Phylogeography and demography of sympatric sister surfperch species, Embiotoca jacksoni and E. lateralis along the California coast: historical versus ecological factors

With 18 closely related endemic species that radiated in a diversity of ecological niches, the California surfperches (Embiotocidae) species flock is a good candidate for the study of sympatric speciation. Resource partitioning has been suggested as an important driving force in the radiation of the surfperch family. Within the family, two congeneric sister species, Embiotoca jacksoni and E. lateralis, are known to compete strongly for a preferred single food resource and may be used as a model of ecological interactions for the family. Along the California coast, the distribution of the two species differs. Embiotoca jacksoni has a continuous range, whereas E. lateralis shows a disjunction with a distribution gap in the Southern California Bight. Two hypotheses may explain this disjunct distribution. Ecological competition may have displaced E. lateralis in favor of E. jacksoni. Alternatively, a common vicariant event may have separated the species into northern and southern populations, followed by secondary contact in E. jacksoni but not in E. lateralis. The two hypotheses predict different phylogeographic and demographic signatures. Using a combined phylogeographic and coalescent approach based on mitochondrial control region data, we show that vicariance can only account for a portion of the observed divergences. Our results are compatible with a significant role played by ecological competition in the southern range of the species.     

Molecular Evidence for High Frequency of Multiple Paternity in a Freshwater Shrimp Species Caridina ensifera

Background

Molecular genetic analyses of parentage provide insights into mating systems. Although there are 22,000 members in Malacostraca, not much has been known about mating systems in Malacostraca. The freshwater shrimp Caridina ensifera blue, is a new species belonging to Malacostraca which was discovered recently in Sulawesi, Indonesia. Due to its small body size and low fecundity, this species is an ideal species to study the occurrence and frequency of multiple paternity and to understand of how the low fecundity species persist and evolve.

Methodology/Principal Findings

In this study, we developed four polymorphic microsatellites from C. ensifera and applied them to investigate the occurrence and frequency of multiple paternity in 20 C. ensifera broods caught from Lake Matano, Sulawesi. By genotyping the mother and all offspring from each brood we discovered multiple paternity in all 20 broods. In most of the 20 broods, fathers contributed skewed numbers of offspring and there was an apparent inverse correlation between reproductive success of sires and their relatedness to mothers.

Conclusions/Significance

Our results in combination with recent reports on multiple paternity in crayfish, crab and lobster species suggests that multiple paternity is common in Malacostraca. Skewed contribution of fathers to the numbers of offspring and inverse correlation between reproductive success of sires and their relatedness to mothers suggest that sperm competition occurred and/or pre- and postcopulatory female choice happen, which may be important for avoiding the occurrence of inbreeding and optimize genetic variation in offspring and for persistence and evolution of low fecundity species.     

Molecular evidence for multiple paternity in a feral population of green swordtails

Genetic parentage analyses provide insights into mating systems and have revealed widespread evidence for polyandry in natural populations. Here, we use 5 microsatellite markers to estimate female mating rates in a feral population of green swordtails, Xiphophorus helleri, a live-bearing poeciliid fish that has become a model system in the study of precopulatory mate choice and mating competition. Although heralded as a potential model for investigating sperm competition as early as 1950, there has been no attempt to explore postcopulatory sexual selection in its mating system. We thus obtained information on the prevalence, and therefore biological relevance, of polyandry from a wild population. We genotyped the offspring from 14 wild-caught gravid females and determined the number of fathers in each brood using allele counting methods and the programs GERUD and PARENTAGE. Our analyses revealed that 57% (allele counts and GERUD) and 71% (PARENTAGE) of the sampled broods had at least 2 sires, with a global mean 1.74 fathers per brood. Paternity skew was generally high in mixed paternity broods so that our analyses almost certainly underestimate actual mating frequencies in the wild. Our data provide a solid underpinning for future studies of postcopulatory sexual selection in this species.     

