Geographical variation in reproductive character displacement in mate choice by male sailfin mollies

Female Amazon mollies, Poecilia formosa, are a unisexual species that reproduce by gynogenesis. They must coexist and mate with males of other species (usually the mollies Poecilia latipinna or Poecilia mexicana) to induce embryogenesis, but inheritance is strictly maternal. We examined the mating preference of the male sailfin molly, P. latipinna, for female sailfin mollies versus Amazon mollies, P. formosa. We compared the mating preferences of sympatric and allopatric populations collected throughout the Gulf Coast of North America. Male P. latipinna from six populations sympatric with Amazon mollies showed a significantly greater strength of preference for conspecific sailfin females than males from five populations that were allopatric with Amazon mollies. These results provide strong evidence for reproductive character displacement of male mate choice in sympatry. Furthermore, the large geographical range of populations that we tested revealed variation among populations within sympatry and allopatry, indicating that it is important to evaluate a large number of populations when examining reproductive character displacement.     

Character displacement in sailfin mollies, <Emphasis Type="Italic">Poecilia latipinna</Emphasis>: allozymes and behavior

Synopsis We analyzed variation in allozymes and mating preferences in 12 populations across much of the range of the sailfin molly, Poecilia latipinna. Sailfin mollies can be sympatric with its sexual parasite Amazon mollies, P. formosa. Amazon mollies must co-exist and mate with bisexual males of closely related species (including sailfin mollies) to induce embryogenesis but inheritance is strictly maternal. Where sailfin and Amazon mollies are sympatric there is evidence of reproductive character displacement as males show a significantly stronger mating preference for sailfin molly females over Amazon mollies compared to preferences of males from allopatric populations. From the allozyme data we found a moderate amount of genetic variation across all populations but this variation did not reveal significant partitioning between sympatric and allopatric populations. Additionally, we found no evidence for isolation by distance as genetic distance was not significantly correlated with geographic distance. While allozyme variation also did not significantly correlate with male mating preferences, there was a significant correlation between male mating preferences and geographic distance. This correlation between mating preferences and geographic distance may have arisen from coevolution with Amazon mollies resulting in reproductive character displacement. Taken together, the distribution of genetic and behavioral variation among sympatric and allopatric populations suggests that behavioral evolution has outpaced evolution at the allozyme loci we examined in P. latipinna.     

Lack of species discrimination based on chemical cues by male sailfin mollies, <Emphasis Type="Italic">Poecilia latipinna</Emphasis>

When making mating decisions, individuals may rely on multiple cues from either the same or multiple sensory modalities. Although the use of visual cues in sexual selection is well studied, fewer studies have examined the role of chemical cues in mate choice. In addition, few studies have examined how visual and/or chemical cues affect male mating decisions. Male mate choice is important in systems where males must avoid mating with heterospecific females, as is found in a mating complex of Poecilia. Male sailfin mollies, Poecilia latipinna, are sexually parasitized by gynogenetic Amazon mollies, P. formosa. Little is known about the mechanism by which male sailfin mollies base their mating decisions. Here we tested the hypothesis that male sailfin mollies from an allopatric and a sympatric population with Amazon mollies use multiple cues to distinguish between conspecific and heterospecific females. We found that male sailfin mollies recognized the chemical cues of conspecific females, but we found no support for the hypothesis that chemical cues are by themselves sufficient for species discrimination. Lack of discrimination based on chemical cues alone may be due to the close evolutionary history between P. latipinna and P. formosa. Males from populations sympatric with Amazon mollies did not differentially associate with females of either of the two species when given access to both visual and chemical cues of the females, yet males from the allopatric population did associate more with conspecific females than with heterospecific females in the presence of both chemical and visual cues. The lack of discrimination by males from the sympatric population between conspecific and heterospecific females based on both chemical and visual cues suggests that these males require more complex combinations of cues to distinguish species, possibly due to the close relatedness of these species.     

Female Preferences for Sailfin and Body Size in the Sailfin Molly, Poecilia latipinna

We tested the mating preference of female sailfin mollies (Poecilia latipinna) by presenting them with pairs of dummy males differing in: (I) sailfin and body size together (holding sailfin : body size ratio constant); (II) body size alone (holding sailfin size constant); (III) sailfin size alone (holding body size constant); and (IV) sailfin : body size ratio (holding total lateral projection area constant). Females spent more time near dummies of greater sailfin or greater body size. The preference functions based on the first three sets of stimuli showed a similar pattern: the preference between any two simultaneously presented dummies increased with the magnitude of the discrepancy in lateral projection area (LPA) between them. However, when LPA was held constant in expt (IV), neither body size, sailfin size, nor any particular dummy (i.e. any particular sailfin + body size combination) was preferred. These findings suggest that increased LPA is more stimulating to sexually receptive females and that females consequently prefer larger males. The sailfin may therefore have evolved as a way for males to exploit this sensory bias and appear larger to prospective mates.     

