Yeison Tolosa1,2, Claudia Molina-Zuluaga1,3, Adriana Restrepo1,4, Juan M. Daza1,5,6


1Grupo Herpetológico de Antioquia, Instituto de Biología, Universidad de Antioquia, AA 1226, Medellín, Colombia

6Corresponding author



Differences in sexual characteristics such as dimorphism and size to maturity may indicate natural selection forces acting on natural populations and will improve parameter estimation in demographic studies. We inferred the minimum size of sexual maturity and the existence of sexual dimorphism in a population of rocket-frog, Colostethus aff. fraterdanieli, through morphology, morphometry and gonadal examination. Female sexual maturity was reached near to 17.90 ± 0.1 mm snout-vent length (SVL) while males reached sexual maturity over the 16.13 ± 0.06 mm. Females were significantly larger than males in snout vent length (SVL), humerus length (LHU), and head width (HW). Males have a dark and marked gular coloration that sometimes extend to the chest, while females lack this feature, with a throat immaculate or weakly stained.

Key words: Amphibian, Andes, gonads, histology, morphometry, Natural Selection.


Diferencias en características tales como el dimorfismo sexual y la edad mínima a la madurez sexual pueden indicar como selección natural actua sobre poblaciones naturales y su identificación pueden ser de gran utilidad para estimar parametros en estudios demográficos. Establecimos el tamaño mínimo de madurez sexual y la existencia de dimorfismo sexual en una población de la ranita cohete, Colostethus aff. fraterdanieli, a través de la morfología, morfometría y observaciones macro y microscópicas de las gónadas. La madurez sexual de las hembras fue alcanzada cerca a los 17.90 ± 0.1 mm Longitud rostro-cloaca (SVL), mientras que los machos alcanzaron la madurez sexual sobre los 16.13±0.06 mm (SVL): Las hembras fueron significativamente más grandes que los machos en la logitud rostro cloaca (SVL), longitud del húmero (LHU) y ancho de la cabeza (HW). Los machos presentan una marcada y oscura coloración gular que algunas veces se extiende hasta el vientre, mientras que las hembras carecen de esta característica, con una garganta inmaculada o débilmente manchada.

Palabras clave: Anfibios, Andes, gónadas, histología, morfometría, Selección Natural.


Reproductive characteristics play a critical role in the species life history, ecology and population dynamics (Watling and Donnelly 2002). The minimum size of sexual maturity (MSSM), the size reached by an individual at which is ready for reproduction (D’ Ancona 1960), and sexual dimorphism (SD), allow both to estimate parameters, that inform us about the population status, such as sex-age structure and the number of mature individuals (Yilmaz et al. 2005).

There are numerous studies about reproductive patterns in neotropical anurans, mainly on lowland species from Brazil and Central America (Bertolucci and Rodrigues 2002, Duellman 1988, Haddad and Prado 2005, Lima and Keller 2003, Watling and Donnelly 2002). Although the northern Andes in Colombia harbors one of the richest frog communities in the Neotropics, few studies have addressed reproductive aspects of montane frog fauna (Arroyo et al. 2008, Valderrama-Vernaza et al. 2010).f

Our purpose here is to describe some aspects of the reproductive ecology of Colostethus aff. fraterdanieli, such as the minimum size of sexual maturity and the evidence of sexual dimorphism. The results will not only indicate if natural selection is acting on these attributes but also will help in a better estimation of demographic parameters.


Study Area. The population is located in the site known as El Edén, a secondary forest in vereda San Antonio, municipio Alejandría, Antioquia, Colombia (6,36664 N, -75,02746 W), in the buffer area of the Jaguas hydroelectric project. The area corresponds to Pre-montane wet forest (Holdridge 1967) and is located at 1400 masl at the northeastern of the Cordillera Central in Colombia. Temperature varies between 18 and 24 ° C, with an average relative humidity of 82.2%, and annual rainfall between 2000 and 4000 mm.

Study species. Colostethus aff fraterdanieli is an undescribed species sister to C. fraterdanieli with 13% of genetic distance in the mitochondrial gene COI (Daza et al., unpublished). The genus Colostethus is a trans-Andean clade, extending from eastern Central America to northwestern Ecuador, with most species occurring in cloud forests in the western Andes (Grant et al. 2006). C. aff fraterdanieli is a leaf-litter dwelling frog inhabiting forest fragments with small streams. Like its congeners, this species is extremely fast and agile, responding to danger by hopping a short distance and hiding underneath the leaf litter. Males call constantly from leaf litter, the advertisement call is composed by one note and in some cases, males emit calls with three notes. Adults seem to be highly territorial and males have been observed carrying ten to eleven tadpoles in their backs.

