Vicariant Speciation of Curassows (Aves, Cracidae): A Hypothesis Based on Mitochondrial DNA

Phylogeny
Author(s): Sérgio Luiz Pereira and Allan J. Baker
Source: The Auk, Vol. 121, No. 3 (Jul., 2004), pp. 682-694
Published by: American Ornithologists' Union
Stable URL: http://www.jstor.org/stable/4090306
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The Auk 121(3):682-694, 2004

VICARIANT SPECIATION OF CURASSOWS (AVES, CRACIDAE):

A HYPOTHESIS BASED ON MITOCHONDRIAL DNA PHYLOGENY
SERGIO Luiz PEREIRA1AND ALLAN J. BAKER
Centrefor Biodiversity and Conservation Biology, Royal Ontario Museum, 100 Queen's Park, Toronto,
Ontario M5S 2C6, Canada;and
Department of Zoology, University of Toronto,Toronto,Ontario M5S lAl, Canada

ABSTRACT. -The curassows comprise 14 species of sedentary Neotropical birds classified in four
genera (Crax, Nothocrax, Mitu, and Pauxi) in the family Cracidae. Congeneric species have a
striking pattern of allopatric distributions that might be attributable to vicariance, dispersal, or a
combination of the two. To test those biogeographic hypotheses, a strongly supported phylogeny
was needed, so that existing problems of taxonomic rank could be solved and a better
understanding of the group's evolutionary history attained. We therefore estimated the
phylogenetic relationships of all 14 species, on the basis of 6,929 sites of six different mito-chondrial
DNA regions, and reassessed the status of the four genera. Sequences from the ND4 gene favored a
tree that was highly incongruent with the tree recovered using the other five gene regions.
However, when the ND4 sequences were concatenated with the sequences of the other genes, the
optimal phylogeny was unchanged from that derived for the other genes. That combined tree was
divided into two well-supported clades: one containing the seven species of Crax and the other
containing the monospecific genus Nothocrax, as sister to a clade of the Mitu and Pauxi species.
Mitu and Pauxi are not reciprocally monophyletic, which appears to be attributable to a distant
hybridization event and a transfer of Mitu mtDNA into P. unicor-
nis. We estimated divergence times; the diversification of curassow seems to have occurred
from the Middle Miocene to the end of the Pliocene (9.5 to 1.6 Ma). Vicariance-following
marine transgressions, the rise of the Andes, and subsequent changes in river basins in South
America-seems to be the major mode of isolation that favored allopatric speciation in the
group. Received20 January 2003, accepted23 February2004.

RESUMEN.-El grupo de los pavones o paujiles incluye 14 especies de aves neotropicales se-
dentarias clasificadas en cuatro generos (Crax, Nothocrax, Mitu y Pauxi) en la familia Cracidae.
Las especies cogenericas presentan un llamativo patron de distribuciones alopAtricas, que
podria ser atribuido a vicarianza, dispersion o a una combinaci6n de los dos procesos. Para
poner a prueba estas hip6tesis biogeograficas, resultaba necesario contar con una filogenia
fuertemente respaldada de modo que los problemas de rango taxon6mico existentes pudieran
resolverse y se alcanzara un mejor entendimiento de la historia evolutiva del grupo. Por lo
tanto, estimamos las relaciones filogeneticas entre las 14 especies con base en 6,929 posiciones
nucleotidicas de seis regiones mitocondriales diferentes, y revaluamos el estatus de los cuatro
generos. Las secuencias del gen ND4 sugirieron un arbol que fue sustancialmente incongruente con
el Arbol recobrado usando las otras cinco regiones. Sin embargo, cuando las secuencias de ND4 se
concatenaron con las de los demas genes, la filogenia 6ptima no difiri6 de la derivada a partir de
estos. Este arbol combinado estuvo dividido en dos clados con buen respaldo, uno incluyendo las
siete especies de Crax y el otro incluyendo al genero monotipico Nothocrax como
hermano de un clado compuesto por las especies de Mitu y Pauxi. Mitu y Pauxi no son grupos
reciprocamente monofileticos, lo que parece ser atribuible a un evento distante de hibridaci6n
y a la transferencia de ADNmt de Mitu a P. unicornis. Tambien estimamos tiempos de diver-
gencia; la diversificaci6n de los pavones parece haber sucedido desde el Mioceno medio hasta el
final del Plioceno (9.5 a 1.6 millones de afios atras). Los procesos de vicarianza causados por
transgresiones marinas, el levantamiento de los Andes y los cambios subsecuentes en las cuencas
de los rios de Sur America parecen representar el principal modo de aislamiento que favoreci6 la
especiaci6n alopAtrica en el grupo.

