization.

The newly described horned iguanaIguanainsularis from the southern Lesser Antilles is separated in two easily recognized subspecies: I.insularissanctaluciae from St. Lucia and I.insularisinsularis from the Grenadines. Its former description is completed by the use of 38 new samples for genetic and morphological analysis. Seventeen microsatellites were used to estimate genetic diversity, population structure and the level of introgression with other Iguana species over nearly the whole range of the species. ND4 and PAC sequences were also used to better characterize hybridization and to complete the description of this lineage. The I.insularis population of St. Vincent shows a high level of introgression from I.iguana whereas in the Grenadines, most islands present pure insularis populations but several show evidence of introgressions. Of the two remaining populations of I.insularissanctaluciae, only one is still purebred. The recent identification of this and other distinct insular species and subspecies in the eastern Caribbean, and evaluation of where hybridization has occurred, are timely and important because the native iguanas are in urgent need of conservation action. Among the greatest threats is the ongoing human-mediated spread of invasive iguanas from Central and South America, which are destroying the endemic insular lineages through multiple diachronic introgression events.

Development and characterization of microsatellite markers for the French endemic Angelica heterocarpa (Apiaceae) and congeneric sympatric species.

OBJECTIVE: Angelica heterocarpa (Apiaceae) is a wild endemic French species with special conservation interest in the European Union. It belongs to Angelica complex genus which is widespread throughout the north temperate zone, and is sympatric with other congeneric species. The objective of this work is to develop and characterize microsatellite markers as a new tool for understanding the ecology and evolution of Angelica species complex. RESULTS: We identified simple sequence repeat (SSR) regions in a microsatellite-enriched library from A. heterocarpa and A. sylvestris. All 16 selected SSR regions were found to amplify in these species and were highly polymorphic. Marker transferability was validated in A. razulii and A. archangelica. These markers will help us to better understand the evolutionary dynamic between rare endemics and widespread sister species, and be useful for conservation of the endemic species. Moreover, they can provide new tools for studying the numerous traditional medicinal herbs of the Angelica genus.

Population genomic and historical analysis suggests a global invasion by bridgehead processes in Mimulus guttatus.

Imperfect historical records and complex demographic histories present challenges for reconstructing the history of biological invasions. Here, we combine historical records, extensive worldwide and genome-wide sampling, and demographic analyses to investigate the global invasion of Mimulus guttatus from North America to Europe and the Southwest Pacific. By sampling 521 plants from 158 native and introduced populations genotyped at >44,000 loci, we determined that invasive M. guttatus was first likely introduced to the British Isles from the Aleutian Islands (Alaska), followed by admixture from multiple parts of the native range. We hypothesise that populations in the British Isles then served as a bridgehead for vanguard invasions worldwide. Our results emphasise the highly admixed nature of introduced M. guttatus and demonstrate the potential of introduced populations to serve as sources of secondary admixture, producing novel hybrids. Unravelling the history of biological invasions provides a starting point to understand how invasive populations adapt to novel environments.

Fast sequence-based microsatellite genotyping development workflow.

Application of high-throughput sequencing technologies to microsatellite genotyping (SSRseq) has been shown to remove many of the limitations of electrophoresis-based methods and to refine inference of population genetic diversity and structure. We present here a streamlined SSRseq development workflow that includes microsatellite development, multiplexed marker amplification and sequencing, and automated bioinformatics data analysis. We illustrate its application to five groups of species across phyla (fungi, plant, insect and fish) with different levels of genomic resource availability. We found that relying on previously developed microsatellite assay is not optimal and leads to a resulting low number of reliable locus being genotyped. In contrast, de novo ad hoc primer designs gives highly multiplexed microsatellite assays that can be sequenced to produce high quality genotypes for 20-40 loci. We highlight critical upfront development factors to consider for effective SSRseq setup in a wide range of situations. Sequence analysis accounting for all linked polymorphisms along the sequence quickly generates a powerful multi-allelic haplotype-based genotypic dataset, calling to new theoretical and analytical frameworks to extract more information from multi-nucleotide polymorphism marker systems.

Variable outcomes of hybridization between declining Alosa alosa and Alosa fallax.

