DNA metabarcoding suggests adaptive seasonal variation of individual trophic traits in a critically endangered fish.

Dietary studies are critical for understanding foraging strategies and have important applications in conservation and habitat management. We applied a robust metabarcoding protocol to characterize the diet of the critically endangered freshwater fish Zingel asper (the Rhone streber). We conducted modelling and simulation analyses to identify and characterize some of the drivers of individual trophic trait variation in this species. We found that population density and ontogeny had minor effects on the trophic niche of Z. asper. Instead, our results suggest that the majority of trophic niche variation was driven by seasonal variation in ecological opportunity. The total trophic niche width of Z. asper seasonally expanded to include a broader range of prey. Furthermore, null model simulations revealed that the increase of between-individual variation in autumn indicates that Z. asper become more opportunistic relative to summer and spring, rather than being associated with a seasonal specialization of individuals. Overall, our results suggest an adaptive variation of individual trophic traits in Z. asper: the species mainly consumes a few ephemeropteran taxa (Baetis fuscatus and Ecdyonurus) but seems to be capable of adapting its foraging strategy to maintain its body condition. This study illustrates how metabarcoding data obtained from faeces can be validated and combined with individual-based modelling and simulation approaches to explore inter- and intrapopulational individual trophic traits variation and to test hypotheses in the conventional analytic framework of trophic ecology.

Microbiota Diversity Within and Between the Tissues of Two Wild Interbreeding Species.

Understanding the role of microbiota as reproductive barriers or sources of adaptive novelty in the fundamental biological phenomenon of speciation is an exciting new challenge necessitating exploration of microbiota variation in wild interbreeding species. We focused on two interbreeding cyprinid species, Chondrostoma nasus and Parachondrostoma toxostoma, which have geographic distributions characterized by a mosaic of hybrid zones. We described microbiota diversity and composition in the three main teleost mucosal tissues, the skin, gills and gut, in the parental parapatric populations. We found that tissue type was the principal determinant of bacterial community composition. In particular, there was strong microbiota differentiation between external and internal tissues, with secondary discrimination between the two species. These findings suggest that specific environmental and genetic filters associated with each species have shaped the bacterial communities, potentially reflecting deterministic assemblages of bacteria. We defined the core microbiota common to both Chondrostoma species for each tissue, highlighting the occurrence of microbe-host genome interactions at this critical level for studies of the functional consequences of hybridization. Further investigations will explore to what extend these specific tissue-associated microbiota signatures could be profoundly altered in hybrids, with functional consequences for post-mating reproductive isolation in relation to environmental constraints.

Non-homogeneous combination of two porous genomes induces complex body shape trajectories in cyprinid hybrids.

INTRODUCTION: Hybridization is a common phenomenon in fish and is considered to be a major source of diversification. Deciphering the remoulding of genomic regions and phenotypes in zones where hybrid specimens occur is of particular interest to elucidate the emergence of evolutionary novelties. This approach is particularly challenging because the first step of hybridization seems to be the most important stage in the emergence of hybrid lineages. However, the signal can be significantly altered after only a few generations. RESULTS: We studied 41 microsatellites and partial cytochrome b gene sequences in 970 specimens belonging to two fish species (Chondrostoma nasus and Parachondrostoma toxostoma) in allopatric/parapatric zones, hybrids between them in a natural sympatric zone: the Ardèche basin. We showed that the genomic architecture in hybrids presented pattern heterogeneity of selection for the different loci. Indeed, the upstream part of the river (Rosières and Labeaume) presented an overdominant fitness of heterozygotes (12.20%) corresponding to a genomic compatibility, and underselection was observed for 4.88%-7.32% of the loci tested indicating a genomic incompatibility. Moreover the upstream station (Rosières) presented a positive selection of invasive C. nasus homozygotes (17.07% to 21.95%) indicating that hybridization may increase the fitness of admixed individuals.We showed that hybrid morphology (body shape based on 21 landmarks) correlated with genomic dilution indicating a species fingerprint. However, we demonstrated that the hybrid morphology was not a linear modification between the two parental species but a trade-off between several correlated traits. CONCLUSIONS: Hybrid specimens present a mosaic of genomic combination, showing regions with genomic compatibility and others with genomic incompatibility between the two species. Positive selection (invasive advantage ranging from 9.76% to 21.95% of the loci) was evidenced in the upstream part of the Ardèche indicating that environmental selection makes a substantial contribution. Although the presence of a dam is known to impose heterogeneous hybrid zones between these two species, we demonstrated in this study that a natural environment can also generate a hybrid zone with a large number (and diversity) of hybrids. The combination of the two genomes in the hybrids results in complex ontogenetic trajectories (with different morphological traits evolving at different rates) that correspond to novel developmental pathways.

