ABSTRACT
Lymph nodes and other secondary lymphoid organs play critical roles in immune surveillance and immune activation in mammals, but the deep internal locations of these organs make it challenging to image and study them in living animals. Here, we describe a previously uncharacterized external immune organ in the zebrafish ideally suited for studying immune cell dynamics in vivo, the axillary lymphoid organ (ALO). This small, translucent organ has an outer cortex teeming with immune cells, an inner medulla with a mesh-like network of fibroblastic reticular cells along which immune cells migrate, and a network of lymphatic vessels draining to a large adjacent lymph sac. Noninvasive high-resolution imaging of transgenically marked immune cells can be carried out in the lobes of living animals, and the ALO is readily accessible to external treatment. This newly discovered tissue provides a superb model for dynamic live imaging of immune cells and their interaction with pathogens and surrounding tissues, including blood and lymphatic vessels.
ABSTRACT
Advances in technology have increased our knowledge of the processes that effect genomic changes and of the roles of RNA networks in biocommunication, functionality, and evolution of genomes. Natural genetic engineering and genomic inscription occur at all levels of life: cell cycles, development, and evolution. This has implications for phylogenetic studies and for biogeography, particularly given the general acceptance of using molecular clocks as arbiters between vicariance and dispersal explanations in biogeography. Léon Croizat's development of panbiogeography and his explanation for the distribution patterns of organisms are based on concepts of dispersal, differential form-making, and ancestor that differ from concepts of descent used broadly in phylogenetic and biogeographic studies. Croizat's differential form-making is consistent with the extensive roles ascribed to RNAs in development and evolution and recent discoveries of genome studies. Evolutionary-developmental biology (evo-devo), including epigenetics, and the role of RNAs should be incorporated into biogeography.
Subject(s)
Biological Evolution , PhylogenyABSTRACT
Teleost fishes show an enormous diversity of parental care, ranging from no care to viviparity with maternal provisioning of embryos. External brooders carry their developing eggs attached to their bodies. This requires the formation of novel morphological structures to support attachment. The pelvic brooding ricefish Oryzias eversi evolved such a structure, called the "plug." The plug anchors attaching filaments from the fertilized eggs inside the female reproductive system, allowing the female to carry the embryos until hatching. Using histological sections and µ-computed tomography scanning, we show that the plug is formed by several types of interstitial cells, blood capillaries, and collagen fibrils that encapsulate the end of the attaching filaments in the anterior part of the gonoduct. Even 15 days after the loss of the protruding attaching filaments, the plug remains. In addition, the developed plug contains multinucleated giant cells that are derived from fusing macrophages. We thus hypothesize that the ricefish plug, which is vital for egg attachment in O. eversi, evolved due to an inflammatory reaction. We assume that it forms similar to a foreign body granuloma, as a reaction to irritation or injury of the gonoduct epithelium by the attaching filaments. Our study further corroborates that pelvic brooding entails a complex set of adaptations to prolonged egg-carrying in the female reproductive system. During brooding, for instance, ovulation in the ovary is suppressed and the anterior part of the gonoduct is characterized by an intricate, recessed folding.
Subject(s)
Beloniformes , Oryzias , Female , Animals , Reproduction , Ovary/anatomy & histology , CollagenABSTRACT
In the oviparous medaka fish, Oryzias latipes, mature spermatozoa that were artificially introduced into the ovarian cavity retaining ovulated eggs could internally fertilize these eggs. This enabled us to examine the effect of ovarian gestation on the ovulation cycle. Most freshly ovulated eggs could be internally fertilized in the ovarian cavity. Yet eggs ovulated 24 h after single insemination remained unfertilized in the ovarian cavity. Artificially pregnant females persisted in a daily cycle of ovulation, which occurred shortly before the onset of light under the present reproductive conditions. Females continuously ovulated a certain number of eggs despite ovarian gestation, that is, the presence of embryos within the ovarian cavity. Repeated cycles of ovulation led to crowding in the ovarian cavity because the group of fertilized eggs, with their hardened egg envelope (chorion or zona radiata), plugged the genital orifice. The development of fertilized eggs was retarded and ceased around the initiation stage of blood circulation, but when they were transferred from the ovarian cavity into regular saline, they regained their ability to develop normally up to hatching. These results show that in oviparous female medaka, ovarian gestation exerted little effect on the time of ovulation and the number of ovulated eggs.
