2012 Detecting cryptic speciation in the widespread and morphologically conservative carpet chameleon (Furcifer lateralis) of Madagascar

doi: 10.1111/j.1420-9101.2012.02528.x
Detecting cryptic speciation in the widespread and morphologically
conservative carpet chameleon (Furcifer lateralis) of Madagascar
A. M. FLORIO*, C. M. INGRAM , H. A. RAKOTONDRAVONYà, E. E. LOUIS JR§
& C. J. RAXWORTHY*
*Richard Gilder Graduate School, Department of Herpetology, American Museum of Natural History, New York, NY, USA
Division of Vertebrate Zoology, American Museum of Natural History, New York, NY, USA
àDépartement de Biologie Animale, Université d’Antananarivo, Antananarivo, Madagascar
§Center for Conservation and Research, Henry Doorly Zoo, Omaha, NE, USA
Keywords:
Abstract
canonical variates analysis;
cryptic species;
ecological niche modelling;
phylogeography;
species delimitation.
Species delimitation within recently evolved groups can be challenging
because species may be difficult to distinguish morphologically. Following the
General Lineage Concept, we apply a multiple evidence approach to assess
species limits within the carpet chameleon Furcifer lateralis, which is endemic
to Madagascar and exported in large numbers for the pet trade. Cryptic
speciation within F. lateralis was considered likely because this species (1) has a
vast distribution, (2) occupies exceptionally diverse habitats and (3) exhibits
subtle regional differences in morphology. Phylogenetic trees reconstructed
using nuclear and mitochondrial genes recovered three well-supported clades
corresponding with geography. Morphological results based on canonical
variates analysis show that these clades exhibit subtle differences in head
casque morphology. Ecological niche modelling results found that these
phylogenetic groups also occupy unique environmental space and exhibit
patterns of regional endemism typical of other endemic reptiles. Combined,
our findings provide diverse yet consistent evidence for the existence of three
species. Consequently, we elevate the subspecies F. lateralis major to species
rank and name a new species distributed in northern and western Madagascar.
Initial ecological divergence, associated with speciation of F. lateralis in humid
eastern habitat, fits the Ecographic Constraint model for species diversification
in Madagascar. By contrast, the second speciation event provides some support
for the Riverine Barrier model, with the Mangoky River possibly causing
initial isolation between species. These findings thus support two contrasting
models of speciation within closely related species and demonstrate the utility
of applying a combined-evidence approach for detecting cryptic speciation.
Introduction
Identifying species limits is challenging for recently
evolved groups due to the stochastic nature of gene
sorting (Knowles & Carstens, 2007), difficulties in developing robust diagnoses (Shaffer & Thomson, 2007) and
Correspondence: Antonia M. Florio, Richard Gilder Graduate School,
Department of Herpetology, American Museum of Natural History,
Central Park West at 79th Street, New York, NY, USA.
Tel.: +1 212 769 5859; fax: +1 212 769 5031; e-mail: [email protected]
branch length heterogeneity (Edwards, 2009). However,
multiple-locus approaches, along with new methods that
utilize morphological characters and niche differences,
can overcome these constraints (Raxworthy et al., 2007;
Rissler & Apodaca, 2007; Hickerson et al., 2010; Glor &
Warren, 2011). This integrated approach has been
promoted to reconcile differences in species delimitation
approaches among disciplines and to strengthen the
validity of species hypotheses (Dayrat, 2005; DeSalle
et al., 2005; Padial et al., 2010). In addition, integrative
taxonomy offers new opportunities for improving the
ª 2012 THE AUTHORS. J. EVOL. BIOL. 25 (2012) 1399–1414
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A . M. FL O R I O E T A L .
sensitivity of species recognition, especially in groups that
have undergone recent or cryptic speciation (Padial & De
La Riva, 2009).
The identification of cryptic species is critically important for many reasons, including accurate assessment of
biodiversity estimates, facilitating disease and crop plant
pathogen control, and directing conservation efforts
towards vulnerable endemic species (Bickford et al.,
2007). In addition, cryptic speciation may result from
recently evolved sibling species where species limits can
be ambiguous because morphological differences have
not yet accumulated (Knowlton, 1993). These recently
evolved species are good candidates for speciation
research because these groups more closely meet the
assumption that the species’ geographic range has not
changed over time (Losos & Glor, 2003). Further,
research into cryptic species limits can help to both
identify and conserve the processes currently driving
speciation in various groups and regions (Carnaval et al.,
2009).
Situated off the south-eastern coast of Africa, the
island of Madagascar is a model region for cryptic species
identification and studies of speciation (Vences et al.,
2010). Madagascar and Greater India first broke away
from Africa as early as 165 Ma, and Madagascar has
been isolated since separating from Greater India about
88 Ma (Storey et al., 1995). This island continent has a
complicated and poorly understood paleoclimatic history, but some information is known. Madagascar
experienced a generally dry environment when first
separated from India (Wells, 2003), but the overall
climate became more humid as it drifted northwards
towards the equator. However, the climate again became
drier and cooler during glacial periods, with areas at
lower elevations experiencing more pronounced aridification than those at higher elevations (Haffer, 1969).
