The corneal surface of aquatic vertebrates: microstructures with optical and nutritional function?

The anterior surface of the mammalian cornea plays an important role in maintaining a smooth optical interface and consequently a sharp retinal image. The smooth surface is produced by a tear film, which adheres to a variety of microprojections, which increase the cell surface area, improve the absorbance of oxygen and nutrients and aid in the movement of metabolic products across the outer cell membrane. However, little is known of the structural adaptations and tear film support provided in other vertebrates from different environments. Using field emission scanning electron microscopy; this study examines the density and surface structure of corneal epithelial cells in representative species of the classes Cephalaspidomorphi, Chondrichthyes, Osteichthyes, Amphibia, Reptilia, Aves and Mammalia, including some Marsupialia. Variations in cell density and the structure and occurrence of microholes, microridges, microplicae and microvilli are described with respect to the demands placed upon the cornea in different aquatic environments such as marine and freshwater. A progressive decrease in epithelial cell density occurs from marine (e.g. 29348 cells mm(-2) in the Dover sole Microstomius pacficus) to estuarine or freshwater (e.g. 5999 cells mm(-2) in the black bream Acanthopagrus butcheri) to terrestrial (e.g. 2126 cells mm(-2) in the Australian koala Phascolarctos cinereus) vertebrates, indicating the reduction in osmotic stress across the corneal surface. The microholes found in the Southern Hemisphere lampreys, namely the pouched lamprey (Geotria australis) and the shorthead lamprey (Mordacia mordax) represent openings for the release of mucus, which may protect the cornea from abrasion during their burrowing phase. Characteristic of marine teleosts, fingerprint-like patterns of corneal microridges are a ubiquitous feature, covering many types of sensory epithelia (including the olfactory epithelium and the oral mucosa). Like microplicae and microvilli, microridges stabilize the tear film to maintain a smooth optical surface and increase the surface area of the epithelium, assisting in diffusion and active transport. The clear interspecific differences in corneal surface structure suggest an adaptive plasticity in the composition and stabilization of the corneal tear film in various aquatic environments.     

Corneal microprojections in coleoid cephalopods

The cornea is the first optical element in the path of light entering the eye, playing a role in image formation and protection. Corneas of vertebrate simple camera-type eyes possess microprojections on the outer surface in the form of microridges, microvilli, and microplicae. Corneas of invertebrates, which have simple or compound eyes, or both, may be featureless or may possess microprojections in the form of nipples. It was previously unknown whether cephalopods (invertebrates with camera-type eyes like vertebrates) possess corneal microprojections and, if so, of what form. Using scanning electron microscopy, we examined corneas of a range of cephalopods and discovered nipple-like microprojections in all species. In some species, nipples were like those described on arthropod compound eyes, with a regular hexagonal arrangement and sizes ranging from 75 to 103?nm in diameter. In others, nipples were nodule shaped and irregularly distributed. Although terrestrial invertebrate nipples create an antireflective surface that may play a role in camouflage, no such optical function can be assigned to cephalopod nipples due to refractive index similarities of corneas and water. Their function may be to increase surface-area-to-volume ratio of corneal epithelial cells to increase nutrient, gas, and metabolite exchange, and/or stabilize the corneal mucous layer, as proposed for corneal microprojections of vertebrates.     

Comparative study of the corneal epithelium in some reptiles inhabiting different environments