High prevalence of multiple paternity in the invasive crayfish species,Procambarus clarkii

Reproductive strategy is a central feature of the ecology of invasive species as it determines the potential for population increase and range expansion. The red swamp crayfish, Procambarus clarkii, has invaded many countries and caused serious problems in freshwater ecosystems. However, little is known about the effects of environmental conditions on crayfish paternity and offspring traits in the wild. We studied these reproductive characteristics of P. clarkii in wild populations from two different habitats (ponds and ditches) in three locations with different environmental conditions in China. Genotyping of 1,436 offspring and 30 mothers of 30 broods was conducted by using four microsatellites. An analysis of genotyping results revealed that gravid females were the exclusive mother of the progeny they tended. Twenty-nine of 30 mothers had mated with multiple (2-4) males, each of which contributed differently to the number of offspring in a brood. The average number of fathers per brood and the number of offspring per brood were similar (P > 0.05) among six sampling sites, indicating that in P. clarkii multiple paternity and offspring number per brood are independent of environmental conditions studied. Indirect benefits from increasing the genetic diversity of broods, male and sperm competition, and cryptic female choice are a possible explanation for the high level multiple paternity and different contribution of fathers to offspring in this species.     

Multiple paternity is a shared reproductive strategy in the live‐bearing surfperches (Embiotocidae) that may be associated with female fitness

According to Bateman's principle, female fecundity is limited relative to males, setting the expectation that males should be promiscuous, while females should be choosy and select fewer mates. However, several surfperches (Embiotocidae) exhibit multiple paternity within broods indicating that females mate with multiple males throughout the mating season. Previous studies found no correlation between mating success and reproductive success (i.e., a Bateman gradient). However, by including samples from a broader range of reproductive size classes, we found evidence of a Bateman gradient in two surfperch species from distinct embiotocid clades. Using microsatellite analyses, we found that 100% of the spotfin surfperch families sampled exhibit multiple paternity (Hyperprosopon anale, the basal taxon from the only clade that has not previously been investigated) indicating that this tactic is a shared reproductive strategy among surfperches. Further, we detected evidence for a Bateman gradient in H. anale; however, this result was not significant after correction for biases. Similarly, we found evidence for multiple paternity in 83% of the shiner surfperch families (Cymatogaster aggregata) sampled. When we combine these data with a previous study on the same species, representing a larger range of reproductive size classes and associated brood sizes, we detect a Bateman gradient in shiner surfperch for the first time that remains significant after several conservative tests for bias correction. These results indicate that sexual selection is likely complex in this system, with the potential for conflicting optima between sexes, and imply a positive shift in fertility (i.e., increasing number) and reproductive tactic with respect to the mating system and number of sires throughout the reproductive life history of females. We argue that the complex reproductive natural history of surfperches is characterized by several traits that may be associated with cryptic female choice, including protracted oogenesis, uterine sac complexity, and sperm storage.     

Variation in multiple paternity in natural populations of a free-spawning marine invertebrate

For free-spawning marine invertebrates, fertilization processes control the genetic diversity of offspring. Each egg can potentially be fertilized by a sperm from a different male, and hence genetic diversity within a brood varies with levels of multiple paternity. Yet, few studies have characterized the frequency of multiple paternity in natural spawns. We analysed patterns of multiple paternity in two populations of the colonial ascidian Botryllus schlosseri using microsatellites. Because previous studies have shown that at moderate to high population densities, competition among male-phase B. schlosseri colonies results in the nearest male dominating the paternity of a brood, we specifically tested the effect of population density on patterns of paternity. Paternity was estimated using three multilocus indices: minimum number of fathers, counts of sperm haplotypes, and effective paternity (K(E)). Multiple paternity was evident in more than 92% of the broods analysed, but highly variable, with a few broods displaying unequal contributions of different males. We found no effect of population density on multiple paternity, suggesting that other factors may control paternity levels. Indirect benefits from increasing the genetic diversity of broods are a possible explanation for the high level of multiple paternity in this species.     

Same school,different conduct: rates of multiple paternity vary within a mixed‐species breeding school of semi‐pelagic cichlid fish (Cyprichromis spp.)

