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Hoxa2 genes provide critical patterning signals during development, and their regulation and function have been extensively studied. We report a previously uncharacterized significant sequence divergence of a highly conserved hindbrain hoxa2b enhancer element in the family syngnathidae (pipefishes, seahorses, pipehorses, seadragons). We compared the hox cis-regulatory element variation in the Gulf pipefish and two species of seahorse against eight other species of fish, as well as human and mouse. We annotated the hoxa2b enhancer element binding sites across three species of seahorse, four species of pipefish, and one species of ghost pipefish. Finally, we performed in situ hybridization analysis of hoxa2b expression in Gulf pipefish embryos. We found that all syngnathid fish examined share a modified rhombomere 4 hoxa2b enhancer element, despite the fact that this element has been found to be highly conserved across all vertebrates examined previously. Binding element sequence motifs and spacing between binding elements have been modified for the hoxa2b enhancer in several species of pipefish and seahorse, and that the loss of the Prep/Meis binding site and further space shortening happened after ghost pipefish split from the rest of the syngnathid clade. We showed that expression of this gene in rhombomere 4 is lower relative to the surrounding rhombomeres in developing Gulf pipefish embryos, reflecting previously published functional tests for this enhancer. Our findings highlight the benefits of studying highly derived, diverse taxa for understanding of gene regulatory evolution and support the hypothesis that natural mutations can occur in deeply conserved pathways in ways potentially related to phenotypic diversity.

Within Syngnathidae (pipefish, seadragons and seahorses), male pregnancy often results in choosy males and competitive females. Females in these species often evolve secondary sexual traits and engage in courtship displays that make their ornaments more noticeable to males. Most syngnathids probably continue to grow larger throughout their lives, but we know little about the relationship between age and mating competition in these taxa. Here, we use the Gulf pipefish, Syngnathus scovelli, to investigate the roles of ornament size, courtship activity level, age and fecundity in female mating competition. We conducted male choice trials that allowed males to choose between similarly sized females of different ages. We also measured age and size at maturity. We found that females with larger ornaments were deeper bodied and engaged in longer courtship displays, yet males chose females based on body depth and display rather than ornamentation. This result suggests that ornamentation serves to help males assess female quality. Female age plays no role in male choice or in female ornamentation. Our finding that males care more about female phenotype than female age considerably simplifies the interpretation of mating patterns in natural populations of Gulf pipefish, which are characterized by considerable age structure.

How organisms adapt to the novel challenges imposed by the colonization of a new habitat has long been a central question in evolutionary biology. When multiple populations of the same species independently adapt to similar environmental challenges, the question becomes whether the populations have arrived at their adaptations through the same genetic mechanisms. In recent years, genetic techniques have been used to tackle these questions by investigating the genome-level changes underlying local adaptation. Here, we present a genomic analysis of colonization of freshwater habitats by a primarily marine fish, the Gulf pipefish (Syngnathus scovelli). We sample pipefish from four geographically distinct freshwater locations and use double-digest restriction site associated DNA sequencing to compare them to 12 previously studied saltwater populations. The two most geographically distant and isolated freshwater populations are the most genetically distinct, although demographic analysis suggests that these populations are experiencing ongoing migration with their saltwater neighbours. Additionally, outlier regions were found genome-wide, showing parallelism across ecotype pairs. We conclude that these multiple freshwater colonizations involve similar genomic regions, despite the large geographical distances and different underlying mechanisms. These similar patterns are probably facilitated by the interacting effects of intrinsic barriers, gene flow among populations and ecological selection in the Gulf pipefish.

A major goal of evolutionary biology is to identify the genome-level targets of natural and sexual selection. With the advent of next-generation sequencing, whole-genome selection components analysis provides a promising avenue in the search for loci affected by selection in nature. Here, we implement a genome-wide selection components analysis in the sex role reversed Gulf pipefish, Syngnathus scovelli. Our approach involves a double-digest restriction-site associated DNA sequencing (ddRAD-seq) technique, applied to adult females, nonpregnant males, pregnant males, and their offspring. An FST comparison of allele frequencies among these groups reveals 47 genomic regions putatively experiencing sexual selection, as well as 468 regions showing a signature of differential viability selection between males and females. A complementary likelihood ratio test identifies similar patterns in the data as the FST analysis. Sexual selection and viability selection both tend to favor the rare alleles in the population. Ultimately, we conclude that genome-wide selection components analysis can be a useful tool to complement other approaches in the effort to pinpoint genome-level targets of selection in the wild.

