Male Intromittent Organ Research Articles

Quantifying Shape Variation in an Antisymmetrical Trait in the Tropical Fish Xenophallus umbratilis

Antisymmetry is a striking, yet puzzling form of biological asymmetry. The livebearing fish Xenophallus umbratilis exhibits antisymmetry in the male intromittent organ and provides a system that is well-suited for studying the nature of variation in antisymmetrical traits. Using geometric morphometrics, we test the hypothesis that because the gonopodium is critical to fitness there will not be significant differences in gonopodium shape between the two gonopodial morphs in this species. Our results are consistent with this prediction, though we found that gonopodium shape differed with gonopodium size.

First evidence of hemiclitores in snakes.

Female genitalia are conspicuously overlooked in comparison to their male counterparts, limiting our understanding of sexual reproduction across vertebrate lineages. This study is the first complete description of the clitoris (hemiclitores) in female snakes. We describe morphological variation in size and shape (n = 9 species, 4 families) that is potentially comparable to the male intromittent organs in squamate reptiles (hemipenes). Dissection, diffusible iodine contrast-enhanced micro-CT and histology revealed that, unlike lizard hemiclitores, the snake hemiclitores are non-eversible structures. The two individual hemiclitores are separated medially by connective tissue, forming a triangular structure that extends posteriorly. Histology of the hemiclitores in Australian death adders (Acanthophis antarcticus) showed erectile tissue and strands/bundles of nerves, but no spines (as is found in male hemipenes). These histological features suggest the snake hemiclitores have functional significance in mating and definitively show that the hemiclitores are not underdeveloped hemipenes or scent glands, which have been erroneously indicated in other studies. Our discovery supports that hemiclitores have been retained across squamates and provides preliminary evidence of differences in this structure among snake species, which can be used to further understand systematics, reproductive evolution and ecology across squamate reptiles.

External reproductive structures of male Surfperches (Embiotocidae): Urogenital papilla.

Fishes exhibit a dynamic array of reproductive diversity. Among the various forms of reproduction, internal fertilization has evolved at least 13 times and has been observed in 2-3% of teleost species, half of which are marine. Internal fertilization has led to the evolution of male intromittent organs to aid in sperm transfer during copulation. The intromittent organs of poeciliids have been widely investigated, but little research has focused on the intromittent organs of marine fishes. The surfperches are internally fertilizing, viviparous fishes with a ventrally located papilla. In this article, the external morphology of 19 Embiotocidae species was examined. Additionally, paraffin histology was used to investigate the walleye surfperch (Hyperprosopon argenteum), black perch (Embiotoca jacksoni), and dwarf perch (Micrometrus minimus) papilla to aid in the identification of tissue composition to determine its function. External morphology was documented via macro photography and males were dissected to observe the relationship between the testes, spermatic duct, and papilla. We provide histological evidence that the urogenital papilla is directly connected to the spermatic duct and testis and likely involved in spermatozeugmata transfer. Our research also determined that the papillae were composed of both urinary and reproductive tissues, thus we propose the papilla be referred to as a urogenital papilla. Our study contributes to an overall understanding of intromittent organs of marine fishes, particularly in the surfperches, which has not been previously documented.

Penetration mechanics of elongated female and male genitalia of earwigs

We unveiled the penile penetration mechanics of two earwig species, Echinosoma horridum, whose intromittent organ, termed virga, is extraordinarily long, and E. denticulatum, whose virga is conversely short. We characterised configuration, geometry, material and bending stiffness for both virga and spermatheca. The short virga of E. denticulatum has a material gradient with the stiffer base, whereas the long virga of E. horridum and the spermathecae of both species are homogeneously sclerotised. The long virga of E. horridum has a lower bending stiffness than the spermatheca. The virga of E. denticulatum is overall less flexible than the spermatheca. We compared our results to a previous study on the penetration mechanics of elongated beetle genitalia. Based on the comparison, we hypothesised that the lower stiffness of the male intromittent organ comparing to the corresponding female structure is a universal prerequisite for the penetration mechanics of the elongated intromittent organ in insects.

Does Asymmetrical Gonopodium Morphology Predict Lateralized Behavior in the Fish Xenophallus umbratilis?