IE-mail: sergio.pereira@utoronto.ca

682
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July 2004] Curassow Phylogeny and Biogeography
BIOLOGISTS HAVE LONG debated the relative
importance of dispersal and vicariance in ex-
plaining disjunct geographic distributions and
speciation of organisms (Zink et al. 2000, Crisci
2001). Proponents of dispersal (e.g. Udvardy
1969) hypothesize that new species originated
after individuals of an ancestral population
crossed geographic barriers and colonized an area
isolated from the original population. Proponents
of vicariance (e.g. Nelson and Platnick 1981)
argue that speciation commonly occurred when a
continuous ancestral popula-tion was fragmented
by a geographic event, such as uplift of a
mountain chain or change in a river's course;
provided that gene flow was restricted between
ancestral and derived populations, the latter
accumulated genetic incompatabilities and
eventually became new species. Vicariance and
dispersal are not mutu-ally exclusive propositions,
however, and both are likely to have occurred in
the historical biogeography of major clades of
organisms (e.g. Ronquist 1997).
Availability of DNA sequences has provided a
new opportunity to test the roles of dispersal and
vicariance in biological diversification; sequences
not only can be used to construct hy-potheses of
phylogenetic relationships but also allow
estimation of approximate times of diver-gence of
taxa. If allopatric taxa are older than the barrier
that separates them, vicariance is the most
parsimonious explanation for their diver-gence; if
the barrier is older, dispersal is most
parsimonious. Although phylogenies of mul-tiple
lineages can be useful in checking whether
patterns of distribution correspond with major
geographic barriers (Cracraft and Prum 1988,
Prum 1988, Zink et al. 2000, Hovenkamp 2001),
that comparative approach is presently limited by
availability of well-supported phylogenies and
estimates of times of divergence.
Because of their power of flight, dispersal has
often been assumed to play a large role in
speciation of birds, but many examples of
vicariance have also been documented. For
example, Voelker (2002) showed that dispersal
played a strong role in speciation of wagtails
(Motacilla), a group of migratory birds; whereas
Zink et al. (2000), in their comparative analy-sis of
nonmigratory arid-land birds of North America,
concluded that vicariance likely had a major role
in speciation of those birds and that dispersal
needed to be invoked in only a
683
few instances. Comparing vertebrate groups in
South America, Ron (2000) showed that vi-
cariance generally explained speciation in those
groups. However, the group chosen by Voelker
(2002), most of the examples cited by Zink et al.
(2001), and some of those compiled by Ron (2000)
are of passerine birds with putatively higher
dispersal power than nonpasserines. A study
group of relatively sedentary birds, such as the
curassows, should provide some insight on how
important dispersal is to the evolution-ary history
of less-mobile birds.
Curassows are large, heavy-bodied galli-form
birds of the family Cracidae, distributed in the
rainforests and highland forests of the
Neotropical region, where they are generally as-
sociated with rivers, lakes, and other water bod-
ies (del Hoyo 1994). They are usually crested and
have yellow, red, or blue head ornaments, such as
wattles, tubercules, or caruncules (Delacour and
Amadon 1973). Their flight capabilities are
moderate, basically consisting of the ability to fly
away when threatened. Long-distance dispersal is
not known in curassows, though latitudinal
movements related to seasonal changes may oc-
cur in the Helmeted Curassow and in the guans
(del Hoyo 1994).
As a general rule in curassows, congeneric
species have allopatric distributions bounded by
rivers and mountain chains that form major
barriers between them (Fig. 1). Because curra-
sows are mainly sedentary birds, vicariance
attributable to changes in the geological land-
scape of South America may have had a great role
in their speciation (Delacour and Amadon 1973).
Generic limits in curassows have long been
questioned (e.g. Vaurie 1968, Delacour and
Amadon 1973), and a more recent classification
recognizes four genera: Crax, Mitu, Pauxi, and
Nothocrax (del Hoyo 1994). Status of some spe-
cies and subspecies is uncertain (Nardelli 1993, del
Hoyo 1994 and references therein), partly because
some of them appear to represent hy-brids
between different species (Joseph et al. 1999).
Although Vaurie (1968) and Delacour and
Amadon (1973) commented on possible
relationships among cracid genera, only Pereira et
al. (2002) have presented a hypothesis based on
modern phylogenetic methods. The only
phylogenetic hypothesis proposed for the cu-
rassows was based on behavioral and ecological
characters for some species within Crax (Garcia
and Brooks 1997). The goal here is to provide a
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684 PEREIRA AND BAKER [Auk, Vol. 121

