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.

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.

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.

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.

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.

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.

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.

Multiple mating and its relationship to brood size in pregnant fishes versus pregnant mammals and other viviparous vertebrates.

We summarize the literature on rates of multiple paternity and sire numbers per clutch in viviparous fishes vs. mammals, two vertebrate groups in which pregnancy is common but entails very different numbers of embryos (for species surveyed, piscine broods averaged >10-fold larger than mammalian litters). As deduced from genetic parentage analyses, multiple mating by the pregnant sex proved to be common in assayed species but averaged significantly higher in fish than mammals. However, within either of these groups we found no significant correlations between brood size and genetically deduced incidence of multiple mating by females. Overall, these findings offer little support for the hypothesis that clutch size in pregnant species predicts the outcome of selection for multiple mating by brooders. Instead, whatever factors promote multiple mating by members of the gestating sex seem to do so in surprisingly similar ways in live-bearing vertebrates otherwise as different as fish and mammals. Similar conclusions emerged when we extended the survey to viviparous amphibians and reptiles. One notion consistent with these empirical observations is that although several fitness benefits probably accrue from multiple mating, logistical constraints on mate-encounter rates routinely truncate multiple mating far below levels that otherwise could be accommodated, especially in species with larger broods. We develop this concept into a "logistical constraint hypothesis" that may help to explain these mating outcomes in viviparous vertebrates. Under the logistical constraint hypothesis, propensities for multiple mating in each species register a balance between near-universal fitness benefits from multiple mating and species-idiosyncratic logistical limits on polygamy.

Multiple mating and clutch size in invertebrate brooders versus pregnant vertebrates.

We summarize the genetic literature on polygamy rates and sire numbers per clutch in invertebrate animals that brood their offspring and then compare findings with analogous data previously compiled for vertebrate species displaying viviparity or other pregnancy-like syndromes. As deduced from molecular parentage analyses of several thousand broods from more than 100 "pregnant" species, invertebrate brooders had significantly higher mean incidences of multiple mating than pregnant vertebrates, a finding generally consistent with the postulate that clutch size constrains successful mate numbers in species with extended parental care. However, we uncovered no significant correlation in invertebrates between brood size and genetically deduced rates of multiple mating by the incubating sex. Instead, in embryo-gestating animals otherwise as different as mammals and mollusks, polygamy rates and histograms of successful mates per brooder proved to be strikingly similar. Most previous studies have sought to understand why gestating parents have so many mates and such high incidences of successful multiple mating; an alternative perspective based on logistical constraints turns the issue on its head by asking why mate numbers and polygamy rates are much lower than they theoretically could be, given the parentage-resolving power of molecular markers and the huge sizes of many invertebrate broods.

Evaluating fossil calibrations for dating phylogenies in light of rates of molecular evolution: a comparison of three approaches.

Evolutionary and biogeographic studies increasingly rely on calibrated molecular clocks to date key events. Although there has been significant recent progress in development of the techniques used for molecular dating, many issues remain. In particular, controversies abound over the appropriate use and placement of fossils for calibrating molecular clocks. Several methods have been proposed for evaluating candidate fossils; however, few studies have compared the results obtained by different approaches. Moreover, no previous study has incorporated the effects of nucleotide saturation from different data types in the evaluation of candidate fossils. In order to address these issues, we compared three approaches for evaluating fossil calibrations: the single-fossil cross-validation method of Near, Meylan, and Shaffer (2005. Assessing concordance of fossil calibration points in molecular clock studies: an example using turtles. Am. Nat. 165:137-146), the empirical fossil coverage method of Marshall (2008. A simple method for bracketing absolute divergence times on molecular phylogenies using multiple fossil calibration points. Am. Nat. 171:726-742), and the Bayesian multicalibration method of Sanders and Lee (2007. Evaluating molecular clock calibrations using Bayesian analyses with soft and hard bounds. Biol. Lett. 3:275-279) and explicitly incorporate the effects of data type (nuclear vs. mitochondrial DNA) for identifying the most reliable or congruent fossil calibrations. We used advanced (Caenophidian) snakes as a case study; however, our results are applicable to any taxonomic group with multiple candidate fossils, provided appropriate taxon sampling and sufficient molecular sequence data are available. We found that data type strongly influenced which fossil calibrations were identified as outliers, regardless of which method was used. Despite the use of complex partitioned models of sequence evolution and multiple calibrations throughout the tree, saturation severely compressed basal branch lengths obtained from mitochondrial DNA compared with nuclear DNA. The effects of mitochondrial saturation were not ameliorated by analyzing a combined nuclear and mitochondrial data set. Although removing the third codon positions from the mitochondrial coding regions did not ameliorate saturation effects in the single-fossil cross-validations, it did in the Bayesian multicalibration analyses. Saturation significantly influenced the fossils that were selected as most reliable for all three methods evaluated. Our findings highlight the need to critically evaluate the fossils selected by data with different rates of nucleotide substitution and how data with different evolutionary rates affect the results of each method for evaluating fossils. Our empirical evaluation demonstrates that the advantages of using multiple independent fossil calibrations significantly outweigh any disadvantages.

