DNA Fingerprinting in Minyobates minutus, the Minute Poison Frog

Abstract

DNA fingerprinting has become a widely used technique in the study of ecology and evolution (e.g., Parker et al., 1998). However, this technique has rarely been used in research on anurans (e.g., D'Orgeix and Turner, 1995). Here we present DNA fingerprinting data from a study of the minute poison frog, Minyobates minutus, in Panama. The purpose of this study was to investigate whether commonly used DNA fingerprinting probes are useful in investigating genetic relatedness in a wild population of poison frogs. DNA fingerprinting could be a particularly useful tool for research on the ecology and evolution of poison frogs, because these frogs exhibit complex patterns of parental care involving high levels of parental investment (Wells, 1981; Weygoldt, 1987; Zimmermann and Zimmermann, 1988). Such high levels of parental investment make issues of paternity and maternity of critical interest to those investigating the evolution of parental care in members of the family Dendrobatidae (Summers and Earn, 1999). Furthermore, the fact that tadpoles in this group are highly cannibalistic (e.g., Summers, 1990; Caldwell and Araujo, 1998), and frequently placed together in small pools, such as bromeliad tanks or Heliconia leaf axils (e.g., Summers and Amos, 1997; Caldwell and Araujo, 1998), makes knowledge of kinship particularly valuable for investigations of kin recognition and its functional significance (Pfennig, 1997). The clutch size of Minyobates minutus appears to be small, consisting of two eggs on average. Survival rates of clutches in the field are unknown, but once the developing embryo reaches the tadpole stage it is carried to phytotelmata and deposited by a male nurse frog (pers. obs.). These phytotelmata are typically found in bromeliads, which are abundant at the study site. Collections were made in the Nusagandi Biological Reserve, Comarca de San Bias, Republic of Panama. Ten males carrying tadpoles were collected to serve as references for bandsharing values between parents and offspring. Males in this species typically carry a single tadpole on their back. An additional 17 adults were collected in order to investigate levels of bandsharing between putatively unrelated individuals. Comparisons were made among these individuals and among adults or tadpoles from the reference group, for a total of 21 comparisons of putatively unrelated individuals. Each individual was compared only once, and comparisons were made between individuals that were run on the same gel (within four lanes of each other). Frogs and tadpoles were instantaneously frozen in liquid nitrogen in the field, and later stored at -70 degrees in the Smithsonian Tropical Research Institute's molecular evolution laboratory in Panama City, Panama. Specimens were later transported in a dry shipper (liquid nitrogen container) to the molecular ecology laboratory at Queen's University. DNA was extracted from the samples using standard phenol/chloroform methods (Summers et al., 1999): Approximately 0.2 g of leg muscle tissue was homogenized in 500 ,pl of Ix AB (Applied Biosystems) lysis buffer in a 1.5 ml tube and mixed on a rotating wheel overnight at 37 C. The following day 25 units of proteinase K were added to each of the samples, which were mixed overnight on a rotating wheel at 37 C. The following day an equal volume of buffered, equilibrated phenol was added to each sample and shaken for 2-3 min by hand, then centrifuged at 13,000 rpm in a clinical centrifuge. The aqueous (top) layer was removed and transferred to a new tube, and the process was repeated with PCI (phenol: chloroform:isoamyl alcohol: 25:24:1) and then with chloroform: isoamyl alcohol (24:1). Next 50 pLI of 3M sodium acetate (pH 3.5) was added to the resulting supernatant and mixed gently, followed by the addition of two volumes of 95% ethanol. This mixture was