Chaco Chachalaca and three guans (Table 1). Using

alberti daubentoni standard protocols, we extracted DNA from blood
samples (Sambrook et al. 1989). Blood and DNA samples
alector are deposited at the Laborat6rio de Genetica e Evolucao
Molecular de Aves do Departamento de Biologia da
rubraJ Universidade de Sao Paulo, Brazil (except for samples
from Mitu salvini, which are deposited at the Royal
Ontario Museum, Canada). All samples are from birds
globulosa kept in captivity (see Acknowledgments).
Because cracids can easily be hybridized in captiv-ity,
caution was taken to avoid inclusion of possible hybrids;
fasciolata we interviewed keepers and used only birds said to have
been bred from pure parentage.
Polymerase chain reaction amplifications and
blumenbachDl sequencing.-We used the protocol described in
Hagelberg et al. (1994) for polymerase chain reac-tion
(PCR) amplifications. Buffer solution contained 10 mM
pauxi tomentosa Tris-HCl, pH 8.3, 50 mM KCl, 2.5 mM MgCl2, 0.01%
gelatin, 160 'g mL-UBSA. We recovered ampli-fied
salvini fragments from agarose gel by excising the band and
centrifuging it to retain agarose on a filtered tip that was
unicooris later discarded. We cycle-sequenced recov-ered DNA
samples and obtained sequences on a Li-Cor 4200 bi-
directional automated DNA sequencer, according to
tuberosa mltu protocol suggested by the manufacturer. Mitochondrial
gene regions sequenced were ND2-tRNA Trp, ND4-
tRNA His, ND5, ATP8-ATP6-CO3, CO1, and
cytochrome (cyt) b. Except for cyt b, primers used were
designed by Oliver Haddrath: MetL-AAG CTA TCG
GGC CCA TAC CCG, AsnH-GAT CRA GGC CCA
urumutum TCT GTC TAG, L11474-AAC CTA AAC CTN CTA
CAA TGC TAA A, ND4SerH-GAT GCA GGA RTT
AGC AGT TCT TG, L14105-GCC TTC TCY ACA TCN
AGY CAA CTW GGN YTM AT, H14910-ATT TTN
AGY AGN GGG TGG GWT
TTT CG, LysL-CAG CAC TAG CCT TTT AAG CT,
FIG. 1. Approximate historical geographic range for COIIIRH-ATT ATT CCG TAT CGN AGN CCY TTT
the 14 species of curassows in tropical America, based TG, Ltyr-TGT AAA AAG GWC TAC AGC CTA ACG
on Delacour and Amadon (1973) and del Hoyo (1994). C, and H8205-GGT TCG ATT CCT TCC TTT CTT G.
Crax species are shown at the top, Mitu and Pauxi in For cyt b, we used primers bl-CCA TCC AAC ATC
the middle, and Nothocrax urumutum at the bottom. TCA GCA TGA TGA AA (Kocher et al. 1989) and b6-
Note the allopatricdistributionof congeneric GTC TTC AGT TTT TGG TTT ACA AGA C (T. Birt
species. pers. comm.).
Sequence alignment and base-composition hetero-
well-supported phylogeny of the 14 currently geneity-L- and H-strand sequences were checked for
recognized species of curassows, based on ambiguities in SEQUENCHER 4.1.2 for Macintosh
(GeneCodes Corporation, Ann Arbor, Michigan).
sequences of six mitochondrial DNA regions and
Sequences can be retrieved from GenBank (accession
modern methods of tree inference. We also
numbers AF165466-AF165470, AF165472-AF165474,
propose a time-frame for their taxonomic AF165490-AF165494,AF165496-AF165498,AY140740-
diversification and relate it to relevant geologi-cal AY140750, AY141900-AY141957, AY143680-AY143684,
(vicariant) events and subsequent dispersal across AY143686-AY143688). Alignments were performed by
barriers in South America. eye in MACCLADE 4.0 (Maddison and Maddison 2000).
The final concatenated alignment contained 6,929 sites.
METHODS Significant differences in base composi-tion among taxa
can seriously mislead tree construc-tion methods (Foster
Taxa.-We selected one representative each of the and Hickey 1999, Mooers and Holmes 2000, Haddrath
14 species of curassows and four outgroups: the and Baker 2001). Therefore,
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July 2004] Curassow Phylogeny and Biogeography 685

TABLE 1. Curassow species represented here, and geographic distribution based on Delacour and Amadon (1973)
and del Hoyo (1994). For outgroups, only countries are indicated.

Species Common name Distribution

Crax alberti Blue-knobbed Curassow Northern Colombia
C. alector Black Curassow Northern Brazil, eastern Colombia, Guyana, French
Guyana, Suriname, eastern Venezuela
C. blumenbachii Red-billed Curassow Southeastern Brazil
C. daubentoni Yellow-billed Curassow Eastern Colombia, northern Venezuela
C. fasciolata Bare-faced Curassow Northern Argentina, eastern Bolivia, Brazil, Paraguay
C. globulosa Wattled Curassow Northern Bolivia, Amazonian Brazil, southeastern
Colombia, eastern Ecuador, eastern Peru
C. rubra Great Curassow Central America, west of the Andes in Colombia and
Ecuador, southeastern Mexico
Mitu mitu Alagoas Curassow Northeastern Brazil
M. salvini Salvin's Curassow South-central Colombia, eastern Ecuador,
northeastern Peru
M. tomentosa Crestless Curassow Northwestern Brazil, eastern Colombia, northern
Guyana, Venezuela south of Orinoco River
M. tuberosa Razor-billed Curassow Bolivia, Brazil, southeastern Colombia, Ecuador, Peru
Nothocrax urumutum Nocturnal Curassow Western and central Amazonian Brazil, southeastern
Colombia, eastern Ecuador, northern Peru, southern
Venezuela
Pauxi pauxi Helmeted Curassow Eastern Colombia, north-central and western
Venezuela
P. unicornis Horned Curassow Eastern slope of Andes in central Bolivia and Peru
Ortalis canicollis Chaco Chachalaca Argentina, Bolivia, Brazil, Paraguay
Penelope obscura Dusky-legged Guan Argentina, Bolivia, Brazil, Uruguay
Aburria aburri Wattled Guan Colombia, Ecuador, Peru, Venezuela
Chamaepetesgoudotii Sickle-winged Guan Bolivia, Colombia, Ecuador, Peru