In vitro analysis of gene depression in goldfish choroidal melanophores.

The common goldfish depigments its larval choroidal melanophores as well as the skin melanophores for about two to three months following hatching. Thereafter it begins to show a characteristic xanthic coloration. This depigmentation is under the control of depigmentation genes Dp1 and Dp2. The larval choroidal melanophores of the common goldfish were cultured in vitro for more than four months without showing any sign of depigmentation. During the same period the skin and choroidal melanophores in the ACTH to the culture medium had no effect on depigmentation. The mechanisms of depigmentation in the goldfish are discussed.     

Pigmentary system of the adult alpine salamander Salamandra atra atra (Laur., 1768).

The pigmentary system of the skin from adult specimens of the black alpine salamander Salamandra atra atra was investigated by light microscope, electron microscope, and biochemical studies. Results were compared with those obtained in previous study of the subspecies Salamandra atra aurorae. Unlike Salamandra atra aurorae, which presents epidermal xanthophores and iridophores, Salamandra atra atra is completely melanized, presenting only epidermal and dermal melanophores. The melanosomes in both the epidermis and the dermis appear to derive from a multivesicular premelanosome similar to that in the goldfish, and the epidermal melanosomes are smaller than those in the dermis. Premelanosomes with an internal lamellar matrix were not observed. The biochemical results have shown that in the ethanol extracts obtained from the skin in toto and from the melanosomes, pteridines and flavins are always present and are the same as those extracted from the black skin areas of Salamandra atra aurorae.     

Separating the effects of intra- and interspecific age-structured interactions in an experimental fish assemblage

We documented patterns of age-structured biotic interactions in four mesocosm experiments with an assemblage of three species of co-occurring fishes from the Florida Everglades, the eastern mosquitofish (Gambusia holbrooki), sailfin molly (Poecilia latipinna), and bluefin killifish (Lucania goodei). These species were chosen based on their high abundance and overlapping diets. Juvenile mosquitofish and sailfin mollies, at a range of densities matching field estimates, were maintained in the presence of adult mosquitofish, sailfin mollies, and bluefin killifish to test for effects of competition and predation on juvenile survival and growth. The mesocosms held 1,200 l of water and all conditions were set to simulate those in Shark River Slough, Everglades National Park (ENP), USA. We placed floating mats of periphyton and bladderwort in each tank in standard volumes that matched field values to provide cover and to introduce invertebrate prey. Of 15 possible intra- and interspecific age-structured interactions, we found 7 to be present at the densities of these fish found in Shark River Slough marshes. Predation by adult mosquitofish on juvenile fish, including conspecifics, was the strongest effect observed. We also observed growth limitation in mosquitofish and sailfin molly juveniles from intra- and interspecific competition. When maintained at high densities, juvenile mosquitofish changed their diets to include more cladocerans and fewer chironomid larvae relative to low densities. We estimated size-specific gape limitation by adult mosquitofish when consuming juvenile mosquitofish and sailfin mollies. At high field densities, intraspecific competition might prolong the time period when juveniles are vulnerable to predation by adult mosquitofish. These results suggest that path analysis, or other techniques used to document food-web interactions, must include age-specific roles of these fishes.     

Male Sailfin mollies (Poecilia latipinna) copy the mate choice of other males

Female mate-copying has been shown to occur between heterospecifics:female sailfin mollies Poecilia latipinna copy the choice oftheir gynogenetic associates, Amazon mollies P. formosa. Femalemate-copying thus contributes to the maintenance of this asexual-sexualspecies complex by providing an advantage to male sailfin molliesthat mate with Amazon females; because of mate-copying thesemales increase their attractiveness to conspecific females.Here we show that male mate-copying, an unreported phenomenon,also occurs and that it can reverse male preferences for conspecificfemales. Male mate-copying should also contribute to the maintenanceof gynogens and might be advantageous in allowing males a meansto rapidly assess female receptivity although sometimes resultingin heterospedfic matings.     

