Tissue sampling and DNA extraction

2.1 Field activity

The data used in the present study were collected during three field seasons from 2008 to 2010 for a total of seven months. The work was carried out between April and July in the first half of the rainy season when the frogs calling was intense. According to the frogs activity field work was performed in most cases from 5:00 am in the morning to 1:00 pm when temperature increased and the calling activity became weak. Data collection was also performed during minor activity time of Oophaga granulifera in late afternoon until sunset. In case of precedent very abundant rainfall or slight rain during the day, frogs remain active throughout the day enabling continuous data collection.

2.2 Tissue sampling and DNA extraction

Tissues samples used for genetic analyses were obtained in the field by toe-clipping and stored in 96% ethanol in 1.5 ml plastic tubes with screw cap. Toe-clipping is the removal of one or more toes and is a common practice in amphibian studies (Hero, 1989; Waichman, 1992; Halliday, 1996). Other than for obtaining tissue for genetic analyses it is used to allow individual recognition in behavioural studies removing several toes in specific combinations and to obtain bone for estimating age using skeletochronology (Friedl & Klump, 1997;

Driscoll, 1998; McGuigan et al., 1998). Some studies revealed possible deleterious effects of removing several toes in amphibians (Clarke, 1972; Humphries, 1979; Golay & Durrer, 1994;

Reaser & Dexter, 1996) while others did not (Lemckert, 1996; Williamson & Bull, 1996;

Parris & McCarthy, 2001). Nonetheless, it is recognized that the removal of one toe is likely not to provoke any negative effect on survival (McCarthy & Parris, 2004). In the present

study only a portion of one toe (2-3 mm) was removed and no adverse effects were observed after release. The sample was stored for some weeks in the 1.5 ml screw cap plastic tubes filled with 96% ethanol. The preservation of tissue for DNA extraction in ethanol for a short time is appropriate because ethanol inhibits the proteolytic enzymes responsible for tissue degradation. Once in the laboratory, the samples were stored in the fridge for few days until DNA extraction. The DNA was extracted in the genetic laboratory of the Institute of Zoology at the TiHO (co-responsible Prof. Dr. Heike Pröhl) in Hannover and at the Department of Zoology of Cambridge University (evolutionary genetics group, responsible Dr. Nicholas Mundy) using a Qiagen DNeasy extraction kits and a Qiagen QIAquick purification kit respectively (Qiagen, Hilden, Germany) following the manufacturer's instructions. The general procedure involved digestion of the tissue in 1.5 ml tubes containing a proteinase K solution and incubation at 37°C overnight. The proteinase K releases the DNA from the tissue and inhibits the nucleases enzymes responsible for nucleic acid degradation. Then a binding buffer was added and the samples were passed through filtering mini-columns embedded in 1.5 ml centrifuge tubes. After centrifugation, a washing buffer was added to the columns to eliminate the impurities from the filter that binds the DNA and the flow-through discarded. As a final step the DNA was eluted by adding an elution buffer in two successive aliquots each followed by centrifugation. The eluate was stored at -20° C. Small known aliquots of purified DNA mixed with a loading dye were run on 1% agarose gel stained with ethidium bromide to quantify the DNA yield and prepare proper dilution for subsequent amplification steps.

2.3 Phylogeography

2.3.1 Mitochondrial DNA sequencing

Two fragments of mitochondrial DNA were considered for the phylogeographic analysis. I amplified a 600 bp fragment of the cytochrome b gene (cyt b) from 28 individuals (10

populations) using primers MTAL (Hauswaldt et al., 2011) and cyt b-c (Bossuyt &

Milinkovitch, 2000). A portion (527 bp) of the 16S rRNA gene (16S) was amplified in a subset of samples (N = 16; 9 population) using universal primers 16SA and 16SB (Palumbi et al., 1991) (Table 1). PCRs were performed in 25 µl volume using Taq DNA-polymerase (Eppendorf, Hamburg, Germany). The thermocycling conditions for cyt b comprised an initial denaturation at 95°C for 2 min followed by 35 cycles of denaturation (95°C for 45s), annealing (52°C for 45s), elongation (72°C for 1min), and a final elongation at 72°C for 10 min. The profile for 16S was identical except for an annealing temperature of 55°C. PCR products were purified using Qiagen purification kits and filtering columns system described above. The purified PCR product was then precipitated and prepared for sequencing according to standard procedure. The fragments were sequenced in both directions in 10 µl volume using BigDye 3.1. The sequences were run on an ABI sequencer 3500 (Applied Biosystems, Darmstadt, Germany).

2.3.2 Mitochondrial DNA analyses

Sequences were aligned and edited with MEGA 4.1 (Tamura et al., 2007). Haplotypes were extracted using the on–line tool “DNA to haplotype collapser and converter” available in FABOX 1.40 (http://www.birc.au.dk/software/fabox/). I calculated the number of variable sites as well as uncorrected pairwise distances between populations (p-distance) using MEGA 4.1. I performed Maximum Likelihood and Bayesian phylogenetic analyses with PAUP* 4.0 (Swofford, 2002) and MrBayes 3.1 (Ronquist & Huelsenbeck, 2003), respectively. All the cyt b sequences of comparable size published for O. granulifera were included in the analyses (Wang, 2011; see Additional Information for all accession numbers). The populations considered in Wang (2011) were Corcovado (CO), Drake Bay (DB), Piedras Blancas (PB), Rio Terribe (TE), Rio Baru (RB), Cerro Nara (CN), Rio Savegre (SV), Rio Damitas (DM) and Fila Chonta (FC). The geographical areas of Costa Rica where the populations were found

and the correspondent colour phenotypes described in Wang (2011) were: south-west, red (CO, DB, PB), central-south-west, red (TE), central-west, red (BR), central-north, yellow (CN, SV), central-north, green (DM, FC) (Fig. 1). Hereafter, these populations will be cited in the text using the abbreviations. I am not aware of any sampled population in common with Wang (2011) (Fig. 1). As outgroups, I included eight 16S sequences from all but one of the other species of the genus Oophaga (nomenclatural authorities) [O. arborea (Myers et al., 1984), O. histrionica (Berthold, 1845), O. lehmanni (Myers & Daly, 1976), O. pumilio, O.

speciosa (Schmidt, 1857), O. sylvatica (Funkhouser, 1956) and O. vicentei (Jungfer et al., 1996); two sequences for O. pumilio northern and southern lineages, one sequence for each other species; O. occultator (Myers & Daly, 1976) was not included because there were no sequences available], two 16S sequences of Dendrobates leucomelas and D. tinctorius and two cyt b sequences of O. histrionica and O. sylvatica, (see Additional Information for all accession numbers). For ML analyses, I selected the model of evolution that best fitted the data using jMODELTEST 0.1 (Posada, 2008). Analyses were carried out using a heuristic search with 10 random additions per replicate and TBR branch swapping. Node confidence was assessed using 1000 bootstrap replicates. Bayesian posterior probabilities were calculated using MrBayes 3.1 (MCMC sampling) applying the substitution model selected in MrModeltest 3.7. Two markov chains were run from random trees for 4 × 10⁶ generations and monitored to ensure that the average standard deviation of split frequencies was <0.01. The 20% of the initial trees were discarded as burn-in. Finally, I reconstructed the cyt b haplotype network using TCS 1.21 (Clement et al., 2000) and determined the minimum number of mutational steps required to connect different clusters of haplotypes using the fix connection limit option. I included the same published cyt b sequences (Wang, 2011) considered in the phylogenetic analyses.