we tested heterogeneity in base composition using
TREEPUZZLE 5.0 (Strimmer and von Haeseler 1996),
excluding constant sites from analysis, because they
can hinder detection of compositional biases among
taxa (Foster and Hickey 1999).
Combinability analysis.-Before deciding whether
we should combine all mitochondrial gene sequences
for tree reconstruction, we performed phylogenetic
analysis for each region individually and compared
the topologies obtained in those analyses. All parti-
tions, except for ND4, recovered similar tree topolo-
gies (not shown). Analysis of ND4 alone resulted in
a tree in which none of the curassow genera were
monophyletic, as well as other topological changes
among well-supported clades obtained when all gene
regions were combined (hereafter the "congruent"
data set) (not shown). Therefore, we decided to per-
form subsequent analyses with and without the ND4
gene and check its influence on tree topology when
the conguent data set was used. A likelihood ratio
test (LRT) that accounts for statistical uncertainty in
estimating the resulting tree topologies from the two
combined data sets was then applied to determine
whether addition of ND4 significantly reduced the
likelihood of the tree.
Phylogenetic analysis. -We used 6,457 sites (or 5,128,
excluding the ND4 sequences) for phylogenetic recon-
struction; sites with alignment gaps and overlapping
bases between ND2 and tRNA Trp, ATPase 8 and
ATPase 6, and ATPase 6 and COIII were excluded from
all analyses. Tree searches were carried out in PAUP*
4.0 b8.0 (Swofford 2001), using the branch-and-bound
and heuristic algorithms under maximum parsimony
(MP) and maximum likelihood (ML) crite-ria,
respectively. Prior to ML analysis, a search for the best
model of DNA substitution for the data set was
performed with a hierarchical LRT and the Akaike's
Information Criterion (AIC) score in MODELTEST 3.0
(Posada and Crandall 1998). Parameters estimated from
MODELTEST were used as initial estimates to speed up
all ML searches. Bootstrap proportions were obtained in
MP and ML analyses using 1,000 and 100 replicates,
respectively, with random addition of taxa in each
replicate.
Bayesian analysis with Markov Chain Monte Carlo
(MCMC) sampling was performed with MRBAYES3.0
(Ronquist and Huelsenbeck 2003) using partitioned
likelihood, assuming six classes of DNA substitution,
and estimating proportion of invariable sites and shape
parameter of gamma distribution for each gene parti-
tion. Analysis was run for 3,000,000 generations, with
one cold and three heated chains and a burn-in time of
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686 PEREIRA
500,000 generations. One tree in every thousand was
sampled. The MCMC sampling was run three times
and recovered the same tree topology in each run.
Hypothesis testing using parametricbootstrapping-.We
performed a parametric bootstrap and an a posteriori
significance test (i.e. SOWH test; Huelsenbeck and Bull
1996, Swofford et al. 1996, Goldman et al. 2000) to test the
significance of the differences between MP and ML trees.
The MP tree was used as the model tree for parameter
estimation using PAUP* 4.0 b8.0, and 100 replicate data sets
were generated with SEQ-GEN 1.2.3 (Rambaut and Grassly
1997) to maintain uniform base composition among taxa.
Differences in log likelihoods of the ML tree and the
alternative tree were compared with the null distribution of
differences in log likeli-hood over the replicates for the two
trees.
Molecular dating of divergence time.-The assump-
tion of rate constancy of DNA substitution through
time within the 14 curassow species was checked
by an LRT of the likelihood of the "congruent" ML tree
estimated with and without the clock constraint.
Difference in likelihoods was compared with a chi-
square distribution, with the number of taxa minus 2 as
the degree of freedom to determine the significance of
the test. Estimates of divergence time and their 95%
confidence intervals were then obtained by the method
of Haubold and Wiehe (2001). In that method, it is
assumed that the number of nucleotide substitu-tions
and the substitution rate for a particular pair of taxa
may be different from any other pair, and they are
modeled according to a gamma distribution. The oldest
fossil record for cracids seems to represent stem groups
rather than crown cracids (Crowe and Short 1992),
which makes those data inappropriate for calibration
purposes for the curassows.
Alternatively, Pereira et al. (2002) estimated di-
vergence time for all cracid genera with a combined
approach using several nuclear and mitochondrial DNA
sequences. Their calibration point was based on the split
between Galliformes and Anseriformes at 85 mya, as
estimated by Haddrath and Baker (2001) on the basis of
complete mitochondrial DNA se-quences, and calibrated
with the fossil record of ratite birds. Haddrath and
Baker's estimate is in agreement with other independent
estimates using nuclear DNA sequences, and calibrated
with both fossil-record and molecular estimates of non-
avian taxa-90 mya as es-timated by van Tuinen and
Hedges (2001). Therefore, we assumed divergence of
Crax from the other cu-rassow genera at 10 mya
(Pereira et al. 2002) as our calibration point.
As a check on our molecular calibration, we as-
sumed that C. alberti, C. daubentoni, and C. rubra split
at 6 mya when the major uplift of the northern end of
the Andes in South America (Hooghiemstra and van
der Hammen 1998) occurred, creating the Andean
Cordilleras Occidental and Oriental, which serve as the
species' geographic barriers.
AND BAKER [Auk, Vol. 121
RESULTS
Sequences and base composition.-The PCR
amplifications resulted in a single product
of the expected size. The reading frame for
the protein-coding genes and the secondary
structure for the tRNAs were as expected for
functional mitochondrial genes and similar to
other corresponding avian sequences deposited
in GenBank. No signal of saturation of DNA
substitution was detected for the segments se-
quenced here (not shown).
Average base composition of the curassow
sequences (A = 28.8%; C = 34.6%; G = 12.3%; T =
24.3%) was typical for mitochondrial se-quences of
birds. Comparison of base composi-tion across
taxa indicated that the ingroup was homogeneous
for all genes sequenced in the present work.
Although the outgroups failed the stationarity test
for some genes, that did not seem to interfere with
ingroup topology during tree reconstruction.
Failed outgroup taxa and genes were: Aburria
aburri for ND4 (P = 0.028); Ortalis canicollis for
COI (P = 0.015); Chamaepetes goudotii for
ATPase6 (P = 0.037), ND2 (P = 0.047), ND4 (P =
0.034), and ND5 (P = 0.005); and Penelope obscura
for ND2 (P = 0.002) and ND4 (P = 0.046).
Phylogenetic analysis using the "congruent"
genes. -The congruent data set employed in tree
reconstruction has 5,128 sites (3,608 con-stant sites
and 598 parsimony-informative sites). One MP
tree (Fig. 2) of 2,880 steps was recovered
(consistency index [CI] = 0.59; reten-tion index
[RI] = 0.58; rescaled consistency index [RC] =
0.34) in which Nothocrax is sister to all other
curassows, though that relationship is not strongly
supported by bootstrap proportions. Crax forms a
well-supported monophyletic group. However, the
genus Pauxi, having only two recognized species,
appears to be paraphy-letic for those maternally
inherited genes. Pauxi pauxi is a sister group to a
clade containing all Mitu species, with P. unicornis
embedded within it. The exclusion of P. pauxi
from the Mitu-Pauxi clade and the association of
P. unicornis with M. tuberosa are well supported
by bootstrap proportions (Fig. 2).
Model search for the ML analysis showed that
the Tamura-Nei model of DNA substitution
(Tamura and Nei 1993) assuming rate variation
among sites (shape parameter of the gamma
distribution = 0.19) is the best-fitting model for
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 July 2004] CurassowPhylogenyand Biogeography 687

