The Unique Penile Morphology of the Short-Beaked Echidna, Tachyglossus aculeatus.

Monotremes diverged from therian mammal ancestors approximately 184 million years ago and have a number of novel reproductive characteristics. One in particular is their penile morphology. There are differences between echidna and platypus phalluses, but both are somewhat similar in structure to the reptilian phallus. The echidna penis consists of 4 rosette glans, each of which contains a termination of the quadrifurcate urethra, but it appears that only 2 of the 4 glans become erect at any one time. Despite this, only a few historical references describe the structure of the echidna penis and none provides an explanation for the mechanisms of unilateral ejaculation. This study confirmed that the echidna penis contains many of the same overall structures and morphology as other mammalian penises and a number of features homologous with reptiles. The corpus cavernosum is well supplied with blood, extends up to the base of the glans penis and is primarily responsible for erection. However, the echidna possesses 2 distinct corpora spongiosa separated by a septum, each of which surround the urethra only distal to the initial urethral bifurcation in the glans penis. Together with the bifurcation of the main penile artery, this provides a mechanism by which blood flow could be directed to only one corpus spongiosum at a time to maintain an open urethra that supplies 2 of the 4 glans to facilitate unilateral ejaculation.

Immunolabeling indicates that sulfhydryl oxidase is absent in anamniote epidermis but marks the process of cornification in the skin of terrestrial vertebrates.

The passage between keratinization to cornification of the epidermis and skin appendages in vertebrates requires formation of a stratum corneum rich in SS bonds among other cross-linking chemical bonds. A key enzyme, sulfhydryl oxidase (SOXase) catalyzes the oxidation of SH groups present in keratins and in corneous proteins of the epidermis into SS. Presence and distribution of SAXase has been studied by immunohistochemistry in all vertebrates, from fish to mammals. SOXase is immunohistochemically absent in all fish and amphibian species tested with the exception of a thin pre-corneous layer in the epidermis of adult anurans. SOXase is low to absent in corneous appendages such as horny teeth of lamprey or claws and horny beaks of amphibians. Conversely, SOXase is detected in the transitional (pre-corneous) and inner corneous layers of the epidermis of sauropsids and mammals. In lepidosaurian reptiles, SOXase appears in both beta- and alpha-corneous-layers, but is limited to the pre-corneous and corneous layers of the thin soft epidermises of birds and mammals, including the granular layer. SOXase is localized in pre-corneous layers and disappears in external corneous layers of amniote skin appendages such as claws, beaks of turtles and birds, and in developing feathers. This distribution further indicates that the increase activity of epidermal SOXase is/was essential, in addition to other enzymes such as epidermal transglutaminases, for the evolution of the corneous layer and of the different hard skin appendages present in terrestrial vertebrates.

Early Triassic marine reptile representing the oldest record of unusually small eyes in reptiles indicating non-visual prey detection.

The end-Permian mass extinction (EPME) led to reorganization of marine predatory communities, through introduction of air-breathing top predators, such as marine reptiles. We report two new specimens of one such marine reptile, Eretmorhipis carrolldongi, from the Lower Triassic of Hubei, China, revealing superficial convergence with the modern duckbilled platypus (Ornithorhynchus anatinus), a monotreme mammal. Apparent similarities include exceptionally small eyes relative to the body, snout ending with crura with a large internasal space, housing a bone reminiscent of os paradoxum, a mysterious bone of platypus, and external grooves along the crura. The specimens also have a rigid body with triangular bony blades protruding from the back. The small eyes likely played reduced roles during foraging in this animal, as with extant amniotes (group containing mammals and reptiles) with similarly small eyes. Mechanoreceptors on the bill of the animal were probably used for prey detection instead. The specimens represent the oldest record of amniotes with extremely reduced visual capacity, utilizing non-visual cues for prey detection. The discovery reveals that the ecological diversity of marine predators was already high in the late Early Triassic, and challenges the traditional view that the ecological diversification of marine reptiles was delayed following the EPME.

Comparative cranial morphology in living and extinct platypuses: Feeding behavior, electroreception, and loss of teeth.

