Group Title: BMC Evolutionary Biology
Title: Multi-character perspectives on the evolution of intraspecific differentiation in a neotropical hylid frog
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Title: Multi-character perspectives on the evolution of intraspecific differentiation in a neotropical hylid frog
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Language: English
Creator: Lougheed, Stephen
Austin, James
Bogart, James
Boag, Peter
Chek, Andrew
Publisher: BMC Evolutionary Biology
Publication Date: 2006
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Abstract: BACKGROUND:Multi-character empirical studies are important contributions to our understanding of the process of speciation. The relatively conserved morphology of, and importance of the mate recognition system in anurans, combined with phylogenetic tools, provide an opportunity to address predictions about the relative role of each in the process of speciation. We examine the relationship among patterns of variation in morphology, call characters, and 16S gene sequences across seven populations of a neotropical hylid frog, Hyla leucophyllata, to infer their relative importance in predicting the early stages of population differentiation.RESULTS:Multivariate analyses demonstrate that both morphological and call characteristics were significantly variable among populations, characterized by significantly lower intra-population dispersion in call space than morphological space, and significantly greater among-population variation in call structure. We found lack of concordance between a 16S DNA phylogeny of Hyla leucophyllata and the significant population-level differentiation evident in both external morphology and male advertisement call. Comparisons of the reconstructed gene trees to simulated lineages support the notion that variation in call cannot be simply explained by population history.CONCLUSION:Discordance among traits may reflect sampling biases (e.g. single genetic marker effects), or imply a decoupling of evolution of different suites of characters. Diagnostic differences among populations in call structure possibly reflect local selection pressures presented by different heterospecific calling assemblages and may serve as a precursor of species-wide differentiation. Differentiation among populations in morphology may be due to ecophenotypic variation or to diversifying selection on body size directly, or on frequency attributes of calls (mediated by female choice) that show a strong relationship to body size.
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BMC Evolutionary Biology BioMed



Research article

Multi-character perspectives on the evolution of intraspecific
differentiation in a neotropical hylid frog
Stephen C Lougheed*t1, James D Austint2, James P Bogart3, Peter T Boag'
and Andrew A Chekt4


Address: 'Department of Biology, Queen's University, Kingston, Ontario, Canada, K7L 3N6, 2Departments of Wildlife Ecology & Conservation
and Fisheries & Aquatic Sciences (IFAS), University of Florida, Gainesville, FL, USA, 32611, 3Department of Integrative Biology, University of
Guelph, Guelph, Ontario, Canada and 4Organization for Tropical Studies, Box 90630, Durham, NC, USA
Email: Stephen C Lougheed* lougheed@biology.queensu.ca; James D Austin austinj@ufl.edu; James P Bogart boagp@biology.queensu.ca;
Peter T Boag lougheed@biology.queensu.ca; Andrew A Chek achek@tropicalstudies.org
* Corresponding author tEqual contributors


Published: 15 March 2006
BMC Evolutionary Biology2006, 6:23 doi: 10.1186/1471-2148-6-23


Received: 25 October 2005
Accepted: 15 March 2006


This article is available from: http://www.biomedcentral.com/1471-2148/6/23
2006Lougheed et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.



Abstract
Background: Multi-character empirical studies are important contributions to our understanding
of the process of speciation. The relatively conserved morphology of, and importance of the mate
recognition system in anurans, combined with phylogenetic tools, provide an opportunity to
address predictions about the relative role of each in the process of speciation. We examine the
relationship among patterns of variation in morphology, call characters, and I 6S gene sequences
across seven populations of a neotropical hylid frog, Hyla leucophyllata, to infer their relative
importance in predicting the early stages of population differentiation.
Results: Multivariate analyses demonstrate that both morphological and call characteristics were
significantly variable among populations, characterized by significantly lower intra-population
dispersion in call space than morphological space, and significantly greater among-population
variation in call structure. We found lack of concordance between a 16S DNA phylogeny of Hyla
leucophyllata and the significant population-level differentiation evident in both external morphology
and male advertisement call. Comparisons of the reconstructed gene trees to simulated lineages
support the notion that variation in call cannot be simply explained by population history.
Conclusion: Discordance among traits may reflect sampling biases (e.g. single genetic marker
effects), or imply a decoupling of evolution of different suites of characters. Diagnostic differences
among populations in call structure possibly reflect local selection pressures presented by different
heterospecific calling assemblages and may serve as a precursor of species-wide differentiation.
Differentiation among populations in morphology may be due to ecophenotypic variation or to
diversifying selection on body size directly, or on frequency attributes of calls (mediated by female
choice) that show a strong relationship to body size.


Background
The process of geographic speciation may be represented
as the shift from panmixia, through polyphyly and para-


phyly, to reciprocal monophyly of newly emerged sister
species [1]. From this vantage, understanding speciation
requires the study of historical and geographical factors


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Central


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Figure I
Map of sampling locations. Locations from which Hyla leu-
cophyllata and H. triangulum were sampled. AdC = Alter do
Chao, Auk = A-Ukre, Man = Manaus, Obd = Obidos, RB = Rio
Branco, SdN = Serra do Navio, Tab = Tabatinga, Por = Igarap6
Porongaba, NV = Nova Vida, Sac = Sacado.


that may underlie origins and diversification of lineages
within species (e.g. isolation by distance, vicariance), and
also the changes that occur in suites of characters that can
affect survivorship or are important in reproductive isola-
tion (e.g. morphology, mate recognition system). Multi-
character phylogeographic perspectives are particularly
fruitful in this regard, where the evolutionary history
inferred from DNA sequence data provides a baseline for
evaluating the divergence of various phenotypic
attributes. In other words, evolutionarily independent,
reciprocally-monophyletic lineages in similar environ-
ments can theoretically diverge in heritable phenotypic
attributes due to genetic drift, with greater divergence pre-
dicted for more deeply diverged lineages simply because
they have been separated for longer periods of time. Devi-
ation from such expectation implies the action of selec-
tion or constraint, indicating that on some level the
evolution of phenotype is decoupled from history.