100 C. alector 100/10 C. alector

93 C.fasciolata 52/44 C.fasciolata

C. blumenbachii 98/100 C. blumenbackii

so 6 C. globulosa
C. globulosa
C daubentoni
97 C alberti
98/100 C. rubra
C. daubentoni
C. alberti
C. rubra M. tuberosa
95/100
98 M. tuberosa 100/100 100/1 P. unicornis
100 P. unicornis M. salvini
100/100
M. salvini M. mitu
~~~100 64/55
100 M. tomentosa 100/100 81/10M. tontosa
55 M. mitu 95/100 P. pauxi
100 P. pauxi N. urumutum
Ortalis
N. urumutum
98/100 Aburria
Ortalis
Penelope
100 Aburria Chamaepetes
0.1
Penelope
Chamaepetes
10 FIG.3. Maximum likelihood tree for the 14 curassow
species recovered from the "congruent" mitochondri-
FIG.2. Maximumparsimony tree for the 14 species al sequences using the Tamura-Nei + gamma model
of curassows recovered from the "congruent"mito- of DNA substitutions. Aburria, Chamaepetes,Penelope,
chondrial sequences. Aburria,Chamaepetes,Penelope, and Ortalis were used as outgroups. Numbers are
and Ortalis were used as outgroups. Numbers are ML bootstrap proportions higher than 50% for 100
bootstrapproportionsequal to or higher than 50%for replicates/Bayesian posterior probabilities. Bar repre-
1,000 replicates. Bar represents number of substitu- sents number of substitutions per site.
tions per site.
Effects of inclusion of ND4 on tree topology. -
these mitochondrial sequences. The ML and the Analysis of the data set including ND4 se-quences
Bayesian analysis (Fig. 3) recovered the same tree (6,457 sites) produced similar MP and ML trees
topology, which is similar to the MP tree, except to those obtained by the congruent data set,
that Nothocrax is a well-supported sister species to except for the positioning of N. uru-mutum (Fig.
Mitu-Pauxi. As in the MP tree, the monophyly of 5). The MP tree is 3,728 steps long,
Pauxi is not recovered, and the position of P. pauxi with CI = 0.58, RI = 0.56, and RC = 0.33-indexes
as a sister clade to all Mitu species, plus P. similar to those obtained using the sequences of
unicornis, is well supported by bootstrap propor- only the "congruent" genes. The Tamura-Nei
tions and posterior probabilities. Pauxi unicornis model of DNA substitution (Tamura and Nei
and M. tuberosaare again placed as strongly sup- 1993), assuming rate variation among sites (shape
ported sister species. The Crax species form a sis- parameter = 1.40) and a proportion of invariable
ter clade to the Nothocrax-Mitu-Pauxi clade with sites (I = 0.56), is the best-fitting model. Inclusion
high bootstrap support and posterior probability. of the ND4 sequences with the congruent data set
The small changes in topology among Crax spe- did not significantly re-duce the likelihood of the
cies in the ML and MP trees are related to nodes tree (ML tree without ND4 = 20,515.70354; ML
with low bootstrap support or short branches. tree including ND4 = 20,516.74356; A = 1.04002).
However, differences in log likelihoods between Bootstrap propor-tions for the MP and ML trees
MP and ML trees are statistically significant, as obtained for the data set including ND4 are
shown by the parametric bootstrap and a poste- virtually the same as those obtained for the
riori SOWH test, using the MP tree as a model "congruent genes" ML analysis, except for the
tree (Fig. 4). association of C. alberti
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688 PEREIRA AND BAKER [Auk, Vol. 121

50 1 MP tree = 20527.59088
=
AMLtree 20515.70354
Observed difference = 11.88734

20

critical value = 3.74

10

0
0-0.5 0.5-1 1-1.5 1.5-2 2-2.5 2.5-3 3-3.5 3.5-4 4-4.5 4.5-5 5-5.5 5.5-6

FIG.4. Distribution of the SOWH-test statistic generated by parametric bootstrapping of 100 replicates of the
congruent mitochondrial sequence data set using the MP tree of Figure 2 as the model tree. The critical value that
must be exceeded for a significant result at the 5% level is indicated.

and C. daubentoni, now supported by 81% and
59% in the MP and ML analyses, respectively,
with the exclusion of C. rubra. Some nodes had
decreased support when ND4 sequences were
added to the congruent data set (e.g. association of
Nothocrax with the Mitu-Pauxi clade and as-
sociation of C. globulosa with C. alector, C. fascio-
lata, and C. blumenbachii).
Molecular dating of divergence times. -
According to the LRT comparing trees con-
structed with and without a molecular clock,
mitochondrial sequences of curassows accu-
mulate DNA substitutions at a constant rate (log
likelihood of clock tree =-15,324.48155; log
likelihood of non-clock tree =-15,314.31779; 2A = -
20.32752; x2= 21.03; df = 12; P > 0.05). Hence,
divergence times and their confidence interval
were estimated on the basis of rate of substitution
per site per million years, calcu-lated at the
calibration point (see Table 2).
