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Materials and methods

Taxon sampling, amplification and sequencing

We obtained sequence data of 22 taxa sampled across the passeriform radiation (Table 1). Three nuclear gene regions, myoglobin intron 2, ornithine decarboxylase (ODC) introns 6 to 7, and glyceraldehyde-3-phosphodehydrogenase (G3PDH) intron 11, have been sequenced and used to estimate the phylogenetic relationships. For each gene and taxon, multiple sequence fragments were obtained by sequencing with different primers. These sequences were assembled to complete sequences with SeqMan II (DNASTAR Inc.). Positions where the nucleotide could not be determined with certainty were coded with the appropriate IUPAC code. GenBank accession numbers are given in Table 1. See Irestedt et al. (2002), Allen and Omland (2003), and Fjeldså et al. (2003) for extractions, amplifications, and sequencing procedures for fresh tissue/blood samples. Corresponding lab procedures for study skins are detailed in Irestedt et al. (2006). In addition three new primers, Myo-eup157H (5’ – TGA GCC TTT TCT GTG CCT CCT GCT – 3´), Myo-eup309L (5’ – CAT AAG GAC TGT CAG TGA CTG GA – 3´), and Myo-eup549H (5’ – GAG ACA GTG AGG TCT AGT GTG TA – 3´) were designed to be able to amplify myoglobin intron 2 from the study skin extracts from Eupetes and Pachycephalopsis.

We were able to sequence all three gene regions almost completely for all included taxa (Prunella lacks 70 base pairs in the 3´ end in myoglobin, and in ODC all sequences obtained from study skins lack a short 22 bp fragment in exon 7). With the missing base pairs taken into account the sequences obtained varied in length between 708-729 bp for the myoglobin intron 2, between 253-307 bp for the G3PDH intron 11, and between 591-621 bp for the ODC region.

Most indels observed in the introns were autapomorphic and mainly found in certain variable regions. Some indels vary in length between taxa, which makes it difficult to know if these indels are homologous or represent independent evolutionary events. Several apparently synapomorphic indels were also observed when mapping the data onto the tree topology obtained from the Bayesian analyses of the combined data set. A few indels were also found to be incongruent with the phylogenetic tree obtained from the analysis of the combined data set. These were generally found in the most variable regions and some of the single base pair insertions actually consist of different bases. The combined alignment consists of 1742 bp. For more details of indel length and positions see the alignments of the individual gene regions deposited in GenBank.

We also sequenced a short stretch of exon 3 of the nuclear c-myc gene to investigate whether Eupetes or Pachycephalopsis possesses the insertion of one codon which has been proposed to be diagnostic of Passerida (Ericson et al. 2000, Ericson & Johansson 2003). Two new primers were designed both for the amplification and sequencing of this portion of the c-myc gene: cmyc-intF1 (5´– TGA ATA TGA ATC CAG CAC AGA GT – 3´), cmyc-intR1 (5´– AAC CTT AGC CTT TTT GCA GCT GGG TA – 3´).

Phylogenetic inference and model selection

Due to the rather low number of insertions in the introns, the combined sequences could easily be aligned by eye. All gaps have been treated as missing data in the analyses. Bayesian inference (see, e.g., Holder & Lewis 2003; Huelsenbeck et al. 2001) was used to estimate the phylogenetic relationships. The models for nucleotide substitutions used in the analyses were selected for each gene individually by applying the Akaike Information Criterion (AIC, Akaike 1973) and the program MrModeltest 2.2 (Nylander 2005) in conjunction with PAUP* (Swofford 1998).

Posterior probabilities of trees and parameters in the substitution models were approximated with MCMC and Metropolis coupling using the program MrBayes 3.1.1 (Ronquist & Huelsenbeck 2003). Analyses were performed for both the individual gene partitions and the combined data set. In the analysis of the combined data set, the models selected for the individual gene partition were used, but the topology was constrained to be the same. The chains for the individual gene partitions and for the combined data set were all run for 10 million generations, with trees sampled every 100th generations. The trees sampled during the burn-in phase (i.e., before the chain had reached its apparent target distribution) were discarded after checking for convergence and the final inference was made from the concatenated outputs.

The priori selection of nucleotide substitution models suggested that the GTR + Γ model had the best fit for all three gene regions, but as the nucleotide state frequencies and gamma distribution differed between the partitions we still applied a partitioned analysis of the combined data set. After discarding the burn-in phase the inference for the individual genes and the combined data set were based on a total of 95,000 samples each. The posterior distribution of topologies is presented as a majority-rule consensus tree from the combined analysis in Figure 1.