Anurans (frogs) provide excellent systems for multi-char-
acter phylogeographic approaches. They are typically less
vagile than other vertebrates (e.g. mammals and birds),
presumably promoting the development of differentia-
tion among populations and regions. Indeed, many trop-
ical and temperate amphibian species exhibit striking
phylogeographic structure and deep genetic divisions [e.g.
[2,3], implying that significant spans of time have elapsed
over which phenotypic differences could evolve. Many
frog species breed in aggregations facilitating point sam-


pling for geographic surveys. Finally frogs have a well-
studied mate recognition system (hereafter MRS), with
a well-understood neuroethological basis and variation
that is readily quantified and manipulated to test for its
significance [4].

MRS was historically viewed as preventing fitness costs
associated with inter-specific mating [5]. Frogs and their
male-delivered advertisement calls have featured promi-
nently in research illustrating such classical notions of
MRS evolution (e.g. [6-8]. Other explanations for MRS
evolution recently have gained greater acceptance focus-
ing on (i) a role for sexual selection, especially through
female preference for certain male traits [reviewed in [9]],
or (ii) on direct selection on the MRS through predation,
competition, and environmental effects (e.g. selection for
signal transmission in different media) [10]. These
hypotheses share the prediction that MRS evolution may
precede the evolution of other hallmarks or correlates of
species status (including morphological distinctiveness)
and may even initiate speciation [11].

In this study we examined patterns of variation in mor-
phology, phylogeny, and male advertisement call across
"populations" of a widely distributed neotropical frog,
Hyla leucophyllata [12]. Morphology tends to be conserved
in frogs both within and among species (e.g. [13,14]), and
it is distinction in the MRS that is the hallmark of specia-
tion [4,15]. For this reason alone we might predict that the
latter will exhibit greater differentiation among popula-
tions. However, we can embed predictions of among pop-
ulation divergence within a more formal theoretical
framework. For example, in contrast to morphology, the
MRS in frogs may be under intense stabilizing selection
because of the fitness costs of inappropriate mate choice
[e.g. [16]]. Thus, we might expect there to be little range
wide variation in call at least in those characters important
in female choice. Alternatively, differences in call charac-
teristics among populations may mirror phylogeny if drift
alone results in divergence. Further, call characteristics
may not map onto phylogeny because the former diverges
before differences in neutral characters accrue. In the
present study we test: (i) if there are significant differences
in call and morphology among seven populations of H.
leucophyllata, (ii) if differentiation between populations in
these two suites of traits are significantly correlated, and
how each relates to spatial separation between popula-
tions (a potential predictor of both gene flow and degree
of environmental similarity), and (iii) if divergence in
phenotype, and particularly MRS, shows a significant rela-
tionship with genealogical history.







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Table I: Sample sizes of Hyla leucophyllata for each character set. For acronyms see Figure I legend.


Sampling Location


DNA Sequence


Results
Variation
We obtained between 490 and 503 base pairs for all 65
surveyed H. leucophyllata (see Figure 1 for locations), three
H. triangulum, and two outgroup H. elegans. The 24 dis-
tinct ingroup haplotypes differed at between 1 and 46
sites (sequence divergence between 0.2 and 8.8 %, respec-
tively), with an average ti/tv ratio, excluding comparisons
with an undefined quotient, of 2.73. Average base compo-
sition was as follows: 25.3% A, 20.9% C, 24.4% G, and
29.3% T.

Coefficients of variation for morphology variables among
populations (Additional file 2) ranged from 5.4 % (snout
length) to 11.1 % (hand disc diameter). The first two axes
of the CVA accounted for 77% (CV I = 56.7%, CV II =
20.3%) of the total morphological variance. All variables
loaded positively on CV I (Additional file 3), but were not
equal in magnitude suggesting that these axes represented
some element of both size and shape [17]. Loadings on
CV II were almost all negative, and here again magnitudes
were variable suggesting some shape dimension in addi-
tion to size.

Coefficients of variation varied more widely for call varia-
bles than those for morphology varying from 2.2% (sec-
ondary pulse duty cycle) to 143% (secondary note FM
sweep) (Additional file 4). Five characters varied signifi-
cantly with body size (Table 3). The CVA of call variables
accounted for 77% of among-population variance over
the first two axes (CV I = 53.7%, CV = II 23.2%) (Addi-
tional file 5). A few temporally-based variables showed
the heaviest loadings on CVA axes; generally, shorter pri-
mary notes with fewer pulses corresponded to greater
pulse rates and number of secondary notes.

Population distinction in morphology and advertisement
call
All morphological variables differed significantly among
populations, as indicated by highly significant Kruskal-
Wallis tests (Additional file 2). However, separation
among populations in the space defined by the first two


CV axes was low relative to within-population scatter (Fig-
ure 2A). Nonetheless, post-hoc classification by the overall
CV function was quite accurate (94.7%; Table 4) indicat-
ing diagnostic morphological variation among popula-
tions.

After sequential Bonferroni adjustment, only 10 of the 30
call variables showed significant inter-population varia-
tion as judged by Kruskal-Wallis tests (Additional file 4).
Of these 10 variables, five represented the heaviest load-
ing factors on CV I. Separation along CV I and II was much
more obvious in call than in morphological space (Figure
2B), a fact reflected by the increase in post-hoc classifica-
tion success (100%) by the CV call function (data not
shown). The increased discriminatory power of the call
CV function is likely due to comparatively low intra-pop-
ulation dispersion in call space. A difference in intra-pop-
ulation dispersion is reflected in two explicit
comparisons. First, when call and morphology CV matri-
ces are scaled equivalently, median intra-population dis-
persion in call space (diagonal matrix elements) is indeed
significantly lower than that for morphological space
(Mann-Whitney 2 Sample, Zapprox. = -3.066, nI = 7, n2 = 7,
p = 0.0022). Second, there is a difference in the coeffi-
cients of variation: the ten significantly different call vari-
ables had a higher average among-population coefficient
of variation (28.3 16.2 %) than morphological variables
(8.6 1.6 %); a difference that is highly significant
(Mann-Whitney 2 Sample, Zapprox. = 4.24, nI = 10, n2 = 17,
p < 0.0001).

Comparison of morphology, call and geographic distance
Apportioning out the known affects of body size (SVL) on
some call parameters (Table 3) in our call CVA, we found
no relation between level of inter-population differentia-
tion in call and morphology (Table 5). This was true using
both ranked and unranked matrices. Distinctiveness in
morphology between populations estimated from a CVA
showed no relation to geographic distance; however, dif-
ferentiation in advertisement call structure did show a
clear and significant relationship (Table 5).