Assuming the genus Crax to have last shared a
common ancestor with the other three curassow
genera at 10 mya (Pereira et al. 2002), speciation
events in Crax occurred between 1.1 and 8.2 mya,
on the basis of 95% confidence intervals.
Divergence of Nothocrax from the Mitu-Pauxi
clade occurred between 7.2 and 11.7 mya, with a
mean of 9.5 mya. Species diversification within the
Mitu-Pauxi clade was estimated to have occurred
between 1.7 and 9.9 mya. Those ages
correspond to the end of the Middle Miocene
through the end of the Pliocene.
Using geological evidence for the Andean up-lift
in northern South America starting at 6 mya for
the split of C. alberti, C. daubentoni, and C. rubra
produced estimates of divergence time and
confidence intervals very similar to those obtained
from the molecular calibration (Table 2),
therefore independently confirming that the
dating method is reasonable.
DISCUSSION
Inclusion and exclusion of the "incongruent"
ND4 data partition.-A central issue in phyloge-
netic reconstruction has been whether or not to
combine genes, especially when tests of different
data partitions suggest incongruence between them
(Kluge 1989, Miyamoto and Fitch 1995,
Huelsenbeck et al. 1996). Although the sequences
of the ND4 gene were judged to be incongruent on
the basis of phylogenetic relationships re-covered
by the ND4 data set, inclusion of those sequences in
a combined analysis with the se-quences of the
other six genes actually improved agreement
between trees constructed with MP and ML
methods. That is most likely attributable to
improvement in the accuracy of phylogeny
reconstruction methods when longer DNA se-
quences are analyzed (e.g. Cao et al. 1994, Russo
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July 2004] Curassow Phylogeny and Biogeography 689

A 10 C alector 100 B
56 C.fasciolata 78
88 C. blumenbachii 80
70 C. globulosa 98

81~g C alberti
C. daubentoni
C. rubra
62 9 M tuberosa 89 100
100 P. unicornis 10010
100 M. salvini 100

100 me titu 60 100

10063 M. tomentosa 79
P. pauxi
N. urumutum
Ortalis
Aburria 100
Penelope
Chamaepetes
10
* 0.1

FIG. 5. (A) Maximum parsimony and (B) maximum likelihood (Tamura-Nei + gamma + invariable sites) trees
obtained from analysis of the "congruent" data set plus the ND4 sequences. Aburria, Chamaepetes,Penelope, and
Ortalis were used as outgroups. Numbers are bootstrap proportions based on 1,000 (MP) and 100 (ML) repli-cates.
Bars represent numbers of substitutions per site.

TABLE 2. Estimates of divergence time (mya) and confidence intervals (CI) for the curassow species based on
molecular and geological calibrations.

Molecular Geological
Species mya CI mya CI
Crax alector from C.fasciolata 1.6 1.1-2.1 1.4 1.0-1.9
C. blumenbachiifrom above Crax species 4.5 3.6-5.7 4.1 3.2-5.2
C. globulosa from above Crax species 6.1 4.9-7.4 5.5 4.5-6.8
C. daubentoni from above Crax species 6.2 5.0-7.6 6 Calibration
C. rubra from above Crax species 6.7 5.4-8.2 6 Calibration
C. alberti from above Crax species 6.7 5.5-8.2 6 Calibration
(Mitu mitu, M. tomentosa) from
(M. salvini, M. tuberosa,Pauxi unicornis) 5.5 4.5-6.7 5.1 4.1-6.2
M. tuberosafrom P. unicornis 2.2 1.7-2.7 2.0 1.5-2.5
M. salvini from M. tuberosaand P. unicornis 3.4 2.7-4.3 3.2 2.5-4.0
M. mitu from M. tomentosa 5.3 4.3-6.5 4.9 4.0-6.0
P. pauxi from other Pauxi and Mitu species 8.0 6.5-9.9 7.4 5.9-9.0
Nothocrax from Mitu and Pauxi 9.5 7.2-11.7 8.7 7.0-10.7
Crax from (Nothocrax, Mitu, Pauxi) 10 Calibration 9.2 7.5-11.2
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690 PEREIRA AND BAKER
et al. 1996, Pereira et al. 2002). Additionally, topo-
logical agreement was achieved with
no marked drop in bootstrap support
except at nodes that were not well
supported when congruent genes
were analyzed alone.
Systematic implications.-Although
ML trees are very similar, Nothocrax is the sister
group to the other three genera in the MP tree,
and sister to Mitu and Pauxi to the exclusion of
Crax in the ML and Bayesian trees. The a poste-
riori SOWH test determined that the differences
in log likelihoods of the MP and ML topologies
were significant statistically; the MP topology can
be rejected as significantly less likely. Thus, the
ML tree (or the Bayesian tree) is taken as the best
phylogenetic hypothesis for the curassows (Fig. 3).
In addition, the sequences obtained here show a
complex pattern of evolution that can be
accounted for in the explicit statistical framework
of ML. Besides statistical reasons for choosing the
ML tree over the MP tree, morpho-logical
evidence has been adduced in the past that also
supports this topology. The presence of a long
tracheal loop, patterns of plumage coloration, and
bill coloration could be used as evidence that
Nothocrax is more closely related to Mitu than to