Biological Invasion of Fish Parasite Neoergasilus japonicus (Harada, 1930) (Copepoda: Ergasilidae) in Lake Grand Laoucien, France: A Field Study on Life Cycle Parameters and Reasons for Unusual High Population Density.

The fish parasite, Neoergasilus japonicus (Harada, 1930), native to Eastern Siberia and the Amur River catchment area, invaded European water bodies in the middle of the last century, possibly due to the human-mediated distribution of fish in the Amur complex (i.e., the genera Hypophthalmichthys and Ctenopharyngodon). In the deep karst lake, Grand Laoucien (Marseille area, France), this species had an unusually high population density (from 1000 ind./ m3 in zooplankton to 4000 ind./ m3 in the nearshore area) during the free-living period of its life cycle. The annual cycle of N. japonicus includes a 5-month overwintering of fertilized females attached to fish fins and, following this, a five- to six-generation chain from March to November, when the free-living stages in the population alternate with parasite females which attach to their hosts for breeding. The population density of the parasites in zooplankton increased exponentially from spring to autumn, which positively correlated with temperature. We found a strong correlation between N. japonicus density and the community development of microphytobenthos, but not between N. japonicus and phyto- or zooplankton dynamics. The local contributing factors included a seasonal three-fold decrease in water levels and the development of anoxia in profundal waters, which led to a high ambient fish density and thus susceptibility to the parasite. Although the free-living parasite represented only 1% of zooplankton production, it consumed up to 25% of small invertebrate productivity. The maximum intensity of infection reached 140 parasites per fish, or 4.14 per g of weight. The high infection of fish with this parasite, in our opinion, indicated the danger it poses to the local ichthyofauna, which first encountered this new parasite.

Trade-off between morphological convergence and opportunistic diet behavior in fish hybrid zone.

BACKGROUND: The invasive Chondrostoma nasus nasus has colonized part of the distribution area of the protected endemic species Chondrostoma toxostoma toxostoma. This hybrid zone is a complex system where multiple effects such as inter-species competition, bi-directional introgression, strong environmental pressure and so on are combined. Why do sympatric Chondrostoma fish present a unidirectional change in body shape? Is this the result of inter-species interactions and/or a response to environmental effects or the result of trade-offs? Studies focusing on the understanding of a trade-off between multiple parameters are still rare. Although this has previously been done for Cichlid species flock and for Darwin finches, where mouth or beak morphology were coupled to diet and genetic identification, no similar studies have been done for a fish hybrid zone in a river. We tested the correlation between morphology (body and mouth morphology), diet (stable carbon and nitrogen isotopes) and genomic combinations in different allopatric and sympatric populations for a global data set of 1330 specimens. To separate the species interaction effect from the environmental effect in sympatry, we distinguished two data sets: the first one was obtained from a highly regulated part of the river and the second was obtained from specimens coming from the less regulated part. RESULTS: The distribution of the hybrid combinations was different in the two part of the sympatric zone, whereas all the specimens presented similar overall changes in body shape and in mouth morphology. Sympatric specimens were also characterized by a larger diet behavior variance than reference populations, characteristic of an opportunistic diet. No correlation was established between the body shape (or mouth deformation) and the stable isotope signature. CONCLUSION: The Durance River is an untamed Mediterranean river despite the presence of numerous dams that split the river from upstream to downstream. The sympatric effect on morphology and the large diet behavior range can be explained by a tendency toward an opportunistic behavior of the sympatric specimens. Indeed, the similar response of the two species and their hybrids implied an adaptation that could be defined as an alternative trade-off that underline the importance of epigenetics mechanisms for potential success in a novel environment.