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Morphology


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Table 2: Definitions of call variables measured for each Hyla leucophyllata call.


Variable


Description


Prim. Dom. Frequency
Prim. FM Range
Prim. FM Sweep
Sec. Dom. Frequency
Sec. FM Range
Sec. FM Sweep
Call Length
Inter-note Interval
Prim. Note Length
Prim. Note & Inter-Note Inter.
Prim. Note Rise
Prim. Note Shape
Number of Prim. Pulses
Prim. Pulse Length
Prim. Pulse + Inter-pulse Inter.
Prim. Pulse Rise

Prim. Pulse Shape
Prim. Pulse Duty
Prim. Pulse Rate
Sec. Note Length
Sec. Note Rise

Sec. Note Shape
Number of Sec. Notes
Number of Sec. Pulses
Sec. Pulse Length
Sec. Pulse + Inter-pulse Inter.
Sec. Pulse Rise

Sec. Pulse Shape
Sec. Pulse Duty
Sec. Pulse Rate


Frequency containing the most energy over the length of the primary note
Dominant frequency difference between the beginning and end of the primary note
Primary FM range divided by the primary note length
Frequency containing the most energy over the length of the first secondary note
Dominant frequency difference between the beginning and end of the first secondary note
Secondary FM range divided by the first secondary note length
Total length of call including all secondary notes
Time between the end of the primary note and beginning of the first secondary note
Length of primary note
Length of primary note plus the inter-note interval
Time from the beginning of the primary note until the maximum amplitude of the primary note is reached
Primary note rise time divided by primary note length
Number of pulses contained in the primary note
Length of first clearly discernible pulse of the primary note
Primary pulse length plus the time to the onset of the next pulse
Time from the beginning of first clearly discernible pulse of the primary note to that pulse's maximum
amplitude
Primary pulse rise time divided by primary pulse length
Primary pulse length divided by primary pulse +inter-pulse interval
Number of primary pulses divided by the primary note length
Length of first secondary note
Time from the beginning of the first secondary note until the maximum amplitude of the first secondary note is
reached
Secondary note rise time divided by secondary note length
Number of notes following the primary note
Number of pulses contained in the first secondary note
Length of first clearly discernible pulse of the first secondary note
Secondary pulse length plus the time to the onset of the next pulse
Time from the beginning of first clearly discernible pulse of the secondary note to that pulse's maximum
amplitude
Secondary pulse rise time divided by secondary pulse length
Secondary pulse length divided by secondary pulse +inter-pulse interval
Number of secondary pulses divided by the secondary note length


Phylogenetics
Topologies of trees from our maximum likelihood and
Bayesian analyses were identical in almost all aspects and
we present only the former in Figure 3. Both approaches
showed three major well supported clades with > 90%
bootstrap support (maximum likelihood) and posterior
probabilities of 1.00 (Bayesian). One clade was com-


prised of three haplotypes found exclusively in H. triangu-
lum (Clade 2 Figure 3), while the other two contained
only Hyla leucophyllata haplotypes. Clade 1 is distributed
across the entire sampled range of H. leucophyllata with
some well-supported phylogenetic structure within. Clade
3 is comprised of only four H. leucophyllata haplotypes
from two sites (SdN and Man see Figures 1 and 3). Two


Table 3: Regression statistics for call variables that showed a significant relationship to snout-vent-length (SVL). Of the tests for a
relationship between SVL and other call variables, almost all had p-values between 0.3 and 0.8. Adjusted p refers to sequential
Bonferroni correction for multiple tests [54]. Only the variables marked with an asterisk showed significant variation with SVL after
correction. For these variables we used residuals in our CVA.


Adjusted p

0.0016
0.0017
0.0017
0.0018
0.0019
0.0020
0.0020


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Variable

2dom freq*
I dom freq*
lprim pulses*
Ilnote + inter*
2note rise*
2note shape
I length


0.676
0.611
0.278
0.225
0.211
0.183
0.128


F

94.14
73.94
18.11
13.07
12.09
10.09
6.92


p

<0.000 I
<0.000 I
0.0001
0.0008
0.0011 I
0.0027
0.01 15


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CVI


Figure 2
Bivariate CVA plots for morphology and call. Individ-
ual scores of Hyla leucophyllata individuals on first two canon-
ical variates axes with 95% density ellipses shown for each
population. Population codes follow Figure I. A: morphology
B: calls


haplotypes (C and D) are shared among three eastern
sites, and one sampling locale (SdN) contains haplotypes
(C, B and A) from Clades 1 and 3. Pairwise divergence
among haplotypes within clades ranged from 0.4% to
6.5% in Clade 1, 1.4% to 2.5% in Clade 2, and 0.1 to
0.2% for Clade 3. Point estimates of divergence among
clades were 8.3%, 9.3% and 6.2% for Clade 1 verses Clade
2, Clade 1 verses 3, and Clade 2 verses Clade 3, respec-
tively.

Although highly structured phylogenetically, population
history is complicated by the geographic distribution of


deeply divergent, paraphyletic clades. The discordance
was significantly greater for the reconstructed gene tree
than the gene trees simulated under the model of popula-
tion fragmentation from a single ancestral population,
over a number of effective population sizes (Figure 5A).
This suggests that either ancestral populations were very
large and that in part, incomplete lineage sorting pre-
cludes us from inferring population history from 16S
mtDNA sequences. Alternatively, historical fragmentation
with subsequent secondary contact could also explain
some or all of the genetic pattern observed.

Relation of morphology and call to genealogical patterns
A simple, visual comparison of the patterns of differentia-
tion in morphology and advertisement call (from our CV
analyses) to phylogeny suggest no obvious relationship
between our two measures of phenotypic distinction and
genealogical history. For instance, locales Man and Tab
appear little differentiated in call space (Figure 2B), yet all
seven individuals sampled from Tab have haplotypes
embedded within Clade 1 (haplotypes S, T, U, V), whereas
all 10 individuals from Man have haplotypes within
Clade 3 (L, M). Although SdN haplotypes are dispersed
among divergent Clades 1 (haplotype C) and 3 (A, B)
individuals within this population do not exhibit any
greater dispersion in call space. Similar discord is evident
between morphology and phylogeny. For example, Auk
and Obd exhibit the greatest inter-centroid distances on
CV axis 1 in Figure 2A, yet haplotypes from both sites are
dispersed throughout Clade 1 with no evidence for com-
partmentalization. Lineages simulated under a popula-
tion 'stepping stone' model designed to reflect population
similarity based on call parameters (Figure 4B) do not
reduce the disparity in discord between reconstructed
gene and population trees, which remain significantly
higher than expected under neutral coalescence.

