Male pregnancy in seahorses and pipefishes (family Syngnathidae): rapid diversification of paternal brood pouch morphology inferred from a molecular phylogeny

In contrast to the majority of vertebrate species, primary male parental care is common in fishes and encompasses a remarkable diversity of adaptations. Seahorses and pipefishes (Family Syngnathidae) exhibit some of the most specialized forms of paternal care in animals and so are ideally suited to the study of the evolution of male parental care. During mating, female syngnathids transfer eggs to specialized morphological structures that are located on either the abdomen or tail of the male. The male provides all postfertilization parental care and has morphological and physiological adaptations to osmoregulate, aerate, and even nourish the developing embryos. While all syngnathid species are adapted for paternal care, the brooding structure with which this is accomplished varies between species, from simple ventral gluing areas to much more complex structures such as the completely enclosed pouches of the seahorses. Our combined cytochrome b-, 12S rDNA-, and 16S rDNA-based molecular phylogeny of syngnathid fishes demonstrates that rapid diversification of male brooding structures has been associated with the major evolutionary radiation of the group, suggesting that development and diversification of structures involved in paternal care may have been key evolutionary innovations of the Syngnathidae. Molecular analyses also highlight geographical centers of biodiversity and suggest interoceanic migration of Syngnathus pipefishes from their center of origin in the Pacific.     

The dynamics of male brooding,mating patterns,and sex roles in pipefishes and seahorses (family Syngnathidae)

Modern theory predicts that relative parental investment of the sexes in their young is a key factor responsible for sexual selection. Seahorses and pipefishes (family Syngnathidae) are extraordinary among fishes in their remarkable adaptations for paternal care and frequent occurrences of sex-role reversals (i.e., female-female competition for mates), offering exceptional opportunities to test predictions of sexual selection theory. During mating, the female transfers eggs into or onto specialized egg-brooding structures that are located on either the male's abdomen or its tail, where they are osmoregulated, aerated, and nourished by specially adapted structures. All syngnathid males exhibit this form of parental care but the brooding structures vary, ranging from the simple ventral gluing areas of some pipefishes to the completely enclosed pouches found in seahorses. We present a molecular phylogeny that indicates that the diversification of pouch types is positively correlated with the major evolutionary radiation of the group, suggesting that this extreme development and diversification of paternal care may have been an important evolutionary innovation of the Syngnathidae. Based on recent studies that show that the complexity of brooding structures reflects the degree of paternal investment in several syngnathid species, we predicted sex-role reversals to be more common among species with more complex brooding structures. In contrast to this prediction, however, both parsimony- and likelihood-based reconstructions of the evolution of sex-role reversal in pipefishes and seahorses suggest multiple shifts in sex roles in the group, independent from the degree of brood pouch development. At the same time, our data demonstrate that sex-role reversal is positively associated with polygamous mating patterns, whereas most nonreversed species mate monogamously, suggesting that selection for polygamy or monogamy in pipefishes and seahorses may strongly influence sex roles in the wild.     

The genetics and genomics of Syngnathidae: pipefishes, seahorses and seadragons

The goal of this review was to provide a historical overview of how molecular techniques have increased the understanding of the ecology and evolution of the family Syngnathidae (pipefishes, seahorses and seadragons). Molecular studies based primarily on mitochondrial DNA markers have proved their worth by elucidating complex phylogenetic relationships within the family. Phylogeographic studies, which have revealed how life-history traits and past climatic events shape geographic distributions and patterns of genetic variation within syngnathid species, also provide interesting case studies for the conservation and management of threatened species. The application of microsatellite DNA markers has opened a floodgate of studies concerned with the breeding biology of these fishes, which are interesting due to their unique reproductive mode of male pregnancy. Research in this area has contributed significantly to the understanding of mating patterns and sexual selection. Molecular markers may also be employed in studies of demography, migration and local breeding population sizes. Genomic studies have identified genes that are probably involved in male pregnancy and promise additional insights into various aspects of syngnathid biology at the level of the gene. Despite these advances, much more remains to be explored. Goals for future research should include: (1) a more inclusive phylogeny to resolve outstanding issues concerning the relationships within the family and higher order taxa, (2) a broader use of molecular studies to aid management and conservation efforts, (3) the inclusion of more genera in comparative behavioural studies and (4) the continued development of genomic resources for syngnathids to facilitate comparative genomic work.     