Field work. Frogs were collected between March and May of 2013 using active search (Crump and Scott 2001). All individuals were euthanized using Lidocaine 3%, fixed with formaldehyde 10% and stored in the herpetological collection of the Museo de Herpetología de la Universidad de Antioquia (vouchers MHUAA 7878-7898, 7900-7929, 7931-7933, 7940-7984).

Minimum Size of Sexual Maturity (MSSM). Prior to dissection, frogs were photographed, measured (SVL) and classified as male or female using external features such as the presence of vocal slits and spots in the throat area in males (Grant and Castro 1998, Silverstone 1971). All individuals were dissected under a stereomicroscope to examine gonadal characteristics and to confirm the sex and the reproductive stage. We used the presence and condition of oviducts (convoluted oviducts), and the presence of vitellogenic follicles to classify individuals as adult females (Prado et al. 2004, Valderrama-Vernaza et al. 2010, Wake and Dickie 1998). In addition, we prepared histological slides with the hematoxylin-eosin method (Luna 1968) and used the presence of sperm in the seminiferous tubules to identify adult males (Prado et al. 2004, Valderrama-Vernaza et al. 2010, Wake and Dickie 1998). The MSSM was inferred on each sex as the individual having reproductive characters with the lowest SVL (Valderrama-Vernaza et al. 2010).

Sexual Dimorphism (SD). To determine the presence of sexual dimorphism, external characters were measured in all individuals following Felgueiras-Napoli (2005). All measurements were taken three times at the stereomicroscope with a 0.1 mm precision. Selected characters were: snout-vent length (SVL), head length (HL), head width (HW), measured at the level of the posterior part of the tympanum, inter-orbital diameter (IOD), inter-nostril distance (IND), eye diameter (ED), disc diameter of the finger three (DD3), disc diameter of the toe four (DF4), eye-nostril distance (END), tympanum diameter (TD), length of the humerus (LHU), forearm length (FOL), thigh length (THL), tibia length (TL), and foot length (FL) (Figure 1).

A T-test with each variable and a MANOVA using all variable together were used to assess differences between sexes. Then, we used a PCA to evaluate if the differences were due to size or shape. In addition to morphometric characters, we classify all mature individuals according to their throat coloration to test differences between sexes in this feature. The individuals with spots covering less than 20 % of the throat were classified as Immaculate and the remaining individuals were classified as Spotted.


Minimum Size of Sexual Maturity. We examined 100 frogs and based on external observation of the gonads and histological evaluation, we found 24 adult females, 22 immature females, 52 adult males and two immature males. The Minimum Size of Sexual Maturity (MSSM) in females was inferred at 17.90 ± 0.1 mm. All mature females had pre-vitellogenic follicles and underdeveloped oviduct. The reproductive condition of all mature females were gravid with approximately 8-10 brown developed follicles although we did not find eggs in the oviduct (Figure 2A). The MSSM in males was estimated at 16.13 ± 0.06 mm, and all showed seminiferous tubules with cysts that had most of the stages of spermatogenesis (spermatogonia, spermatocytes, spermatids and sperm). All mature males had free sperm in the seminiferous tubules (Figure 2B). The right testis was higher than the left one, and sometimes testes had some dark pigmentation surrounding the seminiferous tubules (Figure 2C).

Sexual dimorphism. Morphometric analysis showed differences in size between males and females (Figure 3). In general, morphometric measurements were larger in females than in males (Table 1). Based on the MANOVA analysis, we found significant differences between sexes (Wilks' Lambda =0.44, p < 0.001). We also found differences between sexes in the coloration on the throat (X2= 41.57, p<0.001). Most of the mature males have the Spotted pattern. The two immature males lacked this coloration. All females have an Immaculate pattern on the throat (Figure 4).


Minimum size of sexual maturity. We are not aware of studies that has evaluated the MSSM in Colostethus species. In fact, there are few studies with amphibians that use the MSSM to classify individuals as mature or inmature. Age and size at sexual maturity are critical components of life history studies because of their importance in determining an organism’s fitness (Roff 2002). Many population studies on frogs arbitrarily divide the individuals in size classes (Cummins and Swan 1995, Donelly 1989b, Lima et al. 2002). For example, Lima et al. (2002) divided the females of one population of Colostethus caeruleodactylus in Brazil, in three classes assuming that the two first were inmature states and the third one were gravid females. In small frogs like Colostethus species splitting the population in more than two classes only provides noise to population analyses, making spurious the inferences in population trends.