Sexual dimorphism often results from hormonally regulated trait differences between the sexes. In sex-role-reversed vertebrates, females often have ornaments used in mating competition that are expected to be under hormonal control. Males of the sex-role-reversed Gulf pipefish (Syngnathus scovelli) develop female-typical traits when they are exposed to estrogens. We aimed to identify genes whose expression levels changed during the development and maintenance of female-specific ornaments. We performed RNA-sequencing on skin and muscle tissue in male Gulf pipefish with and without exposure to estrogen to investigate the transcriptome of the sexually dimorphic ornament of vertical iridescent bands found in females and estrogen-exposed males. We further compared differential gene expression patterns between males and females to generate a list of genes putatively involved in the female secondary sex traits of bands and body depth. A detailed analysis of estrogen-receptor binding sites demonstrates that estrogen-regulated genes tend to have nearby cis-regulatory elements. Our results identified a number of genes that differed between the sexes and confirmed that many of these were estrogen-responsive. These estrogen-regulated genes may be involved in the arrangement of chromatophores for color patterning, as well as in the growth of muscles to achieve the greater body depth typical of females in this species. In addition, anaerobic respiration and adipose tissue could be involved in the rigors of female courtship and mating competition. Overall, this study generates a number of interesting hypotheses regarding the genetic basis of a female ornament in a sex-role-reversed pipefish.

A major goal of molecular ecology is to identify the causes of genetic and phenotypic differentiation among populations. Population genomics is suitably poised to tackle these key questions by diagnosing the evolutionary mechanisms driving divergence in nature. Here, we set out to investigate the evolutionary processes underlying population differentiation in the Gulf pipefish, Syngnathus scovelli. We sampled approximately 50 fish from each of 12 populations distributed from the Gulf coast of Texas to the Atlantic coast of Florida and performed restriction-site-associated DNA sequencing to identify SNPs throughout the genome. After imposing quality and stringency filters, we selected a panel of 6348 SNPs present in all 12 populations, 1753 of which were not physically linked. We identified a genome-wide pattern of isolation by distance, in addition to a more substantial genetic break separating populations in the Gulf of Mexico from those in the Atlantic. We also used several divergence outlier approaches and tests for genotype-environment correlations to identify 400 SNPs putatively involved in local adaptation. Patterns of phenotypic differentiation and variation diverged from the overall genomic pattern, suggesting that selection, phenotypic plasticity or demographic factors may be shaping phenotypes in distinct populations. Overall, our results suggest that population divergence is driven by a variety of factors in S. scovelli, including neutral processes and selection on multiple traits.

The trade-offs of using single-digest vs. double-digest restriction site-associated DNA sequencing (RAD-seq) protocols have been widely discussed. However, no direct empirical comparisons of the two methods have been conducted. Here, we sampled a single population of Gulf pipefish (Syngnathus scovelli) and genotyped 444 individuals using RAD-seq. Sixty individuals were subjected to single-digest RAD-seq (sdRAD-seq), and the remaining 384 individuals were genotyped using a double-digest RAD-seq (ddRAD-seq) protocol. We analysed the resulting Illumina sequencing data and compared the two genotyping methods when reads were analysed either together or separately. Coverage statistics, observed heterozygosity, and allele frequencies differed significantly between the two protocols, as did the results of selection components analysis. We also performed an in silico digestion of the Gulf pipefish genome and modelled five major sources of bias: PCR duplicates, polymorphic restriction sites, shearing bias, asymmetric sampling (i.e., genotyping fewer individuals with sdRAD-seq than with ddRAD-seq) and higher major allele frequencies. This combination of approaches allowed us to determine that polymorphic restriction sites, an asymmetric sampling scheme, mean allele frequencies and to some extent PCR duplicates all contribute to different estimates of allele frequencies between samples genotyped using sdRAD-seq versus ddRAD-seq. Our finding that sdRAD-seq and ddRAD-seq can result in different allele frequencies has implications for comparisons across studies and techniques that endeavour to identify genomewide signatures of evolutionary processes in natural populations.