Hybridization dynamics between co-occurring species in environments where human-mediated changes take place are important to quantify for furthering our understanding of human impacts on species evolution and for informing management. The allis shad Alosa alosa (Linnaeus, 1758) and twaite shad Alosa fallax (Lacépède, 1803), two clupeids sister species, have been severely impacted by human activities across Europe. The shrinkage of A. alosa distribution range along with the decline of the remaining populations' abundance threatens its persistence. The main objective was to evaluate the extent of hybridization and introgression between those interacting species. We developed a set of 77 species-specific SNP loci that allowed a better resolution than morphological traits as they enabled the detection of hybrids up to the third generation. Variable rates of contemporary hybridization and introgression patterns were detected in 12 studied sites across the French Atlantic coast. Mitochondrial markers revealed a cyto-nuclear discordance almost invariably involving A. alosa individuals with an A. fallax mitochondrial DNA and provided evidence of historical asymmetric introgression. Overall, contemporary and historical introgression revealed by nuclear and mitochondrial markers strongly suggests that a transfer of genes occurs from A. fallax toward A. alosa genome since at least four generations. Moreover, the outcomes of introgression greatly depend on the catchments where local processes are thought to occur. Undoubtedly, interspecific interaction and gene flow should not be overlooked when considering the management of those species.

DNA methylation of the promoter region of bnip3 and bnip3l genes induced by metabolic programming.

BACKGROUND: Environmental changes of biotic or abiotic nature during critical periods of early development may exert a profound influence on physiological functions later in life. This process, named developmental programming can also be driven through parental nutrition. At molecular level, epigenetic modifications are the most likely candidate for persistent modulation of genes expression in later life. RESULTS: In order to investigate epigenetic modifications induced by programming in rainbow trout, we focused on bnip3 and bnip3l paralogous genes known to be sensitive to environmental changes but also regulated by epigenetic modifications. Two specific stimuli were used: (i) early acute hypoxia applied at embryo stage and (ii) broodstock and fry methionine deficient diet, considering methionine as one of the main methyl-group donor needed for DNA methylation. We observed a programming effect of hypoxia with an increase of bnip3a and the four paralogs of bnip3l expression level in fry. In addition, parental methionine nutrition was correlated to bnip3a and bnip3lb1 expression showing evidence for early fry programming. We highlighted that both stimuli modified DNA methylation levels at some specific loci of bnip3a and bnip3lb1. CONCLUSION: Overall, these data demonstrate that methionine level and hypoxia stimulus can be of critical importance in metabolic programming. Both stimuli affected DNA methylation of specific loci, among them, an interesting CpG site have been identified, namely - 884 bp site of bnip3a, and may be positively related with mRNA levels.

Genetic architecture of threshold reaction norms for male alternative reproductive tactics in Atlantic salmon (Salmo salar L.).

Alternative mating tactics have important ecological and evolutionary implications and are determined by complex interactions between environmental and genetic factors. Here, we study the genetic effect and architecture of the variability in reproductive tactics among Atlantic salmon males which can either mature sexually early in life in freshwater or more commonly only after completing a migration at sea. We applied the latent environmental threshold model (LETM), which provides a conceptual framework linking individual status to a threshold controlling the decision to develop alternative traits, in an innovative experimental design using a semi-natural river which allowed for ecologically relevant phenotypic expression. Early male parr maturation rates varied greatly across families (10 to 93%) which translated into 90% [64-100%] of the phenotypic variation explained by genetic variation. Three significant QTLs were found for the maturation status, however only one collocated with a highly significant QTL explaining 20.6% of the variability of the maturation threshold located on chromosome 25 and encompassing a locus previously shown to be linked to sea age at maturity in anadromous Atlantic salmon. These results provide new empirical illustration of the relevance of the LETM for a better understanding of alternative mating tactics evolution in natural populations.

Remodelling of the hepatic epigenetic landscape of glucose-intolerant rainbow trout (Oncorhynchus mykiss) by nutritional status and dietary carbohydrates.