Why bilaterally symmetrical organisms express handedness remains an important question in evolutionary biology. In some species, anatomical asymmetries have evolved that accompany behavioral handedness, yet we know remarkably little about causal links between asymmetric morphological traits and behavior. Here, we explore if a dextral or sinistral orientation of the male intromittent organ predicts side preferences in male behaviors. Our study addresses this question in the Costa Rican livebearing fish, Xenophallus umbratilis. This fish has a bilaterally symmetrical body plan, with one exception—the male anal fin (gonopodium), used to inseminate females, terminates with a distinct left- or right-handed corkscrew morphology. We used a detour assay to test males for side biases in approach behavior when exposed to four different stimuli (predator, potential mate, novel object, empty tank control). We found that left morph males preferred using their right eye to view potential mates, predators, and the control, and that right morph males preferred to use their left eye to view potential mates and predators, and their right eye to view the control. Males of both morphs displayed no eye bias when approaching the novel object. Our results suggest that there is a strong link between behavior and gonopodium orientation, with right and left morph males responding with opposite directional behaviors when presented with the same stimuli. This presents the intriguing possibility that mating preferences—in this case constrained by gonopodial morphology—could be driving lateralized decision making in a variety of non-mating behaviors.

Rapid evolution and plasticity of genitalia.

Genital morphology exhibits tremendous variation and is intimately linked with fitness. Sexual selection, nonmating natural selection and neutral forces have been explored as potential drivers of genital divergence. Though less explored, genitalia may also be plastic in response to the developmental environment. In poeciliid fishes, the length of the male intromittent organ, the gonopodium, may be driven by sexual selection if longer gonopodia attract females or aid in forced copulation attempts or by nonmating natural selection if shorter gonopodia allow predator evasion. The rearing environment may also affect gonopodium development. Using an experimental introduction of Trinidadian guppies into four replicate streams with reduced predation risk, we tested whether this new environment caused the evolution of genitalia. We measured gonopodium length after rearing the source and introduced populations for two generations in the laboratory to remove maternal and other environmental effects. We split full-sibling brothers into different rearing treatments to additionally test for developmental plasticity of gonopodia in response to predator cues and food levels as well as the evolution of plasticity. The introduced populations had shorter gonopodia after accounting for body size, demonstrating rapid genital evolution in 2-3years (8-12 generations). Brothers reared on low food levels had longer gonopodia relative to body size than those on high food, reflecting maintenance of gonopodium length despite a reduction in body size. In contrast, gonopodium length was not significantly different in response to the presence or absence of predator cues. Because the plastic response to low food was maintained between the source and introduced populations, there was no evidence that plasticity evolved. This study demonstrates the importance of both evolution and developmental plasticity in explaining genital variation.

Successful mating and hybridisation in two closely related flatworm species despite significant differences in reproductive morphology and behaviour

Reproductive traits are some of the fastest diverging characters and can serve as reproductive barriers. The free-living flatworm Macrostomum lignano, and its congener M. janickei are closely related, but differ substantially in their male intromittent organ (stylet) morphology. Here, we examine whether these morphological differences are accompanied by differences in behavioural traits, and whether these could represent barriers to successful mating and hybridization between the two species. Our data shows that the two species differ in many aspects of their mating behaviour. Despite these differences, the species mate readily with each other in heterospecific pairings. Although both species have similar fecundity in conspecific pairings, the heterospecific pairings revealed clear postmating barriers, as few heterospecific pairings produced F1 hybrids. These hybrids had a stylet morphology that was intermediate between that of the parental species, and they were fertile. Finally, using a mate choice experiment, we show that the nearly two-fold higher mating rate of M. lignano caused it to mate more with conspecifics, leading to assortative mating, while M. janickei ended up mating more with heterospecifics. Thus, while the two species can hybridize, the mating rate differences could possibly lead to higher fitness costs for M. janickei compared to M. lignano.