Discussion
Variation and distinction among populations
Morphological
Hyla leucophyllata does not show sufficient morphological
variation to have provoked the naming of subspecies or
other taxonomic revision (see Methods). However, upon
close examination the species nonetheless demonstrates
diagnostic patterns of morphological variation across
populations. That significant variation has gone unre-
marked is perhaps not surprising when the CVA plot for
morphology (Figure 2A) is examined in detail. Despite a
statistically significant separation in this multivariate
space, and indeed on every univariate morphological
measure, most populations of H. leucophyllata grade into
one another in morphological space rather than showing
obvious gaps. Most of the variation among populations
was in size although there was also a shape component.
Elements of size and shape or general appearance feature


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Table 4: Accuracy of CVA of the morphological data set in the post-hoc prediction of populations membership of Hyla leucophyllata
individuals.


Observed population membership


Predicted pop. AdC


AdC
Aukre
Man
Obd
RB
SdN
Tab


% correct


in most taxonomic descriptions of frogs [18,19]. Thus, in
that sense, the variation found here is significant. How-
ever, the evolutionary significance of this variation is
harder to judge. For example, although size and shape dif-
ferences among populations might reflect evolutionary
divergence in form they could also be due to ontogeny. All
individuals were males and almost all were calling and
thus presumably sexually mature. However, frogs may
undergo some growth-related changes even at this stage
[18], so variation among populations might only reflect
age class differences and thus sampling bias [20]. Equally
problematic would be ecophenotypic variation that is not
heritable and thus not indicative of evolved differences
among populations. Both factors are hard to rule out
without direct experimentation when the taxa studied are
allopatric, the characters continuous, and the differences
subtle or relatively small.

Call
Variation in the advertisement calls of H. leucophyllata was
both greater and more distinctive than that in morphol-
ogy. For significantly variable call characters, among-pop-
ulation coefficients of variation were on average more
than three times as large as those coefficients for morpho-

Table 5: Summary of pairwise Mantel's tests on inter-
populational distance matrices. Matrices used in each test were
derived from CVA of call and morphological variables, and from
straight-line geographic distances between collecting localities.
Top number in each cell is r (correlation coefficient), bottom is
p-value. Comparisons marked by an asterisk were significant
after sequential Bonferroni adjustment. Each matrix uses ranked
values but the results are unchanged with raw (unranked) values


Morph


-0.1286
0.3223


Geog

-0.0104
0.4824
0.6753
*0.0028


logical variables. In addition, within-population variation
was lower and populations were better separated from
one another in call space than in morphological space
(Figure 2B). However, inasmuch as calls here serve as an
indicator of the MRS, it is insufficient to show variation in
structure of calls; rather this variation must also have
some bearing on mate choice.

Significant variation among H. leucophyllata populations
was found in pulse rate and other temporal features like
call length. In addition, the difference among populations
in dominant frequency was significant if it was not
adjusted for body size. All of these features are known to
affect mate choice in other hylid frogs [4,21]. Moreover,
both pulse rate and dominant frequency are known to
function in mate discrimination in another leucophyllata-
group species, H. ebraccata [22]. The number of secondary
notes was also significantly different among populations
of H. leucophyllata and this character is thought important
to mate choice in H. ebraccata (J. Schwartz pers. comm.).
Although there are no direct tests of which characters
influence mate choice in H. leucophyllata, the tendencies of
frogs in general (and a close relationship with H. ebrac-
cata, in particular) suggest that at least some of the above
characters play a role in mate choice in H. leucophyllata.

Significant geographical variation in the call of H. leuco-
phyllata is consistent with other studies on frogs (e.g., [6-
8,23-26]. However, most of these studies involve calls as
corroborating evidence for differences established first on
morphological or genetic grounds, or are an explicit
attempt to illustrate reproductive character displacement/
reinforcement between established species. Few studies
examine the potential for call evolution to initiate inter-
population divergence, although recent biogeographic
and experimental data have demonstrated that reinforce-
ment may act to drive rapid pre-mating isolation in frogs
(Hoskins 27). Regardless, the data presented here for H.


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Aukre

0
26
0
0
0
0
0

96.3


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7911.0


-t0.94 Haplotype 4 [AdC, Auk, Obd]
55/0.56 Haplotype 7 [AdC]
Haplotype 3 [SdN, Auk, Obd]
5810.82 Haplotype 5 [Auk]
Haplotype 19 [Tab]
10 | Haplotype 20 [Tab]
l- Haplotype 21 [Tab]
Haplotype 15 [NV]


9311.00 ,


7510.6
1


Scale
0.1


10011.00


Hyla elegans OUTGROUP
Hyla elegans


Haplotype 16 [Por]


Haplotype 8 [Tab]
971.00 f Haplotype 14 [Tab]
Haplotype 9 [Obd]
-0.66 Haplotype 10 [Obd]
7 64t Haplotype 11 [Obd]
Haplotype 6 [Auk]

9811.00 Haplotype 7 [RBr]
Haplotype 18 [RBr]
Haplotype 23 [Sac]
9311.00 Haplotype 24 [Tab] Clade 2
Haplotype 22 [Tab] Hyla triangulut
8610.96 Haplotype 12 [Man]

Haplotype 13 [Man] Clade 3
10011.00 Haplotype 2 [SdN] Hyla leucophy

Ml Haplotype 1 [SdN]
60/0.96


Clade 1
Hyla leucophyllata


m


llata


Figure 3
Phylogenetic hypothesis derived from maximum likelihood analysis of 16S DNA sequence. ML tree showing rela-
tionship among Hyla leucophyllata and H. triangulum haplotypes. Bootstrap support for ML and Bayesian posterior probabilities
(before and after the forward slash) are indicated where the former exceeds 50% and the latter 0.70.


leucophyllata emphasize the value of considering MRS evo-
lution as a potential cause rather than effect of frog speci-
ation, given that it appears to be under greater selection
pressure than either morphology or DNA sequence.