The modern platypus, Ornithorhynchus anatinus, has an eye structure similar to aquatic mammals; however, platypuses also have a "sixth sense" associated with the bill electro- and mechanoreception that they use without opening their eyes underwater. We hypothesize that Ornithorhynchus and the Miocene taxon Obdurodon have different sensory capacities, which may have resulted from differences in foraging behavior. To estimate differences in foraging, sensory systems, and anatomical divergence between these monotremes, we compared their skull morphologies. Results indicate that the bill of Obdurodon is more dorsally deflected than that of Ornithorhynchus, suggesting a pelagic foraging behavior in Obdurodon compared to the bottom-feeding behavior in Ornithorhynchus. The infraorbital foramen of Obdurodon, through which the maxillary nerve passes sensory data from the bill to the brain, is relatively less developed than that of Ornithorhynchus. Whereas bill-focused sensory perception was likely shared among Mesozoic monotremes, the highly developed electrosensory system of Ornithorhynchus may represent an adaptation to foraging in cloudy water. Computed tomography imagery indicates that the enlarged infraorbital canal of Ornithorhynchus restricts the space available for maxillary tooth roots. Hence, loss of functional teeth in Ornithorhynchus may possibly have resulted from a shift in foraging behavior and coordinate elaboration of the electroreceptive sensory system. Well-developed electroreceptivity in monotremes is known at least as far back as the early Cretaceous; however, there are differences in the extent of elaboration of the feature among members of the ornithorhynchid lineage.

The platypus in Edinburgh: Robert Jameson, Robert Knox and the place of the Ornithorhynchus in nature, 1821-24.

The duck-billed platypus, or Ornithorhynchus, was the subject of an intense debate among natural historians in the late eighteenth and early nineteenth centuries. Its paradoxical mixture of mammalian, avian and reptilian characteristics made it something of a taxonomic conundrum. In the early 1820s Robert Jameson (1774-1854), the professor of natural history at the University of Edinburgh and the curator of the University's natural history museum, was able to acquire three valuable specimens of this species. He passed one of these on to the anatomist Robert Knox (1791-1862), who dissected the animal and presented his results in a series of papers to the Wernerian Natural History Society, which later published them in its Memoirs. This paper takes Jameson's platypus as a case study on how natural history specimens were used to create and contest knowledge of the natural world in the early nineteenth century, at a time when interpretations of the relationships between animal taxa were in a state of flux. It shows how Jameson used his possession of this interesting specimen to provide a valuable opportunity for his protégé Knox while also helping to consolidate his own position as a key figure in early nineteenth-century natural history.

Formation and Dissociation of Sperm Bundles in Monotremes.

Because monotremes are the earliest offshoot of the mammalian lineage, the platypus and short-beaked echidna were studied as model animals to assess the origin and biological significance of adaptations considered unique to therian mammals: epididymal sperm maturation and subsequent capacitation. We show that spermatozoa from both species assemble into bundles of approximately 100 cells during passage through the epididymis and that an epididymal protein-secreted protein, acidic, cysteine-rich (osteonectin; SPARC)-is involved in bundle formation. The bundles persisted during incubation in vitro for at least 1 h under conditions that capacitate therian spermatozoa, and then underwent a time-dependent dissociation to release spermatozoa capable of fertilization. Only after this dissociation could the spermatozoa bind to the perivitelline membrane of a hen's egg, display an altered form of motility reminiscent of hyperactivation, and be induced to undergo an acrosome reaction. It is concluded that the development of sperm bundles in the monotreme epididymis mandates that they require a time-dependent process to be capable of fertilizing an ovum. However, because this functional end point was achieved without overt changes in protein tyrosine phosphorylation (a hallmark of capacitation in therians), it is concluded that the process in monotremes is distinctly different from capacitation in therian mammals.

Internally coupled ears in living mammals.

It is generally held that the right and left middle ears of mammals are acoustically isolated from each other, such that mammals must rely on neural computation to derive sound localisation cues. There are, however, some unusual species in which the middle ear cavities intercommunicate, in which case each ear might be able to act as a pressure-difference receiver. This could improve sound localisation at lower frequencies. The platypus Ornithorhynchus is apparently unique among mammals in that its tympanic cavities are widely open to the pharynx, a morphology resembling that of some non-mammalian tetrapods. The right and left middle ear cavities of certain talpid and golden moles are connected through air passages within the basicranium; one experimental study on Talpa has shown that the middle ears are indeed acoustically coupled by these means. Having a basisphenoid component to the middle ear cavity walls could be an important prerequisite for the development of this form of interaural communication. Little is known about the hearing abilities of platypus, talpid and golden moles, but their audition may well be limited to relatively low frequencies. If so, these mammals could, in principle, benefit from the sound localisation cues available to them through internally coupled ears. Whether or not they actually do remains to be established experimentally.

A Spur to Atavism: Placing Platypus Poison.