Phylogenomics and genital morphology of cave raptor spiders (Araneae, Trogloraptoridae) reveal an independent origin of a flow‐through female genital system

The monotypic family Trogloraptoridae was only recently described from caves and old-growth forest of Oregon and California (Western USA). These enigmatic spiders are characterized by striking raptorial claws, and based on their spinneret morphology, a close relationship to dysderoid spiders, a large clade within Synspermiata, was suggested. Here, we used a phylogenomic framework using transcriptomes to test the phylogenetic position of Trogloraptor marchingtoni. Our analysis placed this taxon within Synspermiata, which is supported by the presence of synspermia. Furthermore, a sister group relationship with Dysderoidea is strongly supported. In a second step, we reinvestigated the female genitalia using a non-destructive approach. Our data revealed that Trogloraptor has a flow-through genital system (entelegyne condition) and is not haplogyne as previously described based on dissections. The Trogloraptor female genital system consists of paired large spermathecae, which connect by a fertilization duct to a wide bursa. The copulatory duct arises from the sclerotized anterior margin of the bursa, and its organization is likely related to the organization of the male intromittent organ. Based on our phylogenetic data, we show that the entelegyne condition evolved at least six times independently within spiders. Moreover, our results indicate that the peculiar organization of the dysderoid female genitalia with an additional posterior sperm storage site is a synapomorphy of this Synspermiata clade.

Evolution of vertebrate postcranial complexity: axial skeleton regionalization and paired appendages in a Devonian jawless fish

AbstractOne of the major events in vertebrate evolution involves the transition from jawless (agnathan) to jawed (gnathostome) vertebrates, including a variety of cranial and postcranial innovations. It has long been assumed that characters such as the pelvic girdles and fins, male intromittent organs independent from the pelvic girdles, as well as a regionalized axial skeleton first appeared in various basal gnathostome groups if not at the origin of gnathostomes. Here we describe the first occurrence of pelvic girdles and intromittent organs in the Late Devonian jawless anaspid‐like fish Euphanerops longaevus Woodward (Miguasha Lagerstätte, eastern Canada), associated with a morphologically differentiated region of the axial skeleton. Morphological differentiation of the axial skeleton is also described for the first time in an extant jawless fish, the sea lamprey Petromyzon marinus Linnaeus. Our data indicate that regionalization of the axial skeleton occurred earlier in vertebrate evolutionary history than previously appreciated. This regionalization is coupled with modifications of the appendicular skeleton in Euphanerops. These new observations combined with a new phylogenetic analysis of early vertebrates provide a more precise understanding of how the appendicular and axial skeletons developed and evolved within vertebrate evolutionary history.

X-ray micro-CT scanning reveals temporal separation of male harm and female kicking during traumatic mating in seed beetles.

In the seed beetle Callosobruchus maculatus, the male intromittent organ is covered in sharp spines that pierce the female copulatory tract wall during mating. Although the fitness consequences of traumatic mating are well studied in this species, we know much less about how the male and female genitalia interact during mating. This is partly due to the fact that genital interactions occur primarily inside the female, and so are difficult to observe. In this study, we use X-ray micro-CT scanning to examine the proximate mechanisms of traumatic mating in C. maculatus in unprecedented detail. We show that this technique can be used to identify female tissue damage before the melanization of wound sites. We visualize the positioning of the male intromittent organ inside the female copulatory tract during mating, and show how this relates to tract wounding in three dimensions. By scanning pairs flash-frozen at different times during mating, we show that significant tract wounding occurs before the onset of female kicking. There is thus some degree of temporal separation between the onset of wounding and the onset of kicking, which supports recent suggestions that kicking is not an effective female counter-adaptation to reduce copulatory wounding in this species. We also present evidence that the sharp teeth protruding from the female tract wall are able to pierce the spermatophore as it is deposited, and may thus function to aid sperm release.

Evolution of the elaborate male intromittent organ of Xiphophorus fishes.

Internally fertilizing animals show a remarkable diversity in male genital morphology that is associated with sexual selection, and these traits are thought to be evolving particularly rapidly. Male fish in some internally fertilizing species have “gonopodia,” highly modified anal fins that are putatively important for sexual selection. However, our understanding of the evolution of genital diversity remains incomplete. Contrary to the prediction that male genital traits evolve more rapidly than other traits, here we show that gonopodial traits and other nongonopodial traits exhibit similar evolutionary rates of trait change and also follow similar evolutionary models in an iconic genus of poeciliid fish (Xiphophorus spp.). Furthermore, we find that both mating and nonmating natural selection mechanisms are unlikely to be driving the diverse Xiphophorus gonopodial morphology. Putative holdfast features of the male genital organ do not appear to be influenced by water flow, a candidate selective force in aquatic habitats. Additionally, interspecific divergence in gonopodial morphology is not significantly higher between sympatric species, than between allopatric species, suggesting that male genitals have not undergone reproductive character displacement. Slower rates of evolution in gonopodial traits compared with a subset of putatively sexually selected nongenital traits suggest that different selection mechanisms may be acting on the different trait types. Further investigations of this elaborate trait are imperative to determine whether it is ultimately an important driver of speciation.