The rainbow trout, a carnivorous fish, displays a 'glucose-intolerant' phenotype revealed by persistent hyperglycaemia when fed a high carbohydrate diet (HighCHO). Epigenetics refers to heritable changes in gene activity and is closely related to environmental changes and thus to metabolism adjustments governed by nutrition. In this study we first assessed in the trout liver whether and how nutritional status affects global epigenome modifications by targeting DNA methylation and histone marks previously reported to be affected in metabolic diseases. We then examined whether dietary carbohydrates could affect the epigenetic landscape of duplicated gluconeogenic genes previously reported to display changes in mRNA levels in trout fed a high carbohydrate diet. We specifically highlighted global hypomethylation of DNA and hypoacetylation of H3K9 in trout fed a HighCHO diet, a well-described phenotype in diabetes. g6pcb2 ohnologs were also hypomethylated at specific CpG sites in these animals according to their up-regulation. Our findings demonstrated that the hepatic epigenetic landscape can be affected by both nutritional status and dietary carbohydrates in trout. The mechanism underlying the setting up of these epigenetic modifications has now to be explored in order to improve understanding of its impact on the glucose intolerant phenotype in carnivorous teleosts.

Sexual selection leads to a tenfold difference in reproductive success of alternative reproductive tactics in male Atlantic salmon.

The precocious maturation of some male Atlantic salmon (Salmo salar L.) has become a textbook example of alternative mating tactics, but the only estimates of reproductive success available so far are either the collective contribution of precocious males to reproduction in the wild or individual reproductive success in oversimplified experimental conditions. Using genetic parentage analysis on anadromous and precocious potential spawners and their offspring, we quantified components of individual reproductive success of both tactics in a natural population. On average, precocious males produced 2.24 (variance 67.62) offspring, against 27.17 (3080) for anadromous males. For both tactics, most of the variance in reproductive success was due to mating success, with 83% of precocious males having no mate, against 50% for anadromous males. Body size increased reproductive success of anadromous males and tended to decrease precocious males' reproductive success. Although these results do not solve the coexistence of alternative reproductive tactics (ARTs) in Atlantic salmon, their inclusion in comprehensive models of lifetime reproductive success should shed light on the evolution of precocious maturation in Atlantic salmon and its effect on the selection of phenotypic traits.

Genetic Evidence of Hybridization between the Endangered Native Species Iguana delicatissima and the Invasive Iguana iguana (Reptilia, Iguanidae) in the Lesser Antilles: Management Implications.

The worldwide increase of hybridization in different groups is thought to have become more important with the loss of isolating barriers and the introduction of invasive species. This phenomenon could result in the extinction of endemic species. This study aims at investigating the hybridization dynamics between the endemic and threatened Lesser Antillean iguana (Iguana delicatissima) and the invasive common green iguana (Iguana iguana) in the Lesser Antilles, as well as assessing the impact of interspecific hybridization on the decline of I. delicatissima. 59 I. delicatissima (5 localities), 47 I. iguana (12 localities) and 27 hybrids (5 localities), who were all identified based on morphological characters, have been genotyped at 15 microsatellites markers. We also sequenced hybrids using ND4 mitochondrial loci to further investigate mitochondrial introgression. The genetic clustering of species and hybrid genetic assignment were performed using a comparative approach, through the implementation of a Discriminant Analysis of Principal Component (DAPC) based on statistics, as well as genetic clustering approaches based on the genetic models of several populations (Structure, NewHybrids and HIest), in order to get full characterization of hybridization patterns and introgression dynamics across the islands. The iguanas identified as hybrids in the wild, thanks to morphological analysis, were all genetically F1, F2, or backcrosses. A high proportion of individuals were also the result of a longer-term admixture. The absence of reproductive barriers between species leads to hybridization when species are in contact. Yet morphological and behavioral differences between species could explain why males I. iguana may dominate I. delicatissima, thus resulting in short-term species displacement and extinction by hybridization and recurrent introgression from I. iguana toward I. delicatissima. As a consequence, I. delicatissima gets eliminated through introgression, as observed in recent population history over several islands. These results have profound implications for species management of the endangered I. delicatissima and practical conservation recommendations are being discussed in the light of these findings.

The concentration of plasma metabolites varies throughout reproduction and affects offspring number in wild brown trout (Salmo trutta).