Comparative Analyses of Reproductive Structures in Harvestmen (Opiliones) Reveal Multiple Transitions from Courtship to Precopulatory Antagonism

Explaining the rapid, species-specific diversification of reproductive structures and behaviors is a long-standing goal of evolutionary biology, with recent research tending to attribute reproductive phenotypes to the evolutionary mechanisms of female mate choice or intersexual conflict. Progress in understanding these and other possible mechanisms depends, in part, on reconstructing the direction, frequency and relative timing of phenotypic evolution of male and female structures in species-rich clades. Here we examine evolution of reproductive structures in the leiobunine harvestmen or “daddy long-legs” of eastern North America, a monophyletic group that includes species in which males court females using nuptial gifts and other species that are equipped for apparent precopulatory antagonism (i.e., males with long, hardened penes and females with sclerotized pregenital barriers). We used parsimony- and Bayesian likelihood-based analyses to reconstruct character evolution in categorical reproductive traits and found that losses of ancestral gift-bearing penile sacs are strongly associated with gains of female pregenital barriers. In most cases, both events occur on the same internal branch of the phylogeny. These coevolutionary changes occurred at least four times, resulting in clade-specific designs in the penis and pregenital barrier. The discovery of convergent origins and/or enhancements of apparent precopulatory antagonism among closely related species offers an unusual opportunity to investigate how major changes in reproductive morphology have occurred. We propose new hypotheses that attribute these enhancements to changes in ecology or life history that reduce the duration of breeding seasons, an association that is consistent with female choice, sexual conflict, and/or an alternative evolutionary mechanism.     

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.     

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.     

The courtship behaviour of the pipefish Nerophis lumbriciformis: reflections of an adaptation to intertidal life

The courtship behaviour of Nerophis lumbriciformis (Pisces: Syngnathidae) consists of three distinct phases (initial courtship, spawning, and embrace) marked by prominent behavioural changes. The first courtship phase is characterised by female quivering. Courtship activity increases from low to high levels of quivering, causing undulatory head movements in the female. In the second phase, the female transfers her eggs onto the male incubating ventral surface. During the final phase, the male wraps his body around the female (embrace). Females actively initiate courtship indicating that these pipefishes are courtship role reversed. The complete lack of swimming and vertical movements in the courtship behaviour of N. lumbriciformis, unlike in the behaviour of other syngnathid species, suggests an adaptation to intertidal conditions.     

Evolution of seahorses' upright posture was linked to Oligocene expansion of seagrass habitats

Seahorses (Syngnathidae: Hippocampus) are iconic marine teleosts that are readily identifiable by their upright posture. The fossil record is inadequate to shed light on the evolution of this trait because it lacks transitional forms. There are, however, extant syngnathid species (the pygmy pipehorses) that look like horizontally swimming seahorses and that might represent a surviving evolutionary link between the benthic seahorses and other, free-swimming members of the family Syngnathidae. Using sequence data from five nuclear loci, we confirm the sister taxon relationship between seahorses and pygmy pipehorses. Molecular dating indicates that the two taxa diverged during the Late Oligocene. During this time, tectonic events in the Indo-West Pacific resulted in the formation of vast amounts of new shallow-water areas and associated expansion of seagrass habitats that would have favoured the seahorses’ upright posture by improving their camouflage while not affecting their manoeuvrability negatively. The molecular techniques employed here provide new insights into the evolution of a taxon whose fossil record is incomplete, but whose evolutionary history is so recent that the major stages of morphological evolution are still represented in extant species.     