Coevolution of female and male genital components to avoid genital size mismatches in sexually dimorphic spiders.

BackgroundIn most animal groups, it is unclear how body size variation relates to genital size differences between the sexes. While most morphological features tend to scale with total somatic size, this does not necessarily hold for genitalia because divergent evolution in somatic size between the sexes would cause genital size mismatches. Theory predicts that the interplay of female-biased sexual size dimorphism (SSD) and sexual genital size dimorphism (SGD) should adhere to the ‘positive genital divergence’, the ‘constant genital divergence’, or the ‘negative genital divergence’ model, but these models remain largely untested. We test their validity in the spider family Nephilidae known for the highest degrees of SSD among terrestrial animals.ResultsThrough comparative analyses of sex-specific somatic and genital sizes, we first demonstrate that 99 of the 351 pairs of traits are phylogenetically correlated. Through factor analyses we then group these traits for MCMCglmm analyses that test broader correlation patterns, and these reveal significant correlations in 10 out of the 36 pairwise comparisons. Both types of analyses agree that female somatic and internal genital sizes evolve independently. While sizes of non-intromittent male genital parts coevolve with male body size, the size of the intromittent male genital parts is independent of the male somatic size. Instead, male intromittent genital size coevolves with female (external and, in part, internal) genital size. All analyses also agree that SGD and SSD evolve independently.ConclusionsInternal dimensions of female genitalia evolve independently of female body size in nephilid spiders, and similarly, male intromittent genital size evolves independently of the male body size. The size of the male intromittent organ (the embolus) and the sizes of female internal and external genital components thus seem to respond to selection against genital size mismatches. In accord with these interpretations, we reject the validity of the existing theoretical models of genital and somatic size dimorphism in spiders.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-016-0734-9) contains supplementary material, which is available to authorized users.

Studying Genital Coevolution to Understand Intromittent Organ Morphology.

Male intromittent organs are exceedingly diverse, yet we know comparatively little about female genital diversity. However, the most direct mechanical interaction between males and females occurs during copulation, and therefore, genital coevolution is expected to be widespread. This means that diversification of male structures must influence diversity of female genital features and vice versa. As we expand our understanding of coevolutionary interactions between the sexes, we need to expand our knowledge of three basic areas: First, we need quantitative data, on morphological variation of female genitalia. Second, we need to study the mechanics of copulatory interactions, and third, we need to use this understanding to determine which features of genital morphology are under selection, and how their variable morphology and function may affect fitness. Though studying coevolution is certainly difficult, this knowledge is crucial to our understanding of diversity in morphology of the male intromittent organ.

Variation in pre- and post-copulatory sexual selection on male genital size in two species of lygaeid bug.

Sexual selection has been shown to be the driving force behind the evolution of the sometimes extreme and elaborate genitalia of many species. Sexual selection may arise before and/or after mating, or vary according to other factors such as the social environment. However, bouts of selection are typically considered in isolation. We measured the strength and pattern of selection acting on the length of the male intromittent organ (or processus) in two closely related species of lygaeid seed bug: Lygaeus equestris and Lygaeus simulans. In both species, we measured both pre- and post-copulatory selection. For L. equestris, we also varied the experimental choice design used in mating trials. We found contrasting pre- and post-copulatory selection on processus length in L. equestris. Furthermore, significant pre-copulatory selection was only seen in mating trials in which two males were present. This selection likely arises indirectly due to selection on a correlated trait, as the processus does not interact with the female prior to copulation. In contrast, we were unable to detect significant pre- or post-copulatory selection on processus length in L. simulans. However, a formal meta-analysis of previous estimates of post-copulatory selection on processus length in L. simulans suggests that there is significant stabilising selection across studies, but the strength of selection varies between experiments. Our results emphasise that the strength and direction of sexual selection on genital traits may be multifaceted and can vary across studies, social contexts and different stages of reproduction.Significance statementAnimal genitalia vary greatly in size and complexity across species, and selection acting on genital size and shape can be complex. In this study, we show that the length of the penis in two species of seed bug is subject to complex patterns of selection, varying depending on the social context and whether selection is measured before or after mating. In one of the species, we show unexpectedly that penis length is correlated with male mating success, despite the fact that the penis does not interact with the female prior to mating. Our results highlight the fact that genitalia may be subject to both direct and indirect selection at different stages of mating and that to fully understand the evolution of such traits we should combine estimates of selection arising from these multiple episodes.Electronic supplementary materialThe online version of this article (doi:10.1007/s00265-016-2082-6) contains supplementary material, which is available to authorized users.

