Population genetics and evolution of the mangrove rivulus Kryptolebias marmoratus, the world's only self-fertilizing hermaphroditic vertebrate.

The mangrove rivulus, Kryptolebias marmoratus (Rivulidae, Cyprinodontiformes), is phylogenetically embedded within a large clade of oviparous (egg laying) and otherwise mostly gonochoristic (separate sex) killifish species in the circumtropical suborder Aplocheiloidei. It is unique in its reproductive mode: K. marmoratus is essentially the world's only vertebrate species known to engage routinely in self-fertilization as part of a mixed-mating strategy of selfing plus occasional outcrossing with gonochoristic males. This unique form of procreation has profound population-genetic and evolutionary-genetic consequences that are the subject of this review.

Extreme homogeneity and low genetic diversity in Kryptolebias ocellatus from south-eastern Brazil suggest a recent foundation for this androdioecious fish population.

This study documents unexpectedly low levels of intra and interpopulation genetic diversity in Kryptolebias ocellatus, an androdioecious and predominantly self-fertilizing killifish from south-eastern Brazil. This finding generally is inconsistent with the established opinion that the K. ocellatus and K. marmoratus clade originated in this geographic region and later dispersed northward into the Caribbean.

Microgeographic population structure of green swordail fish: genetic differentiation despite abundant migration.

Swordtails (Xiphophorus; Poeciliidae) have figured prominently in research on fish mating behaviours, sexual selection, and carcinogenesis, but their population structures and dispersal patterns have been relatively neglected. Using nine microsatellite loci, we estimated genetic differentiation in Xiphophorus helleri within and between adjacent streams in Belize. The genetic data were complemented by a tagging study of movement within one stream. In the absence of physical dispersal barriers (waterfalls), population structure followed an isolation by distance (IBD) pattern. Genetic differentiation (F(ST) up to 0.07) was significant between and within creeks, despite high dispersal in the latter as judged by the tagging data. Such heterogeneity apparently was a result of genetic drift in local demes, due to small population sizes and highly skewed paternity. The IBD pattern was interrupted by waterfalls, boosting F(ST) above 0.30 between adjacent samples across these barriers. Overall, our results are helpful in understanding the interplay of evolutionary forces and population dynamics in a small fish living in a changeable habitat.

Evolutionary diversity and turn-over of sex determination in teleost fishes.

Sex determination, due to the obvious association with reproduction and Darwinian fitness, has been traditionally assumed to be a relatively conserved trait. However, research on teleost fishes has shown that this need not be the case, as these animals display a remarkable diversity in the ways that they determine sex. These different mechanisms, which include constitutive genetic mechanisms on sex chromosomes, polygenic constitutive mechanisms, environmental influences, hermaphroditism, and unisexuality have each originated numerous independent times in the teleosts. The evolutionary lability of sex determination, and the corresponding rapid rate of turn-over among different modes, makes the teleost clade an excellent model with which to test theories regarding the evolution of sex determining adaptations. Much of the plasticity in sex determination likely results from the dynamic teleost genome, and recent advances in fish genetics and genomics have revealed the role of gene and genome duplication in fostering emergence and turn-over of sex determining mechanisms.

Evolutionary perspectives on hermaphroditism in fishes.

Hermaphroditism is a derived and polyphyletic condition in fishes, documented in about 2% of all extant teleost species scattered across more than 20 taxonomic families in 9 orders. It shows a variety of expressions that can be categorized into sequential and synchronous modes. Among the sequential hermaphrodites are protogynous species in which an individual begins reproductive life as a female and later may switch to male, protandrous species in which a fish starts as a male and later may switch to female, and serial bi-directional sex changers. Among the synchronous hermaphrodites (in which an individual can simultaneously produce eggs and sperm) are several outcrossing and one predominantly selfing species. A few species also consist of mixtures of hermaphroditic and single-sex individuals. All of these reproductive categories have been the subject of numerous theoretical and empirical treatments from an evolutionary perspective. Here we highlight some of the major conclusions from these studies, which collectively have been informative on a variety of biological topics related to reproductive modes, gender allocations, sexual conflict and gamesmanship, mating systems, and life-history tradeoffs.

Supertree analyses of the roles of viviparity and habitat in the evolution of atherinomorph fishes.