Background Evolutionary origins of derived morphologies ultimately stem from changes in protein structure, gene regulation, and gene content. A well-assembled, annotated reference genome is a central resource for pursuing these molecular phenomena underlying phenotypic evolution. We explored the genome of the Gulf pipefish (Syngnathus scovelli), which belongs to family Syngnathidae (pipefishes, seahorses, and seadragons). These fishes have dramatically derived bodies and a remarkable novelty among vertebrates, the male brood pouch. Results We produce a reference genome, condensed into chromosomes, for the Gulf pipefish. Gene losses and other changes have occurred in pipefish hox and dlx clusters and in the tbx and pitx gene families, candidate mechanisms for the evolution of syngnathid traits, including an elongated axis and the loss of ribs, pelvic fins, and teeth. We measure gene expression changes in pregnant versus non-pregnant brood pouch tissue and characterize the genomic organization of duplicated metalloprotease genes (patristacins) recruited into the function of this novel structure. Phylogenetic inference using ultraconserved sequences provides an alternative hypothesis for the relationship between orders Syngnathiformes and Scombriformes. Comparisons of chromosome structure among percomorphs show that chromosome number in a pipefish ancestor became reduced via chromosomal fusions. Conclusions The collected findings from this first syngnathid reference genome open a window into the genomic underpinnings of highly derived morphologies, demonstrating that de novo production of high quality and useful reference genomes is within reach of even small research groups. Electronic supplementary material The online version of this article (doi:10.1186/s13059-016-1126-6) contains supplementary material, which is available to authorized users.

Sexual selection is responsible for the evolution of some of the most elaborate traits occurring in nature, many of which play a vital role in competition over access to mates and individual reproductive fitness. Because expression of these traits is typically regulated by sex-steroids there is a significant potential for their expression to be affected by the presence of certain pollutants, such as endocrine disrupting compounds. Endocrine disruptors have been shown to alter primary sexual traits and impact reproduction, but few studies have investigated how these compounds affect secondary sexual trait expression and how that may, in turn, impact mating dynamics. In this study we examine how short-term exposure to a synthetic estrogen impacts secondary sexual trait expression and mating dynamics in the Gulf pipefish, a species displaying sex-role reversal. Our results show that only 10 days of exposure to 17α-ethinylestradiol results in adult male pipefish developing female-like secondary sexual traits. While these males are capable of reproduction, females discriminate against exposed males in mate choice trials. In natural populations, this type of discrimination would reduce male mating opportunities, thus potentially reducing their long-term reproductive success. Importantly, the effects of these compounds on mating dynamics and mating opportunity would not be observed using the current standard methods of assessing environmental contamination. However, disrupting these processes could have profound effects on the viability of exposed populations.

The evolution of complex traits, which are specified by the interplay of multiple genetic loci and environmental effects, is a topic of central importance in evolutionary biology. Here, we show that body and tail vertebral numbers in fishes of the pipefish and seahorse family (Syngnathidae) can serve as a model for studies of quantitative trait evolution. A quantitative genetic analysis of body and tail vertebrae from field-collected families of the Gulf pipefish, Syngnathus scovelli, shows that both traits exhibit significantly positive additive genetic variance, with heritabilities of 0.75 ± 0.13 (mean ± standard error) and 0.46 ± 0.18, respectively. We do not find any evidence for either phenotypic or genetic correlations between the two traits. Pipefish are characterized by male pregnancy, and phylogenetic consideration of body proportions suggests that the position of eggs on the pregnant male's body may have contributed to the evolution of vertebral counts. In terms of numbers of vertebrae, tail-brooding males have longer tails for a given trunk size than do trunk-brooding males. Overall, these results suggest that vertebral counts in pipefish are heritable traits, capable of a response to selection, and they may have experienced an interesting history of selection due to the phenomenon of male pregnancy. Given that these traits vary among populations within species as well as among species, they appear to provide an excellent model for further research on complex trait evolution. Body segmentation may thus afford excellent opportunities for comparative study of homologous complex traits among disparate vertebrate taxa.