Natural breakage of the very long intromittent organ of the seed bug Lygaeus simulans (Heteroptera: Lygaeidae)

The male intromittent organ of the seed bug Lygaeus simulans ends in a long, sclerotized structure which is used to transfer sperm during mating. Observations suggest that this structure becomes brittle and is liable to breakage after being artificially exposed to the air for an extended period of time. In this study we investigate the frequency of intromittent organ breakage in L. simulans. We first examined the intromittent organ of a sample of males that mated once, and found that breakage was rare. We hypothesised that breakages are likely to be more frequent if a male is able to mate multiple times, and so we next paired males with a female for 21 days in order to provide the opportunity for multiple mating. Almost a quarter (22.5%) of these males exhibited signs of genital breakage. The point of breakage varied: for six males only the tip of the structure (around 6% of its length) was missing, whereas for three males over 50% of the structure was missing. However we were unable to locate any fragments of male genitalia in the reproductive tracts of any females that came into contact with these males. This suggests that breakages do not necessarily occur during mating itself, but instead probably occur as the intromittent organ is being retracted into the genital capsule following mating. In this species breakage may not significantly reduce male reproductive fitness as sperm transfer may still be possible.

Paired and complex copulatory organs: do they really impede flexible use?

AbstractAnimals with internal fertilization possess complex male genitalia that are characterized by rapid and divergent evolution. Such complexity and divergence renders male genitalia highly species specific. Furthermore, in species with paired genitalia in males and females as is typically the case in spiders, the genital complexity is considered to restrict usage of each male intromittent organ (pedipalp) to a specific genital opening of the female genitalia. Here, we report cases of flexible pedipalp insertion (16%) in the dwarf spider Oedothorax retusus – a species with complex pedipalps. A flexible insertion mode allows males of this species to react to the mating history of the female by circumventing the highly effective mating plugs and mating into the unused side. Our findings disagree with the species isolating lock‐and‐key hypothesis for explaining divergence in genital morphology as the ‘key’ can be applied to the mirror‐inversed female ‘lock’ in the spider species O. retusus. Rather, our data suggest that sexual selection is an important underlying selective regime for the evolution of highly complex and diverse genitalia.

Experimental reduction of intromittent organ length reduces male reproductive success in a bug.

It is now clear in many species that male and female genital evolution has been shaped by sexual selection. However, it has historically been difficult to confirm correlations between morphology and fitness, as genital traits are complex and manipulation tends to impair function significantly. In this study, we investigate the functional morphology of the elongate male intromittent organ (or processus) of the seed bug Lygaeus simulans, in two ways. We first use micro-computed tomography (micro-CT) and flash-freezing to reconstruct in high resolution the interaction between the male intromittent organ and the female internal reproductive anatomy during mating. We successfully trace the path of the male processus inside the female reproductive tract. We then confirm that male processus length influences sperm transfer by experimental ablation and show that males with shortened processi have significantly reduced post-copulatory reproductive success. Importantly, male insemination function is not affected by this manipulation per se. We thus present rare, direct experimental evidence that an internal genital trait functions to increase reproductive success and show that, with appropriate staining, micro-CT is an excellent tool for investigating the functional morphology of insect genitalia during copulation.