Ultrastructural and biochemical analysis of epidermal xanthophores and dermal chromatophores of the teleost Sparus aurata

We have studied the pigmentary system of the teleost Sparus aurata skin by electron microscopy and chromatographic analysis. Under electron microscopy, we found the dermis to contain the three major types of recognized chromatophores: melanophores, xanthophores and iridophores. Melanophores were more abundant in the dorsal region, whereas the iridophores were more abundant in the ventral region. The most important discovery was that of epidermal xanthophores. Epidermal xanthophores were the only chromatophores in the epidermis, something only found in S aurata and in a teleost species living in the Antartic sea. In contrast, the biochemical analysis did not establish any special characteristics: we found pteridine and flavin pigments located mostly in the pigmented dorsal region. Riboflavin and pterin were two of the most abundant coloured pigment types, but other colourless pigments such as xanthopterin and isoxanthopterin were also detected.     

Pigmentary System of the Adult Alpine Salamander Salamandra atra atra (Laur., 1768)

The pigmentary system of the skin from adult specimens of the black alpine salamander Salamandra atra atra was investigated by light microscope, electron microscope, and biochemical studies. Results were compared with those obtained in previous study of the subspecies Salamandra atra aurorae. Unlike Salamandra atra aurorae, which presents epidermal xanthophores and iridophores, Salamandra atra atra is completely melanized, presenting only epidermal and dermal melanophores. The melanosomes in both the epidermis and the dermis appear to derive from a multivesicular premelanosome similar to that in the goldfish, and the epidermal melanosomes are smaller than those in the dermis. Premelanosomes with an internal lamellar matrix were not observed. The biochemical results have shown that in the ethanol extracts obtained from the skin in toto and from the melanosomes, pteridines and flavins are always present and are the same as those extracted from the black skin areas of Salamandra atra aurorae.     

Changes in the distribution of melanophores and xanthophores inTriturus alpestris embryos during their transition from the uniform to banded pattern

Summary The change in distribution of melanophores from stage 28+ (uniform melanophore pattern) to stage 34 (banded melanophore pattern) and the participation of xanthophores in these changes has been investigated inTriturus alpestris embryos by studying the social behaviour of single cells. While melanophores are clearly visible from outside the embryo at stage 28+, xanthophores cannot be recognized from the outside until after stage 34. In ultrathin sections of stage 34 embryos, xanthophores are observed alternating with melanophores in a zonal distribution (Epperlein 1982). Using detached pieces of dorsolateral trunk skin, which retain their chromatophores after detachment, the entire distribution of melanophores and xanthophores can be visualized in a scanning electron microscope (SEM). In ambiguous cases (early stages), cells were reprocessed for transmission electron microscopy (TEM) and the presence of the characteristic pigment organelles was assessed. In addition, xanthophores were specifically identified by pteridine fluorescence with dilute ammonia. Pteridines were also identified chromatographically in skin homogenates. The combination of these methods allowed precise identification and quantitative determination of melanophores and xanthophores. Both cell types were present as codistributed, biochemically differentiated cells at stage 28+. Changes in the pattern up to stage 34 were due to the rearrangement at the epidermal-mesodermal interface of a relatively fixed number of melanophores which became preferentially localised at the dorsal somite edge and at the lateral plate mesoderm, and to the distribution of an increasing number of xanthophores to subepidermal locations in the dorsal fin and between the melanophore bands. Concomitant was an increase in the thickness of the epidermal basement membrane and a change in shape of chromatophores from elongate via stellate to rosette shaped, which may be correlated with a shift from migratory to sessile phases.     

Development of the pteridine pathway in the zebrafish, Danio rerio

In the zebrafish, the peripheral neurons and the pigment cells are derived from the neural crest and share the pteridine pathway, which leads either to the cofactor tetrahydrobiopterin or to xanthophore pigments. The components of the pteridine pattern were identified as tetrahydrobiopterin, sepiapterin, 7-oxobiopterin, isoxanthopterin, and 2,4,7-trioxopteridine. The expression of GTP cyclohydrolase I activity during the first 24-h postfertilization, followed by 6-pyruvoyl-5,6,7,8-tetrahydropterin synthase and sepiapterin reductase, suggest an early supply of tetrahydrobiopterin for neurotransmitter synthesis in the neurons and for tyrosine supply in the melanophores. At 48-h postfertilization, sepiapterin formation branches off the de novo pathway of tetrahydrobiopterin synthesis. Sepiapterin, via 7,8-dihydrobiopterin and biopterin, serves as a precursor for the formation of 7-oxobiopterin, which may be further catabolized to isoxanthopterin and 2,4,7-trioxopteridine. Neither 7, 8-dihydrobiopterin nor biopterin is a substrate for xanthine oxidoreductase. In contrast, both of these compounds are oxidized at C-7 by a xanthine oxidase variant form, which is inactivated by KCN, but is insensitive to allopurinol. The oxidase and the dehydrogenase form of xanthine oxidoreductase as well as the xanthine oxidase variant have specific developmental patterns. It follows that GTP cyclohydrolase I, the formation of sepiapterin, and the xanthine oxidoreductase family control the pteridine pathway in the zebrafish.     