The gulf pipefish,Syngnathus scovelli, is the dominant syngnathid found in coastal regions of the Gulf of Mexico and is the only species in this region known to occur in both freshwater and saltwater habitats. Relatively little is known about the population and reproductive cycles ofS. scovelli, particularly for those found in low salinity environments. The focus of the current study was to collect population structure and reproductive data forS. scovelli from a low salinity habitat, Meaher Park, located at the mouth of Mobile Bay, Alabama. Sampling was conducted twice a month from January 2003 to January 2004. Environmental parameters, as well as population and reproductive parameters, were collected. Water temperature was the primary abiotic factor associated with both the appearance ofS. scovelli and their breeding cycle. Based on gonadosomatic (GSI) and brood pouch somatic indices (BPSI), females and males were reproductively active throughout the summer. Peaks in male GSI and BPSI were consistent with the subsequent appearance of a large number of juveniles in early fall. These event coincided with the higher temperatures seen during late summer and early fall. Sex ratios (male : female 1 : 1.64) and operational sex ratios (1 : 4.09) indicated that this was a female biased population. Although there was no difference in the average size of males and females in this population, the largest individuals collected were female. The female-biased sex ratio supports previous genetic analyses suggesting thatS. scovelli exhibits true sex-role reversal and that operational sex ratios are a reliable indicator of the intensity of mating competition and sex roles.

Syngnathid fishes (pipefishes, seahorses and seadragons) are characterized by a unique mode of paternal care in which embryos develop on or in the male’s body, often within a structure known as a brood pouch. Evidence suggests that this pouch plays a role in mediating postcopulatory sexual selection and that males have some control over the events occurring within the pouch during the pregnancy. These observations lead to the prediction that males should invest differently in broods depending on the availability of food. Here, we use the Gulf pipefish to test this prediction by monitoring growth rate and offspring survivorship during the pregnancies of males under low- or high-food conditions. Our results show that pregnant males grow less rapidly on average than non-pregnant males, and pregnant males under low-food conditions grow less than pregnant males under high-food conditions. Offspring survivorship, on the other hand, does not differ between food treatments, suggesting that male Gulf pipefish sacrifice investment in somatic growth, and thus indirectly sacrifice future reproduction, in favor of current reproduction. However, a positive relationship between number of failed eggs and male growth rate in our low-food treatments suggests that undeveloped eggs reduce the pregnancy’s overall cost to the male compared to broods containing only viable offspring.

Understanding how selection acts on traits individually and in combination is an important step in deciphering the mechanisms driving evolutionary change, but for most species, and especially those in which sexual selection acts more strongly on females than on males, we have no estimates of selection coefficients pertaining to the multivariate sexually selected phenotype. Here, we use a laboratory-based mesocosm experiment to quantify pre- and post-mating selection on female secondary sexual traits in the Gulf pipefish (Syngnathus scovelli), a sexually dimorphic, sex-role-reversed species in which ornamented females compete for access to choosy males. We calculate selection differentials and gradients on female traits, including ornament area, ornament number and body size for three episodes of selection related to female reproductive success (number of mates, number of eggs transferred and number of surviving embryos). Selection is strong on both ornament area and ornament size, and the majority of selection occurs during the premating episode of selection. Interestingly, selection on female body size, which has been detected in previous studies of Gulf pipefish, appears to be indirect, as evidenced by a multivariate analysis of selection gradients. Our results show that sexual selection favours either many bands or larger bands in female Gulf 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.

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.

This study attempts to assess the relationship between fecundity and fertility of males and females of the gulf pipefish, Syngnathus scovelli. Specimens of this pipefish were collected in detached algae in the inner surf-zone of a dissipative sandy beach in southeastern Brazil. 97 specimens were collected, and they were associates to detached algae. From these, 14 were males, 36 females and 47 immature. Male-female sex ratio was 0.4:1, which was significantly different from the expected 1:1 ratio. Fish ranged from 43 to 98 mm TL (Fig. 1), and males and females showed similar size. Only 7 males were pregnants, while 9 females had hydrated oocytes. Minimum number of eggs/embryos in the pouch was 30 and the maximum 92. Minimum number of hydrated oocytes was 36, and the maximum was 165. The number of egg/embryos, and hydrated oocytes were weakly related to total length. It seems that the gulf pipefish occupy the inner surf-zone of the Praia do Iate are brought to the shallows accidentally when the specimens were inhabiting drifting vegetation. Este estudo avalia a relação entre fecundidade e fertilidade de machos e fêmeas do peixe-cachimbo, Syngnathus scovelli, o qual ocorre em algas destacadas na zona de arrebentação de uma praia arenosa com ondas do tipo dissipativa em Vitória, ES. Foram coletados 97 exemplares, sendo todos associados com algas destacadas. Destes, 14 eram machos, 36 fêmeas e 47 imaturos. A razão sexual macho/fêmea foi de 0,4:1, a qual foi significativamente diferente da esperada razão sexual de 1:1. O peixe-cachimbo variou de 43 a 98 mm de comprimento total, e machos e fêmeas tiveram tamanhos similares. Somente 7 machos tinham ovos na bolsa incubadora, enquanto 9 fêmeas apresentaram ovócitos hidratados. O número mínimo de ovos/embriões na bolsa incubadora foi 30 e o máximo 92. O número mínimo de ovócitos hidratados foi 36 e o máximo 165. O número de ovos/embriões e ovócitos hidratados foram fracamente relacionados ao comprimento total. Parece que os exemplares de S. scovelli que habitam a parte interna da zona de arrebentação da Praia do Iate são trazidos para as partes rasas involuntariamente quando os espécimens estão ocupando algas destacadas.

