Out to sea: ocean currents and patterns of asymmetric gene flow in an intertidal fish species.

Passive dispersal via wind or ocean currents can drive asymmetric gene flow, which influences patterns of genetic variation and the capacity of populations to evolve in response to environmental change. The mangrove rivulus fish (Kryptolebias marmoratus), hereafter "rivulus," is an intertidal fish species restricted to the highly fragmented New World mangrove forests of Central America, the Caribbean, the Bahamas, and Florida. Mangrove patches are biological islands with dramatic differences in both abiotic and biotic conditions compared to adjacent habitat. Over 1,000 individual rivulus across 17 populations throughout its range were genotyped at 32 highly polymorphic microsatellites. Range-wide population genetic structure was evaluated with five complementary approaches that found eight distinct population clusters. However, an analysis of molecular variance indicated significant population genetic structure among regions, populations within regions, sampling locations within populations, and individuals within sampling locations, indicating that rivulus has both broad- and fine-scale genetic differentiation. Integrating range-wide genetic data with biophysical modeling based on 10 years of ocean current data showed that ocean currents and the distance between populations over water drive gene flow patterns on broad scales. Directional migration estimates suggested some significant asymmetries in gene flow that also were mediated by ocean currents and distance. Specifically, populations in the center of the range (Florida Keys) were identified as sinks that received migrants (and alleles) from other populations but failed to export individuals. These populations thus harbor genetic variation, perhaps even from extirpated populations across the range, but ocean currents and complex arrangements of landmasses might prevent the distribution of that genetic variation elsewhere. Hence, the inherent asymmetry of ocean currents shown to impact both genetic differentiation and directional migration rates may be responsible for the complex distribution of genetic variation across the range and observed patterns of metapopulation structure.

Against the Odds: Hybrid Zones between Mangrove Killifish Species with Different Mating Systems.

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.

Filling the gaps: phylogeography of the self-fertilizing Kryptolebias species (Cyprinodontiformes: Rivulidae) along South American mangroves.

Mangrove killifishes of the genus Kryptolebias have been historically classified as rare because of their small size and cryptic nature. Major gaps in distribution knowledge across mangrove areas, particularly in South America, challenge the understanding of the taxonomic status, biogeographical patterns and genetic structuring of the lineages composing the self-fertilizing "Kryptolebias marmoratus species complex." In this study, the authors combined a literature survey, fieldwork and molecular data to fill major gaps of information about the distribution of mangrove killifishes across western Atlantic mangroves. They found that selfing mangrove killifishes are ubiquitously distributed across the Caribbean, Central and South American mangroves and report 14 new locations in South America, extending the range of both the "Central clade" and "Southern clade" lineages which overlap in the Amazon. Although substantial genetic differences were found between clades, the authors also found further genetic structuring within clades, with populations in Central America, north and northeast Brazil generally showing higher levels of genetic diversity compared to the clonal ones in southeast Brazil. The authors discuss the taxonomic status and update the geographical distribution of the Central and Southern clades, as well as potential dispersal routes and biogeographical barriers influencing the distribution of the selfing mangrove killifishes in the western Atlantic mangroves.

The evolution of the placenta in poeciliid fishes.

How and why complex organs evolve is generally lost to history. The mammalian placenta, for example, was derived from a single common ancestor that lived over 100 million years ago.1-3 Therefore, the selective factors favoring this complex trait remain obscure. Species in the live-bearing fish family Poeciliidae have independently evolved placentas numerous times while retaining closely related non-placental sister species.4-7 This provides the raw material to test alternative hypotheses for the evolution of the placenta. We assemble an extensive species-level dataset on reproductive mode, life histories, and habitat, and then implement phylogenetic comparative methods to test adaptive hypotheses for the evolution of the placenta. We find no consistent family-wide associations between placentation and habitat. However, placental species exhibit significantly reduced reproductive allotment and have a higher likelihood of exhibiting superfetation (the ability to gestate multiple broods at different developmental stages). Both features potentially increase body streamlining and enhance locomotor performance during pregnancy, possibly providing selective advantage in performance-demanding environments such as those with high predation or fast water flow. Furthermore, we found significant interactions between body size and placentation for offspring size and fecundity. Relative to non-placental species, placentation is associated with higher fecundity and smaller offspring size in small-bodied species and lower fecundity and larger offspring size in large-bodied species. This pattern suggests that there may be two phenotypic adaptive peaks, corresponding to two selective optima, associated with placentation: one represented by small-bodied species that have fast life histories, and the second by large-bodied species with slow life histories.