For over two centuries, the platypus (Ornithorhynchus anatinus) has been constructed and categorized in multiple ways. An unprecedented mélange of anatomical features and physiological functions, it long remained a systematic quandary. Nevertheless, since 1797, naturalists and biologists have pursued two recurring obsessions. Investigations into platypus reproduction and lactation have focused attention largely upon females of the species. Despite its apparent admixture of avian, reptilian and mammalian characters, the platypus was soon placed as a rudimentary mammal--primitive, naïve and harmless. This article pursues a different taxonomic trajectory, concentrating on a specifically male anatomical development: the crural spur and venom gland on the hind legs. Once the defining characteristic of both the platypus and echidna (Tachyglossus aculeatus), by 1830 this sexed spur had been largely dismissed as inactive and irrelevant. For a creature regularly depicted as a biological outlier, the systematic and evolutionary implications of platypus poison have remained largely overlooked. In Australia, however, sporadic cases of 'spiking' led to consistent homologies being remarked between the platypus crural system and the venom glands of snakes. As with its reproductive reliance upon eggs, possession of an endogenous poison suggested significant reptilian affinities, yet the platypus has rarely been classed as an advanced reptile. Indeed, ongoing uncertainty regarding the biological purpose of the male's spur has ostensibly posed a directional puzzle. As with so many of its traits, however, platypus poison has been consistently described as a redundant remnant, rather than an emergent feature indicating evolutionary advance.

Immunohistochemical analysis of pancreatic islets of platypus (Ornithorhynchus anatinus) and echidna (Tachyglossus aculeatus ssp.).

Monotremes have undergone remarkable changes to their digestive and metabolic control system; however, the monotreme pancreas remains poorly characterized. Previous work in echidna demonstrated the presence of pancreatic islets, but no information is available for platypus and the fine structure has not been described for either monotreme. Based on our recent finding that monotremes lack the ghrelin gene, which is expressed in mouse and human pancreatic islets, we investigated the structure of monotreme islets in more detail. Generally, as in birds, the islets of monotremes were smaller but greater in number compared with mouse. ß-cells were the most abundant endocrine cell population in platypus islets and were located peripherally, while α-cells were observed both in the interior and periphery of the islets. δ-cells and pancreatic polypeptide (PP)-cells were mainly found in the islet periphery. Distinct PP-rich (PP-lobe) and PP-poor areas (non-PP-lobe) are present in therian mammals, and we identified these areas in echidna but not platypus pancreas. Interestingly, in some of the echidna islets, α- and ß-cells tended to form two poles within the islets, which to our knowledge is the first time this has been observed in any species. Overall, monotreme pancreata share the feature of consisting of distinct PP-poor and PP-rich islets with other mammals. A higher number of islets and α- or ß-cell only islets are shared between monotremes and birds. The islets of monotremes were larger than those of birds but smaller compared with therian mammals. This may indicate a trend of having fewer larger islets comprising several endocrine cell types during mammalian evolution.

Development of the spinal cord and peripheral nervous system in platypus (Ornithorhynchus anatinus) and short-beaked echidna (Tachyglossus aculeatus).

The modern monotremes (platypus and echidnas) are characterized by development of their young in a leathery egg that is laid into a nest or abdominal pouch. At hatching, the young are externally immature, with forelimbs capable of digitopalmar prehension, but hindlimbs little advanced beyond limb buds. The embryological collections at the Museum für Naturkunde in Berlin were used to examine the development of the spinal cord and early peripheral nervous system in developing monotremes and to correlate this with known behavioural development. Ventral root outgrowth to the bases of both the fore- and hindlimbs occurs at 6.0 mm crown-rump length (CRL), but invasion of both limbs does not happen until about 8.0-8.5 mm CRL. Differentiation of the ventral horn precedes the dorsal horn during incubation and separate medial and lateral motor columns can be distinguished before hatching. Rexed's laminae begin to appear in the dorsal horn in the first week after hatching, and gracile and cuneate fasciculi emerge during the first two post-hatching months. Qualitative and quantitative comparisons of the structure of the cervicothoracic junction spinal cord in the two monotremes with that in a diprotodont marsupial (the brush-tailed possum, Trichosurus vulpecula) of similar size at birth, did not reveal any significant structural differences between the monotremes and the marsupial. The precocious development of motor systems in the monotreme spinal cord is consistent with the behavioural requirements of the peri-hatching period, that is, rupture of embryonic membranes and egg, and digitopalmar prehension to grasp maternal hair or nest material.

Ultrastructure of the platypus and echidna mandibular glands.

