THE JOURNAL OF TROPICAL BIOLOGY AND CONSERVATION
BIOTROPICA *(*): ***–*** ****
10.1111/j.1744-7429.2008.00416.x
Acoustic and Morphological Differentiation in the Frog Allobates femoralis:
Relationships with the Upper Madeira River and Other Potential Geological Barriers
Pedro Ivo Simões1 , Albertina P. Lima, William E. Magnusson
Coordenação de Pesquisas em Ecologia, Instituto Nacional de Pesquisas da Amazônia, Caixa Postal 478, CEP 69011-970, Manaus,
Amazonas, Brazil
Walter Hödl
Department für Evolutionsbiologie, Fakultät für Lebenswissenschaften, Universität Wien, Althanstrasse 14, 1090, Wien, Austria
and
Adolfo Amézquita
Departamento de Ciencias Biológicas, Universidad de Los Andes, CRA 1 # 18a 10, AA 4976, Bogotá, Colombia
ABSTRACT
We studied patterns of call acoustics and external morphological differentiation in populations of the dart-poison frog Allobates femoralis occurring in forested areas
along a 250-km stretch of the upper Madeira River, Brazil. Multivariate analyses of variance using principal components representing shared acoustic and morphological
parameters distinguished three groups in relation to call structure and external morphology: (1) populations belonging to a two-note call morphotype; (2) populations
with four-note calls inhabiting the left riverbank; and (3) populations with four-note calls inhabiting the right riverbank. Our results report a case of Amazonian
anuran diversity hidden by current taxonomy and provide evidence for the upper Madeira River being a boundary between distinct populations of A. femoralis, and
suggest a new taxonomic interpretation for these groups. Samples that did not fit into the general differentiation pattern and the existence of a well-defined contact
zone between two morphotypes on the left riverbank indicate that mechanisms complementary to river-barrier hypotheses are necessary to explain the phenotypic
differentiation between populations. Our study shows that at least one anuran species shows congruence between population differentiation and separation by a large
Amazonian river, as documented for birds and mammals. Conservation efforts should not consider the taxon now known as A. femoralis as a homogeneous entity.
There is much within-taxon variability, which can be probably explained partly by the existence of cryptic species, partly by geological barriers and part of which
currently has no obvious explanation.
Abstract in Portuguese is available at http://www.blackwell-synergy.com/loi/btp.
Key words: bioacoustics; Brazil; Dendrobatidade; external morphology; Madeira River; phenotypic differentiation; population distribution patterns.
MANY AMAZONIAN SPECIES HAVE THEIR DISTRIBUTIONS LIMITED by
large Amazonian rivers, such as the Solimões, Negro, and Madeira
(Wallace 1852, Hellmayr 1910, Capparella 1987, Ayres & CluttonBrock 1992, Patton et al. 2000, Hayes & Sewlal 2004). With a
more comprehensive data base on Amazonian species occurrences,
contemporary studies of species distributions and phylogeography
revealed a myriad of distribution patterns, some of which indicated population or species boundaries that are not coincident
with large rivers (Haffer 1997a). A considerable number of alternative hypotheses were proposed to explain the origin of those
distribution patterns and their maintenance through time (Haffer
1997b, Moritz et al. 2000). Such hypotheses are still controversial
and largely debated, but it is reasonable to assume that no single
process affected all groups of organisms with the same intensity
(Bush 1994).
Studies undertaken along the Juruá River, a meandering, 100to 400-m wide tributary of the Amazon, did not detect an effect
of the river on allozyme, external morphology, or mitochondrial
DNA differentiation in anuran populations (Gascon et al. 1996,
1998; Lougheed et al. 1999). Anuran-community composition also
Received 26 June 2007; revision accepted 11 December 2007.
1 Corresponding author; e-mail: [email protected]
did not differ between riverbanks, suggesting that the Juruá River
does not prevent species crossing from one interfluve to another
(Gascon et al. 2000). In contrast to the Juruá, the Madeira River is
a distributional boundary to several primate and bird taxa (Wallace
1852; Hellmayr 1910; Haffer 1974; Capparella 1987; Roosmalen
et al. 1998, 2000), but studies of its importance as a geographic
boundary capable of maintaining population differentiation have
not been undertaken for other groups.
