Intra-horn insemination in the alpaca Vicugna pacos: Copulatory wounding and deep sperm deposition.

Alpacas (Vicugna pacos) are reported to be the rare mammal in which the penis enters the uterus in mating. To date, however, only circumstantial evidence supports this assertion. Using female alpacas culled for meat, we determined that the alpaca penis penetrates to the very tips of the uterine horns, abrading the tract and breaking fine blood vessels. All female alpacas sacrificed one hour or 24 hours after mating showed conspicuous bleeding in the epithelium of some region of their reproductive tract, including the hymen, cervix and the tips of each uterine horn, but typically not in the vagina. Unmated females showed no evidence of conspicuous bleeding. Histological examination of mated females revealed widespread abrasion of the cervical and endometrial epithelium, injuries absent in unmated females. Within one hour of mating, sperm were already present in the oviduct. The male alpaca's cartilaginous penis tip with a hardened urethral process is likely responsible for the copulatory abrasion. The entire female reproductive tract interacts with the penis, functioning like a vagina. Alpacas are induced ovulators, and wounding may hasten delivery of the seminal ovulation-inducing factor beta-NGF into the female's blood stream. There is no evidence of sexual conflict in copulation in alpaca, and thus wounding may also be one of a variety of mechanisms devised by mammals to induce a beneficial, short-term inflammatory response that stimulates blastocyst implantation, the uterine remodeling associated with placental development, and thus the success of early pregnancy.

Vaginas.

What is the vagina? This seemingly simple question has a rather complex answer, depending on whether we use a functional or a developmental definition. The terminal portion of the female reproductive tract that opens to the environment initially served as a conduit for eggs to be laid, and in species with external fertilization the distal oviduct may be specialized for oviposition but there is no vagina. In animals with internal fertilization, this terminal section of the oviduct interacts with the sperm and the intromittent organ leading to functional specialization of this region that we often call a vagina in insects and some vertebrates. Here we address the evolution, morphology and diverse functions of the vagina and some of the unknown questions that remain to be addressed in the study of this remarkable structure.

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.

Bird genitalia.

While most birds do not have external genitalia, in a small number of species the males have penises, which can be either intromittent or non-intromittent. Brennan provides an overview of bird genitalia, including speculation about the evolutionary forces involved.

Evolution and Morphology of Genitalia in Female Amniotes.

Despite their evolutionary and biomedical importance, studies of the morphology and function of female genitalia have continued to lag behind those of male genitalia. While studying female genitalia can be difficult because of their soft, deformable and internal nature, recent advances in imaging, geometric analyses of shape and mechanical testing have been made, allowing for a much greater understanding of the incredible diversity of form and function of female genitalia. Here we summarize some of these methods, as well as discuss some big questions in the field that are beginning to be examined now, and will continue to benefit from further work, especially a comparative approach. Topics of further research include examination of the morphology of female genitalia in situ, in-depth anatomical work in many more species, studies of the interplay between natural and sexual selection in influencing features of vaginal morphology, how these diverse functions influence the mechanical properties of tissues, and studies of clitoris morphology and function across amniotes. Many other research topics related to female genitalia remain largely unexplored, and we hope that the papers in this issue will continue to inspire further research on female genitalia.

Testing Morphological Relationships Between Female and Male Copulatory Structures in Bats.

The lower reproductive tract of female mammals has several competing functions including mating, tract health maintenance, and parturition. Diverse vaginal anatomy suggests interactions between natural and sexual selection, yet despite its importance, female copulatory morphology remains under-studied. We undertook a comparative study across the species-rich mammalian order Chiroptera (bats) with a focus on the suborder Yangochiroptera (Vespertilioniformes) to examine how female vaginal features may have coevolved with male penis morphology to minimize mechanical damage to their tissues during copulation. The penis morphology is diverse, presenting great potential for post-copulatory sexual selection and coevolution with the female morphology, but vaginas have not been carefully examined. Here we test the hypotheses that vaginal thickness and collagen density have coevolved with features of the male penis including the presence of spines and a baculum. We present histological data from females of 24 species from 7 families of bats, and corresponding data on male penis anatomy. We also examine the role of phylogenetic history in the morphological patterns we observe. We found evidence that female vaginal thickness has coevolved with the presence of penile spines, but not with baculum presence or width. Collagen density did not appear to covary with male penile features. Our findings highlight the importance of considering interactions between the sexes in influencing functional reproductive structures and examine how these structures have been under selection in bats.

Bridging the Research Gap between Live Collections in Zoos and Preserved Collections in Natural History Museums.