Highly variable microsatellite loci were employed to study the mating system of the sexually dimorphic Gulf pipefish Syngnathus scovelli. In this species, like others in the family Syngnathidae, 'pregnant' males provide all parental care. Gulf pipefish were collected from one locale in the northern Gulf of Mexico, and internally carried broods of 40 pregnant males were analysed genetically. By comparing multilocus microsatellite fingerprints for the inferred mothers against expected genotypic distributions from the population sample, it was determined that: (i) only one male had received eggs from more than a single female; and (ii) on two separate occasions, two different males had received eggs from the same female. Given the high power to detect multiple matings by males, the first finding indicates that only rarely are individual males impregnated by multiple females during the course of a pregnancy. Conversely, given the lower power to detect multiple matings by females due to sampling constraints, the second finding suggests a high frequency of multiple successful matings by females. Thus, this population of Gulf pipefish displays a polyandrous genetic mating system. The relevance of these genetic findings is discussed with regard to the evolution of secondary sex traits in this species, and in other syngnathids.

The male pregnancy of pipefishes and seahorses has led to the inference that females compete most intensely for access to mates, because males limit female reproduction. However, recent work has shown that in different species either sex may be the predominant competitor for mates. In this family, there is an apparent association between the mating pattern and the sex roles: polygamous species show reversed sex roles whereas monogamous species exhibit 'conventional' sex roles. These studies emphasize that sex role reversal is not synonymous with male parental care.

Arising from: K. A. Paczolt & A. G. Jones , 401–404 (2010)10.1038/nature08861 ; Paczolt & Jones reply Sexual differences in the extent and type of parental care lie at the heart of sexual selection theory1, and evolution resulting from parental conflict has produced some striking behavioural and morphological adaptations. In a study of male pregnancy in Gulf pipefish, Paczolt and Jones2 showed that more eggs were transferred to the male’s brood pouch and more offspring survived following mating with large females (preferred by males) than with small (less preferred) females. Although the authors conclude that the lower survival of embryos from small females is most consistent with males actively removing resources from these offspring2,3,4, no data are presented to directly support this hypothesis (ref. 2, and Supplementary Information therein) and the data do not refute the alternative explanation that differential egg survival is caused by female effects mediated by variation in fecundity and egg size or quality. We argue that only by experimentally manipulating female attractiveness separately from the quality of eggs deposited in the brood pouch can the extent of sexual conflict in this role-reversed system be assessed.

Sexual selection theory predicts that members of the choosy sex, usually females, should employ multiple sensory systems to obtain information about potential mates. Such predictions should also apply to systems in which sexual selection acts most strongly on females (i.e. sex-role-reversed species), and males of these taxa should be the individuals employing multiple cues to assess female attractiveness. Very little work has been directed toward mate choice involving multiple sensory systems in sex-role-reversed taxa, but fishes of the family Syngnathidae (pipefishes, seahorses, and sea dragons) provide an excellent opportunity to contribute to this research enterprise. While much is known about visual communication in pipefish, the role of chemical communication has not been investigated. Using dichotomous choice tests, we found that male, but not female, Gulf pipefish attended to chemical cues of opposite sex conspecifics. Given that males distinguished sex on the basis of chemical cues, we also tested whether males could assess female body size, an important trait with respect to mate choice in pipefish, on the basis of chemical cues alone. When given the choice between chemical cues produced by large vs. small females, males exhibited no preference. Our results suggest that male pipefish can use chemical cues to distinguish between males and females but not to differentiate females of different body size.