Our study aimed to evaluate the geographic patterns of call
features and external morphology in the poison-dart frog Allobates
femoralis (Boulenger 1883) on both sides of a 250-km stretch of
the upper Madeira River. Allobates femoralis is a ground-dwelling
diurnal dendrobatid frog, abundant in forested areas of the Madeira
River, which feeds on a wide range of invertebrate taxa (Caldwell
1996). Males vocalize from sites slightly elevated from the forest
floor, such as logs and rocks. Advertisement calls are constituted
by the repetition of groups of short, frequency-modulated notes,
used for territory defense against conspecific males and to attract
females. Courtship and oviposition occurs inside the male’s territory,
which may range from 0.25 to 26.2 m2 . Females lay eggs on leaf
nests and tadpoles are transported later by the male to a temporary
puddle. Territorial behavior results in calling sites being at least a few
meters apart from each other, allowing calls to be recorded without
C 2008 The Author(s)
C 2008 by The Association for Tropical Biology and Conservation
Journal compilation 1
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Simões, Lima, Magnusson, Hödl, and Amézquita
distressing calling neighbors (Hödl 1987; Roithmair 1992, 1994;
Rodrı́guez & Duellman 1994).
The study of interpopulational variation on phenotypic traits,
such as external morphology and call characteristics, is useful for
evaluating divergence patterns, because populations of a single
species presumably developed unique characters in relatively recent
times. A more recent differentiation between groups of interest reduces the probability that groups showing intermediate phenotypes
have become extinct, and it is even possible that those populations
still inhabit the location where the divergence process took place,
providing unique opportunities for the study of the evolutionary
mechanisms that triggered divergence (Foster 1999, Moritz et al.
2000).
Considering their apparent restriction to forested environments, and consequently reduced interpopulation gene flow across
floodplains (Gascon et al. 1998, Lougheed et al. 1999), populations
of A. femoralis are potential models to test the role of the upper
Madeira River in differentiation of anuran populations.
METHODS
STUDY AREA.—We sampled forests along the upper Madeira River,
from the city of Porto Velho (8◦ 43 34 S, 63◦ 55 24 W) to
the mouth of the Abunã River, a western tributary (9◦ 40 41 S,
65◦ 25 58 W), in the State of Rondônia, Brazil. Along this stretch,
the muddy Madeira River is not meandering and flows between narrow floodplains, limited by pronounced slopes. On the right bank,
the landscape is a continuous flat plateau, originated from Pliopleistocenic sediments. On the left bank, the landscape is represented
by tabular formations of the same plateau and by fragments of a
largely eroded plateau associated with pre-Cambrian sediments. The
drainage system constituting this part of the Madeira River basin
runs primarily through tectonic valleys. Slope ruptures attributed to
tectonic faults along the river cause large rapids across the riverbed.
Climate is characterized by well-defined wet and dry seasons, which
directly influence water levels. On both riverbanks, vegetation is
constituted by low rain forest, with open pioneer vegetation on the
river margins (DNPM 1978, Souza Filho et al. 1999). It is not
known if plant composition and forest structure differ between geological domains or between riverbanks in the study area, but large
deforested gaps are found on the right riverbank, near populated
localities (Porto Velho, Jaci-Paraná, Mutum-Paraná).
SAMPLING DESIGN.—Initially, we established 19 sampling sites in
areas of terra firme forest along the upper Madeira River, ten on
the left bank and nine on the right bank (Fig. 1). Populations of
A. femoralis occur in patches in continuous-forest areas, so each
sampling site was searched for vocally active populations. We found
no populations of A. femoralis in two of the sampling sites established on the right bank (corresponding to the localities of Abunã
and Jirau), both of which had sites apparently appropriate for the
species, but no individuals. Therefore, we used samples from 17
sites. Minimum and maximum distances between effective sampling sites were of 1.3 and 168.0 km, respectively. We also searched
for contact zones along the left riverbank, where at least two mor-
FIGURE 1. Location of study area within the State of Rondônia, Brazil, and
relative position of sampling sites along the upper Madeira river, from the city
of Porto Velho (8◦ 43 34 S, 63◦ 55 24 W) to the mouth of the Abunã river
(9◦ 40 41 S, 65◦ 25 58 W). ‘N’ sampling sites correspond to areas where no
Allobates femoralis populations were found. White dots represent populations
of the two-note call morphotype and black dots represent populations of the
four-note morphotype. Relative position of the boundary between geological
domains was adapted from Souza Filho et al. (1999).
photypes of A. femoralis were known to occur. Each site was visited
once in the same reproductive season, from November 2004 to
January 2005.
Distance between sites was represented in analyses by the UTM
co-ordinates. As models used are additive, and each UTM coordinate represents an individual variable, distance was modeled
as City-Block distance rather than straight-line Euclidean distance
between the sites. However, as the system was nearly linear, the CityBlock distances are highly correlated with the Euclidean distances.
Euclidean distances were not used, as that would have required
Mantel-type analyses that would have confounded the effects of the
variables that are not easily represented as distances.
ACOUSTIC DATA COLLECTION AND ANALYSES.—We chose recording
sites distant from roads and boat routes to prevent anthropogenic
sounds from affecting male calling behavior and recording quality.