Male pregnancy in seahorses and pipefish: beyond the mammalian model

Pregnancy has been traditionally defined as the period during which developing embryos are incubated in the body after egg-sperm union. Despite strong similarities between viviparity in mammals and other vertebrate groups, researchers have historically been reluctant to use the term pregnancy for non-mammals in recognition of the highly developed form of viviparity in eutherians. Syngnathid fishes (seahorses and pipefishes) have a unique reproductive system, where the male incubates developing embryos in a specialized brooding structure in which they are aerated, osmoregulated, protected and likely provisioned during their development. Recent insights into physiological, morphological and genetic changes associated with syngnathid reproduction provide compelling evidence that male incubation in these species is a highly specialized form of reproduction akin to other forms of viviparity. Here, we review these recent advances, highlighting similarities and differences between seahorse and mammalian pregnancy. Understanding the changes associated with the parallel evolution of male pregnancy in the two major syngnathid lineages will help to identify key innovations that facilitated the development of this unique form of reproduction and, through comparison with other forms of live bearing, may allow the identification of a common set of characteristics shared by all viviparous organisms.     

Convergent camouflage and the non‐monophyly of ‘seadragons’ (Syngnathidae: Teleostei): suggestions for a revised taxonomy of syngnathids

Wilson, N. G. & Rouse, G. W. (2010). Convergent camouflage and the non‐monophyly of ‘seadragons’ (Syngnathidae: Teleostei): suggestions for a revised taxonomy of syngnathids. —Zoologica Scripta, 39, 551–558. The phylogeny and classification of the charismatic Syngnathidae (e.g. pipefish, seahorses) has not been comprehensively examined to date. In particular, we assessed morphological hypotheses that previously suggested the three ‘seadragon’ genera (Phycodurus, Phyllopteryx, Haliichthys) do not form a monophyletic group. We used three mitochondrial markers to investigate evolutionary relationships within Syngnathidae, and demonstrated that Phycodurus + Phyllopteryx formed a clade that excluded Haliichthys, indicating the elaborate appendages used for camouflage have evolved independently. A time‐calibrated tree revealed the divergence of true seadragons as coincident with other kelp‐associated fauna. We found evidence for the resurrection of neglected subfamily names, and recovered Doryrhampinae, Nerophinae, Soleganthinae, Phyllopteryginae, Sygnathoidinae and Haliichthyinae as clades. Even after removing these groups from what is currently recognized as Syngnathinae, we showed that the remaining members of Syngnathinae are not monophyletic. In the light of this information, some conclusions about the diversity of reproductive strategies found within ‘Syngnathinae’ need to be re‐examined and further revision of syngnathid classification is needed.     

Male pregnancy and the evolution of body segmentation in seahorses and pipefishes

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.     

Sexual signals and mating patterns in Syngnathidae

Male pregnancy in the family Syngnathidae (pipefishes, seahorses and seadragons) predisposes males to limit female reproductive success; sexual selection may then operate more strongly on females and female sexual signals may evolve (sex-role reversal). A bewildering array of female signals has evolved in Syngnathids, e.g. skin folds, large body size, colouration, markings on the body and elaborate courtship. These female sexual signals do not seem quantitatively or qualitatively different from those that evolve in males in species with conventional sex roles where males provide females or offspring with direct benefits. In several syngnathid species, males also evolve ornaments, females are choosy in addition to being competitive and males compete as well as choosing partners. Thus, sex roles form a continuum, spanning from conventional to reversed within this group of fishes. Cases are presented here suggesting that stronger sexual selection on females may be most extreme in species showing classical polyandry (one male mates with several females, such as many species where males brood their eggs on the trunk), intermediate in polygynandrous species (males and females both mate with more than one partner, as in many species where males brood their eggs on the tail) and least extreme, even exhibiting conventional sex roles, in monogamous species (one male mates solely with one female, as in many seahorses and tropical pipefishes). At the same time caution is needed before unanimously establishing this pattern: first, the connection between mating patterns, strength of sexual selection, sex roles and ornament expression is far from simple and straightforward, and second, knowledge of the actual morphology, ecology and behaviour of most syngnathid species is scanty. Basically only a few Nerophis, Syngnathus and Hippocampus species have been studied in any detail. It is known, however, that this group of fishes exhibits a remarkable variation in sex roles and ornamentation, making them an ideal group for the study of mating patterns, sexual selection and sexually selected signals.     