Zoos and natural history museums are both collections-based institutions with important missions in biodiversity research and education. Animals in zoos are a repository and living record of the world's biodiversity, whereas natural history museums are a permanent historical record of snapshots of biodiversity in time. Surprisingly, despite significant overlap in institutional missions, formal partnerships between these institution types are infrequent. Life history information, pedigrees, and medical records maintained at zoos should be seen as complementary to historical records of morphology, genetics, and distribution kept at museums. Through examining both institution types, we synthesize the benefits and challenges of cross-institutional exchanges and propose actions to increase the dialog between zoos and museums. With a growing recognition of the importance of collections to the advancement of scientific research and discovery, a transformational impact could be made with long-term investments in connecting the institutions that are caretakers of living and preserved animals.

Evidence of a functional clitoris in dolphins.

In species that copulate during non-conceptive periods, such as humans and bonobos, sexual intercourse is known to be pleasurable for females. Dolphins also copulate throughout the year, largely to establish and maintain social bonds1. In dolphins, the clitoris is positioned in the anterior aspect of the vaginal entrance2, where physical contact and stimulation during copulation is likely. Clitoral stimulation seems to be important during female-female sexual interactions in common bottlenose dolphins (Tursiops truncatus), which rub each other's clitorises using snouts, flippers, or flukes3. Determining a sexual pleasure response in animals not amenable to neurobehavioral examination is difficult, but investigation of the clitoris may elucidate evidence of functionality. In this study, we assessed macro- and micromorphological features of the clitoris in common bottlenose dolphins to examine functional features, including erectile bodies with lacunae, extensible collagen and/or elastin fibers, and the presence and location of sensory nerves. Our observations suggest the clitoris of dolphins has well-developed erectile spaces, is highly sensitive to tactile stimulation, and is likely functional. VIDEO ABSTRACT.

Intra-horn Penile Intromission in the Alpaca Vicugna pacos and Consequences to Genital Morphology.

Copulatory behavior and genital morphology interact to deliver sperm more effectively during mating, but the nature of this interaction has not been explored in depth in most vertebrates. Alpacas have unusually long copulations lasting 15-20 min, and a unique copulatory behavior, where the penis intromits all the way past the cervix, into the uterine horns. Here we describe the morphology of male and female genitalia and report unique morphological characteristics that may be associated with this unusual insemination mode. Vaginal shape is highly variable, and seemingly not associated with age or parity. The cranial vagina varies between bulbous and cylindrical, while the caudal vagina is typically narrower and always cylindrical. The cervix consists of a series of two to three spirals or rings, and it is often found in a relaxed state that may prevent damage caused by the cartilaginous penis tip as it pushes through the cervix to reach the uterine horns. The uterus and uterine horns have a complex shape with multiple constrictions. The cartilaginous penis tip has a sharp urethral process that may help to push against these constrictions. The diameter of the vaginal lumen is much greater than the diameter of the penis suggesting that there is little direct interaction between them, and that female vaginal shape is not under strong copulatory selection. In effect, the entire female reproductive tract of the female is interacting with the penis during copulation.

3D genital shape complexity in female marine mammals.

Comparisons of 3D shapes have recently been applied to diverse anatomical structures using landmarking techniques. However, discerning evolutionary patterns can be challenging for structures lacking homologous landmarks. We used alpha shape analyses to quantify vaginal shape complexity in 40 marine mammal specimens including cetaceans, pinnipeds, and sirenians. We explored phylogenetic signal and the potential roles of natural and sexual selection on vaginal shape evolution. Complexity scores were consistent with qualitative observations. Cetaceans had a broad range of alpha complexities, while pinnipeds were comparatively simple and sirenians were complex. Intraspecific variation was found. Three-dimensional surface heat maps revealed that shape complexity was driven by invaginations and protrusions of the vaginal wall. Phylogenetic signal was weak and metrics of natural selection (relative neonate size) and sexual selection (relative testes size, sexual size dimorphism, and penis morphology) did not explain vaginal complexity patterns. Additional metrics, such as penile shape complexity, may yield interesting insights into marine mammal genital coevolution. We advocate for the use of alpha shapes to discern patterns of evolution that would otherwise not be possible in 3D anatomical structures lacking homologous landmarks.

Glans inflation morphology and female cloaca copulatory interactions of the male American alligator phallus†.