Crax, though it was not con-sidered close to the
other three genera (Vaurie 1968, Delacour and
Amadon 1973).
The phylogenetic hypothesis proposed here
differs in two major ways from traditional views
based on morphological characters. First, the
genus Crax is placed as a sister group to the clade
containing Nothocrax, Mitu, and Pauxi,
disagreeing with the propositions of Vaurie (1968)
and of Delacour and Amadon (1973), and of many
other authors following them. Vaurie (1968)
considered Nothocrax to be a sister group to the
other three genera. Delacour and Amadon (1973)
merged both Mitu and Pauxi into Crax, with the
claim that they are not as distinct as Nothocrax is
from Crax, and to em-phasize the close proximity
of Crax, Mitu, and Pauxi. Second, the mtDNA
sequences indicate that Pauxi is a paraphyletic
taxon, with P. uni-cornis embedded in the Mitu
clade as a sister to M. tuberosa.Vaurie (1968)
proposed that the dis-tributional gap between the
two species of Pauxi might be attributed to
extinction of other conge-neric species, assuming
both species to be an-cient on the basis of
morphological characters. Incomplete lineage-
sorting and hybridization are the other possible
explanations for the
[Auk, Vol. 121
paraphyly. Given the relatively long period since
Mitu and Pauxi diverged (6.5 mya, on the basis of
P. pauxi), incomplete lineage-sorting seems an
unlikely explanation.
Conversely, it is well known that cracids can
hybridize very easily in captivity (Nogueira-Neto
1973, Pereira et al. 1996, Grau et al. 2003), though
natural hybridization has not been re-ported in
the wild (Vaurie 1968). However, the restricted
geographic distribution of P. unicornis just south
of the range of M. tuberosais consistent with the
hybridization hypothesis, because they are the
most likely taxa to have come in contact. Sequence
divergence between those taxa rules out the recent
transfer of mtDNA genomes in captive birds, and
indicates instead that an an-cient transfer in the
wild might have occurred. Because only one
specimen of each taxon was sequenced, caution is
warranted in making con-clusions about the
generality of that transfer of the mtDNA genome.
Ultimately, the hybridiza-tion hypothesis needs to
be checked with nuclear markers on bigger
samples of P. unicornis from both disjunct
populations before any recommen-dations can be
made for revising generic limits.
Biogeographyand divergencetime of
curassows.-Inferences about the relationships of
areas are frequently made on the basis of
phylogenetic re-lationships of organisms (e.g.
Prum 1988, Marks et al. 2002 and references
therein). However, such relationships can be
obscured, because spe-cies evolution conforms
basically to a divergent pattern, whereas area
cladograms more often follow a reticulate pattern
(Hovenkamp 1997); also, when using taxa as
characters to infer area cladograms, we should
consider that taxa can move around areas, thus
causing the relation-ship between area splits and
speciation to be lost in time. A well-supported
phylogeny allied with known vicariant events
would be an alternative approach. Because those
conditions are satisfied for the curassows, we can
hypothesize how their speciation took place.
The geographic distribution of the 14 spe-cies
of curassows clearly indicates exclusion of
congeneric species (Fig. 1). The barriers that
currently separate the species, combined with the
essentially nonmigratory behavior of curas-sows
(del Hoyo 1994), hint strongly at a primary role
for vicariance. Evidence in support of this
hypothesis comes from both the branching order
in the molecular phylogeny and the age of the
splits. For example, divergence time at the basal
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July 2004] Curassow Phylogeny and Biogeography
nodes among curassows coincides with a time of
extensive marine transgression into low-lying
basins of South America in the Late Miocene at
12 to 8 mya, including the present areas of the
Orinoco and Amazon river basins (Potter 1997,
Lundberg et al. 1998, Monsch 1998, Nores 1999,
Rossetti 2001). This is in agreement with other
studies invoking vicariant events for diversifi-
cation of other birds (Prum 1988, Cracraft and
Prum 1988, Hackett 1993) and primates (Silva
and Oren 1996) for the same period.
The Andean uplift also represents a ma-jor
vicariant event for further diversification among
curassows. At its northern end in South America,
the uplift occurred at -6 mya (Hoorn 1994,
Hooghiemstra and van der Hammen 1998),
preceding or coinciding with divergence times (8.2
to 5.0 mya) of basal species in the Crax clade
(alberti, rubra, daubentoni, and globulosa).
Presence of C. rubra in Central America and
southeastern Mexico can be attributed to range