El‐Bakry, A.M. 2011. Comparative study of the corneal epithelium in some reptiles inhabiting different environments. —Acta Zoologica (Stockholm) 92 : 54–61. The vertebrate cornea functions in either aquatic or aerial environments and in some cases in both. In terrestrial and aerial vertebrates, the cornea contributes most of the refractive powers of the eye because of the large variation in refractive index between the air and the cornea. The present study aimed to examine and compare the main features of the corneal epithelial surface of three reptilian species related to three different families (Caretta caretta, Varanus griseus and Mabuya quinquetaeniata) and inhabiting different environment, by light, scanning (SEM) and transmission electron microscopy. The mean epithelial cell densities of the species of the study were 8.670 ± 3.134, 5.945 ± 2.144 and 2.124 ± 713 respectively. The corneal epithelium of the three species observed by SEM showed a similarity to one another indicating that the apical cell surfaces possess regular polygonal cells with varieties of microprocesses. These microprocesses were represented by microplicae, numerous microvilli and some long microridges in C. caretta, microplicae and minute microholes in V. griseus and microplicae intermingled with short microvilli in M. quinquetaeniata. According to the densities of these microprocesses, three polymorphic cell types (light, medium and dark) appeared in C. caretta, light and medium cell types were observed in V. griseus and medium and dark cell types were noticed in M. quinquetaeniata. Different types of tight adhesions were observed by transmission electron microscopy between the cell borders of the epithelial cells which differ according to environment where the species occupy. In conclusion, variation in the structure of the corneal epithelial cells appears to be related to the living environment, such as aerial, terrestrial and aquatic ones, which is occupied by every species.     

Surface ultrastructure of the cornea and adjacent epidermis during metamorphosis of Rana pipiens A scanning electron microscopic study

The external surface of the cornea and adjacent epidermis of larvae in representative developmental stages and of adult frogs, Rana pipiens, was studied by scanning electron microscopy. Surface cells are polygonal, usually hexagonal, in outline and covered with microprojections. During larval development prior to metamorphic stages, neither eyelids nor Harderian glands have developed; microprojections on the corneal surface are high and branched, and cell boundaries are elevated. On the anterior portion of the cornea and on the epidermis near the eye, the surface pattern is less dense, and ciliated cells are present. During metamorphic stages, corneal cell boundaries become less prominent and the pattern of microprojections more variable and markedly different from that of larvae of earlier stages. Corneal cells have a spongy appearance, are covered by a coating material, or are characterized as light or dark based on their brightness and surface texture. As eyelids develop in metamorphic stages XX–XXI, the numbers of ciliated cells increase dramatically, both on the corneal surface and on the edges of the developing lids. In later metamorphic stages XXII–XXV, lids and Harderian glands become well-developed, and cilia are no longer observed. The adjacent epidermal surface becomes devoid of cilia but perforated by openings of cutaneous glands. Its spongy appearance is similar to that of both the cornea and neighboring epidermis of the mature frog. Changes in corneal surface features are probably metamorphic events associated with development of lids and Harderian glands and a shift from an aqueous to an air environment.     

Three-dimensional and surface structures of rat filiform papillae

The three-dimensional and surface structures of the simple conical papillae of the rat tongue have been demonstrated with scanning electron microscopy. The papillary projection was organized into the anterior, posterior and central core cell populations, whereas the basal region of the papilla which consisted of circularly arranged cells showed no differentiation into three autonomic cell populations. It is considered that the anterior and posterior cell populations around the central core tend to be mutually attached at the bilateral sides, and that the posterior and core cell contacts are rather close than the anterior one. The anterior papillary cells showed relatively smooth surface with little micropits and without microridges. The reticular microridges on the basal cell surface of the posterior papillary cells appear to later develop the micropits and linear microridges on the tip cell surface. These suggest that the anterior cell surface is more highly keratinized than the posterior one. The microridges or micropits on the outer cell surface and the microprojections on the inner cell surface organizing filiform papilla are considered to be the structures for the purpose of cell adhesion.     

Scanning Electron microscopic Study on the Early Development of Silkworm Eggs (Bombyx mori L.)

Morphological changes of the surface of eggs of the silkworm Bombyx mori L. were studied during early developmental stages by scanning electron microscopy. The egg surface was covered with numerous microvilli at least until 4 h after oviposition. At 6 h the microvilli were replaced by ruffle-like microprojections. This suggests that developmental changes of the surface structure may occur without direct influence of cleavage nuclei. Immediately before blastoderm cell formation, microvilli reappeared in the presumptive groove area. The ruffles seen on the apical portion of newly-formed blastoderm cells gradually became flattened, while microvilli developed on the lateral side of the cells. The mode of blastoderm cell formation is different from the typical one seen in most species of insects.     