Phylogenetics
We found striking phylogenetic divisions within H. leuco-
phyllata but with little obvious geographic structure. Previ-
ous phylogenetic and phylogeographic work on the 30
chromosome clade (including H. leucophylata and H. tri-
angulum) suggested that neither population spatial prox-
imity nor colour pattern polymorphism corresponds well
with mitochondrial phylogeny [28]. Expanded sampling
and character evaluation in this study confirms this con-
clusion. Previous studies of frogs have shown similarly
striking phylogenetic divisions within traditionally
regarded species. For example, allozymes and immuno-
logical techniques have tended to find pronounced


genetic divisions within frog species [e.g. [29-31]].
Sequence-based studies of intra-specific variation in frogs
also have typically found pronounced genetic divisions
within species [32-34]. Here, point estimates of clade
divergence times surpass those found in 16S in a temper-
ate hylid [341.

Relation between character types
The presence of variation in morphology, advertisement
call, and DNA sequence permits questions about relation-
ships among these suites of data. The null hypothesis is
that evolution in phylogenetic distinction, form, and
reproductive isolation should be at least rank correlated
during the speciation process; i.e. lineages that are evolu-
tionarily independent will diverge through genetic drift
alone given sufficient time. However, we found no such
relationship between either morphology or call, and phy-
logeny. Even pronounced separation between two popu-


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lations for one suite of characters was not necessarily
accompanied by separation for another. A variety of pos-
sibilities exist to explain departure from the null expecta-
tion of rank correlation. We examine some of these below.

Single Marker Effects
Despite our finding of strongly supported phylogenetic
divisions, this phylogeny is based only on variation of a
single marker, the 16S rDNA molecule. Thus, the lack of a
concordance between phylogeny and morphological or
call divergence found here might then simply reflect a fail-
ure to accurately recover historical affinities among popu-
lations. However, such correlations can also be absent in
studies of frog populations that use a large number of
nuclear markers such as allozyme studies [e.g. [31], but
see [25]], suggesting the phylogenetic estimate presented
here perhaps could accurately reflect the history of con-
nectedness among these populations. Furthermore, mito-
chondrial DNA is more likely than many single nuclear
sequence loci to reflect historical population affinities
given the former's smaller effective population size. Given
this possibility at least, it is reasonable to explore other
explanations for the absence of a correlation between phy-
logeny and measures of phenotypic distinctiveness.

Selection on morphology
Insofar as our 16S phylogeny is an accurate depiction of
population history, then major clades of H. leucophyllata
may have been separated from each other for up to several
million years [28]. If so, the lack of apparent correspond-
ence between phylogeny and morphology would still not
be particularly surprising for frogs. A number of studies
have found pronounced genetic differences among frog
taxa that are not reflected in morphology [e.g. [29,31,35]].
What morphological differences there are among popula-
tions of H. leucophyllata could reflect ecophenotypic or
ontogenetic effects as noted above. By contrast, deviation
from a relation with phylogeny could indicate the action
of selection upon morphology and adaptation to local
environmental conditions as is suggested in some species-
level comparisons [20,36]. Although a correlation of mor-
phological with geographic distance might then be antic-
ipated, the geographic separation among H. leucophyllata
populations is generally large and, so, even spatially prox-
imate populations could experience substantially differ-
ent environmental pressures.

Whether morphological differentiation is selected or not,
the fact remains that large divergences in DNA sequence
relate to seemingly trivial morphological effects in these
frogs. Indeed, species of the leucophyllata-group are sepa-
rated on average by more than twice as much genetic dis-
tance as are clades of H. leucophyllata [28] yet, even at the
species group level, separation on morphological grounds
alone has been historically problematic [37]. Moreover, as


noted above, available evidence suggests that these clades
show levels of sequence divergence greater than that typi-
cal between congeners in other groups like mammals and
birds [38]. Why frogs might exhibit conservative morpho-
logical evolutionary patterns is not clear. However studies
on another group of amphibians with a conserved mor-
phology offers one explanation. Wake et al. [39] suggested
that in plethodontid salamanders, an unpredictable envi-
ronment has selected for a generalized body type coupled
with facultative behavioral adjustment. Behavioural flex-
ibility buffers the effect of environmental variation and
hence minimizes naturally-selected morphological
change over evolutionary time. On this view, genetic
changes continue to accrue within lineages but morpho-
logical evolution is retarded, leading to decoupling of
morphological and molecular evolution [40]. Hyla leuco-
phyllata fits the profile described by Wake et al. [39] for a
species with an evolutionarily "persistent" morphology. It
seems to face and cope with considerable environmental
heterogeneity, occupying and breeding in a variety of hab-
itats even within the same locale, from cattle ponds in
open fields to closed-canopy forest (Chek, pers. obs).
Hence, this hypothesis could explain why apparently deep
evolutionary splits in H. leucophyllata have not led to obvi-
ous and concordant changes in form.

Selection on body size or calls?
The pattern of call evolution provides some of the most
interesting points for consideration. Although call and
morphology distance matrices are uncorrelated (Table 5),
there is a relationship between some aspects of calls and
morphology (body size). A few of the 30 call characters
measured were correlated to snout-vent-length, but this
variation was removed before overall call distance was cal-
culated. Adjustment for body size allowed examination of
call variation that is independent of the most obvious
influence of morphology. However, call variables corre-
lated to body size are far from unimportant to a consider-
ation of the process of call evolution in H. leucophyllata. Of
the call variables correlated to body size, the strongest
relationship was one with dominant frequency (r2 = 0.61;
Table 3): larger males had calls of lower frequency.
Females of several frog species are known to prefer larger
males as mates and independently to prefer lower domi-
nant frequencies tendencies true of H. ebraccata females
[22,41]. As with any correlation, it is difficult to determine
whether one factor is driving the other, or whether other
factors are causal. Interpopulation variation in body size
might be naturally selected, or indeed could be an ecophe-
notypic response. In both cases, differences in dominant
frequency and its reception by females simply would be
"dragged along" over time. However, an intriguing possi-
bility is the reversal of this scenario: females could prefer
the signal itself with body size evolution a correlated
effect. Sexual selection theory provides several models by


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A:
250


200 1 Ne 5000
Ne 10000
U Ne 40000
c 150

Observed s
L 100


50



1 10 11 12 13 14 15 16 17 18 19 20 21 22 23



B: A A S O M R T
250


200
>,
c 150

a)
S100o observed s


50



1 10 11 12 13 14 15 16 17 18 19 20 21 22 23


s of Slat kin and Maddison
Figure 4
Results of coalescent simulations comparing gene versus population histories. Distributions of expected values of
the test statistic, s, derived from 500 coalescent simulations over a variety of demographic histories. A: Eight populations orig-
inating from a common ancestral population representing a fragmentation model (inset). B: Seven populations structured by a
matrix of distances from the CVA of calls, representing an isolation by (call) distance model (first letter of population indicates
position on tree). Observed discord between gene tree and population tree is indicated with an arrow.