The secretory units of the platypus and echidna mandibular glands consist of a single serous cell type. Secretory granules within the cells of the platypus mandibular gland stained intensely with the periodic acid-Schiff staining procedure but failed to stain with Alcian Blue, suggesting the granules contained neutral glycoproteins. Secretory granules within the mandibular glands of the echidna failed to stain with the methods used indicating little if any glycoprotein was associated with the secretory granules. Ultrastructurally, secretory granules of the platypus mandibular gland were electron dense with a central core of less electron-dense material and were membrane bound. In contrast, those of the echidna presented a lamellated appearance and also were limited by a membrane. These secretory granules appeared to form as a result of concentric layering of lamellae within cisternae of the Golgi membranes. The intralobular ductal system of the platypus was more extensively developed than that of the echidna. The striated ducts of both species were characterized by elaborate infoldings of the basolateral plasmalemma and an abundance of associated mitochondria.

Comparative retinal morphology of the platypus.

The purpose of this study is to identify evolutionary origin and fate of anatomic features of the duck-billed platypus eye. Eyes from the duck-billed platypus and four key evolutionary basal vertebrates (Pacific hagfish, north hemisphere sea lamprey, and Australian and South American lungfishes) were prepared for light microscopy. In addition to a standard panel of stains, tissues were immunostained against a variety of rod and cone opsins. Finally, published opsin sequences of platypus and several other vertebrate species were aligned and compared with immunohistochemical results. A complete scleral cartilage similar to that seen in birds, reptiles and amphibians encloses the platypus eye. This feature is present in sharks and rays, and in extant relatives of tetrapods, the lungfishes. The choroid lacks a tapetum. The retina is largely avascular and is rod-dominated, with a minority of red- and blue- cone immunoreactive photoreceptors. Like marsupials and many nonmammalian vertebrates, cones contain clear inner segment droplets. Double cones were present, a feature not found in eutherian mammals or marsupials. Evaluation of opsins indicates that red and blue immunoreactive cone opsins, but not rhodopsin, are present in the most basal of the extant species examined, the Pacific hagfish. Rhodopsin appears in the Australian and South American lungfishes, establishing emergence of this pigment in an extant relative of tetrapods. Unlike eyes of eutherian mammals, the platypus eye has retained morphologic features present in early tetrapods such as amphibians and their evolutionarily basal sister group, the lungfishes. These include scleral cartilage, double cones and cone droplets. In the platypus, as in other mammals, rod rhodopsin is the predominant photoreceptor pigment, at expense of the cone system.

Monotreme ossification sequences and the riddle of mammalian skeletal development.

The developmental differences between marsupials, placentals, and monotremes are thought to be reflected in differing patterns of postcranial development and diversity. However, developmental polarities remain obscured by the rarity of monotreme data. Here, I present the first postcranial ossification sequences of the monotreme echidna and platypus, and compare these with published data from other mammals and amniotes. Strikingly, monotreme stylopodia (humerus, femur) ossify after the more distal zeugopodia (radius/ulna, tibia/fibula), resembling only the European mole among all amniotes assessed. European moles also share extreme humeral adaptations to rotation digging and/or swimming with monotremes, suggesting a causal relationship between adaptation and ossification heterochrony. Late femoral ossification with respect to tibia/fibula in monotremes and moles points toward developmental integration of the serially homologous fore- and hindlimb bones. Monotreme cervical ribs and coracoids ossify later than in most amniotes but are similarly timed as homologous ossifications in therians, where they are lost as independent bones. This loss may have been facilitated by a developmental delay of coracoids and cervical ribs at the base of mammals. The monotreme sequence, although highly derived, resembles placentals more than marsupials. Thus, marsupial postcranial development, and potentially related diversity constraints, may not represent the ancestral mammalian condition.

Morphological and histochemical observations on the crural gland-spur apparatus of the echidna (Tachyglossus aculeatus) together with comparative observations on the femoral gland-spur apparatus of the duckbilled platypus (Ornithorhyncus anatinus).

The echidna and platypus have a crural/femoral gland that is linked by a large duct to a canalized, keratinous spur located on the medial side of the ankle. The echidna crural gland, like the femoral gland of the platypus, exhibits cyclic activity, being prominent in both monotremes when they are sexually active. In the present study, we compared the structure and histochemistry of these glands. During the active phase, the secretory epithelium forming the respective glands of both species increased in height and became packed with secretory granules that differed markedly in structure. Secretory granules of the echidna crural gland were electron dense and characterized by cores or areas of increased electron density. Those of the platypus were initially electron dense, but then became less dense and coalesced into irregular complexes of secretory material. Large cytoplasmic blebs extended from epithelial cell apices and appeared to be shed into the lumen, resulting in an apocrine mode of secretion. Exocytosis was also observed. A similar form of release of secretory product was not observed in the echidna. Secretory granules of both species were periodic acid-Schiff positive and stained for protein, suggesting that much of the secretory product was glycoprotein. Myoepithelial cells enveloped the secretory tubules of the platypus femoral gland, whereas they were not observed surrounding tubules comprising the echidna crural gland. During the quiescent phase, the epithelial cells of both species lost their secretory granules and decreased in height. As a result, the secretory tubules became smaller, intralobular connective tissue increased and the glands decreased in overall size.

