1
Supplementary Information
Below are additional methods, results, and discussion that further document the details
of our analyses and interpretations. Included are three tables: samples included in the
study, special primers designed for amplification and sequencing from museum study
skins, and details of the weighted ancestral area analysis.
Supplementary Methods
Taxon sampling:
Our analysis omitted genera previously considered monarchs, such as Elminia
and Erythrocercus, but which recently were shown to belong to other clades of birds far
removed from the monarchs (Pasquet et al. 2002, Beresford et al. 2005). We lacked
samples of the monotypic genus Lamprolia, endemic to Fiji. Relationships of this taxon
are uncertain, but all possible placements within the Monarchinae would not change the
interpretation of our results. All continental genera of Monarchs were included in our
sampling.
Taxon sampling for the ingroup was directed by results from a previous study of
the relationships of the genus Monarcha, which sampled broadly across the pied and
chestnut-bellied clades (Filardi and Smith in press). We included representatives from
all major sub-clades within Monarcha. Within some of the endemic Pacific island
genera we sampled only a single taxon. Although much denser sampling will be
required to reveal finer details of biogeographic history within archipelagos, these
genera constitute geographically and morphologically cohesive units. Cibois et al.
(2004) sampled all extant and recently extinct taxa of Pomarea, and found strong
support for monophyly of the genus. Clytorhynchus is the most geographically variable
2
taxon within the island monarchs. We sampled two of the four named species, and they
were recovered as sister taxa. Given these results and the relative lack of geographic
variation in other island monarch genera, radical departures from current generic
assignments, of the magnitude that could cause new interpretations of our results, are
extremely unlikely.
Molecular clock analysis:
As is the case for most avian groups, fossil and geological calibration points are
unavailable for monarch flycatchers. Several avian mitochondrial DNA divergence
rates have been published and they generally cluster around 2% per million years, but
the vast majority of these calibrations are not applicable to the current data because they
derive from distant taxa (Shields & Wilson 1987) or different genes (Fleischer et al.
1998). Furthermore, recent studies have cast considerable doubt on the reality of the
2% per million year rate (e.g., Arbogast & Slowinski 1998, Garcia-Moreno 2004).
Analyses using data from recent speciation events, or otherwise corrected to account for
saturation effects, have tended to yield much higher divergence rates for mitochondrial
DNA. These studies underscore the importance of using a rate derived from the same
gene from closely related taxa, under similar evolutionary circumstances, and using
appropriate models to account for multiple superimposed substitutions. A rate recently
published for ND2 in birds fulfils all of these requirements for our study taxa. The
divergence rate was calculated for the ND2 gene in other passerine birds and used island
ages as calibration points (Drovetski et al. 2004). Not only does the use of island ages
provide reliable maximum age estimates, it also presents similar evolutionary
circumstances to those that produced our data set, including small effective population
3
sizes and potential founder effects. To produce a range of estimates, we also employed
the widely used avian mtDNA rate of 2% per million years (see Garcia-Moreno 2004)
as a minimum estimate and the ND2 specific rate (Drovetski et al. 2004) as a maximum.
Supplementary Results
The combined data set contained 1717 bp, of which 454 were parsimony informative
(61 Myo, 393 ND2). Base composition in the ND2 data was biased (A = 0.30, C =
0.33, G = 0.12, T = 0.25) but homogeneous across taxa (p = 0.99) and typical of this
gene in other birds (e.g., Moyle 2005). Base composition in the intron data was less
biased (A = 0.27, C = 0.23, G = 0.24, T = 0.26), and homogeneous across taxa (p = 1.0).
The partition homogeneity test did not indicate incongruence between the two genes (p
= 0.24). For the combined data, parsimony analysis (results not shown) recovered the
same topology as model-based methods. Phylogenetic analysis of each gene separately
(Supplemental Figure 2) produced differences in topology, but no differences received
high support (0.95 bayesian posterior probability). For example, the ND2 data alone
placed Chasiempis sandwichensis basally in the clade containing Metabolus and the
island Monarcha species, rather than the clade containing the endemic island genera,
but this placement received low support (posterior probability = 0.56).
4
Supplementary Table 1. Samples included in the study, including source, voucher
numerber, and GenBank accession numers.