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call length


inter-note int.
I--I
sec. note


final dominant frequency





initial dominant frequency frequency range


100


200
Time (ms)


300


400


Figure 5
Representative waveform and sonogram for Hyla leucophyllata call A: waveform typical of Hyla leucophyllata male
advertisement call showing primary note with two secondary notes B: expanded view of three primary pulses C: sonogram of
call in (A).



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prim. note


> 50
E
0



|-50






> 50

E
0


E -50


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which direct female preferences and associated male traits
could evolve [42]. A potentially generative role for frog
calls in morphological evolution is reflective of generally
increased research interest in the role of the MRS in initi-
ating speciation, especially with respect to sexual selection
[9,11,27]. Moreover, it emphasizes once again the value
of investigating multiple character suites.

Covariation of calls with geography
As with morphology, there was no obvious relation
between phylogeny and distance among populations in
call (e.g. see Figure 4). Again, this may be due to a failure
of the single genetic marker to accurately reflect the his-
tory of gene flow among populations; a recent history of
gene flow could be obscured by lineage sorting effects. If
so, then further study using a suite of nuclear markers
could reveal a correlation of phylogeny with call distance;
a pattern consistent with classical views that see MRS evo-
lution as a largely pleiotropic effect of divergence in other
aspects of the organism [5]. If call divergence among H.
leucophyllata populations is a result of undetected similar-
ity across nuclear loci, then this may explain the only sig-
nificant and strong correlation among matrices, that
between geography and calls. However, even a study that
found a correlation between call divergence and genetic
divergence also found a residual correlation of call dis-
tance with geographic distance [25], which suggests some
other factor that covaries with geographic distance.

One possibility is that locales in closer proximity are more
similar in vegetational composition than those that are
further apart. Among other effects of vegetation is the deg-
radation of acoustic signals broadcast through it [43].
Frog calls with higher pulse rates are degraded more when
transmitted through vegetation than in open habitat for-
mations [44], and there is some evidence that frog species
in open habitats have higher pulse rates than those in for-
est [45]. Thus, slower pulse rates of some H. leucophyllata
populations might be a selected response of denser vege-
tation at those locales. However, the lowest pulse rates
actually come from populations in swampy open areas
(RB, Tab). If current habitat is any guide to the long-term
selective milieu, then this suggests that geographic varia-
tion in vegetation density is not the cause of the observed
pattern of call variation among populations.

Another factor that could covary with geographic distance
between H. leucophyllata populations is the composition
of the sympatric frog assemblage. Distribution of frog spe-
cies in the lowland forest of the Amazon Basin is far from
uniform [46]. Of the approximately 200 species found
there only about 10% are found throughout the Amazon,
with H. leucophyllata being one of these [46]. Thus, there
is geographic variation in the identity of species co-occur-
ring with H. leucophyllata. For example, this species can be


sympatric with three other leucophyllata-group species in
the western Amazon, whereas, in the eastern Amazon, it
is, as far as is known, the only member of its group. If, all
else being equal, sites closer together are more similar in
species composition, then competition among species for
"acoustic space" could explain both call divergence
among sites and covariation with geographic distance.
Call divergence through acoustic competition could occur
either as a means of avoiding mis-mating (reproductive
character displacement sensu 47], or of avoiding destruc-
tive interference of sound waves, or both. Both mecha-
nisms have been invoked to explain the distribution of
call features among species in frog assemblages [reviewed
in [48]]. Like its ecological counterpart, hypotheses of
acoustic partitioning posit that selection acts to carve out
an acoustic niche for the organism such that overlap with
neighbours in syntopic soundspace is minimized. Unfor-
tunately, an inventory of the species at each site and
recordings of all of their calls is not available to test this
hypothesis for H. leucophyllata. Nonetheless, there is some
evidence for acoustic partitioning in frog assemblages of
which H. leucophyllata is a part [49].

Conclusion
Hyla leucophyllata exhibits strikingly deep phylogenetic
divisions, although the species as currently designated is
possibly paraphyletic with H. triangulum. Significant dif-
ferentiation among populations in both external mor-
phology and male advertisement call does not show any
obvious relationship to the 16S DNA genealogy implying
that, if the latter does mirror the evolutionary history of
the species, then in some sense evolution of these differ-
ent characters is decoupled. A strong relation between call
structure and geographic distance may reflect differential
acoustic partitioning of H. leucphyllata populations within
different calling frog assemblages. Some call attributes
(notably dominant frequency) show a highly significant
relationship to body size raising the possibility that (i)
divergence in body size is either ecophenotypic or caused
by diversifying selection in different environments, and
has resulted in divergence in call frequency, or (ii) selec-
tion on call itself [27], mediated by female choice, has
produced divergence in body size.

Methods
Study species
Hyla leucophyllata is a small (20-35 mm) treefrog widely
distributed and common throughout the lowland forests
of the Amazon Basin [50] and associated habitat. Males
usually call above small water bodies from emergent veg-
etation and may be syntopic with a variety of other species
(Chek pers. obs.). Hyla leucophyllata is one of six species in
the leucophyllata-group [50]. Like many such frogs, species
group members are characterized by extreme morpholog-
ical similarity. This leads to the possibility that H. leuco-


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phyllata is simply a morphotype. However, sympatry of
several species [51] with clearly distinct advertisement
calls suggests that these taxa are reproductively isolated.
Call differences among the species are generally paralleled
by large differences at mtDNA loci [28], also supporting
the assumption that these taxa are indeed species. Hyla
leucophyllata previously had not been suggested to com-
prise more than one species, by any criterion. However,
there is some suggestion that at least some lineages within
H. leucophyllata are implicated in a clade containing H. tri-
angulum, and the species as currently designated may be
paraphyletic [28]. In addition, noticeable variation in
phenotype does occur within H. leucophyllata, but is con-
fined to colour-pattern polymorphism. However, particu-
lar morphs are not restricted to given populations and
allozyme analysis has established that even extreme differ-
ences in colour-pattern are not related to species bounda-
ries [521.