Control of pulmonary surfactant secretion: an evolutionary perspective

Pulmonary surfactant, a mixture consisting of phospholipids (PL) and proteins, is secreted by type II cells in the lungs of all air-breathing vertebrates. Virtually nothing is known about the factors that control the secretion of pulmonary surfactant in nonmammalian vertebrates. With the use of type II cell cultures from Australian lungfish, North American bullfrogs, and fat-tailed dunnarts, we describe the autonomic regulation of surfactant secretion among the vertebrates. ACh, but not epinephrine (Epi), stimulated total PL and disaturated PL (DSP) secretion from type II cells isolated from Australian lungfish. Both Epi and ACh stimulated PL and DSP secretion from type II cells of bullfrogs and fat-tailed dunnarts. Neither Epi nor ACh affected the secretion of cholesterol from type II cell cultures of bullfrogs or dunnarts. Pulmonary surfactant secretion may be predominantly controlled by the autonomic nervous system in nonmammalian vertebrates. The parasympathetic nervous system may predominate at lower body temperatures, stimulating surfactant secretion without elevating metabolic rate. Adrenergic influences on the surfactant system may have developed subsequent to the radiation of the tetrapods. Furthermore, ventilatory influences on the surfactant system may have arisen at the time of the evolution of the mammalian bronchoalveolar lung. Further studies using other carefully chosen species from each of the vertebrate groups are required to confirm this hypothesis.     

An examination of surface epithelium structures of the embryo across the genus Poeciliopsis (Poeciliidae)

In viviparous, teleost fish, with postfertilization maternal nutrient provisioning, embryonic structures that facilitate maternal‐fetal nutrient transfer are predicted to be present. For the family Poeciliidae, only a handful of morphological studies have explored these embryonic specializations. Here, we present a comparative morphological study in the viviparous poeciliid genus, Poeciliopsis . Using microscopy techniques, we examine the embryonic surface epidermis of Poeciliopsis species that vary in their level of postfertilization maternal nutrient provisioning and placentation across two phylogenetic clades and three independent evolutionary origins of placentation. We focus on surface features of the embryo that may facilitate maternal‐fetal nutrient transfer. Specifically, we studied cell apical‐surface morphology associated with the superficial epithelium that covers the body and sac (yolk and pericardial) of embryos at different developmental stages. Scanning electron microscopy revealed common surface epithelial cells across species, including pavement cells with apical‐surface microridges or microvilli and presumed ionocytes and/or mucus‐secreting cells. For three species, in the mid‐stage embryos, the surface of the body and sac were covered in microvillus epithelium. The remaining species did not display microvillus epithelium at any of the stages examined. Instead, their epithelium of the body and sac were composed of cells with apical‐surface microridges. For all species, in the late stage embryos, the surface of the body proper was composed of apical‐surface microridges in a “fingerprint‐like arrangement.” Despite the differences in the surface epithelium of embryos across Poeciliopsis species and embryonic developmental stages, this variation was not associated with the level of postfertilization maternal nutrient provisioning. We discuss these results in light of previous morphological studies of matrotrophic, teleost fish, phylogenetic relationships of Poeciliopsis species, and our earlier comparative microscopy work on the maternal tissue of the Poeciliopsis placenta.     