The phallic glans of the American alligator (Alligator mississippiensis) is the distal termination of the semen-conducting sulcus spermaticus and during copulation has the closest, most intimate mechanical interactions with the female urodeum, the middle cloacal chamber that contains the opening to the vaginal passages and oviducts. However, the details of this interface leading to insemination and gamete uptake are unclear. Here, we: (1) histologically characterize the underlying tissue types and morphologically quantify the shape changes associated with glans inflation into the copulatory conformation, (2) digitally reconstruct from MRI the 3D shape of functional tissue compartments, and (3) diffusible iodine-based contrast-enhanced computed tomography image the copulatory fit between male phallus and female cloaca. We discuss these results in relation to tissue type material properties, the transfer on intromittent forces, establishing potential copulatory lock, inflated glans volume scaling with body mass/length, the mechanics of semen targeting and insemination, and potential female cryptic choice impacting multiple clutch paternity. In part, this study further clarifies the phallic morphological variation observed among crocodylians and begins to investigate the role(s) these divergent male forms play during copulation interacting with female cloacal forms to increase reproductive success.

Asymmetric and Spiraled Genitalia Coevolve with Unique Lateralized Mating Behavior.

Asymmetric genitalia and lateralized mating behaviors occur in several taxa, yet whether asymmetric morphology in one sex correlates or coevolves with lateralized mating behavior in the other sex remains largely unexplored. While lateralized mating behaviors are taxonomically widespread, among mammals they are only known in the harbor porpoise (Phocoena phocoena). Males attempt copulation by approaching a female exclusively on her left side. To understand if this unusual lateralized behavior may have coevolved with genital morphology, we quantified the shape of female and male harbor porpoise reproductive tracts using 2D geometric morphometrics and 3D models of the vaginal lumen and inflated distal penis. We found that the vaginas varied individually in shape and that the vaginas demonstrated both significant directional and fluctuating asymmetry. This asymmetry resulted from complex 3D spirals and vaginal folds with deep recesses, which may curtail the depth or direction of penile penetration and/or semen movement. The asymmetric shapes of the vaginal lumen and penis tip were both left-canted with similar angular bends that mirrored one another and correspond with the left lateral mating approach. We suggest that the reproductive anatomy of both sexes and their lateral mating behavior coevolved.

The evolution of genital shape variation in female cetaceans.

Male genital diversification is likely the result of sexual selection. Female genital diversification may also result from sexual selection, although it is less well studied and understood. Female genitalia are complex among whales, dolphins, and porpoises, especially compared to other vertebrates. The evolutionary factors affecting the diversity of vaginal complexity could include ontogeny, allometry, phylogeny, sexual selection, and natural selection. We quantified shape variation in female genitalia using 2D geometric morphometric analysis, and validated the application of this method to study soft tissues. We explored patterns of variation in the shape of the cervix and vagina of 24 cetacean species (n = 61 specimens), and found that genital shape varies primarily in the relative vaginal length and overall aspect ratio of the reproductive tract. Extensive genital shape variation was partly explained by ontogenetic changes and evolutionary allometry among sexually mature cetaceans, whereas phylogenetic signal, relative testis size, and neonate size were not significantly associated with genital shape. Female genital shape is diverse and evolves rapidly even among closely related species, consistent with predictions of sexual selection models and with findings in invertebrate and vertebrate taxa. Future research exploring genital shape variation in 3D will offer new insights into evolutionary mechanisms because internal vaginal structures are variable and can form complex spirals.

Genital interactions during simulated copulation among marine mammals.

Genitalia are morphologically variable across many taxa and in physical contact during intromission, but little is known about how variation in form correlates with function during copulation. Marine mammals offer important insights into the evolutionary forces that act on genital morphology because they have diverse genitalia and are adapted to aquatic living and mating. Cetaceans have a fibroelastic penis and muscular vaginal folds, while pinnipeds have a baculum and lack vaginal folds. We examined copulatory fit in naturally deceased marine mammals to identify anatomical landmarks in contact during copulation and the potential depth of penile penetration into the vagina. Excised penises were artificially inflated to erection with pressurized saline and compared with silicone vaginal endocasts and within excised vaginas in simulated copulation using high-resolution, diffusible iodine-based, contrast-enhanced computed tomography. We found evidence suggestive of both congruent and antagonistic genital coevolution, depending on the species. We suggest that sexual selection influences morphological shape. This study improves our understanding of how mechanical interactions during copulation influence the shape of genitalia and affect fertility, and has broad applications to other taxa and species conservation.

Dynamic egg color mimicry.

Evolutionary hypotheses regarding the function of eggshell phenotypes, from solar protection through mimicry, have implicitly assumed that eggshell appearance remains static throughout the laying and incubation periods. However, recent research demonstrates that egg coloration changes over relatively short, biologically relevant timescales. Here, we provide the first evidence that such changes impact brood parasite-host eggshell color mimicry during the incubation stage. First, we use long-term data to establish how rapidly the Acrocephalus arundinaceus Linnaeus (great reed warbler) responded to natural parasitic eggs laid by the Cuculus canorus Linnaeus (common cuckoo). Most hosts rejected parasitic eggs just prior to clutch completion, but the host response period extended well into incubation (~10 days after clutch completion). Using reflectance spectrometry and visual modeling, we demonstrate that eggshell coloration in the great reed warbler and its brood parasite, the common cuckoo, changes rapidly, and the extent of eggshell color mimicry shifts dynamically over the host response period. Specifically, 4 days after being laid, the host should notice achromatic color changes to both cuckoo and warbler eggs, while chromatic color changes would be noticeable after 8 days. Furthermore, we demonstrate that the perceived match between host and cuckoo eggshell color worsened over the incubation period. These findings have important implications for parasite-host coevolution dynamics, because host egg discrimination may be aided by disparate temporal color changes in host and parasite eggs.