Mating system variability is known to exist between and within species, often due to environmental influences. An open question is whether, vice versa, similar environmental conditions entail congruent mating behavior, for example in terms of multiple paternity, in species or populations sharing largely comparable breeding modes. This study employed microsatellite markers to investigate the incidence of multiple paternity in Cyprichromis coloratus and Cyprichromis leptosoma, two sympatric, closely related, mouthbrooding Lake Tanganyika cichlids with similar ecological and behavioral characteristics including the formation of open‐water schools. Mouthbrooding females of both species were collected from the same mixed‐species breeding school at the same time, minimizing environmental variation during courtship and mating. In C. coloratus, four of 12 broods had more than one sire, with a mean of 1.33 reconstructed sires per brood. C. leptosoma exhibited multiple paternity in 18 of 22 broods, with a mean of 2.59 or 2.86 reconstructed sires per brood according to the programs gerud and colony , respectively. In addition, two broods were found to contain offspring transplanted from another brood. There was no significant difference in brood size between species, but mean sire number did differ significantly. Hence, substantial similarity in reproductive behavior along with shared environmental conditions during courtship and spawning did not lead to equal rates of polyandry or sneaking in the two species.     

The name of the father: conflict between Louis and Alexander Agassiz and the Embiotoca surfperch radiation

The surfperch genus Embiotoca currently comprises two species, Embiotoca jacksoni , the black surfperch, and Embiotoca lateralis , the striped surfperch. Originally, however, Louis Agassiz described a third species in the genus Embiotoca , the rainbow surfperch, Embiotoca caryi . This latter name was changed by Louis' son, Alexander, to Hypsurus caryi , a name that remains valid. In this study, new molecular data (3545 bp of DNA from four mitochondrial and two nuclear DNA regions) indicated that the rainbow surfperch should be retained within the genus Embiotoca , a result consistent with recent morphological data. Adaptive radiation combined with sexual selection resulting in rapid morphological changes in the rainbow surfperch may have contributed to the conflicting position of this species.     

Evidence of multiple paternity in the bluntnose klipfish, <Emphasis Type="Italic">Clinus cottoides</Emphasis> (Blennioidei: Clinidae: Clinini)

Clinids of the tribe Clinini are viviparous fishes occurring in the temperate waters of southern Africa. Females of the genus Clinus exhibit reproductive traits suggesting multiple paternity. These traits include prolonged gestation periods, matrotrophy and superfetation. We tested the hypothesis that broods of the species C. cottoides are sired by multiple males with the use of microsatellites. We genotyped three broods from known mothers and analysed the relationships within each brood using the software program COLONY, we also used allele counting to find evidence of multiple paternity. Our results revealed that multiple paternity occurred in all three broods analysed; two broods likely had two sires and one brood three sires. One male appear to have fathered offspring with separate females. Our study represents the first molecular analysis of parentage in C. cottoides.     

High frequency of multiple paternity in broods of a socially monogamous cichlid fish with biparental nest defence

In several animal taxa, genetic analyses have demonstrated that social monogamy and biparental brood care do not preclude polygamous reproduction. Few studies have been conducted in fish, but in fish species without alternative reproductive phenotypes, social monogamy was largely congruent with genetic parentage. In contrast to these findings, we report an exceptionally high level of multiple paternity in a socially monogamous cichlid fish with biparental nest defence ( Variabilichromis moorii ), inferred from microsatellite and mitochondrial data of 10 broods. Whereas all offspring in a nest shared a common mother, each brood was sired by 2 to > 10 males. None of the inferred sires was assigned a large proportion of the brood. Paternity was estimated as the minimum number of sires required to explain multilocus offspring genotypes, and as the maximum-likelihood number of sires given population allele frequencies. Analysis of simulated brood genotypes suggested that, although these two methods tend to under- and overestimate, respectively, the true number of sires, primary sires with many offspring in a brood would have been detected. Hence, the genetic data indicate that the nest tending males suffer substantial cuckoldry and provide alloparental care for a large number of unrelated fry. We have no data on the social status of the cuckolding males, but due to synchronous spawning of pairs and commitment to brood care of paired males, it is possible that most of the parasitic spawners are solitary males.     