Using supertree phylogenetic reconstructions, we investigate how livebearing and freshwater adaptations may have shaped evolutionary patterns in the Atherinomorpha, a large clade (approximately 1500 extant species) of ray-finned fishes. Based on maximum parsimony reconstructions, livebearing appears to have evolved at least four times independently in this group, and no reversions to the ancestral state of oviparity were evident. With respect to habitat, at least five evolutionary transitions apparently occurred from freshwater to marine environments, at least two transitions in the opposite direction, and no clear ancestral state was identifiable. All viviparous clades exhibited more extant species than their oviparous sister taxa, suggesting that transitions to viviparity may be associated with cladogenetic diversification. Transitions to freshwater were usually, but not invariably associated with increased species richness, but the trend was, overall, not significant among sister clades. Additionally, we investigated whether livebearing and freshwater adaptations are currently associated with elevated risks of extinction as implied by species' presence on the 2004 IUCN Red List. Despite being correlated with decreased brood size, livebearing has not significantly increased extinction risk in the Atherinomorpha. However, freshwater species were significantly more likely than marine species to be listed as endangered.

Sex-linked markers facilitate genetic parentage analyses in knobbed whelk broods.

To explore the potential of sex-linked polymorphisms for genetic parentage analyses in natural populations, we have employed a recently discovered "X-linked" microsatellite marker (in conjunction with polymorphic autosomal loci) to deduce biological paternity and maternity for large numbers of encapsulated embryos within individual broods of the knobbed whelk (Busycon carica). Empirical findings illustrate how such sex-linked genetic tags can in special instances find at least three novel utilities in genetic dissections of large-clutch species: clarification of paternity assignments that had remained ambiguous from di-locus autosomal data alone; elucidation of linkage relationships among pairs of autosomal loci; and illumination of maternity (and thereby paternity also) in broods for which neither biological parent was known from independent evidence.

Microsatellite null alleles in parentage analysis.

Highly polymorphic microsatellite markers are widely employed in population genetic analyses (eg, of biological parentage and mating systems), but one potential drawback is the presence of null alleles that fail to amplify to detected levels in the PCR assays. Here we examine 233 published articles in which authors reported the suspected presence of one or more microsatellite null alleles, and we review how these purported nulls were detected and handled in the data analyses. We also employ computer simulations and analytical treatments to determine how microsatellite null alleles might impact molecular parentage analyses. The results indicate that whereas null alleles in frequencies typically reported in the literature introduce rather inconsequential biases on average exclusion probabilities, they can introduce substantial errors into empirical assessments of specific mating events by leading to high frequencies of false parentage exclusions.

Microsatellite variation and differentiation in North Atlantic eels.

We screened 11 populations of American, European, and Icelandic eels (Anguillidae) for allelic variation and genetic divergence at six polymorphic microsatellite loci. Within either of the two recognized Anguilla species in the North Atlantic (rostrata in the Americas, anguilla in Europe), population genetic structure was statistically significant but weak; fully 95% of the total genetic variation was present within geographic locales rather than distributed among them. The two Anguilla species also overlap greatly in allelic frequencies, so the available data proved ineffective for addressing hypotheses about the possible hybrid origins of some Icelandic eels. The overlapping microsatellite profiles contrast with nearly diagnostic species differences documented previously in allozymes and mtDNA. This and similar empirical findings in several other species support theoretical concerns that homoplasy (convergent evolution) in allelic states can compromise the utility of rapidly mutating microsatellite loci for certain types of microevolutionary questions regarding gene flow and species differences.

Genetic parentage assessment in the crayfish Orconectes placidus, a high-fecundity invertebrate with extended maternal brood care.

Microsatellite data have recently been introduced in the context of genetic maternity and paternity assignments in high-fecundity fish species with single-parent-tended broods. Here we extend such analyses to an aquatic invertebrate, the crayfish Orconectes placidus, in which gravid females carry large numbers of offspring. Genetic parentage analyses of more than 900 progeny from 15 wild crayfish broods revealed that gravid females were invariably the exclusive dams of the offspring they tended (i.e. there was no allomaternal care), and that most of the females had mated with multiple (usually two) males who contributed sometimes highly skewed numbers of offspring to a brood. Within any multiply sired brood, the unhatched eggs (or the hatched juveniles) from different fathers were randomly distributed across the mother's brood space. All of these genetic findings are discussed in the light of observations on the mating behaviours and reproductive biology of crayfish.

A genetic assessment of parentage in a natural population of dollar sunfish (Lepomis marginatus) based on microsatellite markers.

We employ microsatellite markers to assess mating tactics in Lepomis marginatus. Genetic assignments for 1015 progeny in 23 nests indicate that about 95% of the offspring were sired by their respective nest-guardians, a finding consistent with the apparent absence of a brood parasitic morphotype in this species. Allopaternal care was documented in two nests, one resulting from a nest takeover, the other from cuckoldry by an adjoining nest-tender. Clustered de novo mutations also were identified. About 2.5 females (range 1-7) contributed to the offspring pool within a typical nest. Results are compared to those for other Lepomis species.

