A polymorphism in oocyte pigmentation in natural populations of the glass frog

The adaptive role of amphibian oocyte melanic pigmentation and its molecular control are still elusive. Here we present evidence of a polymorphism in egg pigmentation in the emerald glass frog Espadarana prosoblepon. In Ecuadorian natural populations of this species, females can lay dark brown or pale eggs that develop into normal pigmented tadpoles and adults. This trait is a sex-limited phenotype that is inherited like a recessive allele that we called pale eggs like (pel). The pel phenotype is exclusive of oocyte cortical melanic pigmentation, which is reduced in comparison to wild type (wt) dark pigmented oocytes. Consequently, pel early embryos are paler in appearance, with reduced melanic pigmentation distributed to early blastomeres and embryonic ectoderm. However, these embryos form normal melanocyte derived pigmentation. Finally, we discuss the origin of this polymorphism and propose the use of E. prosoblepon as a model to study the adaptive role of egg pigmentation.


INTRODUCTION
Amphibian oocytes are the only vertebrate gametes capable of synthesizing the pigment melanin (Wallace and Selman, 1990). However, almost every extant clade of anurans has species that produce whitish, seemingly unpigmented eggs, even in the most basal groups (Desnitskiy, 2012). This apparent dichotomy of the egg pigmentation character has allowed generalizations regarding its adaptive role; yet very little is known regarding the state of this character. Indeed, from studies in a few species, now we know that the pattern of melanic pigmentation in amphibian eggs could vary among species, and the lack of pigmentation could be the result of multiple distinct cellular phenotypes regarding melanin synthesis and pigment localization (see below). Even less is known regarding variability of egg pigmentation within species. Here we report the existence of a polymorphism in oocyte and early embryo melanic pigmentation within natural populations of an amphibian, the emerald glass frog Espadarana prosoblepon (Centrolenidae).
In pigmented amphibian eggs, melanin is packed in organelles called melanosomes that are actively localized towards the animal hemisphere, leaving a clear, unpigmented and heavier vegetal pole, rich in yolk, oriented downwards due to gravity, (Elinson, 1980). In Xenopus laevis, oocytes accumulate melanosomes across the cortex during vitellogenesis (Dumont, 1972). In this species, the melanogenic pathway is activated independently within the oocytes, through unknown mechanisms. Within the oocyte cytoplasm, melanin is synthesized and packed in melanosomes that are actively transported towards the animal cortex (Kidson and Fabian, 1989). After fertilization, oocyte melanosomes are distributed in every cell that inherit the cytoplasm from the animal pole (mostly ectodermal), to later be found in internalized tissues like the notochord and neural tube after gastrulation (Eppig, 1970;Eppig and Dumont, 1971;Hughes, 1963;Kordylewski, 1983). The oocyte melanosomes can remain in the developing amphibian embryo until later embryo or tadpole stages, when they are actively extruded, as shown in X. laevis (Uehlinger et al., 1971).
In nature, the character state "whitish eggs", may be the result of at least four distinct cellular phenotypes within the oocyte: a completely lack of melanosomes Also, potential tools to analyze this phenomenon have been understudied, like the Xenopus albino embryos, which lose their melanosomes early in development through still unknown mechanisms (Hoperskaya, 1975). The X. laevis "pale eggs" mutation (pe) causes a reduction in oocytes melanic pigmentation in the animal pole; yet, pe embryos developed normally in spite of its whitish appearance. Older pe embryos were indistinguishable from wild types when neural crest-derived melanocytes developed, similar to other species with whitish eggs (Droin and Fischberg, 1984). This mutation was lost without being mapped to a gene. From this limited genetic evidence and a few descriptions of oocyte phenotypes, we could classify whitish eggs in unpigmented eggs, which lack of melanosomes, and pale eggs, with reduced pigmentation. Through this manuscript, we will use the term "whitish" for amphibian eggs with reduced or absent melanic pigmentation on which this phenotype hasn´t been described.
In eggs and early embryos, melanic pigmentation could be playing important adaptive roles as protection against UV irradiation, and as camouflage against predators.
Melanin is a known barrier against UV damage, and it could protect eggs and early embryos from irradiation (Blaustein and Belden, 2003;Blaustein et al., 2003;Licht and Grant, 1997). Melanin in the animal pole seems to protect sensitive molecules, since amphibian embryos irradiated from the vegetal pole fail to form a neural tube and axial structures (Chung and Malacinski, 1975;Manes and Elinson, 1980). Accordingly, it is common to generalize that amphibians with pigmented eggs lay clutches in exposed areas while amphibians with whitish eggs have evolved adaptations to conceal the eggs and avoid irradiation (Altig and McDiarmid, 2007). Xenopus laevis, Rana, and others, lay dark pigmented eggs in exposed aquatic clutches, while Eleutherodactylus and