Presently, the island is composed of striking environmental heterogeneity, with habitat transitions occurring
abruptly. Trade winds and orographic uplift ensure
regular rainfall on the north-east and eastern coast,
and the central mountain chain acts as a barrier causing
a rainfall gradient from the humid north-eastern and
eastern rainforests to the south-western spiny deserts
(Jury, 2003). In addition, there is complex topography
(maximum elevation 2876 m), seasonal rainfall patterns
and drainage systems, all which contribute to the
environmental complexity.
Several hypotheses about the mechanisms driving speciation in Madagascar have been proposed, and many of
these are related to either the paleoclimate or current
climate of the island. The Watershed hypothesis (Wilme
et al., 2006) proposes that glaciation periods resulting in
severe arid conditions at lower elevations caused species to
move to higher elevations towards more humid areas.
Species distributed in lower elevation watersheds
became trapped in arid pockets, and adapted and diversified in isolation. The Montane Endemism hypothesis
(Raxworthy & Nussbaum, 1995; Wollenberg et al., 2008)
proposes that some populations of species broadly distributed during glacial periods became isolated on mountaintops during warmer interglacials. The hypothesis of
ecologically mediated speciation (Raxworthy et al., 2007,
2008) proposes that the niches of sister species become
divergent as they adapt to ecotones under disruptive
selection and assortative mating. The Riverine Boundary
hypothesis (Pastorini et al., 2003; Goodman & Ganzhorn,
2004) proposes that the river systems in Madagascar have
restricted gene flow causing isolation between populations. Lastly, the Ecogeographic Constraint hypothesis
(Yoder & Heckman, 2006) proposes that the abrupt
distinction between the climates, rainfall patterns, and
vegetation of eastern and western Madagascar allows for
initial east–west divergence within widely distributed
species, with subsequent speciation constrained within
eastern and western regions. This hypothesis is an
example of ecologically mediated speciation, where initial
speciation can be either allopatric or parapatric.
Understanding the processes driving speciation in
Madagascar is of interest because an exceptional number
of new endemic species continue to be described (Myers
et al., 2000; Vieites et al., 2009). Some of these new
descriptions have resulted from the splitting of previously
recognized monotypic genera. For example, until
recently (see Miller, 1977) mouse lemurs (Microcebus)
were considered to be a single species (Microcebus murinus), but are now split into at least 15 species using
phylogeographic and morphometric analyses (Rasoloarison et al., 2000; Yoder et al., 2000; Louis et al., 2006,
2008). Many other phylogeographic studies have shown
other taxa in Madagascar to represent complexes of
cryptic species (e.g. Raxworthy et al., 2007; Raselimanana et al., 2009). In particular, the identification of
recently evolved species, which are often cryptic, offers
important opportunities for improving our understanding of the processes that drive speciation in Madagascar.
A candidate cryptic species complex is the carpet
chameleon Furcifer lateralis (Gray, 1845), which is
endemic to Madagascar. The carpet chameleon is a
popular and familiar species in the pet trade and
consequently is exported in high numbers. Less known
is that F. lateralis is also an excellent model organism for
understanding the processes driving speciation. Although
most Malagasy chameleons exhibit considerable regional
endemism, paradoxically, F. lateralis is one of the most
widely distributed endemic reptiles in Madagascar (Raxworthy et al., 2003). The species is distributed in nearly
all areas of Madagascar, with the exception of montane
regions above 1780-m in elevation and a small high
precipitation region in the north-east. It is also often
found at high population densities in severely degraded
forests, grasslands, agricultural areas and urban environments (Karsten et al., 2009; Randrianantoandro et al.,
2009). This chameleon exhibits subtle regional variation
in axillary pit development, white line development
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Cryptic species in a Malagasy chameleon
under the tail, scalation and head casque height (Hillenius, 1959; Brygoo, 1971), but for the last 50 years has
been consistently considered a single species. The only
phylogenetic study to date found multiple geographically
structured clades within F. lateralis using the mitochondrial gene 16S for 31 individuals, with strongest support
for a southern clade (Boumans et al., 2007).