The evolutionary origins of Syngnathidae: pipefishes and seahorses

Despite their importance as evolutionary and ecological model systems, the phylogenetic relationships among gasterosteiforms remain poorly understood, complicating efforts to understand the evolutionary origins of the exceptional morphological and behavioural diversity of this group. The present review summarizes current knowledge on the origin and evolution of syngnathids, a gasterosteiform family with a highly developed form of male parental care, combining inferences based on morphological and molecular data with paleontological evidence documenting the evolutionary history of the group. Molecular methods have provided new tools for the study of syngnathid relationships and have played an important role in recent conservation efforts. Despite recent insights into syngnathid evolution, however, a survey of the literature reveals a strong taxonomic bias towards studies on the species-rich genera Hippocampus and Syngnathus, with a lack of data for many morphologically unique members of the family. The study of the evolutionary pressures responsible for generating the high diversity of syngnathids would benefit from a wider perspective, providing a comparative framework in which to investigate the evolution of the genetic, morphological and behavioural traits of the group as a whole.     

Comparative phylogenetic analysis of male alternative reproductive tactics in ray-finned fishes

Using comparative phylogenetic analysis, we analyzed the evolution of male alternative reproductive tactics (MARTs) in ray-finned fishes (Actinopterygii). Numerous independent origins for each type of MART (involving sneaker males, female mimics, pirates, and satellite males) indicate that these behaviors have been highly labile across actinopterygiian evolution, consistent with a previous notion that convergent selection in fishes can readily mold the underlying suites of reproductive hormones into similar behaviors. The evolutionary appearance of MARTs was significantly correlated with the presence of sexually selected traits in bourgeois males (P = 0.001) but not with the presence of male parental care. This suggests that MARTs often arise from selection on some males to circumvent bourgeois male investment in mate monopolization, rather than to avoid male brood care per se. We found parsimony evidence for an evolutionary progression of MARTs wherein sneaking is usually the evolutionary precursor to the presumably more complex MARTs of female mimicry and cooperative satellite behavior. Nest piracy appears not to be part of this evolutionary progression, possibly because its late onset in the life cycle of most ray-finned fishes reduces the effects of selection on this reproductive tactic.     

Sexual size dimorphism predicts the frequency of multiple mating in the sex-role reversed pipefish Syngnathus typhle

The sex-role reversed pipefish Syngnathus typhle is a member of the Syngnathidae, a family of fishes in which males brood embryos on their body surface. As in most ectotherms, embryonic development is highly temperature dependent in syngnathids and male brooding periods are extended when water temperatures are reduced. The influence of temperature on reproduction is expected to effectively truncate the breeding season and reduce fecundity in cold waters, potentially enhancing the opportunity for both fecundity and sexual selection. We studied spatial variation in the morphology and reproductive biology of S. typhle in five European populations which vary in latitude and water temperature. Microsatellite analyses indicated that the average number of male mates per population ranged between 1.3 and 3.7. The frequency of multiple mating by males was negatively correlated with the degree of sexual size dimorphism in each population, suggesting that disproportionate increases in female fecundity may be able to compensate for increased male brood pouch capacity. Both sexes were larger and males had an increased brood size where water temperatures during the breeding season were lower. Morphological variation among populations may be mediated by differences in fecundity selection associated with different optimal reproductive strategies in cold and warm water environments.     

SEX‐SPECIFIC INBREEDING DEPRESSION DEPENDS ON THE STRENGTH OF MALE–MALE COMPETITION

Inbreeding depression has become a central theme in evolutionary biology and is considered to be a driving force for the evolution of reproductive morphology, physiology, behavior, and mating systems. Despite the overwhelming body of empirical work on the reproductive consequences of inbreeding, relatively little is known on whether inbreeding depresses male and female fitness to the same extent. However, sex‐specific inbreeding depression has been argued to affect the evolution of selfing rates in simultaneous hermaphrodites and provides a powerful approach to test whether selection is stronger in males than in females, which is predicted to be the consequence of sexual selection. We tested for sex‐specific inbreeding depression in the simultaneously hermaphroditic freshwater snail Physa acuta by comparing the reproductive performance of both sex functions between selfed and outcrossed focal individuals under different levels of male–male competition. We found that inbreeding impaired both male and female reproductive success and that the magnitude of male inbreeding depression exceeded female inbreeding depression when the opportunity for sperm competition was highest. Our study provides the first evidence for sex‐specific inbreeding depression in a hermaphroditic animal and highlights the importance of considering the level of male–male competition when assessing sex differences in inbreeding depression.     