Recordings were conducted during daytime, at 0600–1800 h. Advertisement calls of 206 A. femoralis males were recorded with a
Sony WM-D6C tape recorder (2004, Sony Corr, Japan) and AKG
Frog Acoustic and Morphological Differentiation
568 EB directional microphone (2003, AKG Acoustics GMBH,
Austria) positioned 1.0 m from calling males. For each individual
recorded, air temperature at the calling site at the time of recording was registered. For each recording session, we selected the three
call samples that showed the least interference by environmental
noise, starting from the most central calls. Thus, we avoided selecting warm-up calls at the beginning of the calling bout, that vary
greatly in duration and frequency, and calls at the end of the bout,
which could show temporal and spectral variations due to fatigue
(Gerhardt & Huber 2002).
For each of the three call samples, we obtained temporal and
spectral variables using sonograms generated by Raven 1.2. software
(Charif et al. 2004). Spectral analysis was done by a fast Fourier
transform with a frequency resolution of 82 Hz and 2048 points.
Because two acoustic morphotypes were found in the study area,
the number of variables obtained for each group depended on the
number of notes that constituted its advertisement calls. Means,
standard deviations, and maximum and minimum values of all
acoustic variables for each sampling site are given in Table S1.
We used a principal component analysis (PCA) to reduce dimensionality and produce a smaller number of independent acoustic
variables. The first and second components generated by the analysis were used as dependent variables in a multivariate analysis of
variance (MANOVA) model, to test whether three groups defined
by the number of notes of advertisement calls and origin (i.e., right
or left bank) were significantly different from each other in terms of
acoustic characteristics. Body weight was included as a covariate in
the MANOVA, as body size can affect spectral features of anuran
calls (Ryan 1988, Keddy-Hector et al. 1992). Air temperature at the
time of recording was included as another covariate, as variation in
temperature can influence temporal features of advertisement calls
(Ryan 1988, Keddy-Hector et al. 1992, Gerhardt & Huber 2002).
Sampling-site co-ordinates in UTM units were also included as covariates, to represent distance between sites (Legendre et al. 2002),
because morphological and behavioral characteristics could vary
with geographic distance, independent of physical barriers. Early
components produced by PCA usually recover important patterns
of similarity between samples, while later components tend to accumulate variation related to noise (Gauch 1982). For the present
analysis and for other analyses described below, we used the minimum number of components necessary to explain at least 50 percent
of the total variation in our data sets.
Because two acoustic morphotypes were found in the study
area, the analysis described above was done twice. First, we used
a set of nine variables obtained only from the first and second
notes of the advertisement calls of all individuals sampled. In both
acoustic morphotypes, the first and second notes showed similar
spectrographic patterns, with first note always narrowly modulated
in frequency and short in duration, followed by a longer, broadly
modulated second note. Therefore, we assumed first and second
notes to be homologous in calls of both morphotypes. A second
analysis was carried out using 24 acoustic variables obtained from
the four notes constituting the advertisement calls of one of the
morphotypes that inhabited both riverbanks, to test the acoustic
divergence between populations of a single acoustic morphotype
living on opposite sides of the river.
3
MORPHOLOGICAL DATA COLLECTION AND ANALYSES.—Males were
collected after recording procedures, euthanized, and weighed after
the removal of the digestive tracts, because gut content represented
approximately 10 percent of total body mass in our samples (mean
gut content mass was 0.16 ± 0.08 g for an average body mass of
1.68 ± 0. 27 g, considering all individuals collected). Our measure
of body mass therefore reflects long-term differences in morphology
and not short-term differences due to the time an individual encountered a prey item. For each of the 209 individuals used, 19 external
morphological variables were measured using a digital caliper and
a stereoscopic microscope with a graduated ocular lens (precisions
0.01 and 0.10 mm). All variables were obtained from the left side
of the specimen. Means, standard deviations, and maximum and
minimum values of all morphological variables for each sampling
site are presented in Table S2. Voucher specimens were deposited in
the Herpetology Collections of the Instituto Nacional de Pesquisas
da Amazônia, Manaus, Brazil, accession numbers INPA-H16541
to INPA-H16823.
The 19 morphological variables were log 10 transformed for
scale adjustment. The first and second components generated by a
PCA were included in a MANOVA model as dependent variables
to test whether the three groups identified in the acoustic analysis
presented significant morphological differences.
Principal component analysis on external morphological variables usually produces a first principal component carrying heavy
size effects while the remaining components contain more information on body shape. Therefore, because direct measurements usually
vary continuously with body size, individual body weight was included in the MANOVA model as a covariate representing body
size (Reis et al. 1990, Strauss & Bond 1990). All morphological and
acoustic statistical procedures were done in SYSTAT 8.0 (Wilkinson
1990).