One-locus-several-primers: A strategy to improve the taxonomic and haplotypic coverage in diet metabarcoding studies.

In diet metabarcoding analyses, insufficient taxonomic coverage of PCR primer sets generates false negatives that may dramatically distort biodiversity estimates. In this paper, we investigated the taxonomic coverage and complementarity of three cytochrome c oxidase subunit I gene (COI) primer sets based on in silico analyses and we conducted an in vivo evaluation using fecal and spider web samples from different invertivores, environments, and geographic locations. Our results underline the lack of predictability of both the coverage and complementarity of individual primer sets: (a) sharp discrepancies exist observed between in silico and in vivo analyses (to the detriment of in silico analyses); (b) both coverage and complementarity depend greatly on the predator and on the taxonomic level at which preys are considered; (c) primer sets' complementarity is the greatest at fine taxonomic levels (molecular operational taxonomic units [MOTUs] and variants). We then formalized the "one-locus-several-primer-sets" (OLSP) strategy, that is, the use of several primer sets that target the same locus (here the first part of the COI gene) and the same group of taxa (here invertebrates). The proximal aim of the OLSP strategy is to minimize false negatives by increasing total coverage through multiple primer sets. We illustrate that the OLSP strategy is especially relevant from this perspective since distinct variants within the same MOTUs were not equally detected across all primer sets. Furthermore, the OLSP strategy produces largely overlapping and comparable sequences, which cannot be achieved when targeting different loci. This facilitates the use of haplotypic diversity information contained within metabarcoding datasets, for example, for phylogeography and finer analyses of prey-predator interactions.

A from-benchtop-to-desktop workflow for validating HTS data and for taxonomic identification in diet metabarcoding studies.

The main objective of this work was to develop and validate a robust and reliable "from-benchtop-to-desktop" metabarcoding workflow to investigate the diet of invertebrate-eaters. We applied our workflow to faecal DNA samples of an invertebrate-eating fish species. A fragment of the cytochrome c oxidase I (COI) gene was amplified by combining two minibarcoding primer sets to maximize the taxonomic coverage. Amplicons were sequenced by an Illumina MiSeq platform. We developed a filtering approach based on a series of nonarbitrary thresholds established from control samples and from molecular replicates to address the elimination of cross-contamination, PCR/sequencing errors and mistagging artefacts. This resulted in a conservative and informative metabarcoding data set. We developed a taxonomic assignment procedure that combines different approaches and that allowed the identification of ~75% of invertebrate COI variants to the species level. Moreover, based on the diversity of the variants, we introduced a semiquantitative statistic in our diet study, the minimum number of individuals, which is based on the number of distinct variants in each sample. The metabarcoding approach described in this article may guide future diet studies that aim to produce robust data sets associated with a fine and accurate identification of prey items.

Challenges and advances for transcriptome assembly in non-model species.