All Features Great and Small-the Potential Roles of the Baculum and Penile Spines in Mammals.

Mammalian penises are morphologically diverse, including a highly variable and taxonomically informative baculum (os penis), and variable penile spines, both of which are possessed by many-but not all-species. To understand the evolution of genital morphologies, as well as the potential role of both the baculum, and penile spines that directly interact with female reproductive tract, we undertook a comparative study of male penile spines and their relationship with the baculum across all mammalian orders. Specifically, we investigated several factors that may explain the presence or absence of penile spines in mammals, including mating system, risk of sperm competition, female reproductive physiology, presence and width of the baculum, and phylogenetic history. We observed that the presence of both the baculum and penile spines is correlated with residual testes size, suggesting a potential role in sexual selection for these traits. We found no association between the presence of spines and mating system, or with the presence/width of the baculum, although relative testes mass was marginally associated with baculum width. We found no relationship between baculum presence or width and mating system. We also noted that spines presence or absence have an order-level distribution, and clear phylogenetic patterns of distribution across mammals. It is likely that spine morphology and distribution, not just presence, play an important role in genital evolution in mammals, but these features are poorly described in most groups. Quantitative data collection in most mammalian taxa would be useful to further elucidate the evolution of the complex genital morphology of this group.

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.

Evolution: One Penis After All.

Amniote penises come in many shapes but are missing from the basal tuatara. This has been taken as evidence for multiple evolutionary origins of the penis. Now, genital swellings have been found in tuatara embryos, arguing for a single origin.

Nature's Palette: Characterization of Shared Pigments in Colorful Avian and Mollusk Shells.

Pigment-based coloration is a common trait found in a variety of organisms across the tree of life. For example, calcareous avian eggs are natural structures that vary greatly in color, yet just a handful of tetrapyrrole pigment compounds are responsible for generating this myriad of colors. To fully understand the diversity and constraints shaping nature's palette, it is imperative to characterize the similarities and differences in the types of compounds involved in color production across diverse lineages. Pigment composition was investigated in eggshells of eleven paleognath bird taxa, covering several extinct and extant lineages, and shells of four extant species of mollusks. Birds and mollusks are two distantly related, calcareous shell-building groups, thus characterization of pigments in their calcareous structures would provide insights to whether similar compounds are found in different phyla (Chordata and Mollusca). An ethylenediaminetetraacetic acid (EDTA) extraction protocol was used to analyze the presence and concentration of biliverdin and protoporphyrin, two known and ubiquitous tetrapyrrole avian eggshell pigments, in all avian and molluscan samples. Biliverdin was solely detected in birds, including the colorful eggshells of four tinamou species. In contrast, protoporphyrin was detected in both the eggshells of several avian species and in the shells of all mollusks. These findings support previous hypotheses about the ubiquitous deposition of tetrapyrroles in the eggshells of various bird lineages and provide evidence for its presence also across distantly related animal taxa.

Mechanisms and Evidence of Genital Coevolution: The Roles of Natural Selection, Mate Choice, and Sexual Conflict.

Genital coevolution between the sexes is expected to be common because of the direct interaction between male and female genitalia during copulation. Here we review the diverse mechanisms of genital coevolution that include natural selection, female mate choice, male-male competition, and how their interactions generate sexual conflict that can lead to sexually antagonistic coevolution. Natural selection on genital morphology will result in size coevolution to allow for copulation to be mechanically possible, even as other features of genitalia may reflect the action of other mechanisms of selection. Genital coevolution is explicitly predicted by at least three mechanisms of genital evolution: lock and key to prevent hybridization, female choice, and sexual conflict. Although some good examples exist in support of each of these mechanisms, more data on quantitative female genital variation and studies of functional morphology during copulation are needed to understand more general patterns. A combination of different approaches is required to continue to advance our understanding of genital coevolution. Knowledge of the ecology and behavior of the studied species combined with functional morphology, quantitative morphological tools, experimental manipulation, and experimental evolution have been provided in the best-studied species, all of which are invertebrates. Therefore, attention to vertebrates in any of these areas is badly needed.