Although F. lateralis has long been recognized as a single
species, Angel (1921) described Furcifer lambertoni,
collected from Tananarive (=Antananarivo), which he
distinguished from F. lateralis using the following
F. lambertoni features: (1) absence of gular crest, (2) homogeneous squamation on the body and limbs, (3) 70 scales
between dorsal ridge and midventral line, and (4) tail
shorter than snout-vent length (SVL). Hillenius (1959)
found that three of these states (2–4) were found within
F. lateralis, decided that the absence of the gular crest was
insignificant and considered F. lambertoni a junior synonym of F. lateralis. This view was later supported by
Brygoo (1971) and Klaver & Böhme (1997). A geographic
large-sized variant, ‘forme major’ from the arid south-west
region of Madagascar, was described by Brygoo (1971)
and subsequently treated as the subspecies F. lateralis
major by Klaver & Böhme (1997).
In this study, we use the General Lineage Concept
(GLC), which uses the the term ‘species’ for separately
evolving metapopulation lineages that can be recognized
using diverse secondary recognition criteria (De Queiroz,
2007). We here apply three secondary recognition
criteria to assess species limits within F. lateralis, using
the following approach: (1) identify lineages based
on clades from analysis of multiple molecular loci,
(2) employ canonical variates analysis (CVA) to determine potential morphological variation associated with
phylogenetic groups and (3) use ecological niche modelling to determine the environmental and geographic
space occupied by groups supported by covariation of
genetic and morphological evidence.
Materials and methods
Focal species and sampling
A total of 111 F. lateralis individuals, collected between
1990 and 2011, were included for phylogenetic analysis.
The close outgroup species Furcifer labordi, Furcifer oustaleti and Furcifer verrucosus were included to test the
monophyly of F. lateralis (Raxworthy et al., 2002; Townsend and Larson, 2002), resulting in a total matrix of 121
terminals. Furcifer campani was used as the far outgroup
taxon to root all phylogenetic trees. In most cases,
chameleons were collected during night surveys during
the rainy season (approximately December through
April) using headlamps to find individuals roosting on
vegetation; a detailed description of collection methods is
provided in Raxworthy & Nussbaum (2006). Date, time
and longitude ⁄ latitude of each individual (using GPS,
1401
altimeter or 1 : 100 000 topographic maps) were recorded at time of collection. Voucher specimens were
euthanized and fixed in 10% buffered formalin and then
later transferred to 70% ethanol. Liver and ⁄ or thigh
muscle was preserved in 95% ethanol or tissue buffer for
DNA extraction. Voucher specimens and tissues are
deposited at the American Museum of Natural History
(AMNH), the University of Michigan Museum of Zoology
(UMMZ), Duke University Department of Biology and
the University of Antananarivo Department of Animal
Biology (UADBA). Abbreviations for field series are RAN
(Ronald A. Nussbaum), RAX (Christopher J. Raxworthy)
and HER (Hery A. Rakotondravony). Localities, sample
numbers, coordinates, and Genbank accession numbers
for all samples are provided in the Table S1.
Phylogenetic analysis
DNA was extracted from all tissue samples using the
QIAGEN DNeasy Blood & Tissue kit (Valencia, CA, USA)
following the manufacturer’s instructions. Three mitochondrial genes – 16S ribosomal RNA, NADH dehydrogenase subunit 2 (ND2) and NADH dehydrogenase 4
(ND4) – and two nuclear genes – recombination-activating gene-1 (RAG1) and matrix remodelling-associated
gene (MXRA5) – were amplified. Polymerase chain
reaction was carried out under locus-specific optimal
annealing temperatures (see Table S2). PCR products
were cleaned using MultiScreen PCRl96 Filter plates
(Millipore, Billerica, MA, USA) and sequenced in both
directions using BI G DY E v.3.0 (Applied Biosystems, Foster
City, CA, USA) on an ABI 3730 automated DNA
sequencer. Sequences were edited in G E N E I O U S v.4.8.3
(Biomatters, Auckland, New Zealand). Multiple sequence
alignments were generated using MUSCLE (Edgar, 2004),
with 1000 iterations and default gap opening cost of )1.
Leading and lagging ends were trimmed to remove any
missing data at the alignment edges.
Phylogenetic analyses were conducted using maximum parsimony (MP) and maximum likelihood (ML).
MP was carried out with T N T v1.1 (Goloboff et al., 2008)
and WI N C L A D A v1.0 (Nixon, 2002) with equal weighing
of all characters, and the heuristic search option set at
500 random addition replicates using the New Technology search option. Bootstrap support values were calculated for MP with 500 random addition replicates under a
full heuristic search with 10 random addition sequences
for each. ML was carried out in RAX ML (Stamatakis,
2006) with the RAX MLG U I 0.93 (Silvestro & Michalak,
2010) using the ML + thorough bootstrap analysis option
with 10 runs and 500 repetitions. Due to the large
number of individuals included in the analysis and the
low genetic divergence found between individuals, the
GTR + CAT algorithm was used as it allows for a rapid
navigation into a search space in which trees score well
under GTR + G but at significantly lower computational
costs and memory consumption (Stamatakis, 2006).