Phylogenetic origins of early alterations in brain region proportions.

Adult galliform birds (e.g. chickens) exhibit a relatively small telencephalon and a proportionately large optic tectum compared with parrots and songbirds. We previously examined the embryonic origins of these adult species differences and found that the optic tectum is larger in quail than in parakeets and songbirds at early stages of development, prior to tectal neurogenesis onset. The aim of this study was to determine whether a proportionately large presumptive tectum is a primitive condition within birds or a derived feature of quail and other galliform birds. To this end, we examined embryonic brains of several avian species (emus, parrots, songbirds, waterfowl, galliform birds), reptiles (3 lizard species, alligators, turtles) and a monotreme (platypuses). Brain region volumes were estimated from serial Nissl-stained sections. We found that the embryos of galliform birds and lizards exhibit a proportionally larger presumptive tectum than all the other examined species. The presumptive tectum of the platypus is unusually small. The most parsimonious interpretation of these data is that the expanded embryonic tectum of lizards and galliform birds is a derived feature in both of these taxonomic groups.

Molecules, morphology, and ecology indicate a recent, amphibious ancestry for echidnas.

The semiaquatic platypus and terrestrial echidnas (spiny anteaters) are the only living egg-laying mammals (monotremes). The fossil record has provided few clues as to their origins and the evolution of their ecological specializations; however, recent reassignment of the Early Cretaceous Teinolophos and Steropodon to the platypus lineage implies that platypuses and echidnas diverged >112.5 million years ago, reinforcing the notion of monotremes as living fossils. This placement is based primarily on characters related to a single feature, the enlarged mandibular canal, which supplies blood vessels and dense electrosensory receptors to the platypus bill. Our reevaluation of the morphological data instead groups platypus and echidnas to the exclusion of Teinolophos and Steropodon and suggests that an enlarged mandibular canal is ancestral for monotremes (partly reversed in echidnas, in association with general mandibular reduction). A multigene evaluation of the echidna-platypus divergence using both a relaxed molecular clock and direct fossil calibrations reveals a recent split of 19-48 million years ago. Platypus-like monotremes (Monotrematum) predate this divergence, indicating that echidnas had aquatically foraging ancestors that reinvaded terrestrial ecosystems. This ecological shift and the associated radiation of echidnas represent a recent expansion of niche space despite potential competition from marsupials. Monotremes might have survived the invasion of marsupials into Australasia by exploiting ecological niches in which marsupials are restricted by their reproductive mode. Morphology, ecology, and molecular biology together indicate that Teinolophos and Steropodon are basal monotremes rather than platypus relatives, and that living monotremes are a relatively recent radiation.

The pretectal nuclei in two monotremes: the short-beaked echidna (Tachyglossus aculeatus) and the platypus (Ornithorhynchus anatinus).

We have examined the organization of the pretectal area in two monotremes (the short beaked echidna-Tachyglossus aculeatus, and the platypus-Ornithorhynchus anatinus) and compared it to that in the Wistar strain rat, using Nissl staining in conjunction with enzyme histochemistry (acetylcholinesterase and NADPH diaphorase) and immunohistochemistry for parvalbumin, calbindin, calretinin and non-phosphorylated neurofilament protein (SMI-32 antibody). We were able to identify distinct anterior, medial, posterior (now called tectal gray) and olivary pretectal nuclei as well as a nucleus of the optic tract, all with largely similar topographical and chemoarchitectonic features to the homologous regions in therian mammals. The positions of these pretectal nuclei correspond to the distributions of retinofugal terminals identified by other authors. The overall size of the pretectum in both monotremes was found to be at least comparable in size, if not larger than, the pretectum of representative therian mammals of similar brain and body size. Our findings suggest that the pretectum of these two monotreme species is comparable in both size and organization to that of eutherian mammals, and is more than just an undifferentiated area pretectalis. The presence of a differentiated pretectum with similar chemoarchitecture to therians in both living monotremes lends support to the idea that the stem mammal for both prototherian and therian lineages also had a differentiated pretectum. This in turn indicates that a differentiated pretectum appeared at least 125 million years ago in the mammalian lineage and that the stem mammal for proto- and eutherian lineages probably had similar pretectal nuclei to those identified in its descendants.