Are you more than the sum of your parents’ genes? Phenotypic plasticity in a clonal vertebrate and F1 hybrids of its parental species

All known vertebrate clones have originated from hybridization events and some have produced distinct evolutionary lineages via hybrid speciation. Amazon mollies (Poecilia formosa) present an excellent study system to investigate how clonal species have adapted to heterogeneous environments because they are the product of a single hybridization event between male sailfin mollies (Poecilia latipinna) and female Atlantic mollies (Poecilia mexicana). Here, we ask whether the hybrid species differs from the combination of its parental species’ genes in its plastic response to different environments. Using a three-way factorial design, we exposed neonates produced by Amazon mollies and reciprocal F1 hybrid crosses to different thermal (24°C and 29°C) and salinity (0/2, 12, and 20 ppt) regimes. We measured various ontogenetic and life history characteristics across the life span of females. Our major results were as follows: (1) Reaction norms of growth and maturation to temperature and salinity are quite similar between the two hybrid crosses; (2) Amazon molly reaction norms were qualitatively different than the P. latipinna male and P. mexicana female (L×M) hybrids for the ontogenetic variables; (3) Amazon molly reaction norms in reproductive traits were also quite different from L×M hybrids; and (4) The reaction norms of net fertility were very different between Amazon mollies and L×M hybrids. We conclude that best locale for Amazon mollies is not the best locale for hybrids, which suggests that Amazon mollies are not just an unmodified mix of parental genes but instead have adapted to the variable environments in which they are found. Hybridization resulting in asexuality may represent an underappreciated mechanism of speciation because the unlikely events required to produce such hybrids rarely occur and is dependent upon the genetic distance between parental species.     

The pteridine pathway in zebrafish: regulation and specification during the determination of neural crest cell-fate

This review describes pteridine biosynthesis and its relation to the differentiation of neural crest derivatives in zebrafish. During the embryonic development of these fish, neural crest precursor cells segregate into neural elements, ectomesenchymal cells and pigment cells; the latter then diversifying into melanophores, iridophores and xanthophores. The differentiation of neural cells, melanophores, and xanthophores is coupled closely with the onset of pteridine synthesis which starts from GTP and is regulated through the control of GTP cyclohydrolase I activity. De novo pteridine synthesis in embryos of this species increases during the first 72-h postfertilization, producing H4biopterin, which serves as a cofactor for neurotransmitter synthesis in neural cells and for tyrosine production in melanophores. Thereafter, sepiapterin (6-lactoyl-7,8-dihydropterin) accumulates as yellow pigment in xanthophores, together with 7-oxobiopterin, isoxanthopterin and 2,4,7-trioxopteridine. Sepiapterin is the key intermediate in the formation of 7-oxopteridines, which depends on the availability of enzymes belonging to the xanthine oxidoreductase family. Expression of the GTP cyclohydrolase I gene (gch) is found in neural cells, in melanoblasts and in early xanthophores (xanthoblasts) of early zebrafish embryos but steeply declines in xanthophores by 42-h postfertilization. The mechanism(s) whereby sepiapterin branches off from the GTP-H4biopterin pathway is currently unknown and will require further study. The surge of interest in zebrafish as a model for vertebrate development and its amenability to genetic manipulation provide powerful tools for analysing the functional commitment of neural crest-derived cells and the regulation of pteridine synthesis in mammals.     