Localization of glycoconjugates on the surfaces of pronephric tumor cells in vitro

Differential localization of glycoconjugates was detected on microvilli and microridges of the intact cell surface of frog pronephric tumor cells in tissue culture. Alcian blue and Alcian blue/PAS staining showed a heavy concentration of dye limited to the unique short microvilli and extensive microridges of the tumor cells as previously seen with SEM (Tweedell and Williams 1976). Staining was absent or greatly reduced on microvilli of the normal pronephric cell surface. Previous exposure of each kind of cells to neuraminidase or extraction by mild hydrolysis removed the active staining sites but Alcian blue uptake was unaffected by prior digestion with testicular hyaluronidase. Fluorescein isothiocyanate (FITC) bound wheat germ agglutinin (WGA) produced a similar pattern of fluorescence on the microvilli of the tumor cells and a limited distribution on the normal cells. Digestion with neuraminidase preferentially removed but did not completely eliminate the surface binding of WGA on both the normal and tumor cells. Exposure of tumor cell monolayers to FITC bound limulin, a lectin specific for sialic acid, also produced an intense surface fluorescence on the microvilli and ridges of tumor cells. Prior treatment with neuraminidase prevented the surface fluorescence but not internal binding. Normal pronephric cells gave sparse surface fluorescence but extensive internal binding. Each procedure indicates a preferential localization of complex carbohydrates, including sialic acid, on the unique microvilli of the tumor cells. Concurrent assays for sialic acid recovered from the tumor cells indicated that lectin bound surface sialic acid was removable with neuraminidase.     

Cartilage,bone, and intermandibular connective tissue in the australian lungfish,Neoceratodus forsteri (Osteichthyes: Dipnoi)

The connective tissue that links the bones of the mandible in the Australian lungfish, Neoceratodus forsteri, has been described as an intermandibular cartilage, and as such has been considered important for phylogenetic analyses among lower vertebrates. However, light and electron microscopy of developing lungfish jaws demonstrates that the intermandibular tissue, like the connective tissue that links the bones of the upper jaw, contains fibroblasts and numerous bundles of collagen fibrils, extending from the trabeculae of the bones supporting the tooth plates. It differs significantly in structure and in staining reactions from the cartilage and the bone found in this species. In common with the cladistian Polypterus and with actinopterygians and some amphibians, lungfish have no intermandibular cartilage. The connective tissue linking the mandibular bones has no phylogenetic significance for systematic grouping of lungfish, as it is present in a range of different groups among lower vertebrates. J. Morphol. 274:1085–1089, 2013. © 2013 Wiley Periodicals, Inc.     

Localization of glycoconjugates on the surfaces of pronephric tumor cells in vitro

Summary Differential localization of glycoconjugates was detected on microvilli and microridges of the intact cell surface of frog pronephric tumor cells in tissue culture. Alcian blue and Alcian blue/PAS staining showed a heavy concentration of dye limited to the unique short microvilli and extensive microridges of the tumor cells as previously seen with SEM (Tweedell and Williams 1976). Staining was absent or greatly reduced on microvilli of the normal pronephric cell surface. Previous exposure of each kind of cells to neuraminidase or extraction by mild hydrolysis removed the active staining sites but Alcian blue uptake was unaffected by prior digestion with testicular hyaluronidase. Fluorescein isothyocyanate (FITC) bound wheat germ agglutinin (WGA) produced a similar pattern of fluorescence on the microvilli of the tumor cells and a limited distribution on the normal cells. Digestion with neuraminidase preferentially removed but did not completely eliminate the surface binding of WGA on both the normal and tumor cells. Exposure of tumor cell monolayers to FITC bound limulin, a lectin specific for sialic acid, also produced an intense surface fluorescence on the microvilli and ridges of tumor cells. Prior treatment with neuraminidase prevented the surface fluorescence but not internal binding. Normal pronephric cells gave sparse surface fluorescence but extensive internal binding. Each procedure indicates a preferential localization of complex carbohydrates, including sialic acid, on the unique microvilli of the tumor cells. Concurrent assays for sialic acid recovered from the tumor cells indicated that lectin bound surface sialic acid was removable with neuraminidase.This study was supported by a grant from the Cancer Society of Saint Joseph County, Indiana and from the Phi Beta Psi Sorority     

Morphological changes in frog pronephric cell surfaces after transformation by herpes virus.