Subject(s)
Beloniformes , Oryzias , Animals , Female , Fertilization , Male , Oryzias/physiology , Oviparity/physiology , Ovulation , PregnancyABSTRACT
Rhyacichthys aspro is a "basal" taxon in the Suborder Gobioidei of the teleost order Gobiiformes. We provide detailed descriptions of the reproductive morphology of adult males and females to assess the diagnostic reproductive morphological characters of this speciose clade of bony fishes. Female R. aspro are asynchronous spawners: they are able to spawn more than once in a breeding season. Oocytes are inferred to have short attachment filaments. A conspicuous feature of the external anatomy of the reproductive system (RSy) of female R. aspro is an ornate fimbriate pad upon which the urogenital papilla rests. The male reproductive system is characterized by an intralobar collection system in both the testicular and secretory lobes, termed the "sperm-collecting canal" and "milt-collecting canal," respectively. These may provide additional storage for sperm and milt. The spermatogenic lobe, or testis, is that portion of the male gobioid RSy comprising seminiferous lobules and separate from other RSy components. The secretory lobe is that portion of the male gobioid reproductive system that consists of secretory lobules and is separated from other components of the male RSy. The secretory lobe has also been called, in English, the sperm-duct gland, accessory gonadal structure, or seminal vesicle, and is endorsed as a synapomorphy of gobioid fishes.
Subject(s)
Perciformes , Animals , Female , Fishes/anatomy & histology , Gonads , Male , Spermatozoa , Testis/anatomy & histologyABSTRACT
Ichthyological surveys have traditionally been conducted using whole-specimen, capture-based sampling with varied but conventional fishing gear. Recently, environmental DNA (eDNA) metabarcoding has emerged as a complementary, and possible alternative, approach to whole-specimen methodologies. In the tropics, where much of the diversity remains undescribed, vast reaches continue unexplored, and anthropogenic activities are constant threats; there have been few eDNA attempts for ichthyological inventories. We tested the discriminatory power of eDNA using MiFish primers with existing public reference libraries and compared this with capture-based methods in two distinct ecosystems in the megadiverse Amazon basin. In our study, eDNA provided an accurate snapshot of the fishes at higher taxonomic levels and corroborated its effectiveness to detect specialized fish assemblages. Some flaws in fish metabarcoding studies are routine issues addressed in natural history museums. Thus, by expanding their archives and adopting a series of initiatives linking collection-based research, training and outreach, natural history museums can enable the effective use of eDNA to survey Earth's hotspots of biodiversity before taxa go extinct. Our project surveying poorly explored rivers and using DNA vouchered archives to build metabarcoding libraries for Neotropical fishes can serve as a model of this protocol.
Subject(s)
Biodiversity , DNA, Environmental/analysis , Fishes/genetics , Museums , Animals , DNA Barcoding, Taxonomic , Data Analysis , Databases, Genetic , Fishes/classification , Phylogeny , Rivers , South America , Species Specificity , Surveys and QuestionnairesABSTRACT
This study presents novel findings on the dynamics of growing oocytes in the ovary of the medaka fish, Oryzias latipes. In the ovary of mature females, all follicles are anchored tightly to the abdominal ovarian rete via fibrous follicular stalks on the follicle surface. The follicular stalks lie at the end of the follicle opposite the site of its attachment to the ovarian wall. Various lengths of the follicular stalks reflect the spatial arrangement of follicles in the ovary. Herein, a line that connects the follicular stalk to the opposite side of the follicle toward or attaching to the ovarian wall is called the follicle axis. The animal-vegetal axis in late stage III oocytes is already recognizable as a line that connects the center of the oocyte nucleus and the vegetal pole area; this is ascertainable by the morphological landmark of a compact distribution of granulosa cells or rudimentary attachment filaments. In growing oocytes later than stage V, the beginning of vitellogenesis, the tufted attachment filaments are located on a discrete region of the vegetal pole area. Our observations reveal that during growth, oocytes are arranged randomly between the animal-vegetal axis and the follicle axis, whereas the vegetal pole area of full-grown oocytes in preovulatory follicles turns close to the inner surface of the ovarian wall, from which mature oocytes subsequently ovulate into the ovarian lumen. It is suggested that in the O. latipes ovary, mature oocytes always transpose the vegetal pole area to the ovulatory site of the ovarian wall prior to ovulation. The expulsion of mature oocytes from the vegetal pole appears to be the regular mode of ovulation in O. latipes.