MORPHOLOGICAL AND BIOCHEMICAL CHARACTERIZATION OF GOLDFISH ERYTHROPHORES AND THEIR PTERINOSOMES

The fine structure of integumental erythrophores and the intracellular location of pteridine and carotenoid pigments in adult goldfish, Carassius auratus, were studied by means of cytochemistry, paper and thin-layer chromatography, ionophoresis, density-gradient centrifugal fractionation, and electron microscopy. The ultrastructure of erythrophores is characterized by large numbers of somewhat ellipsoidal pigment granules and a well-developed system of tubules which resembles endoplasmic reticulum. The combined morphological and biochemical approaches show that pteridine pigments of erythrophores are located characteristically in pigment granules and are the primary yellow pigments of these organelles. Accordingly, this organelle is considered to be the "pterinosome" which was originally found in swordtail erythrophores. Major pteridines obtainable from goldfish pterinosomes are sepiapterin, 7-hydroxybiopterin, isoxanthopterin, and 6-carboxyisoxanthopterin. Density-gradient fractions indicate that carotenoids are mostly associated with the endoplasmic reticulum. Both tyrosinase and possibly a tyrosinase inhibitor containing sulfhydryl groups are present in the pterinosome. The possible existence of a tyrosinase inhibitor is suggested by the marked increase of tyrosinase activity upon the addition of iodoacetamide or p-chloromercuribenzoic acid. In the light of their fine structure, pigmentary composition, and enzymatic properties, the erythrophores and pterinosomes are discussed with respect to their probable functions and their relationship to melanophores.     

Localization of sepiapterin reductase in pigment cells of Oryzias latipes

Body colors of poikilothermal vertebrates are derived from three distinct types of pigment cells, melanophores, erythro/xanthophores and irido/leucophores. It is well known that melanin in melanophores is synthesized by tyrosinase within a specific organelle termed the melanosome. Although sepiapterin reductase (SPR) is an important enzyme involved in metabolizing biopterin and sepiapterin (a conspicuous pteridine as a coloring pigment in xanthophores) the distribution of SPR has not been shown in pigment cells. An antibody raised in rabbits against rat SPR was used to demonstrate the presence of SPR in pigment cells of Oryzias latipes. This study, which used immunohistochemistry with fluorescence or peroxidase/diaminobenzidine as markers, revealed that SPR could be detected readily in xanthophores, but only faintly in melanophores. These results suggest that sepiapterin is metabolized within xanthophores. Moreover, these experiments show that a protein sharing immunological cross-reactivity with rat SPR is located in teleost O. latipes xanthophores, which is significant considering the relationship of pteridine metabolism between poikilothermal vertebrates and mammals. Further progress in investigations of the roles of pteridines in vertebrates will be promoted by using these fish which can be bred in mass rather easily in the laboratory.     

The Pteridine Pathway in Zebrafish: Regulation and Specification during the Determination of Neural Crest Cell‐Fate

This review describes pteridine biosynthesis and its relation to the differentiation of neural crest derivatives in zebrafish. During the embryonic development of these fish, neural crest precursor cells segregate into neural elements, ectomesenchymal cells and pigment cells; the latter then diversifying into melanophores, iridophores and xanthophores. The differentiation of neural cells, melanophores, and xanthophores is coupled closely with the onset of pteridine synthesis which starts from GTP and is regulated through the control of GTP cyclohydrolase I activity. De novo pteridine synthesis in embryos of this species increases during the first 72‐h postfertilization, producing H₄biopterin, which serves as a cofactor for neurotransmitter synthesis in neural cells and for tyrosine production in melanophores. Thereafter, sepiapterin (6‐lactoyl‐7,8‐dihydropterin) accumulates as yellow pigment in xanthophores, together with 7‐oxobiopterin, isoxanthopterin and 2,4,7‐trioxopteridine. Sepiapterin is the key intermediate in the formation of 7‐oxopteridines, which depends on the availability of enzymes belonging to the xanthine oxidoreductase family. Expression of the GTP cyclohydrolase I gene (gch) is found in neural cells, in melanoblasts and in early xanthophores (xanthoblasts) of early zebrafish embryos but steeply declines in xanthophores by 42‐h postfertilization. The mechanism(s) whereby sepiapterin branches off from the GTP‐H₄biopterin pathway is currently unknown and will require further study. The surge of interest in zebrafish as a model for vertebrate development and its amenability to genetic manipulation provide powerful tools for analysing the functional commitment of neural crest‐derived cells and the regulation of pteridine synthesis in mammals.     

Pigment cell localizations in anuran ventral skin at climactic metamorphosis.