In wild populations, measuring energy invested in the reproduction and disentangling investment in gametes versus investment in reproductive behavior (such as intrasexual competition or intersexual preference) remain challenging. In this study, we investigated the energy expenditure in brown trout reproductive behavior by using two proxies: variation in weight and variation of plasma metabolites involved in energy production, over the course of reproductive season in a semi natural experimental river. We estimated overall reproductive success using genetic assignment at the end of the reproductive season. Results show that triglycerides and free fatty acid concentrations vary negatively during reproduction, while amino-acids and glucose concentrations remain stable. Decrease in triglyceride and free fatty acid concentrations during reproduction is not related to initial concentration levels or to weight variation. Both metabolite concentration variations and weight variations are correlated to the number of offspring produced, which could indicate that gametic and behavioral reproductive investments substantially contribute to reproductive success in wild brown trout. This study opens a path to further investigate variations in reproductive investment in wild populations.

Two are better than one: combining landscape genomics and common gardens for detecting local adaptation in forest trees.

Predicting likely species responses to an alteration of their local environment is key to decision-making in resource management, ecosystem restoration and biodiversity conservation practice in the face of global human-induced habitat disturbance. This is especially true for forest trees which are a dominant life form on Earth and play a central role in supporting diverse communities and structuring a wide range of ecosystems. In Europe, it is expected that most forest tree species will not be able to migrate North fast enough to follow the estimated temperature isocline shift given current predictions for rapid climate warming. In this context, a topical question for forest genetics research is to quantify the ability for tree species to adapt locally to strongly altered environmental conditions (Kremer et al. ). Identifying environmental factors driving local adaptation is, however, a major challenge for evolutionary biology and ecology in general but is particularly difficult in trees given their large individual and population size and long generation time. Empirical evaluation of local adaptation in trees has traditionally relied on fastidious long-term common garden experiments (provenance trials) now supplemented by reference genome sequence analysis for a handful of economically valuable species. However, such resources have been lacking for most tree species despite their ecological importance in supporting whole ecosystems. In this issue of Molecular Ecology, De Kort et al. () provide original and convincing empirical evidence of local adaptation to temperature in black alder, Alnus glutinosa L. Gaertn, a surprisingly understudied keystone species supporting riparian ecosystems. Here, De Kort et al. () use an innovative empirical approach complementing state-of-the-art landscape genomics analysis of A. glutinosa populations sampled in natura across a regional climate gradient with phenotypic trait assessment in a common garden experiment (Fig. ). By combining the two methods, De Kort et al. () were able to detect unequivocal association between temperature and phenotypic traits such as leaf size as well as with genetic loci putatively under divergent selection for temperature. The research by De Kort et al. () provides valuable insight into adaptive response to temperature variation for an ecologically important species and demonstrates the usefulness of an integrated approach for empirical evaluation of local adaptation in nonmodel species (Sork et al. ).

SimRAD: an R package for simulation-based prediction of the number of loci expected in RADseq and similar genotyping by sequencing approaches.

Application of high-throughput sequencing platforms in the field of ecology and evolutionary biology is developing quickly with the introduction of efficient methods to reduce genome complexity. Numerous approaches for genome complexity reduction have been developed using different combinations of restriction enzymes, library construction strategies and fragment size selection. As a result, the choice of which techniques to use may become cumbersome, because it is difficult to anticipate the number of loci resulting from each method. We developed SimRAD, an R package that performs in silico restriction enzyme digests and fragment size selection as implemented in most restriction site associated DNA polymorphism and genotyping by sequencing methods. In silico digestion is performed on a reference genome or on a randomly generated DNA sequence when no reference genome sequence is available. SimRAD accurately predicts the number of loci under alternative protocols when a reference genome sequence is available for the targeted species (or a close relative) but may be unreliable when no reference genome is available. SimRAD is also useful for fine-tuning a given protocol to adjust the number of targeted loci. Here, we outline the functionality of SimRAD and provide an illustrative example of the use of the package (available on the CRAN at http://cran.r-project.org/web/packages/SimRAD).

High genetic diversity and distinctiveness of rear-edge climate relicts maintained by ancient tetraploidisation for Alnus glutinosa.