The trees obtained from the Bayesian analyses of the individual genes (not shown) are overall topologically congruent, and all gene trees support a monophyletic origin of the Eupetes, Chaetops and Picathartes clade. In fact there are no topological conflicts that are supported by posterior probabilities above 0.95, and the combined tree is also in good topological agreement with other molecular studies of major relationships among oscine passerines.

Parsimony and maximum-likelihood analyses were also performed for the combined data set using Paup* (Swofford, 1998). Searches for the most parsimonious trees were performed under the heuristic search option with ten random additions of taxa, and bootstrap support values were calculated from 1000 replicates. The maximum-likelihood analysis of the combined dataset was performed with the TVM+G model selected by AIC for the combined data set by MrModeltest. The trees obtained from the parsiomony and maximum-likelihood analyses, respectively, were both almost totally congruent with the MrBayes tree, and all nodes with posterior probability support values above 0.95 in the MrBayes tree received bootstrap support values above 80 in the parsimony analysis.



Estimation of divergence dates

Fjeldså et al. (online) calculated that the mean rate of divergence in myoglobin in passerines is 0.145% Ma-1. We used this rate to estimate the timing of the split between the EupetesChaetopsPicathartes-Eopsaltria-Pachycephalopsis clade and the rest of Passerida. The mean of the 25 pairwise observations is 8.07%, which corresponds to 55.6 Ma. We also calculated the timings of the splits within the EupetesChaetopsPicathartes clade. Picatarthes was the first to separate from the others at 44.25 Ma, while Eupetes and Chaetops split ca 38.4 Ma.


References

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insertions in the nuclear gene c-myc: A novel gene in avian phylogenetics. Auk 117, 1077-1086.



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nuclear and mitochondrial sequence data. Mol. Phyl. Evol. 29, 126-138.



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Table 1 Taxon names (family/subfamily names follow Sibley and Monroe, 1990), samples used in the study. Acronyms are PFI, Percy FitzPatrick Institute, Cape Town; ZMUC Zoological Museum of Copenhagen; AM, Australian Museum, Sydney; MV, Museum Victoria, Melbourne; NHMT, Natural History Museum, Tring; NRM, Swedish museum of Natural History; LSU, Louisiana State University.


species

Family/subfamily

Source

G3P

ODC

Myo

Chaetops frenatus

Picathartidae

PFI uncat.

XXXXX

XXXXX

AY228289

Colluricincla sanghirensis

Pachycephalinae

ZMUC123921

XXXXX

XXXXX

XXXXX

Corcorax melanorhamphos

Corcoracinae

AM LAB 1059

XXXXX

XXXXX

AY064737

Cormobates placens

Climacteridae

MV E309

XXXXX

XXXXX

AY064731

Eopsaltria australis

Petroicidae

MV 1390

XXXXX

XXXXX

AY064732

Eupetes macrocerus

Cinclosomatinae

NHMT 1936.4.12.58

XXXXX

XXXXX

XXXXX

Hirundo rustica

Hirundinidae

NRM 976238

XXXXX

XXXXX

AY064258

Malurus amabalis

Maluridae

MV C803

XXXXX

XXXXX

AY064729

Menura novaehollandiae

Menuridae

MV F722

XXXXX

XXXXX

AY064744

Oriolus flavocinctus

Corvinae

MV1603

XXXXX

XXXXX

XXXXX

Orthonyx temminckii

Orthonychidae

MV B831

XXXXX

XXXXX

AY064728

Pachycephala albiventris

Pachycephalinae

ZMUC117176

XXXXX

XXXXX

XXXXX

Pachycephalopsis hattamensis

Petroicidae

NRM552153

XXXXX

XXXXX

XXXXX

Picathartes gymnocephalus

Picathartidae

LSU B-19213

XXXXX

XXXXX

AY228314

Pomatostomus temporalis

Pomatostomidae

MV D257

XXXXX

XXXXX

AY064730

Prunella modularis

Prunellinae

NRM976138

XXXXX

XXXXX

AY228318

Ptilonorhynchus violaceus

Ptilonorhynchidae

MV B836

XXXXX

XXXXX

AY064742

Ptiloprora plumbea

Meliphagidae

MV C173

XXXXX

XXXXX

AY064736

Ptilorrhoa leucosticte

Cinclosomatinae

NRM 84405

XXXXX

XXXXX

XXXXX

Saltator atricollis

Emberizinae

NRM 966978

XXXXX

XXXXX

AY228320

Sturnus vulgaris

Sturnidae

NRM966615

XXXXX

XXXXX

AY228322

Sylvia atricapilla

Sylviidae

NRM 976380

XXXXX

XXXXX

AY228323


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