RESULTS
Two distinct morphotypes of A. femoralis were found in the study
area. One has reddish posterior ventral coloration and advertisement
calls constituted by two notes (Fig. 2A). This morphotype occurs
only on the upper left riverbank, and has a contact zone with the
other morphotype on the same bank, where there are no apparent
barriers to dispersal of individuals at the present time. The second
morphotype has black and white ventral coloration and advertisement calls constituted by four notes (Fig. 2B,C). The distribution
of this morphotype is split by the Madeira River (Fig. 1).
ACOUSTIC ANALYSES.—Principal component analysis using six spectral variables and two temporal variables present in the first and
second notes produced a first and a second component explaining
together 82.2 percent of the acoustic variation. The first component
(PC1) explained 58.9 percent of the total acoustic variation, and
spectral variables had high loadings on that component. The second
component (PC 2) explained 23.3 percent of the acoustic variation, and temporal variables had high loadings on that component
(Table S3). Both components were used as dependent variables in
a MANOVA to test if acoustic characters were statistically different
4
Simões, Lima, Magnusson, Hödl, and Amézquita
FIGURE 2. Spectrographic patterns of advertisement calls of individuals of the
two-note morphotype (A) and four-note morphotype recorded on the left (B)
and right (C) riverbanks. Calls were digitized from tape recordings using Raven
1.2 software, at 22,050 Hz and 16 bits.
among the three groups defined a priori: (1) two-note advertisement
call morphotype; (2) four-note advertisement call morphotype from
the left riverbank; and (3) four-note advertisement call morphotype
from the right riverbank.
MANOVA indicated that body mass (Pillai trace = 0.225;
P < 0.001, df = 192) and air temperature (Pillai trace = 0.043;
P = 0.015, df = 192) significantly affected the acoustic features
of the two notes. However, the three groups defined a priori were
significantly different from each other in relation to acoustic characteristics, independent of covariate effects (Pillai trace = 0.354;
P < 0.001, df = 192). Geographic distance between sampling sites
did not influence call features (Pillai trace = 0.035; P = 0.144,
df = 192).
We plotted the variable means per site on PC1 and PC2,
which represented spectral and temporal characters, respectively.
Populations from the right and left bank formed distinct groups
along PC1 (Fig. 3) as a result of the lower frequencies of calls and
notes of the populations inhabiting the right bank (Table S1). Sampling site No. 17 (St.Antonio-L), on the left bank, was closer in
multidimensional space to sampling sites on the right bank, showing low values for spectral characteristics. This site was located ca
30 km from another sampling site on the left bank, where individuals showed acoustic characteristics typical of that riverbank
(Fig. 3). Two-note morphotype populations (sites from no. 1–5)
tended to be different in temporal characteristics of calls, presenting
shorter notes in relation to four-note call populations from the left
riverbank.
It was expected that the two-note call morphotype on the left
side of the river would have sufficiently different acoustic features
to form a vocally distinct group. Thus, a second PCA was carried
out, using only samples of the four-note call morphotype. Besides
providing a larger number of acoustic variables (15 spectral and
9 temporal), the four-note call morphotype was present on both
riverbanks, so the river effect on acoustic traits could be tested for a
single morphotype.
The first and second components explained 51.2 and 18.0 percent of the acoustic variation, respectively. Again, spectral variables
had high loadings on PC 1 and temporal variables had high loadings
on PC 2 (Table S4). MANOVA showed that the covariates body
mass (Pillai trace = 0.293; P < 0.001, df = 140) and air temperature (Pillai trace = 0.227; P < 0.001, df = 140) significantly
affected acoustic features of the four notes. Nonetheless, acoustic
characteristics differed statistically between riverbanks, independent
of covariate effects (Pillai trace = 0.099; P = 0.001, df = 140). Distances between sampling sites did not influence call features (Pillai
trace = 0.028; P = 0.410, df = 140). Distribution of sampling sites
along the first and second PCA axes showed that samples from opposite riverbanks were separated mainly along PC1, which described
the spectral variation of calls (Fig. 3), reflecting lower frequencies of
calls recorded on the right bank (Table S1). A PC3 was related to the
variation in silent interval duration, but variation in this acoustic
trait occurred between individual sampling sites, and not between
groups of sites defined by riverbanks.
Variation explained by other components was never greater
than expected for components constituted by random variables,
and inclusion increases the chance of type II errors. However, analyses using more components, not reported here, produced similar
qualitative results to analyses with two components.
MORPHOLOGICAL ANALYSES.—A principal component analysis of 19
transformed morphological variables generated a first component
explaining 43.9 percent of the external morphological variation. All
variables used had positive loadings on that component. The second
principal component explained only 6.8 percent of the morphological variation.
