Discovery of skin alkaloids in a miniaturized eleutherodactylid frog from Cuba
Ariel Rodríguez, Dennis Poth, Stefan Schulz, Miguel Vences
Electronic Supplementary Materials
Extended methods
Fieldwork and sampling. Specimens of Eleutherodactylus iberia were collected on 25 January
2010 by A. Rodríguez and J. A. Rodríguez at Bahía de Taco, Guantánamo province in eastern
Cuba (-74.66508, 20.512139, 76 m a.s.l.). Specimens of E. orientalis and of one specimen of
the remotely related E. atkinsi were collected on the same day by the same collectors at the
Yunque de Baracoa, Guantánamo (-74.56284, 20.34242, 188 m). Specimens were obtained by
intensively searching the leaf litter during the day in areas where the typical male
advertisement calls of these miniaturized frogs (Alonso et al. 2007) were heard. Geographic
coordinates and altitude of each locality visited were recorded in the field using a Garmin
ETREX GPS receiver with WGS84 projection. Specimens were anesthetized and killed with
chlorobutanol. For alkaloid determination, several specimens of each species were skinned
and the skins individually preserved in glass vials with methanol and a teflon-lined cap.
Subsequently, all specimens were preserved as vouchers in 70% ethanol, labelled with field
numbers of Ariel Rodríguez (AR) for its deposition in the Zoological Collection of the
Institute of Ecology and Systematics of Cuba (CZACC). In a previous expedition to the same
sites in October 2009 by A. Rodríguez and M. Vences, skins of the same two species were
preserved in cryotubes filled with 99% ethanol, and used for preliminary alkaloid screening.
From four of the collected adult specimens of E. iberia and three adults of E. orientalis we
extracted and analyzed the stomach contents in order to get a first overview of their prey. The
stomach contents were analyzed under a stereomicroscope and identified to major taxonomic
groups. Special interest was paid to the identification of oribatid mites to the family level,
given their known importance as an alkaloid source for poison frogs (Saporito et al. 2007).
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Indolizidines from E. iberia skin extracts
The mass spectra of the indolizidines did not correspond to known frog alkaloid mass spectra
(Daly et al. 2005). The ions at m/z 70 and m/z 154 are typical for indolizidines and fit the
structures of the other identified alkaloids. Nevertheless, the exact nature of the side chain of
these new alkaloids remains elusive until more material for proper identification is available.
Figure S1. Mass spectra of two unknown indolizidine alkaloids from E. iberia. Note that the
denotion of the structure (1) and (2) is not meant to replace the alkaloid nomenclature as used
by J.W. Daly and colleagues (e.g., Daly et al. 2005).
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Figure S2. Phylogenetic relationships among families of neobatrachian frogs, including those
reported to contain lipophilic alkaloids (in bold and underlined; the newly reported
eleutherodactylid lineage in red). Families according to Frost (2010) with some modifications;
phylogenetic relationships largely compiled from Frost et al. (2006) and Hedges et al. (2008).
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