Analyses of high-throughput transcriptome sequences of non-model organisms are based on two main approaches: de novo assembly and genome-guided assembly using mapping to assign reads prior to assembly. Given the limits of mapping reads to a reference when it is highly divergent, as is frequently the case for non-model species, we evaluate whether using blastn would outperform mapping methods for read assignment in such situations (>15% divergence). We demonstrate its high performance by using simulated reads of lengths corresponding to those generated by the most common sequencing platforms, and over a realistic range of genetic divergence (0% to 30% divergence). Here we focus on gene identification and not on resolving the whole set of transcripts (i.e. the complete transcriptome). For simulated datasets, the transcriptome-guided assembly based on blastn recovers 94.8% of genes irrespective of read length at 0% divergence; however, assignment rate of reads is negatively correlated with both increasing divergence level and reducing read lengths. Nevertheless, we still observe 92.6% of recovered genes at 30% divergence irrespective of read length. This analysis also produces a categorization of genes relative to their assignment, and suggests guidelines for data processing prior to analyses of comparative transcriptomics and gene expression to minimize potential inferential bias associated with incorrect transcript assignment. We also compare the performances of de novo assembly alone vs in combination with a transcriptome-guided assembly based on blastn both via simulation and empirically, using data from a cyprinid fish species and from an oak species. For any simulated scenario, the transcriptome-guided assembly using blastn outperforms the de novo approach alone, including when the divergence level is beyond the reach of traditional mapping methods. Combining de novo assembly and a related reference transcriptome for read assignment also addresses the bias/error in contigs caused by the dependence on a related reference alone. Empirical data corroborate these findings when assembling transcriptomes from the two non-model organisms: Parachondrostoma toxostoma (fish) and Quercus pubescens (plant). For the fish species, out of the 31,944 genes known from D. rerio, the guided and de novo assemblies recover respectively 20,605 and 20,032 genes but the performance of the guided assembly approach is much higher for both the contiguity and completeness metrics. For the oak, out of the 29,971 genes known from Vitis vinifera, the transcriptome-guided and de novo assemblies display similar performance, but the new guided approach detects 16,326 genes where the de novo assembly only detects 9,385 genes.

When Anthropogenic River Disturbance Decreases Hybridisation between Non-Native and Endemic Cyprinids and Drives an Ecomorphological Displacement towards Juvenile State in Both Species.

Understanding the impact of non-native species on native species is a major challenge in molecular ecology, particularly for genetically compatible fish species. Invasions are generally difficult to study because their effects may be confused with those of environmental or human disturbances. Colonized ecosystems are differently impacted by human activities, resulting in diverse responses and interactions between native and non-native species. We studied the dynamics between two Cyprinids species (invasive Chondrostoma nasus and endemic Parachondrostoma toxostoma) and their hybrids in 16 populations (from allopatric to sympatric situations and from little to highly fragmented areas) corresponding to 2,256 specimens. Each specimen was assigned to a particular species or to a hybrid pool using molecular identification (cytochrome b and 41 microsatellites). We carried out an ecomorphological analysis based on size, age, body shape, and diet (gut vacuity and molecular fecal contents). Our results contradicted our initial assumptions on the pattern of invasion and the rate of introgression. There was no sign of underperformance for the endemic species in areas where hybridisation occurred. In the unfragmented zone, the introduced species was found mostly downstream, with body shapes similar to those in allopatric populations while both species were found to be more insectivorous than the reference populations. However, high level of hybridisation was detected, suggesting interactions between the two species during spawning and/or the existence of hybrid swarm. In the disturbed zone, introgression was less frequent and slender body shape was associated with diatomivorous behaviour, smaller size (juvenile characteristics) and greater gut vacuity. Results suggested that habitat degradation induced similar ecomorphological trait changes in the two species and their hybrids (i.e. a transition towards a pedomorphic state) where the invasive species is more affected than the native species. Therefore, this study reveals a diversity of relationships between two genetically compatible species and emphasizes constraints on the invasion process in disturbed areas.

Molecular phylogeny of French Guiana Hylinae: implications for the systematic and biodiversity of the Neotropical frogs.

