ELECTRONIC SUPPLEMENT:
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 Eupetes–Chaetops–
Picathartes-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 Eupetes–Chaetops–Picathartes clade. Picatarthes was the first to separate from
the others at 44.25 Ma, while Eupetes and Chaetops split ca 38.4 Ma.
References
Akaike, H. 1973 Information theory as an extension of the maximum likelihood principle. In
Second International Symposium of Information Theory B. (eds. N. Petrov & F.
Csaki), pp. 267-281. Budapest: Akademiai Kiado.
Allen, E. S. & Omland, K. E. 2003 Novel intron phylogeny (ODC) supports plumage
convergence in orioles (Icterus). Auk 120, 961-969.
Barker, F.K., Cibois, A., Schikler, P.A., Feinstein, J. & Cracraft, J. 2004. Phylogeny and
diversification of the largest avian radiation. Proc. Natl. Acad. Sci. USA 101, 1104011045.
Ericson, P.G.P., Johansson, U.S. & Parsons, T.J. 2000 Major divisions of oscines revealed by
insertions in the nuclear gene c-myc: A novel gene in avian phylogenetics. Auk 117,
1077-1086.
Ericson, P.G.P. & Johansson, U.S. 2003 Phylogeny of Passerida (Aves: Passeriformes) based on
nuclear and mitochondrial sequence data. Mol. Phyl. Evol. 29, 126-138.
Fjeldså, J., Zuccon, D., Irestedt, M., Johansson, U.S. & Ericson, P.G.P 2003 Sapayoa aenigma:
a New World representative of 'Old World suboscines'. Proc. R. Soc. B (Suppl.).
Fjeldså, J., Irestedt, M., Jønsson, K.A., Ohlson, J. I. Ericson, P.G.P. (online) Phylogeny of the
ovenbird genus Upucerthia: a case of independent adaptations for terrestrial life.
Zoologica Scripta.
Holder, M. & Lewis, P. O. 2003 Phylogeny estimation: Traditional and Bayesian approaches.
Nature Genetics 4, 275-284.
Huelsenbeck, J.P., Ronquist, F. & Hall, B. 2001 MRBAYES: Bayesian inference of phylogeny.
Bioinformatics 17, 754-755.
Irestedt, M., Fjeldså, J., Johansson, U.S. & Ericson, P.G.P. 2002 Systematic relationships and
biogeography of the tracheophone suboscines (Aves: Passeriformes). Mol. Phyl. Evol.
23, 499-512.
Irestedt, M., Ohlson, J.I., Zuccon, D., Källersjö, M. & Ericson, P.G.P. 2006 Nuclear DNA from
old collections of avian study skins reveals the evolutionary history of the Old World
suboscines (Aves, Passeriformes). Zoologica Scripta 35, 567-580.
Nylander, J. A. A. 2005 MrModeltest v.2.2. [Program distributed by the author]. Uppsala
University, Uppsala: Department of Systematic Zoology.
Peters J. L. 1940 A genus for Eupetes caerulescens Temminck. Auk 57, 94
Ronquist, F. & Huelsenbeck, J. P. 2003 MRBAYES 3: Bayesian phylogenetic inference under
mixed models. Bioinformatics 19, 1572-1574.
Swofford, D.L. 1998 Paup*. Phylogenetic analysis using parsimony (* and other methods), v.4.
Sunderland, MA: Sinauer.
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
Colluricincla sanghirensis
Corcorax melanorhamphos
Cormobates placens
Eopsaltria australis
Eupetes macrocerus
Hirundo rustica
Malurus amabalis
Menura novaehollandiae
Oriolus flavocinctus
Orthonyx temminckii
Pachycephala albiventris
Pachycephalopsis hattamensis
Picathartes gymnocephalus
Pomatostomus temporalis
Prunella modularis
Ptilonorhynchus violaceus
Ptiloprora plumbea
Ptilorrhoa leucosticte
Saltator atricollis
Sturnus vulgaris
Sylvia atricapilla
Picathartidae
Pachycephalinae
Corcoracinae
Climacteridae
Petroicidae
Cinclosomatinae
Hirundinidae
Maluridae
Menuridae
Corvinae
Orthonychidae
Pachycephalinae
Petroicidae
Picathartidae
Pomatostomidae
Prunellinae
Ptilonorhynchidae
Meliphagidae
Cinclosomatinae
Emberizinae
Sturnidae
Sylviidae
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AY228289
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AY064737
AY064731
AY064732
XXXXX
AY064258
AY064729
AY064744
XXXXX
AY064728
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AY228314
AY064730
AY228318
AY064742
AY064736
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AY228320
AY228322
AY228323
PFI uncat.
ZMUC123921
AM LAB 1059
MV E309
MV 1390
NHMT 1936.4.12.58
NRM 976238
MV C803
MV F722
MV1603
MV B831
ZMUC117176
NRM552153
LSU B-19213
MV D257
NRM976138
MV B836
MV C173
NRM 84405
NRM 966978
NRM966615
NRM 976380