Electronic Supplementary Material (S1) For

Electronic Supplementary Material (S1) for:

Phylogenetic position of the monotypic des Murs’ wiretail (Sylviorthorhynchus desmursii, Aves: Furnariidae) based on mitochondrial and nuclear DNA

Javier Gonzalez () and Michael Wink

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Samples, DNA extraction, PCR amplification and sequencing

Birds were captured with mist-nets. Blood samples were obtained by puncturing the brachial vein from at least two individuals per species. Total DNA was isolated from 100 µl of blood using standard Proteinase K (Merck, Darmstadt) and phenol/chloroform procedures (Sambrook et al. 1989) or from feathers (Bello et al. 2001). PCR amplifications were performed in 50 µl reaction volumes containing 1.5 mM MgCl2, 10 mM Tris (pH 8.5), 50 mM KCl, 100 µM dNTPs, 0.8 units Taq DNA polymerase (Pharmacia Biotech, Freiburg), 200 ng DNA and 5 pmol of primers. The primers L14854, mtA1, mtC, and mtFsH (Dietzen et al. 2003) were used to amplify the mitochondrial cytochrome b gene (cytb). The intron 2 of the myoglobin gene (myo) was amplified with the following primers: myo2, myo3f, myoint.nc, myoint.c (Heslewood et al. 1998; Slade et al. 1993), myoint.H1, myoint.H2, and myoint.L1 (Irestedt et al. 2002). Finally, the intron 11 of the glyceraldehyde-3-phosphodehydrogenase gene (g3pdh) was amplified with the primers: g3p13b, g3p14b and g3pintL1 (Fjeldså et al. 2003).

Optimal annealing temperatures were found by gradient PCR in a Tgradient thermocycler (Biometra). The cycle protocol consisted of (1) an initial denaturation at 94°C for 8 min, (2) 32 cycles including denaturation at 94°C for 45 s, annealing at 48–58°C for 1 min and extension at 72°C for 1 min followed by (3) a final extension period at 72°C for 10 min. PCR products were precipitated with 4 M NH4Ac and ethanol (1:1:6) and a centrifugation for 15 min (13,000 rpm).

Sequencing was performed using the DYEnamic ET Terminator Cycle Sequencing Kit (Amersham Pharmacia Biotech) and a cycle sequencing protocol as follows: 31 cycles of 20 s at 96°C, 15 s at 52°C and 1 min at 60°C. SephadexTM G-50 columns (Amersham Biosciences) and MultiScreen filter plates (Millipore Corporation) were used for sequencing purification products. Sequences were analyzed with capillary electrophoresis using a MegaBACETM 1000 sequencer (Molecular Dynamics Inc., Amersham Pharmacia).

Sequence analysis and phylogenetic reconstruction

The sequences were aligned by BioEdit version 7.0.5 (Hall 1999). Alignments are available from the authors on request. Basic statistics and p-distances were calculated with MEGA version 3.1 (Kumar et al. 2004). Phylogenetic trees were reconstructed using maximum likelihood (ML) and maximum parsimony (MP) in PAUP* version 4.0b10a (Swofford 2002), and Bayesian inference (BI) in MPIMrBayes version 3.1.2. (Ronquist and Huelsenbeck 2003; Altekar et al. 2004). The partition homogeneity test implemented in PAUP* version 4.0b10a (Swofford 2002) was used to test the degree of incongruence between nuclear and mitochondrial DNA using 1,000 replicates. Phylogenetic analyses were performed for all genes separately, both nuclear (myo + g3pdh) genes combined and nuclear + mitochondrial (cytb) genes concatenated. MP and ML heuristic searches were performed with 10 random stepwise additions, tree-bisection-reconnection branch-swapping, and ‘multrees’ option. In MP analyses, indels were treated as missing characters. We explored the model of sequence evolution that fits the data best with Modeltest version 3.7 (Posada and Crandall 1998). The best model was then used with the ML analyses. Robustness of nodes was assessed by 1,000 bootstrap replicates. For BI analyses, two independent runs of 8,000,000 generations each were performed along with four Markov chains. The evolutionary model selected for BI analysis was the GTR + Γ + I. Trees were sampled every 500 generations and the first 4,000 samples were discarded as 'burn in'. Two partitions, nuclear (myo + g3pdh) and mitochondrial (cytb) DNA, were considered in BI analysis in the combined data set.