Phylogenetic analyses and ancestral character reconstruction
We carried out two phylogenetic analyses. The first one was done for samples of Espadarana prosoblepon and closely related species, to test the hypothesis that the pel phenotype might belong to a cryptic or species living in sympatry. The second analysis was done at the family level to trace the evolution of egg pigmentation.

DNA extractions, sequencing and alignment
Total genomic DNA was extracted from 15 specimens of E. prosoblepon (Table   S2)

Ancestral character reconstruction
The selection of taxa for ancestral character reconstruction and phylogenetic analyses was based on availability of sequences in GenBank, phylogenetic relations, and existing information about egg coloration and ovipositional data (58 species and 11 outgroups, Table S2). First, we generated a phylogeny using 16S sequences, a relaxed

RESULTS
In 2014, during a field trip to "Los Cedros Biological Reserve" we collected two amplectant pairs of E. prosoblepon that later laid eggs in plastic containers. Both couples were found on vegetation over streams. Embryos in early cleavage stage showed drastic differences in the amount of pigmentation. One of the couples laid dark pigmented eggs (Fig. 1A). Within 24 hrs, while still being kept in the container, all the embryos reached to tailbud stage maintaining their dark appearance. Hatched tadpoles were transported to BDLS where only few survived and metamorphosed to froglets like the one shown in Figure 1C. The other couple laid eggs with dark pigmentation only in the most animal region (Fig. 1D). Importantly, after 24 hours this pigment had banished or diluted, and tailbud stage embryos where completely pale (Fig. 1E). The few metamorphosed froglets from this clutch that survived had the normal pattern of pigmentation described for E. prosoblepon adults (see discussion). The presence of melanic pigmentation in embryos (Fig. 1D), tadpoles (not shown), and the presence of melanic pigmentation in the skin and eyes of adult frogs discarded the possibility that the pale embryo phenotype had its origin on an albinism allele (Fig. 1C, 1F). This also suggested that the phenotype derived from reduced pigmentation in the oocyte, similar to the pe mutation previously recovered in Xenopus (Droin and Fischberg, 1984).
Because of this, we called this phenotype pale egg like (pel) and started to consider this a polymorphism of the dark pigmented eggs (wt).

The pel phenotype is a sex-limited genetic trait possibly caused by a recessive allele
While searching for pel individuals in other populations in Ecuador, we were able to perform a pedigree analysis in a family from the Manta Real location (Fig. 1G).
A single couple of founders collected in the field laid two egg clutches (C1 and C2) with dark pigmented embryos. These embryos were incubated until hatching and reared under homogeneous laboratory conditions to obtain F1 metamorphs and adults. Out of approximately 50 eggs laid in total, we were able to rear successfully eight F1 (I) fertile adults, six females and two males. Females carrying wt or pel oocytes could be easily distinguished due to the characteristic skin transparency of E. prosoblepon frogs, which allowed us to see egg color in the abdomen of live females. We identified three pel females (II4, II6 and II8), and three wt females (II1, II3 and II10). We could not find any clear trait associated to the pel phenotype in males; hence, we concluded the pel phenotype is a sex-limited genic trait. Family II9-II10 (female with wt eggs), produced one female with wt eggs. Family II6-II7, a pel female and a founder male from the same location, produced one female with pel oocytes (III-4), which could confirm the presence of multiple male carriers of the pel allele in this population.