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Gene tree concordance and divergence between
putative species
Gene tree concordance was assessed by analysing each
locus individually using ML. The three mitochondrial
genes were also combined and analysed using both ML
and MP. Haplotypes for nuclear sequences were phased
using an approach employed by P H A S E v2.1 (Stephens
et al., 2001; Stephens & Sheet, 2005) and implemented
using DN A SP v5 (Librado & Rozas, 2009). Runs consisted
of 1000 main iterations with an initial 1000 iterations for
burn-in and a thinning interval of 1. S P L I T S T R E E v4.12.13
(Hudson and Bryant, 2006) was used to identify identical
haplotypes and to reconstruct haplotype median-joining
networks for each nuclear locus. To assess the divergence
between and within potential species, the mean number
of mitochondrial nucleotide substitutions between
groups (Dxy) and nucleotide diversity (Pi) within groups
(Takahata & Nei, 1985) was assessed also using DN A SP
v5, with individuals with ambiguity codes in their
sequences excluded from analysis.
Morphological analysis
A total of 87 adult males, including Gray’s (1854) adult
syntype of F. lateralis BMNH 1946.8.22.12 (the other
syntype is a juvenile), and 53 adult females were
subjected to morphometric analysis. A list of the examined specimens is provided in the Table S3. Adults were
defined as exceeding 60 mm SVL, and chameleons sexed
based on the presence of everted hemipenes (males) or
the presence or absence of hemipenal bulges at the tail
base. High-resolution photographs of a lateral view of the
left side of each specimen’s head were obtained using a
Nikon D5000 Digital SLR Camera with a Nikon AF-S DX
18–55 mm lens on graph paper with a ruler visible to
record scale. Ten landmarks (see Fig. S1) were defined
that could be consistently placed across individuals and
provide adequate coverage of form (Zelditch et al., 2004);
these landmarks were placed on the images using the
digital images of the specimen and tpsUtil (Rohlf, 2004)
with tpsDIG (Rohlf, 2001). Specimens with preparation
irregularities that affect landmark placement (i.e. open
mouths or contortions caused by preservation) were
excluded from the study.
The program Morpho-J (Klingenberg, 2011) was used
to apply a Procrustes superimposition allowing standardization of size and landmark configurations, to generate
covariance matrices and to perform CVA. CVA finds
shape values that maximize group means relative to
variation within groups, by assuming that within-group
covariates matrices are identical (Klingenberg, 2010).
Groups for CVA were predefined based on the major
clades recovered from the phylogenetic analyses. At least
one individual from each population included in this
study was sequenced for molecular data, and morphological specimens lacking molecular data were predefined
based on their population assignment. The male F.
lateralis syntype was not predefined to a group, but
instead included as a separate classification to assess its
relationship to the other groups. Significance for differences across these groups was determined using permutation tests (50000) with Procrustes and Mahalanobis
distances, using Morpho-J. Both tests were used to assess
significance because P-values can differ due to the
anisotropy (direction dependency) of shape variation
(Klingenberg & Monteiro, 2005). Male and female specimens were analysed independently as F. lateralis exhibits
sexual dimorphism in head morphology (Brygoo, 1971).
The morphology of the specimens was described using
standard morphological terms and methods (see Raxworthy & Nussbaum, 2006). The following measurements and scale counts were used for species
identification: head casque height was measured as the
distance on the lateral crest, from where it began turning
vertical to the top of the head casque; head height was
measured as in Hopkins & Tolley (2011 – see Fig. 2) by
measuring the distance from the back of the lower jaw to
the tip of the casque; parietal crest scale counts were
taken by counting the tubercles on the raised parietal
crest.
Distribution mapping and ecological niche models
After deletion of duplicate records, 110 unique localities
were included for the development of the ecological
niche models (ENM) for the F. lateralis complex. As with
the morphometric analysis, localities were partitioned
based on phylogenetic results. Climate data was taken
from the WorldClim database (Hijmans et al., 2005;
http://worldclim.org/), with the 19 bioclimatic variables
used for ENM analyses in MA X E N T V 3 .3.31 (Phillips et al.,
2006). All occurrence localities and environmental variables were resampled to an oblique Mercator projection
at 1 km2 resolution (Pearson et al., 2007) using AR C MA P
(ESRI, 2011). Default values were used for the maximum
number of iterations (500) and for the convergence
threshold (10)5). The minimum training presence [or
lowest predicted value (LPT) of environmental suitability] was chosen for each model as the decision threshold.
The ENM was visualized in AR C MA P by reclassifying the
continuous data to create a binary prediction, and all
values above the LPT were reclassified as suitable
environment.
Model validation was assessed using k-fold partitioning of the data with six replicates (k = 6), as implemented in Maxent using the cross-validation option.