Populations located at the rear-edge of a species' distribution may have disproportionate ecological and evolutionary importance for biodiversity conservation in a changing global environment. Yet genetic studies of such populations remain rare. This study investigates the evolutionary history of North-African low latitude marginal populations of Alnus glutinosa Gaertn., a European tree species that plays a significant ecological role as a keystone of riparian ecosystems. We genotyped 551 adults from 19 populations located across North Africa at 12 microsatellite loci and applied a coalescent-based simulation approach to reconstruct the demographic and evolutionary history of these populations. Surprisingly, Moroccan trees were tetraploids demonstrating a strong distinctiveness of these populations within a species otherwise known as diploid. Best-fitting models of demographic reconstruction revealed the relict nature of Moroccan populations that were found to have withstood past climate change events and to be much older than Algerian and Tunisian populations. This study highlights the complex demographic history that can be encountered in rear-edge distribution margins that here consist of both old stable climate relict and more recent populations, distinctively diverse genetically both quantitatively and qualitatively. We emphasize the high evolutionary and conservation value of marginal rear-edge populations of a keystone riparian species in the context of on-going climate change in the Mediterranean region.

Thirteen microsatellites developed by SSR-enriched pyrosequencing for Solanum rostratum (Solanaceae) and related species.

PREMISE OF THE STUDY: Microsatellite markers were developed using second-generation sequencing in Solanum rostratum as a tool to study the reproductive biology and genetic structure of this invasive species. METHODS AND RESULTS: Thirteen microsatellites were successfully discovered and amplified in a single multiplexed PCR. All loci showed genetic variation in S. rostratum. Cross-amplification in five closely related taxa was successful for a subset of loci. CONCLUSIONS: The set of 13 microsatellite markers developed here provides a time-effective and cost-effective genetic tool to study the reproductive biology of S. rostratum. The demonstrated transferability of the PCR multiplex to related taxa also highlights its usefulness for evolutionary studies across Solanum sect. Androceras.

De novo discovery and multiplexed amplification of microsatellite markers for black alder (Alnus glutinosa) and related species using SSR-enriched shotgun pyrosequencing.

Recent developments in sequencing technologies and bioinformatics analyses provide an unprecedented opportunity for cost and time effective high quality microsatellite marker discovery in nonmodel organisms for which no genomic information is available. Here, we use shotgun pyrosequencing of a microsatellite-enriched library to develop, for the first time, microsatellite markers for Alnus glutinosa, a keystone tree species of European riparian woodland communities. From a total of 17 855 short sequences, we identified 590 perfect microsatellites from which 392 had designed primers. A subset of 48 loci were tested for amplification, 12 of which were polymorphic in A. glutinosa. These 12 loci were successfully coamplified in a single multiplex polymerase chain reaction experiment and validated for population genetics applications. In addition, 10 and 8 of these microsatellites were found to be transferable to the related A. incana and A. cordata species. The developed multiplex of 12 microsatellite markers therefore provides new opportunities for experimental evolutionary and forest genetics research in Alnus.

Comparison of random and SSR-enriched shotgun pyrosequencing for microsatellite discovery and single multiplex PCR optimization in Acacia harpophylla F. Muell. Ex Benth.

Streamlining the development and genotyping of microsatellites in species for which no genetic information is available represents an important technical challenge to overcome in order to enable mainstream application of state-of-the-art population genetic analysis techniques in nonmodel organisms. Using the example of Acacia harpophylla, an acacia tree endemic of north-eastern Australia, we show that high-throughput shotgun pyrosequencing technology, so-called second-generation sequencing, reduces time and cost of microsatellite marker discovery in nonmodel organisms and of their large-scale typing in natural populations. We found that 0.5% of short sequence reads generated on 454 Genome Sequencer FLX Titanium from random genome sampling and 2.2% of reads generated with prior microsatellite enrichment yielded microsatellite markers with designed polymerase chain reaction (PCR) primers, suggesting that enrichment increases efficiency of pyrosequencing when microsatellite discovery is the primary goal. Using stringent selection criteria to facilitate downstream PCR multiplex design, we identified 1435 microsatellite loci with designed primers from a total of 200,908 short sequence reads. From a subset of 96 loci tested for amplification, 38 were validated for population genetics applications, leading to the optimization of a cost-effective multiplex PCR protocol for the simultaneous typing of nine microsatellites in natural populations of A. harpophylla.