expansion during the formation of Central
America or even after the rise of the Panama
isthmus ~3 mya, though that did not result in
subsequent speciation. Moreover, other changes in
South American physiography followed the uplift
of the Andes, including formation of the modern
river basins (Hoorn 1994, Hoorn et al. 1995,
Hooghiemstra and van der Hammen 1998,
Lundberg et al. 1998). For example, the Amazon
River changed its course to the present one and
may have formed a barrier, again indicating a
vicariant origin for species of Mitu and Pauxi at
around 6.7 to 4.5 mya. The Andean uplift also
resulted in aridification of central South America
by blocking the Pacific winds from the continent
(Hooghiemstra and van der Hammen 1998)-a good
example of a concordant vi-cariant event among
groups of curassows. The split-off of the Atlantic
forest representatives of Crax and Mitu (C.
blumenbachiiand M. mitu) from their Amazonian
sister clades at 6.5 to 3.6 mya may be attributable
to that process. Furthermore, uplift of the
mountains along the southeast coast of Brazil,
especially during the Pliocene (de Almeida 1976,
Riccomini et al. 1989, Safford 1999), may have
reinforced a bar-rier between them and their
sister clade, present in the Amazon. Range
contraction of their an-cestral population forced
by aridification is also a plausible explanation, and
might even have happened in conjunction with the
isolation of both forests in South America.
691
Although no vicariant event in the Pliocene is
known to coincide with the time of speciation of
M. salvini at around 4.3-2.7 mya and of M.
tuberosa and P. unicornis at 2.7-1.7 mya, their
present adjacent distribution is delimited by the
Andes and by rivers in the Amazon Basin,
which suggests a possible vicariant event for
their diversification-though dispersal cannot
be ruled out. The Amazon River is the major
barrier for the current distribution of sister
species C. alector and C. fasciolata, which oc-
cur, respectively, in its north and south banks.
They last shared a common ancestor at around
2.1-1.1 mya. For that period, a sea-level rise of
-100 m is documented (Haq et al. 1987, Nores
1999) and may be the vicariant event leading to
speciation in that case.
In conclusion, our findings point to an im-
portant role for vicariance in the diversification of
curassows, and such may also be the case for
other less-mobile birds. Basically, marine
transgression throughout the Miocene and
Pliocene and uplift of the Andes in northern
South America by the end of the Miocene, which
caused changes in the river basins of South
America, seem to be the major events that
isolated formerly continuous popula-tions of
curassows. Estimates of divergence time obtained
here, combined with evidence of a continuous
Amazon forest through the Cenozoic
(Hooghiemstra and van der Hammen 1998,
Colinvaux and de Oliveira 2001), rule out any
influence of Quaternary events commonly
invoked to explain speciation in systems such as
the Amazon (i.e. the refugia theory of Haffer
[1969, 1974]). However, Quaternary events may
have influenced formation of subspecies and
should be further investigated in cracids, using
phylogeographic approaches. Finally, it seems
that the Neotropical rainforest fauna, exempli-
fied by curassows as well as by other avian
(Miyaki et al. 1998, Pereira et al. 2002, Nahum et
al. 2003) and non-avian groups (Moritz et al. 2000
and references therein), represent old lineages
that diversified long before the Pleistocene climate
oscillations.
ACKNOWLEDGMENTS
Blood samples were kindly provided by Brazilian
private conservationist V. Fasano and by C. Keller,
Criadouro Tropicus, Pirassununga; M. de C. Dias,
Criadouro Pocos de Caldas; R. Azeredo, Fundaqo
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692 PEREIRA AND BAKER
Crax; M. Santos, Criadouro Chaparral; Companhia
Energetica de Sao Paulo (CESP) Paraibuna and CESP
Porto Primavera; and by K. Frank and J. J. Estudillo-
Lopez, Granja La Siberia, Mexico; and are deposited at
the Laborat6rio de Genetica e Evoluc,o Molecular de
Aves do Depto de Biologia da Universidade de Sao
Paulo, Brazil. F. Angulo Pratolongo, Zoocriadero
Barbara D'Achille, Peru, provided the M. salvini sam-
ples, deposited at the Royal Ontario Museum. We are
thankful to G. Ibarguchi for discussions on cracid sys-
tematics. K. G. Smith, R. Fleischer, and an anonymous
reviewer provided valuable suggestions. This project
was supported by grants to A.J.B. from the National
Science and Engineering Research Council (NSERC)
and the Royal Ontario Foundation, Canada. S.L.P. was
a post-doc fellow of Fundac,o de Amparo a Pesquisa do
Estado de Sao Paulo (FAPESP), Brazil (grant no.
00/06435-9).
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