Name
Monarcha manadensis
Monarcha guttula
Monarcha castaneiventris
Monarcha chrysomela
Monarcha frater
Monarcha melanopsis
Monarcha cinerascens
Monarcha barbatus
Monarcha axillaris
Monarcha leucotis
Monarcha takatsukasae*
Monarcha godefroyi*
Monarcha mundus*
Monarcha rubiensis*
Mayrornis lessoni*
Metabolus rugensis*
Clytorhynchus hamlini
C. pachycephaloides
Hypothymis azurea
Chasiempis
sandwichensis
Neolalage banksiana
Machaerirynchus
flaviventer
Arses telescopthalmus
Pomarea iphis
Myiagra alecto
Rhipidura rufifrons
Grallina cyanoleuca
Terpsiphone viridis
Locality
Papua New Guinea
Papua New Guinea
Solomon Islands
Papua New Guinea
Papua New Guinea
Queensland, Australia
Solomon Islands
Solomon Islands
Papua New Guinea
Queensland, Australia
Tinian Island
Yap Island
Lesser Sundas
Papua New Guinea
Fiji
Truk Island
Rennell Island
Vanuatu
Indonesia
Oahu
Sourcea
UWBM
UWBM
UWBM
KU
VM
UWBM
UWBM
UWBM
KU
VM
AMNH
AMNH
AMNH
AMNH
AMNH
AMNH
UWBM
LSUMNS
UWBM
UHH
Voucher
67928
67904
63168
AM1021
Eo34
62890
60238
58767
AM838
C526
332278
348863
655386
835580
251270
608465
69806
B45762
67496
Mohi1
ND2
DQ084090
DQ084084
DQ084085
DQ084081
DQ084082
DQ084083
DQ084087
DQ084086
DQ084094
DQ084093
DQ084089
DQ084088
DQ084091
DQ084092
DQ084098
DQ084095
DQ084079
DQ084080
DQ084075
DQ084096
Myo
DQ084115
DQ084111
DQ084112
DQ084108
DQ084109
DQ084110
DQ084114
DQ084113
DQ084117
DQ084116
DQ084106
DQ084107
DQ084102
DQ084118
Vanuatu
Papua New Guinea
LSUMNS
KU
b45765
AM949
DQ084076
DQ084072
DQ084103
DQ084099
Papua New Guinea
Marquesas
Papua New Guinea
Solomon Islands
Australia
KU
MNHN
UWBM
UWBM
AMNH
Genbank
AM910
D42
68058
CEF239
LAB1144
DQ084077
DQ084097
DQ084078
DQ084073
DQ084074
AF407058
DQ084104
DQ084119
DQ084105
DQ084100
DQ084101
AY529939
* denotes taxa for which DNA was extracted from a museum study skin.
a
Abbreviations of sample sources: American Museum of Natural History (AMNH),
University of Washington Burke Museum (UWBM), University of Kansas Museum of
Natural History (KU), Museum of Victoria, Australia (VM), University of Michigan
Museum of Zoology (UMMZ), Louisiana State University Museum of Natural Science
(LSUMNS), Field Museum of Natural History (FMNH), University of Hawaii,
Honolulu (UHH), Muséum national d'Histoire naturelle, Paris (MNHN). Thanks to
Andy Mack (KU), Les Christidis (VM), Jean-Claude Thibault and Société d'Orthologie
de la Polynésie (MNHN), Sarah Burgess (UHH), and Andy Kratter (Florida Museum of
Natural History and LSUMNS).
5
Supplementary Table 2. Primers for amplification and sequencing of ND2 and
Myoglobin intron-2.
Primer name
ND2:
L5215
MonND2-150L
MonND2-350L
ND2-500L
MonND2-572L
MonND2-776L
MonND2-900L
ND2-160H
MonND2-385H
ND2-H5766
MonND2-590H
MonND2-780H
MonND2-925H
H6313
Myoglobin intron-2:
Myo2
Myo3f
Sequence (5’ – 3’)
Source
TATCGGGCCCATACCCCGAAAAT
TAATCTCAAAATCTCACCACCC
CCATTCCAYTTYTGATTYCCAG
GAATAGGACTAAACCAAACAC
TCACAYCTRGGYTGAATGGC
GCCGGCCTGCCCCCCYTRACAGG
GCATACTGCGCAACAATCAC
TTGGTTGCMGCTTCAATGGC
TTTTATGGCTGTTGANAGGTG
GGATGAGAAGGCTAGGATTTTKCG
TTRAGTARAGTRAGTTTTGGG
ATAATGAGTCACTTYGGGAGG
CATTGTTTTATGTGGTTTGT
CTCTTATTTAAGGCTTTGAAGGC
Hackett 1996
this study
this study
AMNH lab primer
this study
this study
this study
AMNH lab primer
this study
Johnson and Sorenson 1998
this study
this study
this study
Johnson and Sorenson 1998
GCCACCAAGCACAAGATCCC
GCAAGGACCTTGATAATGACTT
Heslewood et al. 1998
Slade et al. 1993
6
Supplementary Table 3. Divergence times and ancestral areas for nodes of interest.