A scanning electron-microscope study has established that there are major modifications in the surface membranes of malignant cells transformed from the pronephric kidney of the frog. Tumours were induced in vivo after exposure of embryos Lucké Herpes virus. A pronephric cell line from normal embryos and 3 tumour cell lines derived from experimentally induced adenocarcinomas of the pronephros were observed. The normal embryonic kidney cells have their surfaces covered with long fingerlike microvilli throughout the cell cycle. In contrast, the surface of the pronephric tumour cells are mainly covered by multiple, ruffled or parallel straight microridges and short, stubby microvilli. These comparisons were made on cells subject to similar culture conditions. The topographic changes in the tumour cells are considered significant since the characteristic microridges and stubby villi were consistently found in all 3 pronephric tumour lines.     

Ultrastructural and histochemical differences in cell surface properties of strain-specific and nonstrain-specific TA3 adenocarcinoma cells

Transmission and scanning electron microscopy and histochemical and biochemical methods were used to investigate differences in cell structure and cell surface properties between the strain-specific TA3- St and nonstrain-specific TA3-Ha ascites sublines of the TA3 murine mammary adenocarcinoma. The TA3-St subline is lethal only to the syngeneic strain A mouse (the strain of origin), whereas the TA3-Ha subline is lethal even to foreign species. In contrast to the TA3-St cell surface, which has numerous folds and irregular microprojections, the TA3-Ha cell has abundant long microvilli of uniform dimensions. An extensive cell surface coat which resembles the "fuzz" coat found on microvilli of normal epithelium was present on the TA3-Ha, but not on the TA3-St cells. After routine fixation, the surface coat of the TA3- Ha cell usually appeared as a filamentous network extending 30-50 nm from the plasmalemma; occasionally, longer filamentous or rod-like structures were found extending 200-400 nm from the plasmalemma. The cell coat material was more extensive on the microvilli than on the intermicrovillous membranes. Free virus-like particles associated with TA3-Ha cells have a similar-appearing surface coat on their outer membranes. The density of surface anionic sites, determined with polycationic ferritin, was greater on the TA3-Ha than on the TA3-St cell surface, consistent with the presence at the TA3-Ha cell surface of several-fold more neuraminidase-susceptible sialic acid groups. The observed surface features of the nonstrain-specific TA3-Ha cell, in comparison to the strain-specific TA3-St cell, are consistent with the suggestion that sialic acid-rich glycoproteins at the TA3-Ha cell surface mask histocompatibility antigens and enhance the ability of malignant cells to invade foreign species.     

Cranial neural crest cell migration in the Australian lungfish, Neoceratodus forsteri

SUMMARY A crucial role for the cranial neural crest in head development has been established for both actinopterygian fishes and tetrapods. It has been claimed, however, that the neural crest is unimportant for head development in the Australian lungfish ( Neoceratodus forsteri   ), a member of the group (Dipnoi) which is commonly considered to be the living sister group of the tetrapods. In the present study, we used scanning electron microscopy to study cranial neural crest development in the Australian lungfish. Our results, contrary to those of Kemp, show that cranial neural crest cells do emerge and migrate in the Australian lungfish in the same way as in other vertebrates, forming mandibular, hyoid, and branchial streams. The major difference is in the timing of the onset of cranial neural crest migration. It is delayed in the Australian lungfish in comparison with their living sister group the Lissamphibia. Furthermore, the delay in timing between the emergence of the hyoid and branchial crest streams is very long, indicating a steeper anterior-posterior gradient than in amphibians. We are now extending our work on lungfish head development to include experimental studies (ablation of selected streams of neural crest cells) and fate mapping (using fluoresent tracer dyes such as DiI) to document the normal fate as well as the role in head patterning of the cranial neural crest in the Australian lungfish.     