In anuran amphibians, the specific color pattern of the skin is expressed after metamorphosis, and its formation involves pigment cell migrations. Pigment cells are differently distributed in the tadpole, larval, and froglet skin. To learn more about their fate during metamorphic climax and in the young froglet, we focused our attention on the different localizations of larval melanophores and iridophores in the ventral skin of Rana esculenta before and during skin homing. Localizations of melanophores and iridophores can be elucidated at the developmental stages suggested by Taylor and Kollros (TK stages). At TK stage II (during early premetamorphosis), large melanophores beneath the larval skin are detected. At TK stage X, dispersed melanophores lie under bundles of muscular striated fibrils near the larval skin; they are also observed at the vascular level. At TK stage XVII (prometamorphosis), melanophores are extended on the inner side of the basement lamellar collagen. At the end of prometamorphosis, iridophores are located with melanophores in the separating space between attached basement collagen and derived basement collagen. At TK stage XX (earlier climax), melanophores and iridophores are detected inside the upper extremities of fractures opened in the derived basement collagen. At TK stage XXIV (later climax), both types of larval pigment cells are observed in the inner extremities of breaks derived from the fractures. During climax, these pigment cells occupy the well-formed breaks. At TK stage XXV in young froglet, the pigment cells remain alone in the breaks formed in the derived basement collagen. Briefly, breaks in the basement lamellar collagen are opened by invading cell processes of mesenchymal cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Patterns of Inheritance of Mating Signals in Sailfin and Shortfin Mollies (Poeciliidae: Poecilia: Mollienesia)

Divergence in mating signals is a primary factor leading to reproductive isolation, and thus, speciation. However, the genetic changes underlying such divergence are poorly understood, especially in vertebrates. We used two species of poeciliid fishes, Poecilia velifera and P. mexicana , to explore the link between genes and mating behaviors that has resulted in pre-mating reproductive isolation between these species. Using backcross hybrids created from the F1 male offspring of reciprocal interspecific crosses between a sailfin molly ( P. velifera ) and a shortfin molly ( P. mexicana ), we examined the effects of Y-linkage and autosomal contributions on the expression of two male mating behaviors: courtship displays and gonopodial thrusts. The F1 hybrid males displayed a strong influence of sire on courtship display rates, with F1 males sired by the sailfin species showing courtship display rates that were up to three times higher than the rates of displays performed by F1 males sired by the shortfin species. These results suggest a Y-linked genetic effect on the expression of courtship display behavior. Comparisons between backcross hybrid males with sailfin Y-chromosomes or shortfin Y-chromosomes suggested that the interaction of autosomal genes also influences the inheritance of courtship display rates. Sailfin autosomal genes significantly increased the probability of performing courtship displays for hybrid males, and increased display rate for males from the sailfin Y-chromosome line. Autosomal genes had less of an impact on gonopodial thrusting behavior, however, thrust rates did significantly decrease with an increasing proportion of sailfin autosomes in males from the shortfin Y-chromosome line. These results suggest that the inheritance of species differences in mating signals between shortfin and sailfin mollies involves both genes found on the Y-chromosome and autosomal gene influences on their expression.     

Pigment cell differentiation in the fire-bellied toad,Bombina orientalis. I. Structural,chemical, and physical aspects of the adult pigment pattern

Wild-collected adults of Bombina orientalis are bright green dorsally and red to red-orange ventrally. As a prelude to an analysis of the differentiation of pigment cells in developing B. orientalis, we describe structural and chemical aspects of the fully differentiated pigment pattern of the “normal” adult. Structurally, differences between dorsal green and ventral red skin are summarized as follows: (1) Dorsal green skin contains a “typical” dermal chromatophore unit comprised of melanophores, iridophores, and xanthophores. Red skin contains predominantly carotenoid-containing xanthophores (erythrophores), and skin from black spot areas contains only melanophores. (2) In ventral red skin, there is also a thin layer of deep-lying iridophores that presumably are not involved in the observed color pattern. (3) Xanthophores of red and green skin are morphologically distinguishable from each other. Dorsal skin xanthophores contain both pterinosomes and carotenoid vesicles; ventral skin xanthophores contain only carotenoid vesicles. Carotenoid vesicles in dorsal xanthophores are much larger but less electron dense than comparable structures in ventral xanthophores. The presence of carotenes in ventral skin accounts for the bright red-orange color of the belly of this frog. Similar pigments are also present in green skin, but in smaller quantities and in conjunction with both colored (yellow) and colorless pteridines. From spectral data obtained for xanthophore pigments and structural data obtained from the size and arrangement of reflecting platelets in the iridophore layer, we attempt to explain the phenomenon of observed green color in B. orientalis.