Male mate choice and female-female competition for access to mates were studied in the pipefish Nerophis ophidion , with the aim of evaluating the function and importance of female size and ornamental skin folds. Nerophis ophidion is sex role-reversed in the sense that paternal care limits female reproductive success. Furthermore, females are larger, possess a sexual coloration, and develop an ornamental skin fold during the spawning season. Females are also more active than males during courtship. In mate-choice experiments males actively preferred both larger females and females with larger skin folds. When several females were kept together, only one female in each group developed a large skin fold. Females interacting only with males invariably developed skin folds whereas isolated females did not. The skin fold affects choice of females by males, and since females are socially stimulated to develop their skin fold, interaction with conspecifics indirectly affects male mate choice. Female-female interactions may suppress the development of the skin fold. Hence, both active male mate choice based upon female secondary sexual characters and female-female interactions determine the non-random mating pattern observed.

Publisher Summary The chapter presents the problem in two sex role-reversed pipefish species. The chapter discusses why potential reproductive rates differ and the consequences of this “reversed” mating competition on sexually selected traits in females. Potential reproductive rates—that is, how fast males and females can potentially remate, given that partner availability is not limiting, and operational sex ratios, that is, the proportion of males and females willing to mate are instrumental in understanding this pattern. In two sex role-reversed pipefish species, Syngnathus typhle and Nerophis ophidion, males devoted more time, but not more energy, than females to offspring production. The chapter outlines the costs and benefits of these sexually selected traits and mating preferences to allow for a better understanding of sexual selection processes in sex role-reversed species and understands the very same processes in species with conventional sex roles. The chapter explains that sex role reversal and female ornamentation seem intimately linked with paternal care and how that paternal care may have evolved in syngnathids and how sex role reversal in pipefish is ultimately a consequence of an extreme level of paternal care in combination with a polygamous mating pattern are discussed.

In the dusky pipefish Syngnathus floridae, like other species in the family Syngnathidae, 'pregnant' males provide all post-zygotic care. Male pregnancy has interesting implications for sexual selection theory and the evolution of mating systems. Here, we employ microsatellite markers to describe the genetic mating system of S. floridae, compare the outcome with a previous report of genetic polyandry for the Gulf pipefish S. scovelli, and consider possible associations between the mating system and degree of sexual dimorphism in these species. Twenty-two pregnant male dusky pipefish from one locale in the northern Gulf of Mexico were analyzed genetically, together with subsamples of 42 embryos from each male's brood pouch. Adult females also were assayed. The genotypes observed in these samples document that cuckoldry by males did not occur; males often receive eggs from multiple females during the course of a pregnancy (six males had one mate each, 13 had two mates, and three had three mates); embryos from different females are segregated spatially within a male's brood pouch; and a female's clutch of eggs often is divided among more than one male. Thus, the genetic mating system of the dusky pipefish is best described as polygynandrous. The genetic results for S. floridae and S. scovelli are consistent with a simple model of sexual selection which predicts that for sex role-reversed organisms, species with greater degrees of sexual dimorphism are more highly polyandrous.

Replying to: D. T. Gwynne, K. A. Judge & C. D. Kelly , 10.1038/nature09275 (2010) Our recent study of pre- and post-copulatory sexual selection in the sex-role-reversed Gulf pipefish indicates that males prefer to mate with larger females compared to smaller females, that larger females transfer more eggs per copulation than smaller females, that the eggs from larger females are more likely to result in viable offspring than eggs from smaller females, and that males experience fitness trade-offs between broods1. We suggest that the most likely explanation for this suite of results is that males exert choice before and after copulation in a way that favours larger females over smaller females during every phase of pre- and post-copulatory sexual selection. Gwynne et al.2 have challenged the interpretation of one facet of our results by suggesting that males do not differentially allocate resources to offspring originating from different females. Rather, they suggest that our results can be explained entirely by differences among females in fecundity, egg size and egg quality2.