In the surf zone: Reproductive strategy of the calico surfperch (Amphistichus koelzi) in a comparative context

We examined the reproductive life history of calico surfperch (Amphistichus koelzi), including mating season, pregnancy, gestation and multiple paternity utilizing restriction site-associated DNA sequencing. Furthermore, we compared the mating season of calico with barred (Amphistichus argenteus), walleye (Hyperprosopon argenteum) and silver (Hyperprosopon ellipticum) surfperches to determine if the timing of reproduction is divergent within and between the genera. In calico surfperch, the mating season occurs from October to November, and females gestate from December to May. All broods exhibit multiple paternity with a range of four to seven sires per brood. The mating season of calico overlaps completely with barred surfperch; however, barred surfperches have a protracted mating season which extends until the beginning of December, which may be due to differences in reproductive strategy such as size at first reproduction. In the genus, the Hyperprosopon mating season begins earlier than Amphistichus, with divergence in the onset of mating between Hyperprosopon congeners of approximately 1 month.     

Multiple paternity in an aggregate breeding amphibian: the effect of reproductive skew on estimates of male reproductive success

Aggregate, or explosive, breeding is widespread among vertebrates and likely increases the probability of multiple paternity. We assessed paternity in seven field-collected clutches of the explosively breeding spotted salamander (Ambystoma maculatum) using 10 microsatellite loci to determine the frequency of multiple paternity and the number of males contributing to a female's clutch. Using the Minimum Method of allele counts, multiple paternity was evident in 70% of these egg masses. Simple allele counts underestimate the number of contributing males because this method cannot distinguish multiple fathers with common or similar alleles. Therefore, we used computer simulations to estimate from the offspring genotypes the most likely number of contributing fathers given the distributions of allele frequencies in this population. We determined that two to eight males may contribute to A. maculatum clutches; therefore, multiple paternity is a common strategy in this aggregate breeding species. In aggregate mating systems competition for mates can be intense, thus differential reproductive success (reproductive skew) among males contributing to a female's clutch could be a probable outcome. We use our data to evaluate the potential effect of reproductive skew on estimates of the number of contributing males. We simulated varying scenarios of differential male reproductive success, ranging from equal contribution to high reproductive skew among contributing sires in multiply sired clutches. Our data suggest that even intermediate levels of reproductive skew decrease confidence substantially in estimates of the number of contributing sires when parental genotypes are unknown.     

Microsatellite Evidence for High Frequency of Multiple Paternity in the Marine Gastropod Rapana venosa

Background

Inferring of parentage in natural populations is important in understanding the mating systems of a species, which have great effects on its genetic structure and evolution. Muricidae, a large group (approximately 1,600 species) of marine gastropods, are poorly investigated in patterns of multiple paternity and sperm competition based on molecular techniques. The veined Rapa whelk, Rapana venosa, a commercially important muricid species with internal fertilization, is an ideal species to study the occurrence and frequency of multiple paternity and to facilitate understanding of their reproductive strategies.

Methodology/Principal Findings

We developed five highly polymorphic microsatellites in R. venosa and applied them to identify multiple paternity in 19 broods (1381 embryos) collected from Dandong, China. Multiple paternity was detected in 17 (89.5%) of 19 broods. The number of sires per brood ranged from 1 to 7 (4.3 on average). Of the 17 multiply sired broods, 16 (94.1%) were significantly skewed from equal paternal contributions, and had a dominant sire which was also dominant in each assayed capsule.

Conclusions

Our results indicate that a high level of multiple paternity occurs in the wild population of R. venosa. Similar patterns of multiple paternity in the 2–6 assayed capsules from each brood imply that fertilization events within the body of a female occur mostly (but not entirely) as random draws from a “well-but-not-perfectly blended sperm pool” of her several mates. Strongly skewed distributions of fertilization success among sires also suggest that sperm competition and/or cryptic female choice might be important for post-copulatory paternity biasing in this species.     