First and second principal components were used as dependent variables in a MANOVA to test for morphological differences
between the three groups as defined in the acoustic analysis. Body
mass (Pillai trace = 0.507; P < 0.001, df = 199) and geographic
distance between sites (Pillai trace = 0.086; P = 0.002, df = 199)
had significant effects on morphological traits, but there were statistically significant differences among groups (Pillai trace = 0.208;
P < 0.001, df = 199), after accounting for the effects of covariates.
Groups were generally separated along the principal components, especially along PC1. As this component relates mostly to
body size, separation of populations on this axis is probably due to
larger individuals inhabiting the right riverbank (Table S2). Considering only the sampling sites on the left riverbank, samples of
the two-note call morphotype tended to form a group distinct from
the two-note morphotype along PC 2, which accounted mostly
Frog Acoustic and Morphological Differentiation
5
FIGURE 3. Distribution of population means along the original principal components scores generated from acoustic variables of the two notes present in calls of
both morphotypes (A) and variables obtained from the four notes of a single morphotype (B). Spectral divergence between populations of the right (R) and left (L)
riverbanks were evident in both analyses. On the left riverbank, populations of the two-note call morphotype (in gray) significantly differed in call characteristics from
populations of the four-note call morphotype. See text for statistical comparisons between groups.
FIGURE 4. Distribution of population means along the original scores of the
first and second principal components using external morphological variables.
PC1 accounts for great part of the morphological variation related to size.
Individuals taken from the right bank populations (R) were larger, on average,
than individuals from the left riverbank (L).
for head and limb measurements, except for sampling site no. 3,
Mutum-L (Fig. 4).
DISCUSSION
Many studies suggested that large Amazonian rivers represent distribution boundaries for species and distinct populations of primates
(Wallace 1852; Ayres & Clutton-Brock 1992; Peres et al. 1996;
Roosmalen et al. 1998, 2000) and birds (Hellmayr 1910, Capparella
1987, Hayes & Sewlal 2004). However, the only studies evaluating river boundaries on anurans in Amazonian lowlands were restricted to the Juruá River, in Brazil (Gascon et al. 1996, 1998,
2000; Lougheed et al. 1999). Those studies found no evidence of
differentiation between populations or communities of anurans on
opposite sides of the river, but some authors argue that shifts in the
river course and sediment deposit dynamics, typical of Amazonian
meandering rivers, such as the Juruá, can cause terrain to move
from one riverbank to another, passively transporting individuals
between riverbanks from time to time and homogenizing populations via interbreeding (Ayres & Clutton-Brock 1992, Moritz et al.
2000).
Our results clearly suggest that the upper Madeira River limits distinct populations of the poison-dart frog A. femoralis. The
two-note call morphotype is restricted to the left riverbank and
there is significant differentiation in acoustic and morphological
traits between populations of the four-note morphotype inhabiting
opposite riverbanks. In the study area, the Madeira River has an
average width of 1.0 km and certainly represents a considerable
barrier to forest-specialist anurans. The river runs mostly along an
incisive tectonic valley, predominantly crystalline and geologically
ancient (DNPM 1978), so it is improbable that changes of rivercourse orientation occurred in geologically recent times. A system
of successive tectonic faults (altitude decreases from west to east)
also results in a system of rapids along this section of the river, causing water velocity to be particularly fast. Passive transportation of
individuals from one riverbank to another by means of sedimentary
island dynamics is unlikely due to slow rates of sediment deposit.
Although populations of the four-note call on opposite banks
of the Madeira River had statistically significant differences, those
were greatly influenced by size-related characteristics of morphology
and calls. Body size affects calls due to physical relationships between
frequency wavelength and the dimensions of primary and secondary
oscillators (i.e., larynx, vocal cords, nostrils and vocal sacs; Gerhardt
6
Simões, Lima, Magnusson, Hödl, and Amézquita
& Huber 2002). Therefore, even within the same species, bigger
individuals tend to produce calls and notes with lower frequencies.
In at least one case on the left riverbank (sample site no. 17, Sto.
Antônio-L), we found individuals as large as the ones found on the
right riverbank, resulting in pronounced acoustic and morphological variation between localities separated by only a few kilometers,
on the same side of the river. Such exceptions to geographic patterns
are sometimes hypothesized to result from rare migration events and
weak selection pressures after a phenotypic divergence pattern was
developed under vicariance (Sokal & Oden 1978). Events like that
could occur downstream from the city of Porto Velho, where the
Madeira River reaches the Amazonian plains and strong sedimentation produces extremely dynamic fluvial islands (DNPM 1978).
Thus, unfixed differences between populations of the four-note
morphotype inhabiting opposite riverbanks could be a result of migration of individuals across the downstream section, where there is
greater potential for gene flow between riverbanks.