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.

Colloquium paper: footprints of nonsentient design inside the human genome.

Intelligent design (ID)-the latest incarnation of religious creationism-posits that complex biological features did not accrue gradually via natural evolutionary forces but, instead, were crafted ex nihilo by a cognitive agent. Yet, many complex biological traits are gratuitously complicated, function poorly, and debilitate their bearers. Furthermore, such dysfunctional traits abound not only in the phenotypes but inside the genomes of eukaryotic species. Here, I highlight several outlandish features of the human genome that defy notions of ID by a caring cognitive agent. These range from de novo mutational glitches that collectively kill or maim countless individuals (including embryos and fetuses) to pervasive architectural flaws (including pseudogenes, parasitic mobile elements, and needlessly baroque regulatory pathways) that are endogenous in every human genome. Gross imperfection at the molecular level presents a conundrum for the traditional paradigms of natural theology as well as for recent assertions of ID, but it is consistent with the notion of nonsentient contrivance by evolutionary forces. In this important philosophical sense, the science of evolutionary genetics should rightly be viewed as an ally (not an adversary) of mainstream religions because it helps the latter to escape the profound theological enigmas posed by notions of ID.

Multiple mating and its relationship to alternative modes of gestation in male-pregnant versus female-pregnant fish species.

We construct a verbal and graphical theory (the "fecundity-limitation hypothesis") about how constraints on the brooding space for embryos probably truncate individual fecundity in male-pregnant and female-pregnant species in ways that should differentially influence selection pressures for multiple mating by males or by females. We then review the empirical literature on genetically deduced rates of multiple mating by the embryo-brooding parent in various fish species with three alternative categories of pregnancy: internal gestation by males, internal gestation by females, and external gestation (in nests) by males. Multiple mating by the brooding gender was common in all three forms of pregnancy. However, rates of multiple mating as well as mate numbers for the pregnant parent averaged higher in species with external as compared with internal male pregnancy, and also for dams in female-pregnant species versus sires in male-pregnant species. These outcomes are all consistent with the theory that different types of pregnancy have predictable consequences for a parent's brood space, its effective fecundity, its opportunities and rewards for producing half-sib clutches, and thereby its exposure to selection pressures for seeking multiple mates. Overall, we try to fit these fecundity-limitation phenomena into a broader conceptual framework for mating-system evolution that also includes anisogamy, sexual-selection gradients, parental investment, and other selective factors that can influence the relative proclivities of males versus females to seek multiple sexual partners.

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.

Long-term retention of self-fertilization in a fish clade.

Among vertebrate animals, only the mangrove rivulus (Kryptolebias marmoratus) was known to self-fertilize. Here, we use microsatellite analyses to document a high selfing rate (97%) in a related nominal species, Kryptolebias ocellatus, which likewise is androdioecious (populations consist of males and hermaphrodites). In contrast, we find no evidence of self-fertilization in Kryptolebias caudomarginatus (an androdioecious species closely related to the marmoratus-ocellatus clade) or in Kryptolebias brasiliensis (a dioecious outgroup). These findings indicate that the initiation of self-fertilization predated the origin of the marmoratus-ocellatus clade. From mitochondrial DNA sequences and microsatellite data, we document a substantial genetic distance between Kryptolebias marmoratus and K. ocellatus, implying that the selfing capacity has persisted in these fishes for at least several hundred thousand years.