With k-fold partitioning, occurrence points are randomly split in k parts, then one part is used as a test
set for assessing model performance and the remaining
sets (k ) 1) are used to train the model (Fielding &
Bell, 1997). The number of k-fold partitions typically
varies between 3 and 10 depending on the number of
species occurrence records (Hirzel et al., 2006); here,
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Cryptic species in a Malagasy chameleon
k = 6 was chosen so that the data could be evenly
distributed across the partitions. The area under the
receiver operating characteristic curve (AUC; here
reported as a mean AUC calculated among trials) was
also used to evaluate the model as it provides a
measure of accuracy not dependent on a threshold
(Fielding & Bell, 1997).
Results
Phylogenetic analyses
Gene amplification was successful with all 111 ⁄ 111
F. lateralis individuals sequenced for 368-bp 16S, 622-bp
ND4 and 630-bp RAG1. Additionally, 720-bp ND2 were
sequenced for 110 of 111 F. lateralis individuals, and 635bp MXRA were sequenced for 108 of 111 F. lateralis
individuals. Phylogenetic trees from the individual genes
were not well resolved (data not shown). The mitochondrial genes 16S and ND2 were inconclusive with respect
to the relationship of the sister species F. labordi to
F. lateralis. ND4 recovered a monophyletic F. lateralis
Furcifer campani
**/**
1403
complex, but this also received low support (40% bootstrap support), but with some evidence for further genetic
substructuring within the complex. The two nuclear
genes RAG1 and MXRA also provided poor resolution,
although RAG1 provided weak support for the monophyly of the F. lateralis complex, with 54% bootstrap
support. Combined, the three mitochondrial and two
nuclear genes resulted in a 2975 character matrix.
A heuristic search using MP resulted in 403 equally
parsimonious trees (tree length = 3255). The large number of equally parsimonious trees resulted from minor
incongruence between individuals from geographically
close populations, but deeper tree topology was congruent across all trees. ML recovered a tree congruent with
the MP strict consensus. The ML tree is congruent with
the MP tree and is shown in Fig. 1a. Furcifer lateralis is
supported as a monophyletic group (bootstrap support
values: MP 84%, ML 82%), and there are three obvious
and major clades recovered within the complex that are
well-supported and correspond with geography:
(1) eastern (MP 100%, ML 100%), (2) southern (MP
93%, ML 86%) and (3) north-west (MP 100%, ML
Furcifer verrucosus
Furcifer oustaleti
Furcifer labordi
**/**
**/99
**/**
Furcifer lateralis
‘eastern’
84/82
**/**
Furcifer lateralis
‘north-west’
99/**
North-west
ENM
93/86
Southern
ENM
Furcifer lateralis
‘southern’
Eastern
ENM
(a)
0.02
(b)
Overlap
between
models
Fig. 1 (a) Phylogenetic relationships within the Furcifer lateralis complex and near outgroups resulting from the analysis of 121 individual
chameleons and 2975 characters (partial 16S, ND2, ND4, RAG1 and MXRA5) reconstructed on the maximum likelihood (ML) tree (congruent
with MP). Three well-supported clades (eastern = blue, southern = yellow and north-west = green) are recovered within the F. lateralis
complex (bootstrap support values for MP ⁄ ML, **100%). (b) Ecological niche models for each clade of the F. lateralis complex, projected onto
Madagascar, with collecting localities. MP, maximum parsimony.
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(a)
Outgroups
Furcifer labordi
**/**
**/99
**/99
-/64
(b)
Furcifer lateralis
‘eastern’
RAX11575-2
RAX11302-2 RAX11118-2 TYPE11
TYPE13
HER2651-2
TYPE7
RAX11063-2 RAX11102-2
MAD300-2
RAX11159-2 RAX11135-2
TYPE9
TYPE10
RAX11063-1
TYPE8
TYPE6
RAX11050-2
TYPE12
RAX11183-2
TYPE14
RAX10908-2
RAX11036-2
RAX11211-2
RAX11183-1
TYPE4
TYPE2
TYPE5
TYPE3
RAX8221-2
RAN43137-2
RAX10972-2
TYPE1
RAX11200-2
RAX11350-2
**/**
Furcifer lateralis
‘north-west’
(c)
TYPE11
TYPE7
TYPE9
TYPE12
TYPE6
RAX11302-2
RAX11684-2 RAX8484-1
RAX11748-2
RAX11013-1
TYPE4
RAX11304-1
RAX11159-2
TYPE10 TYPE1
RAN68720-2
TYPE15
RAN43453-2
TYPE3
RAX10838-2
MOR2-2
RAX11354-2
RAX11405-2
RAX9941-2
TYPE13
HER4101-2
RAX11817-2
RAX11314-2
TYPE16
TYPE14
TYPE8
RAX8244-2
RAX9977-2
RAX9978-2
RAX10999-2 MAD300-2
RAX7246-2
**/**
78/87
Furcifer lateralis
‘southern’
TYPE5
RD459-2
RAX10908-2
TYPE2
RAX11037-2
Fig. 2 (a) Phylogenetic relationships within the Furcifer lateralis complex and near outgroups based only on the partial mitochondrial genes
16S, ND2 and ND4 reconstructed on the MP tree. Three well-supported clades (eastern = blue, southern = yellow and north-west = green)
are recovered corresponding to those in Fig. 1a (bootstrap support values for MP ⁄ ML, **100%), but the relationship of Furcifer labordi to
the F. lateralis complex is unresolved with ML. (b) Median-joining network reconstructed from the nuclear locus MXRA, with colours
correponding to the mtDNA clades. All individuals are represented by two haplotypes ()1 or )2 after the sample name), and identical
haplotypes are grouped together by ‘TYPEs’. ‘TYPEs’ that are composed of identical haplotypes from multiple mtDNA clades are represented by
black font in the median-joining network. All haplotype assignment information is provided in Table S4. (c) Median-joining network
reconstructed from the nuclear locus RAG1. ML, maximum likelihood; MP, maximum parsimony.