Subject(s)
Beloniformes , Oryzias , Animals , Female , Oocytes , Ovarian Follicle , OvaryABSTRACT
The cyprinodontiform family Goodeidae comprises some 51 species, including subspecies, of freshwater fishes all of which are at risk or are extinct in the wild. It is classified in two allopatric subfamilies: the Goodeinae, endemic to the Mexican Plateau, and the Empetrichthyinae, known only from relict taxa in Nevada and southern California. The 41 species of goodeins are all viviparous and share a set of well-documented reproductive characters. In contrast, the recent species or subspecies of empetrichthyins are all oviparous and relatively poorly known, yet of critical interest in understanding the evolution of livebearing in the family. We previously described ovarian structure and oogenesis in empetrichthyins using archival museum specimens of females and here extend that study to males. Testicular characters of two species of empetrichthyins, Crenichthys baileyi, and Empetrichthys latos, are studied and compared directly with those of one species of viviparous goodeid, Ataeniobius toweri. The testis is a restricted spermatogonial type in both the Empetrichthyinae and the Goodeinae: spermatogonia are found solely at the distal termini of lobules, a diagnostic character of atherinomorph fishes. Morphology of the differentiation of germinal cells during spermatogenesis is similar in both subfamilies. In the oviparous C. baileyi and E. latos spermatozoa are free in the deferent ducts. In contrast, the spermatozoa of viviparous goodeids are organized into numerous bundles called spermatozeugmata, a characteristic of most fishes that practice internal fertilization. Differences between the goodeid subfamilies are interpreted relative to the oviparous versus viviparous modes of reproduction. Archival museum specimens are a reliable source of data on reproductive morphology, including histology, and may be the only specimens available of rare or extinct taxa.
Subject(s)
Cyprinodontiformes/anatomy & histology , Cyprinodontiformes/physiology , Oviparity/physiology , Spermatogenesis , Testis/anatomy & histology , Animals , Male , Spermatozoa/cytology , Testis/cytology , Testis/physiologyABSTRACT
As global biodiversity declines, the value of biological collections increases. Cryopreserved diploid spermatogonial cells meet two goals: to yield high-quality molecular sequence data; and to regenerate new individuals, hence potentially countering species extinction. Cryopreserved spermatogonial cells that allow for such mitigative measures are not currently in natural history museum collections because there are no standard protocols to collect them. Vertebrate specimens, especially fishes, are traditionally formalin-fixed and alcohol-preserved which makes them ideal for morphological studies and as museum vouchers, but inadequate for molecular sequence data. Molecular studies of fishes routinely use tissues preserved in ethanol; yet tissues preserved in this way may yield degraded sequences over time. As an alternative to tissue fixation methods, we assessed and compared previously published cryopreservation methods by gating and counting fish testicular cells with flow cytometry to identify presumptive spermatogonia A-type cells. Here we describe a protocol to cryopreserve tissues that yields a high percentage of viable spermatogonial cells from the testes of Asterropteryx semipunctata, a marine goby. Material cryopreserved using this protocol represents the first frozen and post-thaw viable spermatogonial cells of fishes archived in a natural history museum to provide better quality material for re-derivation of species and DNA preservation and analysis.
Subject(s)
Cryopreservation , Fishes , Spermatogonia , Animals , Cryopreservation/methods , Male , Specimen Handling/methods , Specimen Handling/trends , Spermatogonia/cytology , Testis/cytologyABSTRACT
We provide histological details of the development of oocytes in the cyprinodontid flagfish, Jordanella floridae. There are six stages of oogenesis: Oogonial proliferation, chromatin nucleolus, primary growth (previtellogenesis [PG]), secondary growth (vitellogenesis), oocyte maturation and ovulation. The ovarian lamellae are lined by a germinal epithelium composed of epithelial cells and scattered oogonia. During primary growth, the development of cortical alveoli and oil droplets, are initiated simultaneously. During secondary growth, yolk globules coalesce into a fluid mass. The full-grown oocyte contains a large globule of fluid yolk. The germinal vesicle is at the animal pole, and the cortical alveoli and oil droplets are located at the periphery. The disposition of oil droplets at the vegetal pole of the germinal vesicle during late secondary growth stage is a unique characteristic. The follicular cell layer is composed initially of a single layer of squamous cells during early PG which become columnar during early vitellogenesis. During primary and secondary growth stages, filaments develop among the follicular cells and also around the micropyle. The filaments are seen extending from the zona pellucida after ovulation. During ovulation, a space is evident between the oocyte and the zona pellucida. Asynchronous spawning activity is confirmed by the observation that, after ovulation, the ovarian lamellae contain follicles in both primary and secondary growth stages; in contrast, when the seasonal activity of oogenesis and spawning ends, after ovulation, the ovarian lamellae contain only follicles in the primary growth stage. J. Morphol. 277:1339-1354, 2016. © 2016 Wiley Periodicals, Inc.