The pel phenotype is widespread in natural populations of E. prosoblepon
The inheritability of the pel phenotype led us to test the commonality of this potential allele. To do this, we evaluated other populations of the species by subsequent field trips and by studying dissected ovaries of adult females preserved at the QCAZ museum. Unexpectedly, we found the same polymorphism in egg clutches collected during field trips to Los Cedros and Junín sites ( Fig. 2A, 2B). Also, six out of 17 E.
prosoblepon females preserved at the QCAZ museum, from distinct collection sites in Ecuador, had ovaries with mature pel oocytes (oocyte diameters between 1.9 and 2 mm; 2D). The remaining females had dark pigmented mature oocytes (Fig. 2C).
The phylogenetic relationships among the specimens collected and museum samples confirms that every analyzed specimen belongs to the same species, showing low phylogenetic divergence between populations and no relationship between genetic structure and egg pigmentation (Fig. 2E). Our results support the monophyly of E.
prosoblepon and show that the Ecuadorian populations form a well-supported clade, sister to a clade from Central America. In addition to Junín and Los Cedros, we found polymorphism for egg color in Río Chillayacu, more than 400 km south from Junín.
The pel phenotype was also found in females from Jama Coaque, Manabí Province, a dry shrub coastal locality, environmentally different to the cloud forest characteristic of the other sites (Fig. 2E, 2F). These results suggest pel is a widespread polymorphism within in the Ecuadorian populations of E. prosoblepon.

The pel phenotype in E. prosoblepon is the result of reduced melanic pigment in the oocyte cortex
Through a small, non-lethal, surgical procedure, we were able to extract pieces of ovaries containing mature oocytes to characterize the pel phenotype in oocytes from E. prosoblepon and Hyalinobatrachium fleischmanni females. Mature oocytes were analyzed using paraffin sectioning without counterstain to characterize the pigmentation phenotype. The mature, post-vitellogenic oocytes of E. prosoblepon have a diameter of approximately 2 mm (Fig. 3A, 3D). Wild type (wt) mature oocytes have characteristic dark pigmentation easily distinguishable from previtellogenic oocytes, which lack of pigment, and early vitellogenic oocytes that have some pigment but are much smaller (Fig. 3A). In large vitellogenic oocytes, the vegetal pole is undistinguishable from the animal pole due to the amount of pigmentation. Paraffin sections without a counterstain clearly show the restricted accumulation of dark melanic pigment within the cortex and the lack of pigment in the white, yolk rich, cytoplasm (Fig. 3B). The dark pigment in oocytes apparently is the result of both, the presence of melanosomes with darker pigment and the dense accumulation of these organelles in the outermost region of the cortex (Fig. 3C). Importantly, there seems to be less melanosomes in the vegetal cortex of the wt oocytes compared to the animal cortex (Fig. 3C). The animal cortex in oocytes was defined as the cortex closer to the germinal vesicle in a large vitellogenic oocyte.
Oocytes with the pel phenotype have a brown-yellow appearance, and pigmentation is clearly located towards the animal pole, leaving a less pigmented vegetal pole ( Fig. 3D-3F). In paraffin sections of pel oocytes, dark pigmentation is still restricted to the cortex, although pigmentation was less noticeable (Fig. 3E). The animal cortex in pel oocytes seems to have more pigment, and the brown-yellow appearance seems to be the result of less melanosomes, dispersed within the outer cortex, that carry a clearer melanic pigment (Fig. 3F). To compare, the mature oocytes of the centrolenid H. fleischmanni are completely unpigmented and have no visible pigment during any stage of oogenesis (Fig. 3G), within the oocyte (Fig, 3H), or in the animal cortex (Fig.   3I).
pel embryos have reduced egg pigmentation but produce normal melanocyte derived pigmentation.
Espadarana prosoblepon embryos from Los Cedros and Junín localities, and embryos later obtained from ex situ breeding at BDLS were observed and photographed from early stages of cleavage to the hatching of tadpoles to describe the pel phenotype.
Embryos from wt and pel clutches develop normally and are able to hatch as tadpoles approximately 4 days after oviposition. In wt embryos, dark oocyte pigmentation is retained throughout embryo development (Fig. 4A-4F). Different to mature oocytes, the melanic is concentrated in the animal pole ( Fig. 4A-4B). Animal pole derived blastomeres from these embryos will retain this dark pigmentation, which results in dark heterogeneously pigmented embryonic epidermis in further stages ( Fig. 4C-4E). In later embryos, neural crest derived melanocytes and eye pigmentation are not distinguishable from the epidermal dark pigmentation (oocyte-derived), and newly hatched tadpoles maintain a dark appearance (Fig. 4D-4F).
In pel embryos, dark oocyte pigmentation is only retained in some animal pole blastomeres, leaving an unpigmented vegetal pole (Fig. 4G-4I). This pigment is apparently diluted during subsequent stages, which results in a pale embryonic epidermis (Fig. 4J). During the heart beat stage, a few differentiating melanocytes can be distinguished in the dorsal trunk region, posterior to the head (Fig. 4K). Following the normal progression of melanocyte development in amphibians, next stages in pel embryos show progressive increase of melanocytes, with a higher density in the most dorsal anterior region. Eye retinal pigmentation is also distinguishable in pel embryos, compared to wt embryos (Fig. 4M, 4N). Newly hatched tadpoles have a dark appearance due to melanocyte derived pigmentation but overall paler when compared to wt tadpoles (Fig. 4L).
Altogether, our results provide strong evidence of the existence of a common allele in Ecuadorian populations of E. prosoblepon that affects oocyte pigmentation but not neural-crest derived melanogenesis.