Variation in multiple paternity and sperm utilization patterns in natural populations of a simultaneous hermaphrodite land snail

Mating frequency has important implications for patterns of sexual selection and sexual conflict, and hence for issues such as the maintenance of genetic diversity and speciation. We assessed the level of multiple paternity and sperm utilization patterns in four natural populations of the simultaneous hermaphrodite land snail Arianta arbustorum using four polymorphic microsatellite loci. A total of 1088 offspring from 26 wild‐caught snails were genotyped to determine the number of fathers siring each brood and paternity skew in succeeding clutches. Multiple paternity was detected in the offspring of all 26 mother snails examined with the contribution of two to six fathers. The four populations examined differed in the level of multiple paternity. Snails in the population with the highest density of adults showed the highest level of multiple paternity, whereas snails in the population with the lowest density exhibited the lowest value of multiple paternity. Highly skewed paternity patterns were found in the progeny of 15 (57.7%) of the 26 mother snails. The number and identity of fathers siring the offspring of single mothers also varied among successive clutches. Furthermore, genetic analyses indicate a low level of self‐fertilization in one of the four populations. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 99 , 350–361.     

High levels of multiple paternity in a spermcast mating freshwater mussel

Multiple paternity is an important characteristic of the genetic mating system and common across a wide range of taxa. Multiple paternity can increase within‐population genotypic diversity, allowing selection to act on a wider spectre of genotypes, and potentially increasing effective population size. While the genetic mating system has been studied in many species with active mating behavior, little is known about multiple paternity in sessile species releasing gametes into the water. In freshwater mussels, males release sperm into the water, while eggs are retained and fertilized inside the female (spermcast mating). Mature parasitic glochidia are released into the water and attach to the gills of fish where they are encapsulated until settling in the bottom substrate. We used 15 microsatellite markers to detect multiple paternity in a wild population of the freshwater pearl mussel (Margaritifera margaritifera). We found multiple paternity in all clutches for which more than two offspring were genotyped, and numbers of sires were extremely high. Thirty‐two sires had contributed to the largest clutch (43 offspring sampled). This study provides the first evidence of multiple paternity in the freshwater pearl mussel, a species that has experienced dramatic declines across Europe. Previous studies on other species of freshwater mussels have detected much lower numbers of sires. Multiple paternity in freshwater pearl mussels may be central for maintaining genetic variability in small and fragmented populations and for their potential to recover after habitat restoration and may also be important in the evolutionary arms race with their fish host with a much shorter generation time.     

Multiple paternity in loggerhead turtle (Caretta caretta) nests on Melbourne Beach, Florida: a microsatellite analysis

Many aspects of sea turtle biology are difficult to measure in these enigmatic migratory species, and this lack of knowledge continues to hamper conservation efforts. The first study of paternity in a sea turtle species used allozyme analysis to suggest multiple paternity in loggerhead turtle (Caretta caretta) clutches in Australia. Subsequent studies indicated that the frequency of multiple paternity varies from species to species and perhaps location to location. This study examined fine-scale population structure and paternal contribution to loggerhead clutches on Melbourne Beach, FL, USA using microsatellite markers. Mothers and offspring from 70 nests collected at two locations were analysed using two to four polymorphic microsatellite loci. Fine-scale population differentiation was not evident between the sampled locations, separated by 8 km. Multiple paternity was common in loggerhead nests on Melbourne Beach; 22 of 70 clutches had more than one father, and six had more than two fathers. This is the first time that more than two fathers have been detected for offspring in individual sea turtle nests. Paternal genotypes could not be assigned with confidence in clutches with more than two fathers, leaving the question of male philopatry unanswered. Given the high incidence of multiple paternity, we conclude that males are not a limiting resource for this central Florida nesting aggregate.     

PATTERNS OF PATERNITY SKEW AMONG POLYANDROUS SOCIAL INSECTS: WHAT CAN THEY TELL US ABOUT THE POTENTIAL FOR SEXUAL SELECTION?