The fate of cranial neural crest cells in the Australian lungfish, Neoceratodus forsteri

The cranial neural crest has been shown to give rise to a diversity of cells and tissues, including cartilage, bone and connective tissue, in a variety of tetrapods and in the zebrafish. It has been claimed, however, that in the Australian lungfish these tissues are not derived from the cranial neural crest, and even that no migrating cranial neural crest cells exist in this species. We have earlier documented that cranial neural crest cells do migrate, although they emerge late, in the Australian lungfish. Here, we have used the lipophilic fluorescent dye, DiI, to label premigratory cranial neural crest cells and follow their fate until stage 43, when several cranial skeletal elements have started to differentiate. The timing and extent of their migration was investigated, and formation of mandibular, hyoid and branchial streams documented. Cranial neural crest was shown to contribute cells to several parts of the head skeleton, including the trabecula cranii and derivatives of the mandibular arch (e.g., Meckel's cartilage, quadrate), the hyoid arch (e.g., the ceratohyal) and the branchial arches (ceratobranchials I-IV), as well as to the connective tissue surrounding the myofibers in cranial muscles. We conclude that cranial neural crest migration and fate in the Australian lungfish follow the stereotyped pattern documented in other vertebrates.     

Complete Mitochondrial Genome Sequences of the South American and the Australian Lungfish: Testing of the Phylogenetic Performance of Mitochondrial Data Sets for Phylogenetic Problems in Tetrapod Relationships

We determined the complete nucleotide sequences (16403 and 16572 base pairs, respectively) of the mitochondrial genomes of the South American lungfish, Lepidosiren paradoxa, and the Australian lungfish, Neoceratodus forsteri (Sarcopterygii, Dipnoi). The mitochondrial DNA sequences were established in an effort to resolve the debated evolutionary positions of the lungfish and the coelacanth relative to land vertebrates. Previous molecular phylogenetic studies based on complete mtDNA sequences, including only the African lungfish, Protopterus dolloi, sequence were able to strongly reject the traditional textbook hypothesis that coelacanths are the closest relatives of land vertebrates. However, these studies were unable to statistically significantly distinguish between the two remaining scenarios: lungfish as the closest relatives to land vertebrates and lungfish and coelacanths jointly as their sister group (Cao et al. 1998; Zardoya et al. 1998; Zardoya and Meyer 1997a). Lungfish, coelacanths, and the fish ancestors of the tetrapod lineage all originated within a short time window of about 20 million years, back in the early Devonian (about 380 to 400 million years ago). This short divergence time makes the determination of the phylogenetic relationships among these three lineages difficult. In this study, we attempted to break the long evolutionary branch of lungfish, in an effort to better resolve the phylogenetic relationships among the three extant sarcopterygian lineages. The gene order of the mitochondrial genomes of the South American and Australian lungfish conforms to the consensus gene order among gnathostome vertebrates. The phylogenetic analyses of the complete set of mitochondrial proteins (without ND6) suggest that the lungfish are the closest relatives of the tetrapods, although the support in favor of this scenario is not statistically significant. The two other smaller data sets (tRNA and rRNA genes) give inconsistent results depending on the different reconstruction methods applied and cannot significantly rule out any of the three alternative hypotheses. Nuclear protein-coding genes, which might be better phylogenetic markers for this question, support the lungfish–tetrapod sister-group relationship (Brinkmann et al. 2004).This article contains online supplementary material.Reviewing Editor: Dr. Rafael Zardoya     

Comparison of funding and demand for the conservation of the charismatic koala with those for the critically endangered wombat <Emphasis Type="Italic">Lasiorhinus krefftii</Emphasis>

This study contrasts the actual conservation spending and the Australian public’s demand for conservation funding for two Australian mammal species, the koala and the northern hairy-nosed wombat. It involves a survey of 204 members of the Australian public. Willingness to fund conservation action to protect the northern hairy-nosed wombat was found to be higher than that for the koala despite the koala’s immense popularity. The critically endangered status of the northern-hairy nosed wombat and the more secure conservation status of the koala is a factor likely to have influenced the comparative willingness-to-pay decisions. Actual annual conservation expenditure for both species is lower than the estimated aggregate willingness-to-pay for their conservation. Furthermore, conservation funding for the koala is much more than that for the northern hairy-nosed wombat even though the estimated public willingness-to-pay (demand) for funding koala conservation was less than for this wombat species. Reasons for this are suggested. They may also help to explain misalignment between demand for conservation funding of other species involving differences in charisma and endangerment.     