In this study we used nucleotide sequences from a segment of mitochondrial 16S ribosomal DNA gene to investigate the evolutionary relationships of some French Guiana Hylinae. New sequences, representing the members of different French Guiana frogs-five specimens of the Scinax genus, two Hyla, one Osteocephalus, one Hyalinobatrachium and two Rana as out-group-were examined. In addition, 26 sequences available from GenBank database representing the other subfamilies of the Hylidae were added to our study. This work allowed us to clarify relationships within the four hylids subfamilies (Pelodryadinae, Phyllomedusinae, Hemiphractinae and Hylinae) and the phylogenetic placement of the enigmatic Scinax genus within the Hylidae. We found that: (1) the Scinax genus displays a high level of differentiation in comparison to two other genera (Litoria and Hyla) belonging to 'Hylidae' family; (2) the Hylinae are paraphyletic given the position of the Litoria, which was the sister-group of the Hyla and the Osteocephalus genera; (3) the anterior works and our results (based on two different data sets) showed the paraphyly of the Hylidae questioning the validity of this family; (4) the reassessment of these different taxonomic groups will induce a huge implication on the estimation (past, present and future) of the biodiversity (in Neotropical frogs).

From Late Miocene to Holocene: processes of differentiation within the Telestes genus (Actinopterygii: Cyprinidae).

Investigating processes and timing of differentiation of organisms is critical in the understanding of the evolutionary mechanisms involved in microevolution, speciation, and macroevolution that generated the extant biodiversity. From this perspective, the Telestes genus is of special interest: the Telestes species have a wide distribution range across Europe (from the Danubian district to Mediterranean districts) and have not been prone to translocation. Molecular data (mtDNA: 1,232 bp including the entire Cyt b gene; nuclear genome: 11 microsatellites) were gathered from 34 populations of the Telestes genus, almost encompassing the entire geographic range. Using several phylogenetic and molecular dating methods interpreted in conjunction with paleoclimatic and geomorphologic evidence, we investigated the processes and timing of differentiation of the Telestes lineages. The observed genetic structure and diversity were largely congruent between mtDNA and microsatellites. The Messinian Salinity Crisis (Late Miocene) seems to have played a major role in the speciation processes of the genus. Focusing on T. souffia, a species occurring in the Danube and Rhone drainages, we were able to point out several specific events from the Pleistocene to the Holocene that have likely driven the differentiation and the historical demography of this taxon. This study provides support for an evolutionary history of dispersal and vicariance with unprecedented resolution for any freshwater fish in this region.

Does invasive Chondrostoma nasus shift the parasite community structure of endemic Parachondrostoma toxostoma in sympatric zones?

BACKGROUND: The composition of parasite communities in two cyprinid species in southern France - native and threatened Parachondrostoma toxostoma and introduced Chondrostoma nasus - was investigated. In sympatry, these two species form two hybrid zones in the Durance and Ardeche Rivers. Due to their different feeding preference and habitat positions in allopatry, we supposed a difference in parasite communities between fish species. We expected more similar parasite communities in sympatric zones associated with habitat overlap (facilitating the transmission of ectoparasites) and similar feeding (more generalist behaviour when compared to allopatry, facilitating the transmission of endoparasites) in both fish species. Finally, we investigated whether P. toxostoma x C. nasus hybrids are less parasitized then parental species. METHODS: One allopatric population of each fish species plus two sympatric zones were sampled. Fish were identified using cytochrome b gene and 41 microsatellites loci and examined for all metazoan parasites. RESULTS: A high Monogenea abundance was found in both allopatric and sympatric populations of C. nasus. Trematoda was the dominant group in parasite communities of P. toxostoma from the allopatric population. In contrast, the populations of P. toxostoma in sympatric zones were parasitized by Dactylogyrus species found in C. nasus populations, but their abundance in endemic species was low. Consequently, the similarity based on parasite presence/absence between the sympatric populations of P. toxostoma and C. nasus was high. Sympatric populations of P. toxostoma were more similar than allopatric and sympatric populations of this species. No difference in ectoparasite infection was found between P. toxostoma and hybrids, whilst C. nasus was more parasitized by Monogenea. CONCLUSIONS: The differences in endoparasites between P. toxostoma and C. nasus in allopatry are probably linked to different feeding or habitat conditions, but host-parasite evolutionary associations also play an important role in determining the presence of Chondrostoma-specific monogeneans. Our findings suggest that Dactylogyrus expanded with the source host C. nasus into introduced areas and that P. toxostoma became infected after contact with C. nasus. Although the genotype of P. toxostoma and recombinant genotypes of hybrids are susceptible to Dactylogyrus transmitted from C. nasus, the intensity of infection is low in these genotypes.

