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BMC Evolutionary Biology 2008, 8:56                            http://www.biomedcentral.com/1471-2148/8/56




































            Oscillogram and spectrogram of an advertisement call of a male Bufo siculusFigure 6
            Oscillogram and spectrogram of an advertisement call of a male Bufo siculus. Recorded at 16°C at the type locality
            (recorder "Marantz CP430" with Philips unidirectional electric condenser microphone). a. Oscillogram of a call train of ca. 35 s
            with three calls. b. Zoomed part of the framed (b) section (in Figure 6a) between 16.2 s to 19.95 s (note slight overlay with
            another male calling in the background). c. Spectrogram of the call section shown in b. The fundamental frequency is at around
            1600 Hz and two harmonic vibrations are visible.



            In representatives from all major mitochondrial clades,  pension was applied to small agar plates. After incubation
            we sequenced two nuclear markers. To amplify a fragment  (18 h, 37°C), at least eight white colonies were amplified
            of ~880 bps of RAG-1 (recombination activating gene),  with  vector primers  M13forw./M13rev. Nested vector
            we used the primers MartFL1 and AmpR1 [81] in a touch  primers T7 and Sp6 (Promega) were used as sequencing
            down PCR (95°C, 4 min, denaturation; first cycle [95°C,  primers.
            30s; decreasing annealing temperature from  60°C to
            45°C of -1°C per cycle, 30 s; 72°C, 1:30 min] 15 times;  All  PCR-products  and  clones were sequenced in both
            followed by a second cycle [95°C, 30s; 45°C, 30 s; 72°C,  directions and visualized on an ABI 3730 sequencer.
            1:00 min] 20 times; 72°C, 10 min). We also applied prim-  Sequences were aligned using Sequencher, v. 4.1.2 and
            ers developed by Friesen et al. [82] for birds to amplify an  edited using MacClade 4.06.
            intron of alpha-tropomyosine, situated between the exons
            5 and 6, for the first time (to our knowledge) to anuran  Phylogenetic analyses
            amphibians. PCR  conditions for Friesen's  primers  pro-  For  each  sequence fragment,  the best fitting model  of
            posed for  "frogs" were adapted as follows: 95°C, 1:30  sequence evolution was selected using MrModeltest [83].
            min; cycle [94°C, 30 s; 55.9°, 30 s; 72°C, 45 s] 30 times;  Phylogeny was inferred for each locus separately, using
            72°C, 5 min. All PCR products were sequenced directly  the program MrBayes (v. 3.0b4 [84]), running four chains
            and apparent heterozygote genotypes of tropomyosine  for 5 or 10 million generations, with tree sampling every
                                            ®
            were also cloned  using  the pGEM -T vector  system  1000 generations. The "burnin"-value was selected by vis-
            (Promega). PCR product concentrations were quantified  ualizing the log likelihoods associated with the posterior
                      ®
            (NanoDrop ND-1000 spectrometer) and adjusted to 25  distribution of trees in the program Tracer. All trees gener-
            ng/µl. We mixed 1.5 µl template, 0.075 µl of vector (50  ated before the log likelihood curve flattened out were dis-
            ng/µl), 2.5  µl 2× ligation buffer, 0.5  µl  T4 ligase, and  carded. From the two different control region fragments,
            0.425 µl water and ligated overnight (10°C). Transforma-  we assembled  an overlapping  alignment of  sequences
            tions (2.5 µl ligation plus 12–25 µl competent cells) were  comprising 541 characters from all 148 individuals
            recovered in SOC for 1 h 30 min; 80–100 µl of cell sus-  included in this study; the best fitting model of evolution


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