Extensive hybridization and past introgression between divergent lineages in a quasi-clonal hermaphroditic fish: Ramifications for species concepts and taxonomy.

Extreme inbreeding is expected to reduce the incidence of hybridization, serving as a prezygotic barrier. Mangrove rivulus is a small killifish that reproduces predominantly by self-fertilization, producing highly homozygous lines throughout its geographic range. The Bahamas and Caribbean are inhabited by two highly diverged phylogeographic lineages of mangrove rivulus, Kryptolebias marmoratus and a 'Central clade' closely related to K. hermaphroditus from Brazil. The two lineages are largely allopatric, but recently were found in syntopy on San Salvador, Bahamas, where a single hybrid was reported. To better characterize the degree of hybridization and the possibility of secondary introgression, here we conducted a detailed genetic analysis of the contact zone on San Salvador. Two mixed populations were identified, one of which contained sexually mature hybrids. The distribution of heterozygosity at diagnostic microsatellite loci in hybrids showed that one of these hybrids was an immediate offspring from the K. marmoratus x Central clade cross, whereas the remaining five hybrids were products of reproduction by self-fertilization for 1-3 generations following the initial cross. Two hybrids had mitochondrial haplotypes of K. marmoratus and the remaining four hybrids had a haplotype of the Central clade, indicating that crosses go in both directions. In hybrids, alleles of parental lineages were represented in equal proportions suggesting lack of recent backcrossing to either of the parental lineages. However, sympatric populations of two lineages were less diverged than allopatric populations, consistent with introgression. Results are discussed in terms of applicability of the biological species concept for isogenic, effectively clonal, organisms.

Natural hybridization between divergent lineages in a selfing hermaphroditic fish.

By definition, mating between individuals is infrequent in highly selfing organisms, and so too, therefore, hybridization should be rare between genetically divergent lineages in predominantly self-fertilizing species. Notwithstanding these expectations, here we report a remarkable case of natural hybridization between highly diverged phylogeographic lineages of the mangrove rivulus, a small killifish that reproduces predominantly by self-fertilization and typically is found as highly homozygous lines in most parts of its extensive geographical range. Two distinctive genetic lineages (Kryptolebias marmoratus and a 'Central clade' closely related to K. hermaphroditus) previously were not known in sympatry, but were found by us to co-occur on San Salvador, Bahamas. Genetic analyses of a mitochondrial and multiple nuclear markers determined the direction of a cross producing a hybrid fish. Furthermore, we show that this hybrid individual was viable, as it successfully reproduced by self-fertilization for two generations. Additional sampling of this population will be necessary to determine if backcrossing of hybrids to the parental lineages occurs in nature and to analyse whether such backcross progeny are viable. Application of the biological species concept (BSC) is traditionally difficult in clonally reproducing organisms. Our results show that although mangrove rivulus fish are mostly highly selfing in nature (resulting in isogenic, effectively clonal and homozygous progeny), classification within this taxonomic complex need not be incompatible with the BSC.

Headwater Capture Evidenced by Paleo-Rivers Reconstruction and Population Genetic Structure of the Armored Catfish (Pareiorhaphis garbei) in the Serra do Mar Mountains of Southeastern Brazil.

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.

Complete mitochondrial genome of a self-fertilizing fish Kryptolebias marmoratus (Cyprinodontiformes, Rivulidae) from Florida.

The complete mitochondrial genome was sequenced in a mangrove rivulus Kryptolebias marmoratus from western Florida using next-generation sequencing. The 17 329 bp-long genome was identical in length and 99.8% similar to a previously published genome of this species from a specimen of unknown geographic origin. Gene arrangement in K. marmoratus is similar to other cyprinodontiform fishes, except for the presence of a second copy of the control region inserted upstream of the nad1 gene.

Transcriptomics of diapause in an isogenic self-fertilizing vertebrate.