Biological influences on inter- and intraspecific isotopic variability among paired chondrostome fishes.

This study aimed at examining resource partitioning both at the inter- and intraspecific levels between paired chondrostome fishes: Chondrostoma nasus, the nase, C. toxostoma, the sofie, and their hybrid. The study was performed in the south of France and concerned a main river (the Durance River) and a tributary (the Buech River). In these rivers, C. nasus was an introduced species, originating in central Europe, and C. toxostoma was an endemic congener, in the south of France. Stable isotope analysis was used to analyse trophic and spatial niches. Isotopic differences indicated that individuals from the three taxa (C. nasus, C. toxostoma and their hybrid) have different spatial origins. At the interspecific level, the different chondrostomes originating from the Buech River showed a high level of trophic niche overlap. At the intraspecific level, nase individuals originating from the different spatial origins showed a resource polymorphism; differences in morphology were associated with variation in behaviour and life history traits. Their coexistence was a likely outcome of resource polymorphism. This study provides an example of the importance of considering the link between intra- and interspecific interactions to gain an understanding of the mechanisms driving the coexistence of species-pairs.

A PCR-based method for diet analysis in freshwater organisms using 18S rDNA barcoding on faeces.

The development of DNA barcoding from faeces represents a promising method for animal diet analysis. However, current studies mainly rely on prior knowledge of prey diversity for a specific predator rather than on a range of its potential prey species. Considering that the feeding behaviour of teleosts may evolve with their environment, it could prove difficult to establish an exhaustive listing of their prey. In this article, we extend the DNA barcoding approach to diet analysis to allow the inclusion of a wide taxonomic range of potential prey items. Thirty-four ecological clade-specific primer sets were designed to cover a large proportion of prey species found in European river ecosystems. Selected primers sets were tested on isolated animal, algal or plant tissues and thereafter on fish faeces using nested PCR to increase DNA detection sensitivity. The PCR products were sequenced and analysed to confirm the identity of the taxa and to validate the method. The methodology developed here was applied to a diet analysis of three freshwater cyprinid species that are assumed to have similar feeding behaviour [Chondrostoma toxostoma toxostoma (Vallot 1837), Chondrostoma nasus nasus (Linnaeus, 1758) and Barbus barbus, (Linneaus 1758)]. These three species were sampled in four different hydrographic basins. Principal Component Analysis based on prey proportions identified distinct perilithon grazer and benthophagous behaviours. Furthermore, our results were consistent with the available literature on feeding behaviour in these fish. The simplicity of the PCR-based method and its potential generalization to other freshwater organisms may open new perspectives in food web ecology.

Cross-species amplification of 41 microsatellites in European cyprinids: A tool for evolutionary, population genetics and hybridization studies.

BACKGROUND: Cyprinids display the most abundant and widespread species among the European freshwater Teleostei and are known to hybridize quite commonly. Nevertheless, a limited number of markers for conducting comparative differentiation, evolutionary and hybridization dynamics studies are available to date. FINDINGS: Five multiplex PCR sets were optimized in order to assay 41 cyprinid-specific polymorphic microsatellite loci (including 10 novel loci isolated from Chondrostoma nasus nasus, Chondrostoma toxostoma toxostoma and Leuciscus leuciscus) for 503 individuals (440 purebred specimens and 63 hybrids) from 15 European cyprinid species. The level of genetic diversity was assessed in Alburnus alburnus, Alburnoides bipunctatus, C. genei, C. n. nasus, C. soetta, C. t. toxostoma, L. idus, L. leuciscus, Pachychilon pictum, Rutilus rutilus, Squalius cephalus and Telestes souffia. The applicability of the markers was also tested on Abramis brama, Blicca bjoerkna and Scardinius erythrophtalmus specimens. Overall, between 24 and 37 of these markers revealed polymorphic for the investigated species and 23 markers amplified for all the 15 European cyprinid species. CONCLUSIONS: The developed set of markers demonstrated its performance in discriminating European cyprinid species. Furthermore, it allowed detecting and characterizing hybrid individuals. These microsatellites will therefore be useful to perform comparative evolutionary and population genetics studies dealing with European cyprinids, what is of particular interest in conservation issues and constitutes a tool of choice to conduct hybridization studies.