100%). The southern and north-west clades are recovered as sister groups by both MP and ML analyses (MP
99%, ML 100%).
The north-west clade shows little mitochondrial diversity (Pi = 0.6% – see Table 1) even though this clade has
a large geographic distribution (see Fig. 1b), but phylogeographic structure exists that is correlated with geography. Within this clade, there is a latitudinal divide
between samples, with high support (bootstrap support
in MP and ML > 99%) for a more northern group, with
samples from the most southern localities of this clade’s
range falling out separate. There is more variation within
Table 1 Mitochondrial divergence (Dxy) and nucleotide diversity
(Pi) between and within the three Furcifer lateralis complex clades as
identified in Fig. 1.
Eastern
Southern
North-west
Eastern
Southern
North-west
1.2
6.9
0.9
7.8
3.6
0.6
the southern F. lateralis clade (Pi = 0.9%) but this
variation does not appear geographically structured. In
contrast, the eastern clade shows high mitochondrial
variation (Pi = 1.2%), and this variation is correlated
with geography. The individual from the high altitude
site, Itremo, is genetically distinctive from the rest of the
clade (MP and ML bootstrap > 100%) (see Fig. 1a).
Genetic variation within the rest of the eastern clade is
relatively low, but phylogeographic structure is correlated with geography with high support (bootstrap
support in MP and ML > 95%) for a south-eastern
subgroup. Mitochondrial divergence between the eastern
clade and both the north-west and southern clades is
high (Dxy: 7.8% and 6.9%, respectively). Mitochondrial
divergence between the north-west and southern clades
is Dxy = 3.6% (Table 1).
These three clades were also recovered with high
support after separate phylogenetic analysis of just the
three mitochondrial genes: (1) eastern (MP 99%, ML
100%), (2) southern (MP 87%, ML 78%) and (3) northwest (MP 100%, ML 100%) (Fig. 2a). However, the
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Cryptic species in a Malagasy chameleon
North-west
Southern
Eastern
Syntype
Canonical variate 2 (12.38% variance)
Key
Key
North-west
Southern
Eastern
Canonical variate 2 (22.98% variance)
monophyly of the F. lateralis complex was not supported
by ML, with F. labordi recovered as sister to the the
eastern clade of the F. lateralis complex, but with low
support (56%). The monophyly of the F. lateralis
complex was supported by MP (64%).
Median-joining networks of the phased nuclear genes
are shown in Fig. 2b,c (see Table S4 for information on
haplotype assignment). The median-joining network
recovered from the nuclear gene MXRA (Fig. 2b) recovers similar clades as those recovered from mtDNA, except
for four noteworthy exceptions: (1) one haplotype from
the individual RAX11200 is recovered as part of the
eastern haplotype group with MXRA, but this individual
is recovered as ‘southern’ in mitochondrial analysis;
(2) one haplotype from each of the individuals
RAX10972, RAX10918 and RAX10971 (grouped in
‘TYPE1’) is recovered as part of the southern clade with
MXRA, but they are recovered as in the eastern clade
with mtDNA; (3) the individual TRA143 is recovered as
part of the southern clade in the MXRA haplotype
network, but as ‘eastern’ with mtDNA; and (4) one
haplotype from the southern individual RAX11240 is
identical to several haplotypes from ‘north-western’
individuals (grouped in ‘TYPE9’). The median-joining
network recovered from RAG1 (Fig. 2c) has a less clear
pattern. Individuals recovered as ‘eastern’ in the mito-
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Canonical variate 1 (77.02% variance)
Eastern
North-west
Southern
Fig. 4 Geometric morphometric analysis (CVA) of 53 adult Furcifer
lateralis females, based on the landmarks shown in Fig. S1.