BACKGROUND: Many vertebrate species have the ability to undergo weeks or even months of diapause (a temporary arrest of development during early ontogeny). Identification of diapause genes has been challenging due in part to the genetic heterogeneity of most vertebrate animals. RESULTS: Here we take the advantage of the mangrove rivulus fish (Kryptolebias marmoratus or Kmar)-the only vertebrate that is extremely inbred due to consistent self-fertilization-to generate isogenic lineages for transcriptomic dissection. Because the Kmar genome is not publicly available, we built de novo genomic (642 Mb) and transcriptomic assemblies to serve as references for global genetic profiling of diapause in Kmar, via RNA-Seq. Transcripts unique to diapause in Kmar proved to constitute only a miniscule fraction (0.1 %) of the total pool of transcribed products. Most genes displayed lower expression in diapause than in post-diapause. However, some genes (notably dusp27, klhl38 and sqstm1) were significantly up-regulated during diapause, whereas others (col9a1, dspp and fmnl1) were substantially down-regulated, compared to both pre-diapause and post-diapause. CONCLUSION: Kmar offers a strong model for understanding patterns of gene expression during diapause. Our study highlights the importance of using a combination of genome and transcriptome assemblies as references for NGS-based RNA-Seq analyses. As for all identified diapause genes, in future studies it will be critical to link various levels of RNA expression with the functional roles of the coded products.

A Genetic Test for Whether Pairs of Hermaphrodites Can Cross-Fertilize in a Selfing Killifish.

Kryptolebias marmoratus, a small killifish that lives in mangrove habitat from southern Florida to Brazil, is one of the planet's only known self-fertilizing hermaphroditic vertebrates. Generation after generation, hermaphroditic individuals simultaneously produce sperm and eggs and internally self-fertilize to produce what are, in effect, highly inbred clones of themselves. Although populations are composed primarily of hermaphrodites, they also contain some true males. The frequency of males in a population varies geographically, from <2% in Florida to as high as 25% in Belize. Males are known to mate occasionally with hermaphrodites, thereby releasing genetic variation that has profound consequences for population genetic structure. However, it is unknown whether hermaphrodites can or do sporadically mate with each other also. Here, we test whether hermaphroditic individuals of the killifish Kryptolebias marmoratus are capable of crossing with one another, in addition to their much more common habits of self-fertilization and occasional outcrossing with pure males. We employ an experimental design in which replicate hermaphrodite pairs were housed together and allowed to reproduce naturally. Among 173 embryos screened at diagnostic microsatellite loci, all were found to result from selfing (i.e., no embryos were the product of a hermaphrodite cross). We thus conclude that hermaphrodite pairs are unlikely to cross, or do so exceedingly rarely.

A Bayesian Approach to Inferring Rates of Selfing and Locus-Specific Mutation.

We present a Bayesian method for characterizing the mating system of populations reproducing through a mixture of self-fertilization and random outcrossing. Our method uses patterns of genetic variation across the genome as a basis for inference about reproduction under pure hermaphroditism, gynodioecy, and a model developed to describe the self-fertilizing killifish Kryptolebias marmoratus. We extend the standard coalescence model to accommodate these mating systems, accounting explicitly for multilocus identity disequilibrium, inbreeding depression, and variation in fertility among mating types. We incorporate the Ewens sampling formula (ESF) under the infinite-alleles model of mutation to obtain a novel expression for the likelihood of mating system parameters. Our Markov chain Monte Carlo (MCMC) algorithm assigns locus-specific mutation rates, drawn from a common mutation rate distribution that is itself estimated from the data using a Dirichlet process prior model. Our sampler is designed to accommodate additional information, including observations pertaining to the sex ratio, the intensity of inbreeding depression, and other aspects of reproduction. It can provide joint posterior distributions for the population-wide proportion of uniparental individuals, locus-specific mutation rates, and the number of generations since the most recent outcrossing event for each sampled individual. Further, estimation of all basic parameters of a given model permits estimation of functions of those parameters, including the proportion of the gene pool contributed by each sex and relative effective numbers.

Evolutionary perspectives on clonal reproduction in vertebrate animals.

A synopsis is provided of different expressions of whole-animal vertebrate clonality (asexual organismal-level reproduction), both in the laboratory and in nature. For vertebrate taxa, such clonal phenomena include the following: human-mediated cloning via artificial nuclear transfer; intergenerational clonality in nature via parthenogenesis and gynogenesis; intergenerational hemiclonality via hybridogenesis and kleptogenesis; intragenerational clonality via polyembryony; and what in effect qualifies as clonal replication via self-fertilization and intense inbreeding by simultaneous hermaphrodites. Each of these clonal or quasi-clonal mechanisms is described, and its evolutionary genetic ramifications are addressed. By affording an atypical vantage on standard vertebrate reproduction, clonality offers fresh perspectives on the evolutionary and ecological significance of recombination-derived genetic variety.

Genetic Subdivision and Variation in Selfing Rates Among Central American Populations of the Mangrove Rivulus, Kryptolebias marmoratus.