Agea
Node
Grallina/M. axillaris
Neolalage/M. axillaris
M. guttula/M. axillaris
Neolalage/M. chrysomela
Neolalage/Metabolus
M. frater/Metabolus
M. frater/M. cinerascens
M. frater/M. castaneiventris
M. frater/M. melanopsis
Metabolus/M. takatsukae
Metabolus/M. godefroyi
Clytorhynchus/Chasiempis
Clytorhynchus/Pomarea
Clytorhynchus/Neolalage
Clytorhynchus/Mayrornis
Clytorhynchus/Clytorhynchus
4.98-13.74
3.86-10.65
2.83-8.08
3.01-8.32
2.05-5.65
1.15-2.98
0.66-1.82
0.55-1.53
0.38-1.05
1.08-3.19
1.01-2.79
1.85-5.65
1.79-4.94
1.54-4.26
1.13-3.12
1.01-2.80
Ancestral
stateb
C (0.53)
C (0.53)
C (0.58)
I (0.53)
I (0.96)
I (0.97)
I (0.98)
I (0.96)
C (0.87)
I (0.97)
I (0.97)
I (0.95)
I (0.95)
I (0.95)
I (0.96)
I (0.96)
WAAA (GSW/LSW = PI)c
Island
Continent
0.66/1.80 = 0.37
2.20/0.75 = 2.93
1.00/1.17 = 0.85
1.83/1.00 = 1.83
1.00/1.00 = 1.00
1.50/0.50 = 3.00
1.20/1.20 = 1.00
1.33/1.20 = 1.11
2.00/0.25 = 8.00
0.75/1.75 = 0.43
2.33/0.33 =7.06
0.33/2.33 = 0.14
1.50/0.50 = 3.00
0.50/1.50 = 0.33
1.00/1.00 = 1.00
1.00/1.00 = 1.00
0.00/2.00 = -2.00/0.00 = ∞
2.00/0.00 = ∞
0.00/2.00 = -2.00/0.00 = ∞
0.00/2.00 = -2.00/0.00 = ∞
0.00/2.00 = -2.00/0.00 = ∞
0.00/2.00 = -2.00/0.00 = ∞
0.00/2.00 = -2.00/0.00 = ∞
0.00/2.00 = -2.00/0.00 = ∞
0.00/2.00 = --
Ancestral
aread
Continent
Continent
Continent
Equivocal
Island
Island
Island
Equivocal
Continent
Island
Island
Island
Island
Island
Island
Island
a
Node age estimates in millions of years. The minimum age is calculated with an ND2
specific rate from Drovetski et al. (2004). The maximum rate is a “universal” avian
mitochondrial rate of 2% per million years (see Garcia-Moreno 2004 for discussion).
b
Ancestral state reconstruction using Discrete (Pagel 1994). Inferred states (C =
continental, I = island), and probability of that ancestral state in parenthesis.
c
Weighted ancestral area analysis (WAAA, Hausdorf 1998). Abbreviations: weighted
gain steps (GSW), weighted loss steps (LSW), probability index (PI).
d
Ancestral area inferred from WAAA.
7
Supplementary References
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tree of songbirds (Passeri): molecular systematics of several evolutionary ‘enigmas’.
Proc. R. Soc. Lond. B 272, 1471-2954 (2005).
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(Pomarea): an endemic genus close to extinction. Condor 106, 837-851 (2004).
Drovetski, S.V., et al. Complex biogeographic history of a Holarctic passerine. Proc. R.
Soc. Lond. B 271, 545-551 (2004).
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Monarcha) with emphasis on Solomon endemics. Mol. Phylogenet. Evol. XX, XX-XX
(2005).
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8
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10
Supplementary Figure 1. Maximum likelihood phylogeny of Pacific monarchs
and selected outgroups based on ND2 and Myoglobin intron-2 data. Crosses
(†) indicate taxa for which DNA was extracted from museum study skins and are
represented by mtDNA only. Numbers by nodes indicate Bayesian posterior
probability/maximum likelihood bootstrap proportion. Asterisks (*) indicate
posterior probability < 0.5 or bootstrap support < 50%.
Supplementary Figure 2. Bayesian phylogeny of Pacific monarchs and selected
outgroups based on A) ND2 and B) Myoglobin intron-2 data analyzed
separately. Crosses (†) indicate taxa for which DNA was extracted from
museum study skins and are represented by mtDNA only. Numbers by nodes
indicate Bayesian posterior probability.