Genetic evidence for extreme polyandry and extraordinary sex-role reversal in a pipefish.

Due to the phenomenon of male pregnancy, the fish family Syngnathidae (seahorses and pipefishes) has historically been considered an archetypal example of a group in which sexual selection should act more strongly on females than on males. However, more recent work has called into question the idea that all species with male pregnancy are sex-role reversed with respect to the intensity of sexual selection. Furthermore, no studies have formally quantified the opportunity for sexual selection in any natural breeding assemblage of pipefishes or seahorses in order to demonstrate conclusively that sexual selection acts most strongly on females. Here, we use a DNA-based study of parentage in the Gulf pipefish Syngnathus scovelli in order to show that sexual selection indeed acts more strongly on females than on males in this species. Moreover, the Gulf pipefish exhibits classical polyandry with the greatest asymmetry in reproductive roles (as quantified by variances in mating success) between males and females yet documented in any system. Thus, the intensity of sexual selection on females in pipefish rivals that of any other taxon yet studied.

Evolving genomic metaphors: a new look at the language of DNA.

Recent genome-sequencing efforts have confirmed that traditional "good-citizen" genes (those that encode functional RNA and protein molecules of obvious benefit to the organism) constitute only a small fraction of the genomic populace in humans and other multicellular creatures. The rest of the DNA sequence includes an astonishing collection of noncoding regions, regulatory modules, deadbeat pseudogenes, legions of repetitive elements, and hosts of oft-shifty, self-interested nomads, renegades, and immigrants. To help visualize functional operations in such intracellular genomic societies and to better encapsulate the evolutionary origins of complex genomes, new and evocative metaphors may be both entertaining and research-stimulating.

Genetic markers substantiate long-term storage and utilization of sperm by female painted turtles.

Most studies of genetic parentage in natural populations have been limited to a single breeding season or reproductive episode and, thus, provide only a snapshot of individuals' mating behaviours. Female turtles can store viable sperm in their reproductive tracts for as long as several years, but the extent to which this capacity is utilized in nature has remained unknown. Here, we employ microsatellite markers to assess genetic paternity in successive clutches of individually marked, free-ranging female painted turtles (Chrysemys picta) over a four year period. The genetic data from 113 clutches from this natural population demonstrate that most females (80.5%) remated each year and that each female generally used a single male's sperm to fertilize all clutches laid within a year. However, sperm usage among females varied considerably, and some females apparently used sperm that had been stored for up to three years to fertilize some or all eggs laid in consecutive nesting seasons. Thus, remating by females is not necessary for continued offspring production from a given sire. Furthermore, 13.2% of all clutches examined showed evidence of multiple paternity, and the genetic paternity patterns across years suggest a 'last in, first out' operation of the females' sperm storage tubules.

How cuckoldry can decrease the opportunity for sexual selection: data and theory from a genetic parentage analysis of the sand goby, Pomatoschistus minutus.

Alternative mating strategies are common in nature and are generally thought to increase the intensity of sexual selection. However, cuckoldry can theoretically decrease the opportunity for sexual selection, particularly in highly polygamous species. We address here the influence of sneaking (fertilization thievery) on the opportunity for sexual selection in the sand goby Pomatoschistus minutus, a marine fish species in which males build and defend nests. Our microsatellite-based analysis of the mating system in a natural sand goby population shows high rates of sneaking and multiple mating by males. Sneaker males had fertilized eggs in approximately 50% of the assayed nests, and multiple sneakers sometimes fertilized eggs from a single female. Successful males had received eggs from 2 to 6 females per nest (mean = 3.4). We developed a simple mathematical model showing that sneaking in this polygynous sand goby population almost certainly decreases the opportunity for sexual selection, an outcome that contrasts with the usual effects of cuckoldry in socially monogamous animals. These results highlight a more complex and interesting relationship between cuckoldry rates and the intensity of sexual selection than previously assumed in much of the literature on animal mating systems.

The genetic mating system and tests for cuckoldry in a pipefish species in which males fertilize eggs and brood offspring externally.

Highly variable microsatellite loci were used to study the mating system of Nerophis ophidion, a species of pipefish in which pregnant males carry embryos on the outside of their body rather than in an enclosed brood pouch. Despite this mode of external fertilization and brooding, otherwise rare in the family Syngnathidae, the genotypes of all embryos proved to be consistent with paternity by the tending male, thus indicating that cuckoldry by sneaker males is rare or nonexistent in this species. N. ophidion is a phylogenetic outlier within the Syngnathidae and its reproductive morphology is thought to be close to the presumed ancestral condition for pipefishes and seahorses. Thus, our genetic results suggest that the evolutionary elaboration of the enclosed brood pouch elsewhere in the family was probably not in response to selection pressures on pregnant males to avoid fertilization thievery. With regard to maternity assignments, our genotypic data are consistent with behavioural observations indicating that females sometimes mate with more than one male during a breeding episode, and that each male carries eggs from a single female. Thus, the polyandrous genetic mating system in this species parallels the social mating system, and both are consistent with a more intense sexual selection operating on females, and the elaboration of secondary sexual characters in that gender.