Permutation tests of Mahalanobis distances are significant (P < 0.05)
between the groups, and Procrustes distances are significant between
all groups except the east and north-west clades.
chondrial analysis are split into two groups, with little
geographic pattern evident. Most notably, several RAG1
haplotypes from different mtDNA clades are identical.
Geometric morphometrics analyses
Canonical variate 1 (87.62% variance)
Southern
North-west
Eastern
Fig. 3 Geometric morphometric analysis (CVA) of 87 adult Furcifer
lateralis males, including the syntype described by Gray (1845),
based on the landmarks shown in Fig. S1. Permutation tests of
Procrustes and Mahalanobis distances are significant between the
groups (P < 0.05).
Grouping morphological specimens based on the three
major phylogenetic clades resulted in the following
sample sizes for males: 25 north-west, 33 eastern and
28 southern; and for females: 19 north-west, 18 eastern
and 16 southern (see Table S3 for specimen list). The
CVA recovered significant differences (P < 0.05, Procrustes and Mahalanobis distances) among all three male
groups. For the first and second canonical variates, there
was some overlap between the north-west and southern
groups, but the east group formed a distinct cluster of
individuals (Fig. 3). The CVA for females was significant
(P < 0.05) for Mahalanobis distances among all groups,
and Procrustes distances among all groups except the east
and north-west clades. For the first and second canonical
variates, the three groups formed distinct clusters with
the CVA (Fig. 4). The first canonical variate (CV1)
accounted for 87.6% of the variance in males and
77.0% in females. The second canonical variate (CV2)
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Table 2 Morphometric results for canonical variates analysis. Eigenvalues (EV), percentages of among-group variability explained
(% exp.), canonical factor loadings for each landmark (1–10; see
Fig. S1) by canonical variate (CV).
Males
EV
% exp.
1
2
3
4
5
6
7
8
9
10
Females
CV1
CV2
CV1
CV2
4.35
87.6
)0.42
0.02
)0.29
)0.39
)0.03
)0.12
0.10
0.24
0.43
0.47
0.61
12.4
0.02
)0.20
0.06
)0.11
0.18
)0.03
0.12
)0.02
0.03
)0.10
3.17
77.0
)0.41
0.01
)0.29
)0.39
)0.02
)0.13
0.10
0.25
0.42
0.48
0.95
23.0
0.03
)0.19
0.06
)0.12
0.18
)0.02
0.12
)0.02
0.02
)0.10
accounted for 12.4% in males and 23.0% in females. For
both the male and female analyses, the highest canonical
factor loadings for CV1 were associated with landmarks
7–10, and each of these landmarks is associated with the
head casque (Table 2). The male F. lateralis syntype
grouped within the eastern cluster in the CVA analysis
(Fig. 3). The permutation test of Mahalanobis distances
found the syntype statistically different from both the
north-west and southern groups (P < 0.05), but not the
east group. The permutation test of Procrustes distances
found the syntype statistically different from the southern group (P < 0.05), but not from the east or north-west
group.
Geographic distribution of phylogenetic clades and
ecological niche models
Grouping localities based on the clades resulted in the
following sample sizes: 36 north-west, 32 east and 42
southern (Fig. 1b). The east group falls within the central
plateau and the south-eastern low-elevation rainforest
regions of Madagascar. The north-west group is distributed throughout western and northern Madagascar, with
localities as far north as Ambodiampana in the Sambava
district, as far south as the Mangoky River, and localities
on the western and northern margin of the central
plateau. The southern group is distributed in southern
Madagascar, from as far north as just south of the
Mangoky River, as far east as Andringitra, and as far
south as Faux Cap and Fort Dauphin.
The ENMs are significant for the eastern (mean AUC:
0.9314, 0.894–0.9765; P < 0.05 for 6 ⁄ 6 trials), northwest (mean AUC: 0.8236, 0.7398–0.9068; P < 0.05 for
6 ⁄ 6 trials), and southern group (mean AUC: 0.9322,
0.9107–0.9585; P < 0.05 for 6 ⁄ 6 trials) (Fig. 1b). The
southern group model has a disjunct area of over-
prediction in north-eastern Madagascar (Sambava region) where F. lateralis is not known to occur. The ENM
of the north-west group predicts a distribution across
almost all of western Madagascar, except for the very
southern regions. The eastern ENM predicts a distribution in both the central plateau and some south-eastern
lowlands. Although the ENMs are statistically significant,
there is some overlap among the three group models.
Part of the predicted distribution of the north-west group
overlaps with the southern group, and the eastern and
north-west groups overlap in the central region. There is
almost no overlap between the southern and eastern
groups.
Separate ENMs for all three groups provide a better fit
to the data than the ENM treating F. lateralis as a single
widely distributed species, with lower AUC scores and
lower significance across trials (mean AUC: 0.7605,
0.7135–0.8121; P < 0.05 for 4 ⁄ 6 trials). This lumped
ENM (data not shown) has substantial areas of overprediction in north-eastern Madagascar, where the species complex is known to be absent (see Fig. 1).