Collections, characters, and measurements
Over three rainy seasons (Nov. 1993 Feb. 1994; Jan.
1995 -Apr. 1995; Feb. 1996 -Apr. 1996), we collected H.
leucophyllata (n = 99) from seven sites that span the major-
ity of the species' range. For purposes of phylogeographic
analyses we added tissue samples for two additional sites
on the Rio Jurua in western Amazonas (see Additional file
1). Inter-site distances ranged from 35 to 1948 km. Figure
1 indicates localities sampled and Additional file 1 lists
the disposition of specimens, as well as the approximate
latitude and longitude where they were sampled. All indi-
viduals collected for this study were males, as judged by
calling activity (only males call) or the presence of testes
during tissue sampling. Once tissue had been sampled,
specimens were fixed in 10% formalin and then stored in
70% ethanol. Sample sizes varied for each character set
(Table 1, Additional file). Minimally, most individuals
that were measured for calls were also measured for mor-
phology and most of these had associated sequences.

Morphology and call
Aspects of shape and size are among the most commonly
used descriptors in systematic diagnoses of frogs. Seven-
teen measurements from each individual were collected
following Lee and Crump [53] (see Additional file 2 for a
list of variables). Measurements were made on preserved
specimens to the nearest 0.1 mm using dial calipers.
Because colour-pattern is difficult to quantify and its anal-
ysis in these frogs may require several thousand individu-
als [37], colour-pattern was not included as a character.

Hyla leucophyllata produces a call composed of a pulsed
trill (primary note) that may be followed by one to six
shorter secondary notes of similar form (Chek, pers. obs.).
Calls were recorded in the field using a Sony WM-D3 Pro-
fessional Walkman and Electro-Voice (model 635A)


microphone. Temperature during recording was always
25-26 C, hence no significant effects of temperature on
call characters were expected. Five calls of each individual
were digitized at a sampling rate of (10 KHz) using
CANARY vers. 1.2 (Cornell Bioacoustics). The same pro-
gram was also used to produce waveforms and sonograms
for each call from which variables were measured using
built-in software tools. Variables comprised a mix of spec-
tral and temporal properties of calls, including some that
are known to influence mate choice in other species (e.g.
pulse rate [21]). For each individual, the average of each
variable across its five measured calls was used in all anal-
yses. Variable names and definitions are listed in Table 2;
Figure 5 shows a typical waveform and sonogram from
which call variables were measured.

Statistical analyses of morphology and call data
For morphological and call variables, means and vari-
ances were calculated for raw values of each variable.
However, subsequent analyses employed log-transformed
values. Because some aspects of frog calls are influenced
by body size (e.g. dominant frequency [51,54]), all call
variables were regressed against a measure of overall body
size (snout-vent-length; SVL). Where a significant rela-
tionship between a call variable and SVL was found, the
residuals of the regression were used in subsequent analy-
ses. Wherever multiple tests were performed (e.g. all call
variables regressed on SVL) a sequential Bonferroni
adjustment [55] was made. Most analyses were performed
in JMP vers. 3.1.6 (SAS Institute Inc., Cary, North Caro-
lina) and SYSTAT vers. 5.2.1 (Systat Inc., Evanston, Illi-
nois), although some (e.g. Mantel's test) used R-
PACKAGE vers. 3.0 [55].

Amount and distinctiveness of character variation
Multiple characters were measured for calls and morphol-
ogy to more fully capture the extent and direction of vari-
ation in trait space. We tested for differences among
populations for each character using a Kruskal-Wallace
test. We also used Canonical Variates Analysis (CVA) to
examine differences among populations in call and mor-
phology. A technique commonly used in studies of frog
morphological and call variation [e.g. [24,26,57]], CVA
yields axes that summarize trait variation and produces
corrected distances among groups. While the use of CVA
with small sample sizes is of some concern, we found that
the results were qualitatively similar to those produced by
Principal Components Analysis (PCA), a technique with
less stringent assumptions. PCA, however, is not recom-
mended for an analysis of geographic variation [58]
because it does not allow for the a priori consideration of
groups (populations).

CVA yielded corrected (Mahalanobis) distances of each
individual from each population centroid, including the


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centroid of its own population. Individual distances from
each population to a given centroid were averaged to pro-
duce an inter-population distance. This procedure was
repeated for all pairs of populations to produce a matrix
of pairwise distances. The diagonal of the matrix repre-
sents the average distance of individuals from their own
population centroid. The ability of each CVA to discrimi-
nate diagnostic variation among populations was assessed
by its success at correctly classifying individuals to the
population from which they originally were drawn.

Relation of morphology, call and geography
Mantel's test was used to calculate correlations between all
distance matrices (call, morphological, a matrix of
straight-line geographical geographic distances between
sites) [59]. One thousand permutation replicates were
performed for each test. Distances within matrices were
first ranked because the principal question concerned
whether the relative magnitudes of variation were corre-
lated. However, the use of unranked matrices did not
change any conclusions. Scales of measurement among
character sets were made comparable by dividing each dis-
tance matrix by its maximum value; thus, in rescaled
matrices the maximum value was one.

Phylogeny
For general details on tissue collection and amplification
and sequencing of a fragment of the mitochondrial 16S
rRNA gene see [28] Chek et al. (2001). DNA sequence
data from the 16S rRNA gene were used to estimate the
genealogical history of sampled populations. Gaps were
treated as missing data for all analyses. Point estimates of
divergence among major clades was estimated following
the moment method [60], which corrects for the ancestral
portion ofwithin-clade diversity (Pnet= PAB- 0.5 [pA+PB]).).