Subject(s)
Estrous Cycle , Killifishes/anatomy & histology , Oogenesis , Ovulation , Animals , Female , Killifishes/physiology , Oocytes/cytology , Oogonia/cytologyABSTRACT
The germinal epithelium, i.e., the site of germ cell production in males and females, has maintained a constant form and function throughout 500 million years of vertebrate evolution. The distinguishing characteristic of germinal epithelia among all vertebrates, males, and females, is the presence of germ cells among somatic epithelial cells. The somatic epithelial cells, Sertoli cells in males or follicle (granulosa) cells in females, encompass and isolate germ cells. Morphology of all vertebrate germinal epithelia conforms to the standard definition of an epithelium: epithelial cells are interconnected, border a body surface or lumen, are avascular and are supported by a basement membrane. Variation in morphology of gonads, which develop from the germinal epithelium, is correlated with the evolution of reproductive modes. In hagfishes, lampreys, and elasmobranchs, the germinal epithelia of males produce spermatocysts. A major rearrangement of testis morphology diagnoses osteichthyans: the spermatocysts are arranged in tubules or lobules. In protogynous (female to male) sex reversal in teleost fishes, female germinal epithelial cells (prefollicle cells) and oogonia transform into the first male somatic cells (Sertoli cells) and spermatogonia in the developing testis lobules. This common origin of cell types from the germinal epithelium in fishes with protogynous sex reversal supports the homology of Sertoli cells and follicle cells. Spermatogenesis in amphibians develops within spermatocysts in testis lobules. In amniotes vertebrates, the testis is composed of seminiferous tubules wherein spermatogenesis occurs radially. Emerging research indicates that some mammals do not have lifetime determinate fecundity. The fact emerged that germinal epithelia occur in the gonads of all vertebrates examined herein of both sexes and has the same form and function across all vertebrate taxa. Continued study of the form and function of the germinal epithelium in vertebrates will increasingly clarify our understanding of vertebrate reproduction. J. Morphol. 277:1014-1044, 2016. © 2016 Wiley Periodicals, Inc.
Subject(s)
Biological Evolution , Epithelium/physiology , Ovarian Follicle/cytology , Sertoli Cells/metabolism , Vertebrates/physiology , Amphibians/physiology , Animals , Female , Fishes/physiology , Male , Reptiles/physiology , Testis/cytology , Time FactorsABSTRACT
Anthropogenic land-cover change is driving biodiversity loss worldwide. At the epicenter of this crisis lies Southeast Asia, where biodiversity-rich forests are being converted to oil-palm monocultures. As demand for palm oil increases, there is an urgent need to find strategies that maintain biodiversity in plantations. Previous studies found that retaining forest patches within plantations benefited some terrestrial taxa but not others. However, no study has focused on aquatic taxa such as fishes, despite their importance to human well-being. We assessed the efficacy of forested riparian reserves in conserving freshwater fish biodiversity in oil-palm monoculture by sampling stream fish communities in an oil-palm plantation in Central Kalimantan, Indonesia. Forested riparian reserves maintained preconversion local fish species richness and functional diversity. In contrast, local and total species richness, biomass, and functional diversity declined markedly in streams without riparian reserves. Mechanistically, riparian reserves appeared to increase local species richness by increasing leaf litter cover and maintaining coarse substrate. The loss of fishes specializing in leaf litter and coarse substrate decreased functional diversity and altered community composition in oil-palm plantation streams that lacked riparian reserves. Thus, a land-sharing strategy that incorporates the retention of forested riparian reserves may maintain the ecological integrity of fish communities in oil-palm plantations. We urge policy makers and growers to make retention of riparian reserves in oil-palm plantations standard practice, and we encourage palm-oil purchasers to source only palm oil from plantations that employ this practice.