Monogamy results in high genetic relatedness among offspring and thus it is generally assumed to be favored by kin selection. Female multiple mating (polyandry) has nevertheless evolved several times in the social Hymenoptera (ants, bees, and wasps), and a substantial amount of work has been conducted to understand its costs and benefits. Relatedness and inclusive fitness benefits are, however, not only influenced by queen mating frequency but also by paternity skew, which is a quantitative measure of paternity biases among the offspring of polyandrous females. We performed a large‐scale phylogenetic analysis of paternity skew across polyandrous social Hymenoptera. We found a general and significant negative association between paternity frequency and paternity skew. High paternity skew, which increases relatedness among colony members and thus maximizes inclusive fitness gains, characterized species with low paternity frequency. However, species with highly polyandrous queens had low paternity skew, with paternity equalized among potential sires. Equal paternity shares among fathers are expected to maximize fitness benefits derived from genetic diversity among offspring. We discuss the potential for postcopulatory sexual selection to influence patterns of paternity in social insects, and suggest that sexual selection may have played a key, yet overlooked role in social evolution.     

High paternal diversity in the self-incompatible herb Arabidopsis halleri despite clonal reproduction and spatially restricted pollen dispersal

The number of sires fertilizing a given dam is a key parameter of the mating system in species with spatially restricted offspring dispersal, since genetic relatedness among maternal sibs determines the intensity of sib competition. In flowering plants, the extent of multiple paternity is determined by factors such as floral biology, properties of the pollen vector, selfing rate, spatial organization of the population, and genetic compatibility between neighbours. To assess the extent of multiple paternity and identify ecological factors involved, we performed a detailed study of mating patterns in a small population of a self-incompatible clonal herb, Arabidopsis halleri . We mapped and genotyped 364 individuals and 256 of their offspring at 12 microsatellite loci and jointly analysed the level of multiple paternity, pollen and seed dispersal, and spatial genetic structure. We found very low levels of correlated paternity among sibs ( P _full-sib = 3.8%) indicating high multiple paternity. Our estimate of the outcrossing rate was 98.7%, suggesting functional self-incompatibility. The pollen dispersal distribution was significantly restricted (mean effective pollen dispersal distance: 4.42 m) but long-distance successful pollination occurred and immigrating pollen was at most 10% of all pollination events. Patterns of genetic structure indicated little extent of clonal reproduction, and a low but significant spatial genetic structure typical for a self-incompatible species. Overall, in spite of restricted pollen dispersal, the multiple paternity in this self-incompatible species was very high, a result that we interpret as a consequence of high plant density and high pollinator service in this population.     

Strong male‐biased operational sex ratio in a breeding population of loggerhead turtles (Caretta caretta) inferred by paternal genotype reconstruction analysis

Characterization of a species mating systems is fundamental for understanding the natural history and evolution of that species. Polyandry can result in the multiple paternity of progeny arrays. The only previous study of the loggerhead turtle (Caretta caretta) in the USA showed that within the large peninsular Florida subpopulation, multiple paternity occurs in approximately 30% of clutches. Our study tested clutches from the smaller northern subpopulation for the presence of multiple paternal contributions. We examined mothers and up to 20 offspring from 19.5% of clutches laid across three nesting seasons (2008–2010) on the small nesting beach on Wassaw Island, Georgia, USA. We found that 75% of clutches sampled had multiple fathers with an average of 2.65 fathers per nest (1–7 fathers found). The average number of fathers per clutch varied among years and increased with female size. There was no relationship between number of fathers and hatching success. Finally, we found 195 individual paternal genotypes and determined that each male contributed to no more than a single clutch over the 3‐year sampling period. Together these results suggest that the operational sex ratio is male‐biased at this site.     

Multiple paternity in Littorina obtusata (Gastropoda, Littorinidae) revealed by microsatellite analyses.

Parental identity for juvenile Littorina obtusata was determined from three egg masses by means of microsatellite DNA markers. Results confirm that the attendant adult female in each case was the dam of the offspring and that at least 4-6 males contributed to each brood. This correlates with our behavioral observations that indicated multiple copulations between the female and several males in each experimental aquarium. A significant number of offspring from each brood were sired by non-sampled males (males that had copulated with females before capture) whose sperm had been stored by the female. This is the first direct evidence of multiple paternity in the Littorinidae. Results are discussed in reference to current theories of sperm competition, male precedence, and cryptic female choice.