Histological Investigation of the Adult Alligator Phallic Sulcus

Male intromittent organs serve two primary reproductive functions: the physical entry into the female body during copulation and the effective delivery of gametes resulting in internal fertilization. Here we present a histological examination of the adult male American alligator phallus semen delivery apparatus, the sulcus spermaticus. While the highly collagenous basal crurae and more distal shaft of the alligator penis contain the rigid structures that facilitate cloacal intromission, the sulcus is more functionally intricate. Here we show the sulcus spermaticus (an open groove that runs along the ventral aspect of the phallic shaft) to be a spatially heterogeneous reproductive structure containing a complex architecture of multiple tissue types. Sulcus morphology markedly changes from its proximal origin between the crurae to its distal exit from the phallus tip. At the proximal origin of the sulcus, the ductus deferens vent semen into an expanded lumen lined by a convoluted secretory epithelium. Along the length of the phallic shaft, an arrangement of longitudinally and radially oriented smooth muscle bundles may act via rhythmic contractions to produce peristaltic sperm conveyance through the sulcus. An extensive vascular network of blood and lymph vessels putatively engorges the sulcus tissues during reproductive activity, increasing tension on an internal network of connective tissues and leading to localized inflation and increased tissue rigidity. We hypothesize that this engorgement works to seal the sulcus groove and allow the structure to convey semen through a functionally closed tube. Further, numerous epithelial secretory cells contribute seminal fluids to the ejaculate and may aid in as yet uncharacterized aspects of sulcus functioning. Together, these observations establish that the sulcus spermaticus is far more than a simple furrow in the phallus shaft for sperm conduction: it contains elements that form a complex functional gamete delivery system.

Mating and genital coupling in the primitive earwig species Echinosoma denticulatum (Pygidicranidae): implications for genital evolution in dermapteran phylogeny

Dermaptera (earwigs) shows much diversity in the genital structures, the presence of either one or two male intromittent organs (penes) being one striking aspect. The members of several groups (Karschiellidae, Eudermaptera, Arixeniina, and Hemimerina) possess a single functional penis, while others have a pair of penes. The latter condition is considered to be plesiomorphic in Dermaptera. Despite its importance for inferring the phylogeny of Dermaptera, it is presently unclear how the ancestor of earwigs acquired paired penes. To estimate the mode of mating and sperm transfer in the common ancestor of extant earwigs, this study examines the mating behavior and genital coupling of the primitive earwig species Echinosoma denticulatum Hincks, 1959 as a representative of Pygidicranidae, one of the basal-most assemblages of earwigs. Staged mating experiments, including surgical manipulation of male penes revealed the following characteristics for this species: (1) males use only one of the paired penes for a single genital coupling; (2) both penes are likely functional; (3) there are no consistent biases in usage of the penes; (4) laterality of the penis-use pattern is not related to the direction of rotation of the male abdomen to establish genital coupling; (5) sperm are transferred directly into the spermatheca, a female sperm storage organ; (6) the spined area of the penis inflicts wounds on the vagina around the spermathecal opening, which bears many setae, during copulation. Characteristics (1)–(4) are considered to be plesiomorphic and may represent the condition of the common ancestor. Traumatic penetration during copulation is reported for earwigs for the first time. Together with intermittent acceptance of courting males by females only after a certain interval, these results suggest sexual conflict over mating in this species.

Nuptial gift size, mating duration and remating success in a Neotropical spider

In many species the function and evolution of nuptial gifts have been explained by the male mating effort hypothesis; i.e., males increase their fertilization success by donating gifts to females. The gift size may determine copulation duration and consequently affects male fertilization rate. In the well-known spider Pisaura mirabilis, previous studies suggested that the brightness of the gift (a prey wrapped in silk) affects female acceptance and larger gifts promote longer matings, but little is known from other gift-giving spider species. In this paper we test if gift size affects mating and remating success in the Neotropical and semi-aquatic spider Paratrechalea ornata (Trechaleidae). We exposed virgin females to males offering one fly (OF group) or four flies (FF group) wrapped in silk, and afterwards mated females were exposed to remating. Males from FF showed both longer courtship and mating than males from OF. We found a positive linear relationship between insertion duration of male intromittent organs (during which sperm transfer occurs) and mating duration in both groups. Mated females were more reluctant to mate than virgin females, but contrary to our prediction, gift size and mating duration did not seem to influence remating success. Our findings show that large gifts prolong mating duration in P. ornata, supporting the mating effort hypothesis. Females would favour polyandry to gain direct benefits from the gifts, while males may improve success in sperm competition by inducing female reluctance for remating.