The female minimum size of sexual maturity (MSSM) in C. aff fraterdanieli was 17.9 mm, based on that, 22 were immature and 24 were adults in a gravid state with brown development follicles. We found four immature females with SVL above the MSSM. The age and size at maturity can vary considerably among individuals in a population, because maturity is controlled by a great number of complex processes acting at the individual level (Bernardo 1993). Although all adult females had vitellogenic follicles in advanced stages of development, we did not find eggs in the oviduct probably because at the capture time, females were not been courted by males. In studies that have found eggs in the oviducts, the females were captured during courtship (Valderrama-Vernaza et al. 2010). In contrast, male MSSM was 16.1 mm and only two out of 54 examined were immature. The low number of immature males in our sample can be explained by differences in detectability because these non-reproductive individuals have more cryptic behavior as opposed to adults males that are more conspicuos because its calling activity (Bailey et al. 2002, Mazerolle et al. 2007, de Solla et al. 2005).

Observed differences between sexes in MSSM, where males reached maturity at shorter SVL than females, can be related to patterns of mating and parental effort (Howard 1981). It has been shown that in species with large clutch sizes, the female size is significantly larger than in males (Duellman and Trueb 1994, Mesquita et al. 2004, but see Lima and Keller 2003). The hypothesis behind this pattern is that delayed maturation in females is related to obtain reproductive benefits (Howard 1981) where females with larger sizes have more fitness as they can accommodate more eggs than small females (Collins 1975, Howard 1978).

Sexual dimorphism. Our results suggest a strong size sexual dimorphism in Colostethus aff. fraterdanieli. All morphological measurements were strongly correlated and snout-vent length (SVL) where larger in females than in males. In addition, males have spots on their throats while females lack of that coloration. Sexual dimorphism is the result of divergent selective pressures between sexes that promote morphological and behavioral differences (Howard 1981). Factors influencing such sexual dimorphisms include differences between the sexes in (a) intensity of sexual competition (Selander 1965, 1972), (b) division of labor in parental effort (Rails 1977), and (c) resource utilization (Selander 1966, 1972). Throat coloration resulted in a good character to distinguish males from females and juveniles. This character is considered as a secondary sexual character exhibited by adult males, and it has been suggested that throat coloration is hormonally regulated as it changes with the reproductive activity of the individual (Grant and Castro 1998). Although not quantified here, we also evidenced that the third finger, as in many Colostethus species, is swollen in adult males (see Figure 4).

Even though we collected the individuals during a short period of time representing the rainy season (march through may), a more thorough demographic study including capture-mark-recapture on this species, has evidenced juveniles and also males carrying tadpoles across the entire year. These findings may indicate that this population has continuous reproduction as observed in other forest dwelling dendrobatids.


This study was funded by ISAGEN and the Universidad de Antioquia under the Convenio Interinstitucional 46/4208. We thank S. Cuartas and the Grupo Herpetológico de Antioquia members for comments to an early draft and help during fieldwork.



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Figure 1. Morphometric measurements used for the analysis of sexual dimorphism in Colostethus aff. fraterdanieli. (Felgueiras-Napoli, 2005). Snout-vent Length (SVL), Head Length (HL), Head Width (HW), Inter-Orbital Diameter (IOD), Inter-Nostril Distance (IND), Eye Diameter (ED), Disc Diameter of the Finger Three (DD3), Disc Diameter Toe Four (DF4), Eye-Nostril Distance (END), Tympanum Diameter (TD), Length of the Humerus (LHU), Forearm Length (FOL), Thigh Length (THL), Tibia Length (TL) and Foot Length (FL).

Figure 2. Macroscopic and microscopic view of the gonads of C. aff. fraterdanieli A. Female oviducts with brown developed follicles (F) B. Histological section of the testes showing: spermatogonia (G), spermatocytes (C), spermid (T*) and sperm (Z). C. Male testes with dark pigmentation surrounding the seminiferous tubules: testicle (T), kidney (Rn), fat bodies (Cg).

Figure 3. PCA results showing size difference between males and females of Colostethus aff. fraterdanieli.

Figure 4. Throat coloration in males and females of Colostethus aff. fraterdanieli.