Isolation and characterization of polymorphic microsatellite loci in the freshwater fishes Telestes souffia and Telestes muticellus (Teleostei: Cyprinidae).

Ten novel polymorphic microsatellites (seven with perfect motifs) were isolated from vairone species (Telestes souffia and Telestes muticellus), which are endangered European cyprinid species. Together with 11 previously published cyprinid-specific loci, five multiplex sets were optimized, allowing the genotyping of 21 polymorphic loci. The level of genetic diversity was assessed in 97 individuals from the two species T. souffia and T. muticellus. We also successfully applied the 21 microsatellites to nine related species. These primers will thus be useful in assessing population structure of the vairone and other cyprinid species, with application for conservation issues and phylogeographical approaches.

Isolation and characterization of polymorphic microsatellite loci for the dace complex: Leuciscus leuciscus (Teleostei: Cyprinidae).

Ten novel polymorphic microsatellites were isolated from the dace complex (Leuciscus leuciscus), which is a European cyprinid species. Four multiplex polymerase chain reaction sets were optimized in order to genotype 26 polymorphic loci in all, including 16 previously published cyprinid-specific loci. The level of genetic diversity was assessed in 142 dace individuals. We also successfully applied 26 of the microsatellites to 10 related species. These primers thus will be useful to assess population structure of the dace and other cyprinid species, with application for conservation issues and phylogeographical approaches.

Novelties in hybrid zones: crossroads between population genomic and ecological approaches.

BACKGROUND: Interspecific hybridization is widespread, occurring in a taxonomically diverse array of species. The Cyprinidae family, which displays more than 30% hybridization, is a good candidate for studies of processes underlying isolation and speciation, such as genetic exchange between previously isolated lineages. This is particularly relevant in the case of recent hybridization between an invasive species, Chondrostoma nasus nasus (from Eastern Europe), and C. toxostoma toxostoma (a threatened species endemic to southern France), in which bidirectional introgressive hybridization has been demonstrated. METHODOLOGY/PRINCIPAL FINDINGS: We studied 128 specimens from reference populations and 1495 hybrid zone specimens (two years of sampling and four stations), using five molecular markers (one mitochondrial gene, four nuclear introns), morphology (meristic and plastic characters) and life history traits (weight, size, coefficient of condition, sex, age, shoaling). We identified 65 hybrid combinations and visualized spatial and temporal changes in composition. The direction of mitochondrial introgression was density-dependent in favor of the rarer species and we demonstrate that the sexual selection hypothesis is a preponderant explanation in the asymmetry of introgression. Despite genomic evolution in the hybrid zone, convergence was observed for body shape and coefficient of condition, indicating changes in foraging behavior with respect to reference populations, reflecting strong environmental pressure. CONCLUSIONS/SIGNIFICANCE: The complex rules of hybrid zone dynamics are established very early in the contact zone. We propose "inheritance from the rare species" as a new evolutionary hypothesis for animal models. The endemic species was not assimilated by the invasive species. Survival rates for this species were highest in the middle of the river (the warmest part) due to a trade-off between food availability and fecundity. The environment-independent hybrid combination may result from nuclear-mitochondrial interactions involving the Tpi1b gene or a gene linked to this gene (Chromosome 16). This genomic region is also responsible for shoaling behavior in Danio rerio and is a promising zone for studies of changes in population dynamics and advances in integrated studies of hybrid zones.