The sequences generated in this study were confirmed from at least two individuals per species and deposited at the GenBank under the accession numbers listed in Table 1. We retrieved other sequences from GenBank that represent all major groups of ovenbirds (Furnariinae, Synallaxinae and Philydorinae, and the Dendrocolaptinae, see Irestedt et al. 2006). We selected only those species of ovenbirds where all three genes were available. A previous analysis based on a larger data set (60 species) was performed to determine the effects of sampling (data not shown). A second analysis based on a smaller data set (41 species) was conducted with Pteroptochos tarnii, Sclerurus mexicanus and Geositta cunicularia as outgroups (Irestedt et al. 2006; Olson et al. 2005). See Table 2 below for detailed information about sequences retrieved from GenBank. Models of nucleotide substitutions, variable sites, parsimony informative sites and basic statistics for the individual genes and the concatenated data set are indicated in Table 3. Finally, in order to evaluate alternative phylogenetic hypotheses, we employed the Shimodaira-Hasegawa test (Shimodaira and Hasegawa 1999, see also the recommendations of Goldman et al. 2000) implemented in SHTests version 1.0 (available in along with the Markov general reversible model (REV), the RELL approximation and 1,000 bootstrap replicates. Missing and ambiguous characters were discarded in the Shimodaira-Hasegawa test analysis.

References

Altekar G, Dwarkadas S, Huelsenbeck JP, Ronquist F (2004) Parallel Metropolis-coupled Markov chain Monte Carlo for Bayesian phylogenetic inference. Bioinformatics 20:407–415

Bello N, Francino O, Sánchez A (2001) Isolation of genomic DNA from feathers. J Vet Diagn Invest 13:162–164

Dietzen C, Witt H-H, Wink M (2003) The phylogeographic differentiation of the European robin Erithacus rubecula on the Canary Islands revealed by mitochondrial DNA sequence data and morphometrics: evidence for a new robin taxon on Gran Canaria? Avian Sci 3:115–131

Ericson PGP, Christidis L, Irestedt M, Norman JA (2002) Systematic affinities of the lyrebirds (Passeriformes: Menura), with a novel classification of the major groups of passerine birds. Mol Phylogenet Evol 25:53–62

Fjeldså J, Zuccon D, Irestedt M, Johansson US, Ericson PG (2003) Sapayoa aenigma: a New World representative of 'Old World suboscines'. P Roy Soc Lond B (Suppl) 270:S238–S241

Fjeldså J, Irestedt M, Ericson PGP (2005) Molecular data reveal some major adaptational shifts in the early evolution of the most diverse avian family, the Furnariidae. J Ornithol 146:1–13

Goldman N, Anderson JP, Rodrigo AG (2000) Likelihood-based tests of topologies in phylogenetics. Syst Biol 49:652–670

Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41:95–98

Heslewood MM, Elphinstone MS, Tidemann SC, Baverstock PR (1998) Myoglobin intron variation in the gouldian finch Erythrura gouldiae assessed by temperature gradient gel electrophoresis. Electrophoresis 19:142–151

Irestedt M, Fjeldså J, Ericson PG (2004) Phylogenetic relationships of woodcreepers (Aves: Dendrocolaptinae) - incongruence between molecular and morphological data. J Avian Biol 35:280–288

Irestedt M, Fjeldså J, Ericson PG (2006) Evolution of the ovenbird-woodcreeper assemblage (Aves: Furnariidae) - major shifts in nest architecture and adaptive radiation. J Avian Biol 37:260–272

Irestedt M, Fjeldså J, Johansson US, Ericson PG (2002) Systematic relationships and biogeography of the tracheophone suboscines (Aves: Passeriformes). Mol Phylogenet Evol 23:499–512

Kumar S, Tamura K, Nei M (2004) MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5:150–163

Olson SL, Irestedt M, Ericson PG, Fjeldså J (2005) Independent evolution of two Darwinian Marsh-Dwelling Ovenbirds (Furnariidae: Limnornis, Limnoctites). Ornitol Neotrop 16:347–359

Posada D, Crandall KA (1998) Modeltest: testing the model of DNA substitution. Bioinformatics 14:817–818

Ronquist F, Huelsenbeck JP (2003) MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19:1572–1574

Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New York

Shimodaira H, Hasegawa M (1999) Multiple comparisons of log-likelihoods with applications to phylogenetic inference. Mol Biol Evol 16:1114–1116

Slade RW, Moritz C, Heideman A, Hale PT (1993) Rapid assessment of single-copy nuclear DNA variation in diverse species. Mol Ecol 2:359–373

Swofford DL (2002) PAUP*, phylogenetic analysis using parsimony (and other methods), version 4.0b10a. Sinauer, Sunderland, Massachusetts

Legends of figures

S1-Fig. 1 Bayesian inference tree based on 2,188 bp of cytochrome b, myoglobin and g3pdh genes concatenated. BI posterior probabilities (≥ 0.90) and MP bootstrap values (≥ 50%) indicated above the branches. ML bootstrap values (≥ 50%) indicated below the branches

S1-Fig. 2 Bayesian inference tree based on 1,189 bp of myoglobin and g3pdh genes concatenated. BI posterior probabilities (≥ 0.90) and MP bootstrap values (≥ 50%) indicated above the branches. ML bootstrap values (≥ 50%) indicated below the branches

S1-Fig. 3 Bayesian inference tree based on 999 bp of cytochrome b gene. BI posterior probabilities (≥ 0.90) and MP bootstrap values (≥ 50%) indicated above the branches. ML bootstrap values (≥ 50%) indicated below the branches

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