Although the upper Madeira River represents a clear boundary between the two- and four-note call morphotypes in a NW–
SE direction, there is no obvious barrier between morphs on the
left riverbank, in a NE–SW direction. The contact zone between
them coincides with the limits between two geomorphological domains, evidenced on the surface as a flattened plateau downstream—
Planalto Rebaixado da Amazônia Ocidental—and by an eroded, older
plateau upstream—Planalto Dissecado Sul da Amazônia (DNPM
1978, Souza Filho et al. 1999). This boundary crosses the left bank
of the Madeira River almost at a right angle and causes a slope
rupture, forming a group of rapids, locally known as Cachoeira do
Jirau (Fig. 5). At the present time, the geomorphological contact
zone does not show any physical barriers that could prevent individual A. femoralis from crossing. The recent tectonic history of the
upper Madeira River is poorly known and little can be said about
FIGURE 5. The contact zone between Allobates femoralis morphotypes on the
left riverbank meets the riverbed almost at a right angle and coincides with the
boundary between two geomorphological domains, represented by plateaus of
different origins.
potential vicariant events associated with past tectonic phenomena.
Thus, at this time, it is not possible to determine whether there was
an effective vicariant barrier at the contact zone between A. femoralis
morphotypes or their divergence occurred in sympatry.
Tectonic events have been previously suggested as possible
factors generating genetic population divergence in A. femoralis.
Lougheed et al. (1999) suggested that factors other than the riverbed
could have generated patterns of genetic divergence in populations
along the Juruá River. The tectonic arch of Iquitos (a rocky crest
formed during the genesis of the Andes), was found to cross the
Juruá River at a right angle and coincides with sampling sites showing high genetic divergence. The contact zone between A. femoralis
morphotypes on the left bank of the Madeira River is not coincident with any of the proposed locations of tectonic arches (Räsänen
1990). Also, if a tectonic arch was responsible for the differentiation,
evidence of a contact zone between different groups of populations
should be present on the right riverbank, where only relatively uniform populations of the four-note morphotype occur.
Haffer (1997a) described the occurrence of at least 20 contact
zones between 40 bird taxa in Amazonia. Those zones occur in
continuous rain forest areas and generally cross large Amazonian
rivers at right angles, providing a similar pattern to what is observed
for A. femoralis. He considered them as suture zones between taxa
that diverged in forest refugia during the Pleistocene, supporting his
refuge theory as an explanation for the existence of such lines, and
discarding ecological factors as determinants of those zones. As we
lack detailed information on microhabitat structure and vegetation
composition in our study area, we cannot reject at this point that
an ecological mechanism of adaptation to a discrete environmental
gradient across geological domains (sensu Endler 1982) is the driving
force that originated or maintains the A. femoralis contact zone.
Our results provide scope for a taxonomic reinterpretation of
two- and four-note call morphotypes based on their phenotypic
differences, as it is highly probable that each morphotype represents a well-delimited evolutionary lineage. Ongoing phylogenetic
and population genetic approaches will help to clarify their evolutionary relationships and evaluate current gene flow. Information
provided by those analyses will also be used to test alternative diversification hypotheses (river-barrier, forest refugia, tectonic arches,
among others) that can explain the current distribution pattern
of phenotypic differentiation in A. femoralis along the Madeira
River.
Establishing the taxonomic limits between species is a key step
for planning conservation strategies based on beta diversity. Cryptic
anuran species are thought to be frequent among widespread taxa
in the Amazon region, and lack of detailed data on variation within
species is still a great impediment to the recognition of diversity at
this level (Azevedo-Ramos & Gallati 2002). Mapping population
variability within-species is also an important conservation tool to
maximize potential adaptation and persistence of species in face of
contemporary environmental changes. Our study reports that populations included in what is thought to be a single anuran species
(Allobates femoralis) present significant phenotypic differences, part
of which is concordant with their division by a large Amazonian
river. These findings have implications for both the planning of
Frog Acoustic and Morphological Differentiation
amphibian conservation strategies in the Amazon and the management of conservation units along the Madeira River.
ACKNOWLEDGMENTS
We thank K. Vulinec, J. Nicolás Urbina-Cardona, and one anonymous reviewer for many helpful comments that improved this paper.
We thank R. Lima, E. Vasconcelos, A. Coelho, A. da Silva, F. Barbosa, M. de Oliveira, C. de Miranda, and J. Rocha for helping
us in the field. Collecting permits were provided by RAN-IBAMA
(license no. 131/04-RAN, p. 02010.003199/04-76). We thank L.
