Molecular Sex Determination of Captive Komodo Dragons (Varanus komodoensis) at Gembira Loka Zoo, Surabaya Zoo, and Ragunan Zoo, Indonesia

Abstract

Captive breeding of endangered species is often difficult, and may be hampered by many factors. Sexual monomorphism, in which males and females are not easily distinguishable, is one such factor and is a common problem in captive breeding of many avian and reptile species. Species-specific nuclear DNA markers, recently developed to identify portions of sex chromosomes, were employed in this study for sex determination of Komodo dragons (Varanus Komodoensis). Each animal was uniquely tagged using a passive integrated micro-transponder (TROVAN 100A type transponders of 13 mm in length and 2 mm in diameter). The sex of a total of 81 individual Komodo dragons (44 samples from Ragunan zoo, 26 samples from Surabaya zoo, and 11 samples from Gembira Loka zoo) were determined using primers Ksex 1for and Ksex 3rev. A series of preliminary PCR amplifications were conducted using DNA from individuals of known sex. During these preliminary tests, researchers varied the annealing temperatures, number of cycles, and concentrations of reagents, in order to identify the best protocol for sex determination using our sample set. We thus developed our own PCR protocol for this study, which resulted in the amplification of band A in females and band C in males. Results from band B, however, turned out to be non-determinative in our study because, for females, band B was not always visible, and for males sometimes a similar, but lighter band was also amplified, making interpretation difficult. In this study, sex determination was based mainly on the difference in size between the female-specific 812 bp fragment and the homologous, longer fragment amplified for males.

Sex determination, longevity, and the birth and death of reptilian species

“…; Sulandari et al. ). The resulting dataset contains information for 87 turtle and 303 lizard species (Table S1a) that have been studied across families (Table S1b).…”

“…An initial reptilian SDM database was obtained from (The Tree of Sex Consortium, 2014) and complemented with an extensive literature search (Ota et al 1992;Gamble 2010;Pokorn a et al 2011Pokorn a et al , 2014Trifonov et al 2011;Badenhorst et al 2013;Matsubara et al 2013Matsubara et al , 2014Gamble et al 2014Gamble et al , 2015Koubov a et al 2014;Pokorna et al 2014;Rovatsos et al 2014a,b;Schmid et al 2014;Sulandari et al 2014). The resulting dataset contains information for 87 turtle and 303 lizard species (Table S1a) that have been studied across families (Table S1b).…”

“…Taken together, while our results indicate that asymmetries in transition rates rather than diversification rates lead to the differential SDM diversity observed in squamates, the jury awaits for improved and well-vetted analytical methods plus the collection of additional information on sex determination in reptiles. Fortunately, SDM data are growing at an accelerated pace thanks to the use of a variety of molecular techniques to complement classic incubation experiments (Ota et al 1992;Gamble 2010;Pokorn a et al 2011Pokorn a et al , 2014Trifonov et al 2011;Badenhorst et al 2013;Matsubara et al 2013Matsubara et al , 2014Mu et al 2013;Gamble et al 2014Gamble et al , 2015Koubov a et al 2014;Pokorna et al 2014;Rovatsos et al 2014a,b;Schmid et al 2014;Sulandari et al 2014;Valenzuela et al 2014;Montiel et al 2016). Extensive research over 50 years has uncovered SDM information in all turtle families (except Platysternidae) for at least 1 species per family, whereas 9 of 37 lizard families remain unstudied.…”

Molecular sexing applicable in 4000 species of lizards and snakes? From dream to real possibility

“…Indeed, the Y-specific marker in Anolis carolinensis does not work for molecular sexing in more distantly related species of the genus (Gamble & Zarkower 2014). Similarly, in our experience, the sex-specific marker in Komodo dragons (Halverson & Spelman 2002;Sulandari et al 2014) is not applicable to two other species of monitor lizards (M. Rovatsos, L. Kratochvil, unpublished data). It is also likely that other molecular sexing techniques in reptiles based on microsatellite markers, such as the X-specific marker in the skinks Tiliqua rugosa (Cooper, Bull & Gardner 1997) and Egernia cunninghami (Stow et al 2001), the uncharacterized Y-specific marker in the skink Bassiana duperreyi (Quinn et al 2009) and the uncharacterized, probably non-coding Wspecific marker in the bearded dragon (Pogona vitticeps; Quinn et al 2010), would have equally narrow applicability.…”

“…Similarly, in our experience, the sex‐specific marker in Komodo dragons (Halverson & Spelman ; Sulandari et al . ) is not applicable to two other species of monitor lizards (M. Rovatsos, L. Kratochvil, unpublished data). It is also likely that other molecular sexing techniques in reptiles based on microsatellite markers, such as the X‐specific marker in the skinks Tiliqua rugosa (Cooper, Bull & Gardner ) and Egernia cunninghami (Stow et al .…”