Accuracy and precision of methods to estimate the number of parents contributing to a half-sib progeny array.

Molecular technologies have made feasible large-scale studies of genetic parentage in nature by permitting the genotypic examination of hundreds or thousands of progeny. One common goal of such studies is to estimate the true number of unshared parents who contributed to a large half-sib progeny array. Here we introduce computer programs designed to count the number of gametotypes contributed by unshared parents to each such progeny array, as well as assess the accuracy and precision of various estimators for the true number of unshared parents via computer simulation. These simulations indicate that under most biological conditions (1) a traditional approach (the multilocus MINIMUM METHOD) that merely counts the number of distinct haplotypes in offspring and divides by 2L, where L is the number of loci assayed, often vastly underestimates the true number of unshared parents who contributed to a half-sib progeny array; (2) a recently developed HAPLOTYPES estimator is a considerable improvement over the MINIMUM METHOD when parental numbers are high; and (3) the accuracy and precision of the HAPLOTYPES estimator increase as marker polymorphism and sample size increase, or as reproductive skew and the number of parents contributing to the progeny array decrease. Generally, HAPLOTYPES-based estimates of parental numbers in large half-sib cohorts should improve the characterization of organismal reproductive strategies and mating systems from genetic data.

Mating systems and sexual selection in male-pregnant pipefishes and seahorses: insights from microsatellite-based studies of maternity.

In pipefishes and seahorses (family Syngnathidae), the males provide all postzygotic care of offspring by brooding embryos on their ventral surfaces. In some species, this phenomenon of male "pregnancy" results in a reversal of the usual direction of sexual selection, such that females compete more than males for access to mates, and secondary sexual characteristics evolve in females. Thus the syngnathids can provide critical tests of theories related to the evolution of sex differences and sexual selection. Microsatellite-based studies of the genetic mating systems of several species of pipefishes and seahorses have provided insights into important aspects of the natural history and evolution of these fishes. First, males of species with completely enclosed pouches have complete confidence of paternity, as might be predicted from parental investment theory for species in which males invest so heavily in offspring. Second, a wide range of genetic mating systems have been documented in nature, including genetic monogamy in a seahorse, polygynandry in two species of pipefish, and polyandry in a third pipefish species. The genetic mating systems appear to be causally related to the intensity of sexual selection, with secondary sex characters evolving most often in females of the more polyandrous species. Third, genetic studies of captive-breeding pipefish suggest that the sexual selection gradient (or Bateman gradient) may be a substantially better method for characterizing the mating system than previously available techniques. Finally, these genetic studies of syngnathid mating systems have led to some general insights into the occurrence of clustered mutations at microsatellite loci, the utility of linked loci in studies of parentage, and the use of parentage data for direct estimation of adult population size.

Genetic perspectives on the natural history of fish mating systems.

Molecular analyses of bird and mammal populations have shown that social mating systems must be distinguished from genetic mating systems. This distinction is important in fishes also, where the potential for extrapair spawning and intraspecific brood parasitism is especially great. We review studies on fishes that have used molecular markers to document biological parentage and genetic mating systems in nature, particularly in species with extended parental care of offspring. On average, nest-guarding adults parented about 70-95% of their custodial offspring, and approximately one-third of the nests were cuckolded to some extent. Furthermore, nearly 10% of the assayed nests contained offspring tended by foster fathers either because of nest takeovers or egg thievery. On average, fish that provide parental care on nests spawned with more mates than did fish with internal fertilization and pregnancy. Overall, genetic markers have both confirmed and quantified the incidence of several reproductive and other social behaviors of fishes, and have thereby enhanced our knowledge of piscine natural history.

Turtle mating systems: behavior, sperm storage, and genetic paternity.

As evidenced by the articles in this volume, a recent increase in interest in the mating systems of poikilothermic vertebrates has focused primarily on fishes, a few amphibians, and squamate reptiles. Turtles by contrast have received relatively little attention, yet they display a wide variety of mating behaviors and life-history characteristics that make them excellent candidates for addressing several aspects of genetic parentage that should contribute to a broader understanding of animal reproductive strategies. Here we focus on genetic studies of the mating systems and reproductive patterns of turtles, specifically with respect to multiple paternity and long-term sperm storage. These phenomena highlight the importance of a temporally extended perspective on patterns of individual reproductive success.