K. Erdtmann and S. Victoria Flechas for helping us with the sound
analyses. Furnas Centrais Elétricas S. A. provided funding and logistics for this project. Travel expenses of WH and E. Vasconcelos, and
flight tickets for PIS were provided by the Austrian Science Foundation (project FWF 15345-B03, proj. leader: WH). PIS received
a fellowship from Brazilian CAPES during this study.
SUPPLEMENTARY MATERIAL
The following supplementary material for this article is available
online at: www.blackwell-synergy.com/loi/btp
Table S1. Means, standard deviations and amplitude of acoustic
variables obtained from advertisement calls of Allobates femoralis.
Table S2. Means, standard deviations and amplitude of body
weight, snout-to-vent length and 19 other morphological variables obtained from Allobates femoralis males.
Table S3. Results of PCA using three temporal and six spectral
acoustic variables from the first and second notes present in the advertisement calls of both Allobates femoralis morphotypes.
Table S4. Results of PCA using nine temporal and 18 spectral
variables obtained from the 4-note calls of the morphotype present on
both riverbanks.
LITERATURE CITED
AYRES, J. M., AND T. H. CLUTTON-BROCK. 1992. River boundaries and species
range size in Amazonian primates. Am. Nat. 140: 531–537.
AZEVEDO-RAMOS, C., AND U. GALATTI. 2002. Patterns of amphibian diversity
in Brazilian Amazonia: Conservation implications. Biol. Conserv. 103:
103–111.
BUSH, M. B. 1994. Amazonian speciation: A necessarily complex model. J.
Biogeogr. 21: 5–17.
CALDWELL, J. P. 1996. The evolution of myrmecophagy and its correlates in
poison frogs (Family Dendrobatidae). J. Zool. 240: 75–101.
CAPPARELLA, A. P. 1987. Effects of riverine barriers on genetic differentiation of
Amazonian forest undergrowth birds. PhD Dissertation. Louisiana State
University. Baton Rouge, Louisiana.
CHARIF, R. A., C. W. CLARK, AND K. M. FRISTRUP. 2004. Raven 1.2 User’s
Manual. Cornell Laboratory of Ornithology, Ithaca, New York.
DNPM - DEPARTAMENTO NACIONAL DA PRODUÇÃO MINERAL – BRASIL. 1978.
Projeto RADAMBRASIL—Levantamento dos recursos naturais. DNPM,
Rio de Janeiro, Brazil.
ENDLER, J. A. 1982. Problems in distinguishing historical from ecological factors
in biogeography. Am. Zool. 22: 441–452.
7
FOSTER, S. A. 1999. The geography of behaviour: An evolutionary perspective.
Trends Ecol. Evol. 14: 190–195.
GASCON, C., S. C. LOUGHEED, AND J. P. BOGART. 1996. Genetic and morphological variation in Vanzolinius discodactylus: A test of the river hypothesis
of speciation. Biotropica 28: 376–387.
GASCON, C., S. C. LOUGHEED, AND J. P. BOGART. 1998. Patterns of genetic
population differentiation in four species of Amazonian frogs: A test of
the Riverine Barrier Hypothesis. Biotropica 30: 104–119.
GASCON, C., J. R. MALCOLM, J. L. PATTON, M. N. F. SILVA, J. P. BOGART, S. C.
LOUGHEED, C. A. PERES, S. NECKEL, AND P. T. BOAG. 2000. Riverine
barriers and the geographic distribution of Amazonian species. Proc.
Natl. Acad. Sci. USA 97: 13672–13677.
GAUCH, H. G. 1982. Noise reduction by eigenvector ordinations. Ecology 63:
1643–1649.
GERHARDT, H. C., AND F. HUBER. 2002. Acoustic communication in insects
and anurans: Common problems and diverse solutions. University of
Chicago Press, Chicago, Illinois.
HAFFER, J. 1974. Avian speciation in Tropical South America: With a systematic survey of the toucans (Ramphastidae) and jacamars (Galbulidae).
Publications of the Nuttall Ornithological Club 14: 1–390.
HAFFER, J. 1997a. Contact zones between birds of southern Amazonia. Ornithol.
Monogr. 48: 281–305.
HAFFER, J. 1997b. Alternative models of vertebrate speciation in Amazonia: An
overview. Biodivers. Conserv. 6: 451–476.
HAYES, F. E., AND J. N. SEWLAL. 2004. The Amazon River as a dispersal barrier to
passerine birds: effects of river width, habitat and taxonomy. J. Biogeogr.
31: 1809–1818.
HELLMAYR, C. E. 1910. The birds of the Rio Madeira. Novitates Zoologicae 17:
257–428.
HÖDL, W. 1987. Dendrobates femoralis (Dendrobatidae): A handly fellow for frog
bioacoustics. In Proceedings of the Fourth Ordinary General Meeting of
the Societas Europaea Herpetologica. Nijmegen, 1987, pp. 201–204.