We used 32 polymorphic microsatellite loci to investigate how a mixed-mating system affects population genetic structure in Central American populations (N = 243 individuals) of the killifish Kryptolebias marmoratus (mangrove rivulus), 1 of 2 of the world's only known self-fertilizing vertebrates. Results were also compared with previous microsatellite surveys of Floridian populations of this species. For several populations in Belize and Honduras, population structure and genetic differentiation were pronounced and higher than in Florida, even though the opposite trend was expected because populations in the latter region were presumably smaller and highly selfing. The deduced frequency of selfing (s) ranged from s = 0.39-0.99 across geographic locales in Central America. This heterogeneity in selfing rates was in stark contrast to Florida, where s > 0.9. The frequency of outcrossing in a population (t = 1 - s) was tenuously correlated with local frequencies of males, suggesting that males are one of many factors influencing outcrossing. Observed distributions of individual heterozygosity showed good agreement with expected distributions under an equilibrium mixed-mating model, indicating that rates of selfing remained relatively constant over many generations. Overall, our results demonstrate the profound consequences of a mixed-mating system for the genetic architecture of a hermaphroditic vertebrate.

Spatiotemporal genetic structure in a protected marine fish, the California Grunion (Leuresthes tenuis), and relatedness in the genus Leuresthes.

The genus Leuresthes displays reproductive behavior unique among marine fish in which mature adults synchronously emerge completely out of the water to spawn on beach land. A limited number of sandy beaches, which are suitable for these spawning events, are present in discontinuous locations along the geographic range of the species, potentially limiting gene flow and the degree of genetic homogeneity between intraspecific populations. Here, we tested for molecular genetic differentiation between 363 individuals, representing 3 populations of California grunion, Leuresthes tenuis, by employing 2 mitochondrial and 4 nuclear DNA markers. We include temporally diverse sampling to evaluate contemporary and temporal divergence, and we also analyze 28 individuals from one population of Gulf grunion (restricted to the Gulf of California), Leuresthes sardina, at the same markers to evaluate the molecular evidence for their separate species distinction. We find no significant differences between temporal samples, but small significant differences among all populations of L. tenuis, and unequivocal support for the separate species distinction of L. sardina. Genetic data suggest that the Monterey Bay population of L. tenuis near the species' most northern range likely represents a relatively recent colonization event from populations along the species' more traditional range south of Point Conception, California. We conclude that both the topographic features of the California and Baja California coastlines and the grunions' unique reproductive behavior have influenced the genetic structure of the populations.

Molecular evidence for multiple paternity in a population of the Viviparous Tule Perch Hysterocarpus traski.

Population density might be an important variable in determining the degree of multiple paternity. In a previous study, a high level of multiple paternity was detected in the shiner perch Cymatogaster aggregata, a species with high population density and a high mate encounter rate. The tule perch Hysterocarpus traski is phylogenetically closely related to C. aggregata, but it has relatively lower population density, which may result in distinct patterns of multiple paternity in these 2 species. To test the hypothesis that mate encounter rate may affect the rate of successful mating, we used polymorphic microsatellite markers to identify multiple paternity in the progeny arrays of 12 pregnant females from a natural population of tule perch. Multiple paternity was detected in 11 (92%) of the 12 broods. The number of sires per brood ranged from 1 to 4 (mean 2.5) but with no correlation between sire number and brood size. Although the brood size of tule perch is considerably larger than that of shiner perch (40.7 vs. 12.9, respectively), the average number of sires per brood in tule perch is much lower than that in shiner perch (2.5 vs. 4.6, respectively). These results are consistent with the hypothesis that mate encounter rate is an important factor affecting multiple mating.

Allard's argument versus Baker's contention for the adaptive significance of selfing in a hermaphroditic fish.

Fertilization assurance (Baker's contention) and multilocus coadaptation (Allard's argument) are two distinct hypotheses for the adaptive significance of self-fertilization in hermaphroditic taxa, and both scenarios have been invoked to rationalize isogenicity via incest in various plants and invertebrate animals with predominant selfing. Here we contrast Allard's argument and Baker's contention as applied to the world's only known vertebrate that routinely self-fertilizes. We pay special attention to frequencies of locally most common multilocus genotypes in Floridian populations of the Mangrove Rivulus (Kryptolebias marmoratus). Isogenicity patterns in this fish appear inconsistent with Allard's argument, thus leaving Baker's contention as the more plausible scenario (a result also supported by natural history information for this species). These results contrast with the isogenicity patterns and conclusions previously drawn from several self-fertilizing plants and invertebrate animal species. Thus, the adaptive significance of selfing apparently varies across hermaphroditic taxa.