We use extensive geographical sampling and surveys of nuclear microsatellite and mitochondrial DNA loci to investigate the phylogeographic structure of the only recognized self-fertilizing vertebrates, the mangrove killifishes, currently thought to comprise two cryptic species, Kryptolebias marmoratus and Kryptolebias hermaphroditus. All genetic markers revealed three concordant main clades. The Northern clade includes populations from Florida, northern Cuba, Bahamas, Belize and Honduras and corresponds to K. marmoratus. The Southern clade encompasses populations from Brazil and corresponds to K. hermaphroditus. This species was considered endemic to southeastern Brazil, but molecular data corroborate its occurrence in northeastern Brazil. The Central clade, not previously resolved with genetic data, includes populations from Panama and Antilles. Despite the geographic proximity of the Northern and Central clades, the latter is genetically closer to the Southern clade. The discovery of the Central clade raises some taxonomic issues -it can either be considered a distinct species or united with the Southern clade into a single species with two subspecies. Another possible taxonomic solution is a single selfing species, K. marmoratus, with three subspecies. We show that the Central and Southern clades are highly selfing (97-100%), whereas selfing rates of the Northern clade populations vary geographically (39-99%). Genetic patterns indicate that populations in SE Brazil are recent, contrary to expectations based on the known distributions of related species.

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Paleo-drainage connections and headwater stream-captures are two main historical processes shaping the distribution of strictly freshwater fishes. Recently, bathymetric-based methods of paleo-drainage reconstruction have opened new possibilities to investigate how these processes have shaped the genetic structure of freshwater organisms. In this context, the present study used paleo-drainage reconstructions and single-locus cluster delimitation analyses to examine genetic structure on the whole distribution of Pareiorhaphis garbei, a ‘near threatened’ armored catfish from the Fluminense freshwater ecoregion in Southeastern Brazil. Sequences of two mitochondrial genes (cytochrome b and cytochrome c oxidase subunit 1) were obtained from five sampling sites in four coastal drainages: Macaé (KAE), São João (SJO), Guapi-Macacu [sub-basins Guapiaçu (GAC) and Guapimirim (GMI)], and Santo Aleixo (SAL). Pronounced genetic structure was found, involving 10 haplotypes for cytB and 6 for coi, with no haplotypes shared between localities. Coalescent-based delineation methods as well as distance-based methods revealed genetic clusters corresponding to each sample site. Paleo-drainage reconstructions showed two putative paleo-rivers: an eastern one connecting KAE and SJO; and a western one merging in the Guanabara Bay (GAC, GMI, and SAL). A disagreement was uncovered between the inferred past riverine connections and current population genetic structure. Although KAE and SJO belong to the same paleo-river, the latter is more closely related to specimens from the Guanabara paleo-river. This discordance between paleo-drainage connections and phylogenetic structure may indicate an ancient stream-capture event in headwaters of this region. Furthermore, all analyses showed high divergence between KAE and the other lineages, suggesting at least one cryptic species in the latter, and that the nominal species should be restricted to the Macaé river basin, its type locality. In this drainage, impacts such as the invasive species and habitat loss can be especially threatening for such species with a narrow range. Our results also suggest that freshwater fishes from headwaters in the Serra do Mar mountains might have different biogeographical patterns than those from the lowlands, indicating a complex and dynamic climatic and geomorphological history.

Epigenetic mechanisms generate plastic phenotypes that can become locally adapted across environments. Disentangling genomic from epigenomic variation is challenging in sexual species due to genetic variation among individuals, but it is easier in self-fertilizing species. We analysed DNA methylation patterns of two highly inbred strains of a naturally self-fertilizing fish reared in two contrasting environments to investigate the obligatory (genotype-dependent), facilitated (partially depend on the genotype) or pure (genotype-independent) nature of the epigenetic variation. We found higher methylation differentiation between genotypes than between environments. Most methylation differences between environments common to both strains followed a pattern where the two genotypes (inbred lines) responded to the same environmental context with contrasting DNA methylation levels (facilitated epialleles). Our findings suggest that, at least in part, DNA methylation could depend on the dynamic interaction between the genotype and the environment, which could explain the plasticity of epigenetically mediated phenotypes.

Parental effects influence offspring phenotypes through pre‐ and post‐natal routes but little is known about their molecular basis, and therefore their adaptive significance. Epigenetic modifications, which control gene expression without changes in the DNA sequence and are influenced by the environment, may contribute to parental effects. We investigated the effects of environmental enrichment on the behaviour, metabolic rate and brain DNA methylation patterns of parents and offspring of the highly inbreed mangrove killifish (Kryptolebias marmoratus). Parental fish reared in enriched environments had lower cortisol levels, lower metabolic rates and were more active and neophobic than those reared in barren environments. They also differed in 1,854 methylated cytosines (DMCs). Offspring activity and neophobia were determined by the parental environment. Among the DMCs of the parents, 98 followed the same methylation patterns in the offspring, three of which were significantly influenced by parental environments irrespective of their own rearing environment. Our results suggest that parental environment influences the behaviour and, to some extent, the brain DNA methylation patterns of the offspring.

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Different mating systems are expected to affect the extent and direction of hybridization. Due to the different levels of sexual conflict, the weak inbreeder/strong outbreeder (WISO) hypothesis predicts that gametes from self-incompatible (SI) species should outcompete gametes from self-compatible (SC) ones. However, other factors such as timing of selfing and unilateral incompatibilities may also play a role on the direction of hybridization. In addition, differential mating opportunities provided by different mating systems are also expected to affect the direction of introgression in hybrid zones involving outcrossers and selfers. Here, we explored these hypotheses with a unique case of recent hybridization between two mangrove killifish species with different mating systems, Kryptolebias ocellatus (obligately outcrossing) and K. hermaphroditus (predominantly self-fertilizing) in two hybrid zones in southeast Brazil. Hybridization rates were relatively high (~20%), representing the first example of natural hybridization between species with different mating systems in vertebrates. All F1 individuals were sired by the selfing species. Backcrossing was small, but mostly asymmetrical with the SI parental species, suggesting pattern commonly observed in plant hybrid zones with different mating systems. Our findings shed light on how contrasting mating systems may affect the direction and extent of gene flow between sympatric species, ultimately affecting the evolution and maintenance of hybrid zones.