KEDDY-HECTOR, A. C., W. WILCZYNSKI, AND M. J. RYAN. 1992. Call patterns
and basilar papilla tuning in cricket frogs. II. Intrapopulation variation
and allometry. Brain Behav. Evol. 39: 238–246.
LEGENDRE, P., M. R. T. DALE, M. J. FORTIN, J. GUREVITCH, M. HOHN, AND D.
MYERS. 2002. The consequences of spatial structure for the design and
analysis of ecological field surveys. Ecography 25: 601–615.
LOUGHEED, S. C., C. GASCON, D. A. JONES, J. P. BOGART, AND P. T. BOAG.
1999. Ridges and rivers: A test of competing hypothesis of Amazonian
diversification using a dart-poison frog (Epipedobates femoralis). Proc. R.
Soc. Lond. Ser. B 266: 1829–1835.
MORITZ, C., J. L. PATTON, C. J. SCHNEIDER, AND T. B. SMITH. 2000. Diversification of rainforest faunas: An integrated molecular approach. Annu.
Rev. Ecol. Syst. 31: 533–563.
PATTON, J. L., M. N. F. SILVA, AND J. R. MALCOLM. 2000. Mammals of the Rio
Juruá and the evolutionary and ecological diversification of Amazonia.
Bull. Am. Mus. Nat. Hist. 44: 1–305.
PERES, C. A., J. L. PATTON, AND M. N. F. SILVA. 1996. Riverine barriers and
gene flow in Amazonian Saddle-Back Tamarins. Folia Primatologica 67:
113–124.
RÄSÄNEN, M. E., J. S. SALO, H. JUNGNER, AND L. R. PITTMAN. 1990. Evolution of the Western Amazon lowland relief: Impact of Andean foreland
dynamics. Terra Nova 2: 320–332.
REIS, S. F., L. M. PESSÔA, AND R. E. STRAUSS. 1990. Application of size-free
canonical discriminant analysis to studies of geographic differentiation.
Rev. Bras. Genética 13: 509–520.
RODRÍGUEZ, L. O., AND W. E. DUELLMAN. 1994. Guide of the frogs of the Iquitos
Region, Amazonian Peru. University of Kansas Publications [Natural
History Museum -Special Publications, 22], Lawrence, Kansas.
ROITHMAIR, M. E. 1992. Territoriality and male mating success in the DartPoison Frog, Epipedobates femoralis (Dendrobatidae, Anura). Ethology
92: 331–343.
ROITHMAIR, M. E. 1994. Field studies on reproductive behaviour in two dartpoison frog species (Epipedobates femoralis, Epipedobates trivittatus) in
Amazonian Peru. Herpetol. J. 4: 77–85.
8
Simões, Lima, Magnusson, Hödl, and Amézquita
ROOSMALEN, M. G. M., T. ROOSMALEN, R. A. MITTERMEIER, AND G.
A. B. FONSECA. 1998. A new and distinctive species of marmoset (Callitrichidae, Primates) from the lower river Aripuanã, State
of Amazonas, central Brazilian Amazonia. Goeldiana Zoologia 22:
1–27.
ROOSMALEN, M. G. M., T. ROOSMALEN, R. A. MITTERMEIER, AND G. A. B.
FONSECA. 2000. A taxonomic review of the Titi Monkeys, genus Callicebus, Tomas, 1903, with the description of two new species, Callicebus bernhardi and Callicebus stephennashi, from Brazilian Amazonia.
Neotrop. Primates 10: 1–52.
RYAN, M. J. 1988. Energy, calling, and selection. Am. Zool. 28: 885–
898.
SOKAL, R. R., AND N. L. ODEM. 1978. Spatial autocorrelation in biology 2.
Some biological implication and four applications of evolutionary and
ecological interest. Biol. J. Linn. Soc. 10: 229–249.
SOUZA FILHO, P. W. M., M. L. E. S. QUADROS, J. E. SCANDOLARA, E. P.
SILVA FILHO, AND M. R. REIS. 1999. Compartimentação morfoestrutural e neotectônica do sistema fluvial Guaporé-Mamoré-Alto Madeira,
Rondônia – Brasil. Rev. Bras. Geociências 29: 469–476.
STRAUSS, R. E., AND C. E. BOND. 1990. Taxonomic methods: morphology. In
P. Moyle and C. Schreck (Eds.). Methods for fish biology, pp. 109–140.
American Fisheries Society, Special Publication.
WALLACE, A. R. 1852. On the monkeys of the Amazon. Proc. Zool. Soc. Lond.
20: 107–110.
WILKINSON, L. 1990. SYSTAT: The system for statistics. SYSTAT inc., Evanston,
Illinois.