Given the aforementioned possibility of paraphyly of H.
leucophyllata, we included sequence from both it and H.
triangulum. We approached inferring the genealogical rela-
tionships of H. leucophyllata using both maximum likeli-
hood (ML) and Bayesian approaches. We included Hyla
elegans as an outgroup, as it lies unequivocally outside of
our leucophyllata/triangulum ingroup [28]. For ML, we used
MODELTEST vers. 3.06, [61] to select the best model of
evolution (GTR + I + G; with proportion of invariant sites
(I) = 0.371, y shape parameter = 0.491). We conducted ML
using an exhaustive search and 'as-is' sequence addition,
and evaluated support for resulting topologies using 100
nonparametric bootstrap pseudoreplicates with PAUP*
vers. 4.10 [62]. We also ran two simultaneous Bayesian
analyses (MR.BAYES vers.3.1.1; [63], each beginning with
random starting trees, with Metropolis-coupled MCMC
using four incrementally heated Markov chains sampled
every 100 generations. We estimated stationarity of the
Markov chain by plotting the sampled log likelihood


scores versus generation time. Analyses were run for 1.0 x
106 generations until the average standard deviation of
split frequencies was less than 0.01. Potential scale reduc-
tion factors (PSRFs; see [64] for all estimated parameters
was close to 1.00 and estimated effective sample sizes
(using the program TRACER vers. 1.2.1 [65]) for all
parameters were all > 100, both suggesting that we had
adequately sampled the posterior distribution of trees.
Trees generated before the bum of 250000 generations
were discarded and we used the remaining trees to gener-
ate 50% majority rule consensus trees.

Coalescent simulations of molecular evolution
We conducted coalescent simulations of molecular evolu-
tion and applied a gene-tree/population-tree approach
[66] to estimate whether the 24 haplotypes in the recon-
structed 16S gene tree was concordant with two popula-
tion models representing 1) divergence from a single
ancestral population, and 2) population history as pre-
dicted by the significant correlation between call variables
and geographic distance (see results). All simulations and
measures of discordance were done in Mesquite version
1.05 [67]. Reconstructed haplotypes were contained
within populations under both population models listed
above. We used s [68] to measure the discord between the
gene tree and its subdivision into populations (treating
later as categorical variables), with s used to infer time
since divergence assuming no gene flow. For population
divergence from a single ancestral population we apply a
star model; appropriate because there we found no evi-
dence of genetic isolation by distance. The second popu-
lation model represents a stepping stone model reflecting
the strong relationship between population similarity in
call variables and geographic proximity of populations.
Specifically, a distance matrix based on the CVA of meas-
ured call parameters was used to construct a population
tree, which was then used to represent population history
that follows a stepping stone model of divergence.

Simulated gene-trees were created using the Genesis pack-
age of Mesquite vers. 1.05. For each simulation we created
500 gene trees for each of three temporal scenarios: Ne =
5000, 10,000, and 40,000, where Ne represents time since
populations splitting measured in generations [67]. Given
that small sample sizes make estimating theta from a sin-
gle marker problematic, and the difficulty in obtaining
accurate mutation rate estimates for 16S in treefrogs, we
chose to simplify the model by assuming each historical
population had similar effective population sizes. Within
Mesquite, this is equivalent to holding the branch widths
of the population tree equal across populations (branch
widths equal 1). Parameters were modelled using empiri-
cally derived nucleotide frequencies, proportion of invar-
iant characters, gamma shape parameter, and six-
parameter rate matrix model (see above). A scaling factor


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was selected through preliminary runs that provided pair-
wise sequence divergence rates similar to our observed Additional File 4
16S data (scaling factor = 2.0 x 10-6) [67]. The 500 simu- Summary statistics and univariate tests for call variables. Population
lated gene-trees for each generation time provided an summaries for 30 call measurements of Hyla leucophyllata for all indi-
of random coalescent genetrees viduals from each population. Localities are shown in Figure 1; variable
expected distribution of random coalescent gene-trees descriptions in Table 2. Variables; .m ... i ,i by Kruskal-
from which to compare observed values of s under the two Wallis tests (p < 0.05) after sequential Bonferroni adjustment of alpha
population hypotheses. level are indicated by an asterisk (*). A t-bar (t) indicates that the resid-
uals on the snout-vent-length were used in the Kruskal-Wallis test and
Authors' contributions CVA (see Table 3).
This manuscript evolved from a portion ofAAC's PhD the- Click here for file
[http://www.biomedcentral.com/content/supplementary/1471-
sis, under the supervision of JPB. The field measurements 2148-6-23-S4 pdf]
and samples were collected by AAC and JPB. SCL JDA and
AAC conducted the majority of the laboratory work, sta- Additional File 5
tistical analyses and writing of the manuscript. JPB and Variable loadings and eigenvalues for call CVA. Correlation,.. i,
PTB contributed ideas, and financial assistance through- clients of call variables with canonical axes (loadings) and associated
out the project. All authors commented on and approved eigenvalues for each axis of the CVA.
the final draft of this manuscript. Click here for file
[http://www.biomedcentral.com/content/supplementary/1471-

Additional material 2148-6-23-S5.pdf]


Additional File 1
Voucher specimens and locality information. All individuals were meas- Acknowledgements
ured for morphological variables. Individuals in bold type were measured Many people kindly made tissues available for this study, provided help in
for call variables. Asterisks indicate individuals that were sequenced. the field, or aided in a variety of ways: Dave Britton, Claude Gascon, Dante
Genebank accession numbers are indicated in square brackets. All indi- Pavam, Moises de Souza, and many others too numerous to mention. The
viduals have been deposited with the Museum of Zoology of the University authors are particularly indebted to Miguel Rodrigues of the Departamento
of Sao Paulo (MZUSP) or the Instituto Nacional de Pesquisas da Ama- de Zoologia, Universidade de Sao Paulo and Barbara Zimmerman of Con-
zonia (INPA). servation International, without whom this study would not have been pos-
Click here for file sible. AAC also thanks the many people the length and breadth of Brasil
[http://www.biomedcentral.com/content/supplementary/1471- who were curious, helpful, and hospitable, and who made his time in their
2148-6-23-Sl.pdf] country so personally rewarding. This work was supported through
NSERC operating grants to JPB, SCL and PTB, and through an NSERC
Additional File 2 scholarship, University of Guelph International Field Studies Grant, and
Summary statistics and univariate tests for morphology. Population Sigma Xi grant to AAC.
means (above) and standard deviations (below) for 17 morphometric
measurements of Hyla leucophyllata for all individuals from each popu- References
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