DISCUSSION
This manuscript presents the first evidence of polymorphisms in egg pigmentation in an amphibian. Espadarana prosoblepon has previously been described as a species that produces dark eggs . From museum specimens and field collected clutches, we could conclude that egg pigmentation in E. prosoblepon is polymorphic, and that most females produce dark-brown eggs. However, we don´t have enough field data to quantify the commonality of the pel phenotype in comparison to wt eggs. The same occurs with most amphibian species on which egg pigmentation is generally thought to be a monomorphic and stable character.

The evolution of egg pigmentation in Centrolenidae and other anurans
There is a possibility that the dark pigmented character might not be fixed in the Here we have shown that pale eggs in E. prosoblepon are the result of a genetic trait, pel, which causes a sex-limited phenotype, since males don't produce oocytes. The fact that 50% of the females carried the pel phenotype leads us to conclude that the founders are possible carriers of a recessive allele, assuming pel is caused by a single allele inherited through mendelian mechanisms. This also allowed us to discard, preliminarily, the possibility of a maternal effect gene or an unknown environmental factor, since the pel phenotype appeared in 50% of an F1 progeny reared under the same controlled laboratory conditions.
A similar allele was recovered once before, in a genetic analysis of mutations arisen from nuclear transplantation in X. laevis. Droin and Fischberg (1984) called this mutation "pale eggs" and determined that this mutation was caused by a single recessive autosomal allele. We can only hypothesize that the pel phenotype is caused by a single allele that follow a mendelian pattern of inheritance since we don't have enough crosses to identify homozygotes or genotype the founders. This is a complicated task, given the long time required to obtain mature adults and the poor success during tadpole rearing. We are currently working on genomic approaches to genotype these individuals and address potential genes and pathways involved in this phenotype.
The potential genes and molecules related to the pel phenotype could be part of pathways involved in melanogenesis, in melanosome biogenesis and transport, or in melanosome stability. In amphibian oocytes, the melanogenic pathway is activated independently from other pigmented tissues through mechanisms that are yet to be Much less evidence is available regarding egg pigmentation and the protection against predators. It has been hypothesized that dark pigment would act as camouflage, since predators would not be able to distinguish the eggs from a dark background . This hypothesis could help explain the ubiquitous tendency in To conclude, the adaptive role of egg pigmentation in amphibians remains elusive due to incomplete experimental evidence and the complex interactions between phenotype and behavior that have evolved to ensure the survival of early embryos.
Research on predation, survival and behavior related to the pel phenotype in E.
prosoblepon could help us understand the true adaptive role of egg pigmentation.       (Table S3). An exposed oviposition site implies mostly the upper side of leaves; however, for a few species there were other exposed sites reported, such as branches, rocks and moss surfaces. The only other reported oviposition site was the underside of leaves. For some species, both sites were reported, either in the same or in distinct publications.