In order to understand the causes of sexual dimorphism, mate choice and size-related fecundity were studied in two pipefish species, Syngnathus typhle and Nerophis ophidion. Sexual dimorphism is more pronounced in N. ophidion; females are larger, have sexual colourings, and are more active during courtship. In S. typhle the sexes are alike in all these respects. Males brood their offspring in both species. In N. ophidion fecundity was positively correlated with both body size and the amount of sexual colouring in females. In males no correlation between body size and fecundity, or between body size and embryo size existed. Predictably, in mate choice experiments with equal-sized females, males chose females with more extensive sexual colourings. We explain sexual dimorphism in this species as a consequence of both natural selection (fecundity increases with size in females but not in males) and sexual selection (males prefer larger females). We argue that sexual size dimorphism did not evolve by selection minimizing overlap in food niches between the sexes, because food production is high in the Zostera beds where the fishes live, and no size dimorphism was found in the sympatrically occurring S. typhle. Furthermore, in N. ophidion dimorphism is not greater in a particular mouth character than in overall body size. In S. typhle egg size and the average number of eggs transferred per spawning were positively correlated with female body size. Apparently more energy per offspring was provided by larger males than by smaller males, and larger males also carried more offspring. As predicted, large mates were preferred by both sexes in mate choice experiments. This is explicable in terms of both natural selection (fecundity increases with size in both sexes) and sexual selection (both sexes prefer large mates). As a consequence of selection acting in the same direction in both sexes, sexual dimorphism is absent in S. typhle.

The fundamental difference between the sexes is one of the size of the gametes and of the energy allocated to them. The female produces the larger gamete, the egg, and the male the smaller gamete, the sperm. This difference has often been used to explain other differences between the sexes. Traditionally, the female has been viewed as a scarce and limiting resource for the male, thus promoting the evolution of traits in males that attract females or that enhance their ability to compete for mates. Thus, sexual dimorphism and "sex roles" are believed to stem from the unequal investment in offspring by the two sexes (Bateman 1948; Trivers 1972; Williams 1975; Wilson 1975; Dawkins 1976; Wittenberger 1981; Daly and Wilson 1983). In some cases, however, males are believed to limit the reproductive success of females, and we would then expect a reversal of the "usual" gender roles (e.g., Knowlton 1982): females may be larger and more ornamented, and they may compete for access to males. Such cases offer a test of the hypothesis regarding the relationship between the limiting sex and sex roles or secondary sexual characters. Role reversals have been documented in a number

Repetitive seine and monthly drop net samples taken in a seagrass bed in the Indian River, Florida, indicate that significant relationships exist between the abundances of both the code goby Gobiosoma robustum and the gulf pipefish Syngnathus scovelli and drift algae biomass. We suggest that drift algae provides a refuge from predation which increases in effectiveness with increased algal biomass, and that this interaction, in concert with habitat preference and food availability, is responsible for the relationships observed.

Reversed sex-roles and parental energy investment in zygotes of two pipefish (Syngnathidae) species

Changes in available food and utilized foods for a densely populated guild of animals can uncover periods of niche displacement among particular consumer species or their separate size classes. Dense populations of small fishes and their foods were censussed for 13 months and studied experimentally in shoalgrass and turtlegrass meadows of Redfish Bay, Texas. Feeding habits were determined with respect to prey availability, and illustrated the extent to which seasonal partitioning of food corresponded to food depletion among these abundant consumers in seagrass meadow food webs. The darter goby, code goby, pinfish, and Gulf pipefish were the most common species throughout the year. Although the darter goby did not show distinct ontogenetic changes in food habits, the code goby, Gulf pipefish, and pinfish demonstrated major ontogenetic progressions of foods selected. Food availability in the seagrass meadows changed seasonally. When major prey such as amphipods were abundant, during spring, many fish species showed high overlap in food use. Regardless of food availability, the code goby and Gulf pipefish fed mainly on amphipods or copepods. The more common darter goby and pinfish seasonally changed their diets with changes in food availability; the darter goby and pinfish were more carnivorous during spring, but they largely consumed epiphytic algae during summer. Cage experiments were used to monitor foods confined with elevated densities of pinfish and darter gobies, relative to control cages at natural overall densities. Prey items in the former cages decreased sharply, with corresponding dietary shifts by these common fishes. During resource depletion, these changes in resource use by these naturally concentrated consumers appeared as temporary partitioning of available resources. This shift occurred during both natural (seasonal) and experimental depletions of food, and appears to result from increased interspecific and intraspecific competition during periods of depleted preferred foods.

1. 1. Male pipefish, Syngnathus fuscus , with embryos were injected with a radioactively labelled non-metabolizable amino acid and accumulation of the acid monitored in the developing embryos. 2. 2. Evidence is presented for the transfer of the amino acid from paternal tissue to developing young.