Subject(s)
Agriculture , Arecaceae , Biodiversity , Conservation of Natural Resources , Fishes/physiology , Animals , Borneo , Fresh Water , IndonesiaABSTRACT
The cyprinodontiform family Goodeidae comprises two biogeographically disjunct subfamilies: the viviparous Goodeinae endemic to the Mexican Plateau, and the oviparous Empetrichthyinae, known only from relict taxa in Nevada and California. Ovarian characteristics of two oviparous species of goodeid, Crenichthys baileyi and Empetrichthys latos, studied using museum collections, are compared with those of viviparous species of goodeids. Both subfamilies have a single, cystovarian ovary. The ovary in the viviparous Goodeinae has an internal septum that divides the ovarian lumen into two compartments, and it may possess oogonia. There is no ovarian septum in the oviparous C. baileyi and E. latos. Oogenesis is similar in both subfamilies with regard to the proliferation of oogonia, initiation of meiosis, primary growth and development of an oocyte during secondary growth in which fluid yolk progressively fuses into a single globule. Notably, eggs of C. baileyi and E. latos are approximately double the size of those of the viviparous Goodeinae in which embryos develop inside the ovarian lumen and are nourished, in part, by nutrients transferred from the maternal tissues, a mode of embryo development called matrotrophy. Egg envelopes of the two subfamilies differ in that those of C. baileyi and E. latos have a relatively thick zona pellucida, attachment fibrils or filaments that develop between the follicle cells during oogenesis, and a micropyle observed only in E. latos. In contrast, viviparous goodeid eggs have a relatively thin zona pellucida, but lack adhesive fibrils, and a micropyle was not observed. These reproductive characters are compared with those of species of the eastern North American Fundulus, a representative oviparous cyprinodontiform. One newlyrecognized shared, derived character, a single, median ovoid ovary with no obvious external evidence of fusion, supports monophyly of the Goodeidae. Differences among the goodeid subfamilies and Fundulus are interpreted relative to the oviparous versus viviparous modes of reproduction.
Subject(s)
Cyprinodontiformes/anatomy & histology , Oogenesis/physiology , Oviparity/physiology , Viviparity, Nonmammalian/physiology , Animals , Cyprinodontiformes/embryology , Cyprinodontiformes/growth & development , Cytoplasm , Female , Humans , Meiosis , Oocytes/cytology , Ovary/cytology , Ovary/growth & development , Ovum , ReproductionABSTRACT
Tomeurus gracilis is a species long considered pivotal in understanding the evolution of livebearing in atherinomorph fishes. Tomeurus gracilis is a zygoparous or embryoparous poeciliid: internal fertilization is followed by females laying fertilized eggs singly or retaining fertilized eggs until or near hatching. Tomeurus was hypothesized as the sister group of the viviparous poeciliids until it was proposed as a close relative of a derived viviparous poeciliid, Cnesterodon, hence nested among viviparous taxa rather than near the root of the tree. Here, we describe and compare reproductive morphological characters of the little-known Tomeurus with those of representative atherinomorphs. In Tomeurus and Cnesterodon, sperm are packaged in naked sperm bundles, or spermatozeugmata, in a configuration considered here diagnostic of viviparous poeciliids. Testes are single and free sperm are stored in the ovary in both taxa in contrast to oviparous atherinomorphs in which testes are paired and sperm are not packaged and not stored in the ovary. Efferent ducts in Cnesterodon testes and other viviparous poeciliids have a PAS-positive secretion demonstrating presence of a glycoprotein that inactivates sperm or prevents final sperm maturation. No PAS-positive staining secretion was observed in Tomeurus or oviparous atherinomorphs. Tomeurus shares apomorphic reproductive characters, such as sperm bundle and testis morphology and a gonopodium, with viviparous poeciliids and plesiomorphic characters, such as a thick zona pellucida with filaments, with oviparous taxa. We do not postulate loss or reversal of viviparity in Tomeurus, and we corroborate its phylogenetic position as sister to the viviparous poeciliids.