Ophthalmologic examination of the normal eye of the koala.

The ophthalmic examination of the koala (Phascolarctos cinereus) was undertaken to describe the normal in vivo structure. Twenty-two of 28 koalas examined were found to have normal eyes. Slit-lamp examination revealed unusual lamellae in the cornea, and mean keratometric readings were 59.3 +/- 3 diopters, corneal endothelial cell area of 381.4 microns2 +/- with a standard deviation of 46.8 microns2. No particular features were seen which would explain the susceptibility of this animal to ocular infection by Chlamydia psittaci.     

Vicariance and dispersal effects on phylogeographic structure and speciation in a widespread estuarine invertebrate

Vicariance and dispersal can strongly influence population genetic structure and allopatric speciation, but their importance in the origin of marine biodiversity is unresolved. In transitional estuarine environments, habitat discreteness and dispersal barriers could enhance divergence and provide insight to evolutionary mechanisms underlying marine and freshwater biodiversity. We examined this by assessing phylogeographic structure in the widespread amphipod Gammarus tigrinus across 13 estuaries spanning its northwest Atlantic range from Quebec to Florida. Mitochondrial cytochrome c oxidase I and nuclear internal transcribed spacer 1 phylogenies supported deep genetic structure consistent with Pliocene separation and cryptic northern and southern species. This break occurred across the Virginian-Carolinian coastal biogeographic zone, where an oceanographic discontinuity may restrict gene flow. Ten estuarine populations of the northern species occurred in four distinct clades, supportive of Pleistocene separation. Glaciation effects on genetic structure of estuarine populations are largely unknown, but analysis of molecular variance (AMOVA) supported a phylogeographic break among clades in formerly glaciated versus nonglaciated areas across Cape Cod, Massachusetts. This finding was concordant with patterns in other coastal species, though there was no significant relationship between latitude and genetic diversity. This supports Pleistocene vicariance events and divergence of clades in different northern glacial refugia. AMOVA results and private haplotypes in most populations support an allopatric distribution across estuaries. Clade mixture zones are consistent with historical colonization and human-mediated transfer. An isolation-by-distance model of divergence was detected after we excluded a suspected invasive haplotype in the St. Lawrence estuary. The occurrence of cryptic species and divergent population structure support limited dispersal, dispersed habitat distribution, and historical factors as important determinants of estuarine speciation and diversification.     

Morphology of rabbit collecting duct.

Recently the assumed structural and functional homogeneity of the collecting duct (CD) has been questioned. The objective of this study was to determine if heterogeneity occurs in luminal surface membrane structure or in cytoplasmic configuration of cells in the collecting duct or both. Straight segments of cortical and medullary CD were examined in perfusion-fixed rabbit kidneys with scanning electron microscopy (SEM), light (LM) and transmission electron microscopy (TEM). Principal cells were the most abundant cells in all CD regions; intercalated cells comprised 37% of the cell population on the cortex, 18% in the outer medulla, and less than 1% in the inner medulla. SEM revealed two surface patterns among the ciliated principal cells: 1, located in the cortex and outer medulla, with few surface microvilli, and 2, located in the inner medulla, with abundant microvilli. Intercalated cells exhibited four distinctive luminal surface configurations: I, numerous short microvilli; II, both short and elongate microvilli; III, microplicae alone; and IV, both microvilli and microplicae. Intercalated cells with patterns I and II were predominant in the cortex, while cells with patterns III and IV were most common at the corticomedullary junction. TEM confirmed that marked variation existed in cytoplasmic structures of both principal and intercalated cells. These findings may either indicate the presence of several specific types of principal and intercalated cells or reflect different functional states of the principal and intercalated cells. Regardless of their significance, their presence must be considered in studies seeking to establish precise structural-functional relationships in this region of the rabbit renal tubule.