Toward a New Sexual Selection Paradigm: Polyandry, Conflict and Incompatibility (Invited Article)

Darwin's recognition that male–male competition and female choice could favor the evolution of exaggerated male traits detrimental to survival set the stage for more than a century of theoretical and empirical work on sexual selection. While this Darwinian paradigm represents one of the most profound insights in biology, its preoccupation with sexual selection as a directional evolutionary force acting on males has diverted attention away from the selective processes acting on females. Our understanding of female reproduction has been further confounded by discreet female mating tactics that have perpetuated the illusion of the monogamous female and masked the potential for conflict between the sexes. With advances in molecular techniques leading to the discovery that polyandry is a pervasive mating strategy, recognition of these shortcomings has brought the study of sexual selection to its current state of flux. In this paper, we suggest that progress in two key areas is critical to formulation of a more inclusive, sexual selection paradigm that adequately incorporates selection from the female perspective. First, we need to develop a better understanding of male × female and maternal × paternal genome interactions and the role that polyandry plays in providing females with non‐additive genetic benefits such as incompatibility avoidance. Consideration of these interaction effects influencing natural selection on females is important because they can complicate and even undermine directional sexual selection on males. Secondly, because antagonistic coevolution maintains a balance between opposing sides that obscures the conflict itself, many more experimental evolution studies and interventionist investigations (e.g. gene knockouts) are needed to tease apart male manipulative adaptations and female counter‐adaptations. It seems evident that the divisiveness and controversy that has plagued sexual selection theory since Darwin first proposed the idea has often stalled progress in this important field of evolutionary biology. What is now needed is a more pluralistic and integrative approach that considers natural as well as sexual selection acting on females, incorporates multiple sexual selection mechanisms, and exploits advances in physiology and molecular biology to understand the mechanisms through which males and females achieve reproductive success.     

Diet‐dependent female evolution influences male lifespan in a nuptial feeding insect

Theory predicts that lifespan will depend on the dietary intake of an individual, the allocation of resources towards reproduction and the costs imposed by the opposite sex. Although females typically bear the majority of the cost of offspring production, nuptial feeding invertebrates provide an ideal opportunity to examine the extent to which reproductive interactions through gift provisioning impose a cost on males. Here we use experimental evolution in an Australian ground cricket to assess how diet influences male lifespan and how the costs of mating evolve for males. Our findings show that males had significantly shorter lifespans in populations that adapted to a low‐quality diet and that this divergence is driven by evolutionary change in how females interact with males over reproduction. This suggests that the extent of sexual conflict over nuptial feeding may be under‐realized by focusing solely on the consequences of reproductive interactions from the female’s perspective.     

Genetic covariance between components of male reproductive success: within‐pair vs. extra‐pair paternity in song sparrows

The evolutionary trajectories of reproductive systems, including both male and female multiple mating and hence polygyny and polyandry, are expected to depend on the additive genetic variances and covariances in and among components of male reproductive success achieved through different reproductive tactics. However, genetic covariances among key components of male reproductive success have not been estimated in wild populations. We used comprehensive paternity data from socially monogamous but genetically polygynandrous song sparrows (Melospiza melodia) to estimate additive genetic variance and covariance in the total number of offspring a male sired per year outside his social pairings (i.e. his total extra‐pair reproductive success achieved through multiple mating) and his liability to sire offspring produced by his socially paired female (i.e. his success in defending within‐pair paternity). Both components of male fitness showed nonzero additive genetic variance, and the estimated genetic covariance was positive, implying that males with high additive genetic value for extra‐pair reproduction also have high additive genetic propensity to sire their socially paired female's offspring. There was consequently no evidence of a genetic or phenotypic trade‐off between male within‐pair paternity success and extra‐pair reproductive success. Such positive genetic covariance might be expected to facilitate ongoing evolution of polygyny and could also shape the ongoing evolution of polyandry through indirect selection.