Subject(s)
Biological Evolution , Fishes , Reproduction/physiology , Viviparity, Nonmammalian , Animals , Female , Fishes/anatomy & histology , Fishes/physiology , Male , Ovary/anatomy & histology , Ovum/ultrastructure , Spermatozoa/ultrastructure , Testis/cytology , Viviparity, Nonmammalian/physiologyABSTRACT
The rainbow trout, Oncorhynchus mykiss (Walbaum, 1792), is a salmoniform fish that spawns once per year. Ripe females that had ovulated naturally, and those induced to ovulate using salmon gonadotropin-releasing hormone, were studied to determine whether follicles were forming at the time of spawning and to describe the process of folliculogenesis. After ovulation, the ovaries of postspawned rainbow trout were examined histologically, using the periodic acid-Schiff procedure, to stain basement membranes that subtend the germinal epithelium and to interpret and define the activity of the germinal epithelium. After spawning, the ovary contained a few ripe oocytes that did not ovulate, numerous primary growth oocytes including oocytes with cortical alveoli, and postovulatory follicles. The germinal epithelium was active in postspawned rainbow trout, as determined by the presence of numerous cell nests, composed of oogonia, mitotic oogonia, early diplotene oocytes, and prefollicle cells. Cell nests were separated from the stroma by a basement membrane continuous with that subtending the germinal epithelium. Furthermore, follicles containing primary growth oocytes were connected to the germinal epithelium; the basement membrane surrounding the follicle joined that of the germinal epithelium. After ovulation, the basement membrane of the postovulatory follicle was continuous with that of the germinal epithelium. We observed consistent separation of the follicle, composed of an oocyte and surrounding follicle cells, from the ovarian stroma by a basement membrane. The follicle is derived from the germinal epithelium. As with the germinal epithelium, follicle cells derived from it never contact those of the connective tissue stroma. As with epithelia, they are always separated from connective tissue by a basement membrane.
Subject(s)
Epithelium/growth & development , Oncorhynchus mykiss/embryology , Oncorhynchus mykiss/physiology , Organogenesis , Ovarian Follicle/embryology , Ovulation/physiology , Animals , Basement Membrane/cytology , Female , Oocytes/cytology , Ovarian Follicle/cytologyABSTRACT
Gonad morphology at the gross anatomical or histological levels has long been studied by fisheries biologists to identify annual reproductive cycles and length of breeding season, among other goals. Comparative surveys across vertebrate taxa have not been detailed enough, however, to describe fully the differences and similarities among gonads of bony fishes and other vertebrates, and to use gonad morphology in phylogenetic systematic analyses. An emerging constant among vertebrates is the presence of a germinal epithelium composed of somatic and germ cells in both males and females. In females, the germinal epithelium lines the ovarian lamellae. In males, arrangement of the germinal epithelium into compartments varies among osteichthyans: basal taxa have an anastomosing tubular testis, whereas derived taxa have a lobular testis. The lobular testis is proposed as a synapomorphy of the Neoteleostei. The annual reproductive cycle is hypothesized to be the source of morphological variation among testis types. Elongation of germinal compartments during early maturation may result in a transition from anastomosing tubular to lobular testes. In all male atherinomorphs surveyed, spermatogonia are restricted to the distal termini of lobules rather than being distributed along the lobule; there is an epithelioid arrangement of Sertoli and germ cells rather than a germinal epithelium. Arrest of the maturation-regression phases is hypothesized to lead to formation of the atherinomorph testis. Atherinomorphs also have a distinctive egg with fluid, rather than granular, yolk. Variation among germinal epithelia is interpreted in a developing phylogenetic framework to understand evolution of gonad morphology and to propose gonad characters for phylogenetic analyses.
ABSTRACT
An extremely large number of fifth ceratobranchial teeth, with highly modified, striated, and hooked tips were observed in the central and western Pacific sicydiine goby genus Stiphodon.A scanning electron microscopic study of the form and arrangement of fifth ceratobranchial teeth was conducted to assess the distribution of these modifications in sicydiine gobies and their putative close relatives. Our goals were to explore a new set of characters in gobioid systematics, to test sicydiine monophyly, and to test hypotheses of relationships of sicydiine gobies. Sicydiines are hypothesized herein to be most closely related to the western Pacific Tukugobius and Rhinogobius,freshwater genera with which they share thickened pelvic-fin rays, no teeth on the anterior portion of the fifth ceratobranchial bones, fifth ceratobranchial teeth with differentiated and striated tips, and overlapping anterior rami of the fifth ceratobranchial bones. The latter two characters occur in some, but not all, sicydiines. The pantropical freshwater goby Awaous,often classified with sicydiines, is not considered the closest relative of the subfamily. The highly modified fifth ceratobranchials of Stiphodon are similar to, and concluded here to be homoplasious with, those of the mudflat-dwelling New World goby Evorthodus and the Indo-west Pacific oxudercine gobies, represented in this study by Pseudapocryptes. J. Morphol. 237:257-274, 1998. Published 1998 Wiley-Liss, Inc.