Discussion
Phylogeographic patterns
The multilocus phylogenetic trees recovered from both
optimality criteria (MP and ML) provide robust support
for the monophyly of (1) the F. labordi + F. lateralis clade,
(2) the F. lateralis complex, and (3) F. labordi clade.
However, phylogenetic trees resulting from the analysis
of individual loci are not well resolved. The mitochondrial genes 16S and ND2 are inconclusive with regard to
the relationship of F. lateralis with its sister species
F. labordi. Additionally, the individually analysed nuclear
loci do not recover much phylogeographic structuring
within the F. lateralis complex, supporting the perspective
that a single locus will often be unable to accurately
facilitate species identification, even for DNA barcoding
(Roe et al., 2010; Niemiller et al., 2011).
Separate analysis of the mitochondrial and nuclear
data reveals several interesting patterns. For MXRA, the
resulting median-joining network reveals a similar result
as the mitochondrial data, with some exceptions. Most
surprising is that individual TRA143 is homozygous for a
MXRA haplotype also seen in north-west individuals
(Fig. 2b), yet this specimen is recovered as part of the
eastern mtDNA clade and has only ‘eastern’ haplotypes
with the other nuclear gene RAG1. TRA143 is a
F. lateralis specimen collected at the very southern range
of the eastern clade, lending evidence that this individual
could be a hybrid. However, it is difficult to distinguish
between hybridization and incomplete lineage sorting in
recently evolved groups (Polihronakis, 2009), and either
of these are possible causes of discrepancies in mtDNA
and nDNA presented in this study. The median-joining
network for MXRA revealed several other differences
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between this locus and the mtDNA. However, these
discrepancies did not result from individuals distributed
at the extremes of their ranges, suggesting that this might
instead be an example of incomplete lineage sorting.
In the RAG1 median-joining network, there is some
support for the mtDNA clades, but the pattern is much
less clear. Particularly interesting is that the mtDNA
eastern clade is broken into two sections, but these do not
appear geographically correlated (Fig. 2c). In addition,
the identical haplotype group ‘TYPE3’ contains a mixture
of individuals from the three different mtDNA clades.
While most belong to the north-west mtDNA clade, this
group also includes a large number of southern mtDNA
individuals and one eastern mtDNA individual. Because
these individuals are not found where the clade distributions meet and because these results contradict both
the mtDNA tree and MXRA median-joining network, it is
unlikely that this represents continued gene flow
between clades. Instead we suspect that this represents
a case of incomplete lineage sorting. However, as is the
case above with the discrepencies found with MXRA and
the mtDNA clades, more loci, samples and analyses are
needed to test between these possibilities.
The combined molecular data support three distinct
north-west, eastern and southern clades within a
F. lateralis species complex with the north-west and
southern clades recovered as sister groups. This supports
an initial divergence in the F. lateralis complex between
populations in the more humid east and those in the
drier south and west. These clades are also highly
differentiated with mitochondrial Dxy values as high as
7.8%. Taken alone, these data suggest that these clades
warrant species-level recognition, as this has been shown
to be characteristic of sister species in other taxa using a
multimethod approach similar to this study (Brown et al.,
2002; Rissler & Apodaca, 2007) and also across diverse
species criterion (see Bradley & Baker, 2001; Hart &
Sunday, 2007). The lower level of divergence between
the southern and north-west clades (Dxy = 3.6%) is also
highly suggestive that these two genetic clades are
different cryptic species.
There is some genetic structure within each clade that
corresponds with geography, but faster-evolving loci and
more samples will be needed to further elucidate these
patterns. The eastern and southern clades have a higher
mitochondrial diversity than the north-west clade. On
the basis of the isolation-by-distance (Slatkin, 1993), this
result is surprising because the north-west clade is the
most widely distributed. The low genetic diversity in this
clade may be indicative of recent population expansion
northwards. We hypothesize expansion northwards
based on greater genetic structuring in the samples
distributed in the most southern regions of this clade’s
range (with Makay populations clustering together – see
Fig. 1a).
These results demonstrate that Brygoo’s (1971) morphologically distinct form in the south-west deserves
1407
species recognition, as also discussed by Boumans et al.
(2007) based on their 16S mitochondrial data. Boumans
et al. (2007) also found evidence for several other
genetically structured clades within the complex, but
overall support values were low. The early east ⁄ west
divergence found in our study is also congruent with
several other taxa. The geckos, Phelsuma madagascariensis
and Phelsuma kochi, are closely related species that have
disjoint distributions in eastern and western Madagascar,
respectively (Raxworthy et al., 2007). In addition, the
lemur Eulemur fulvus rufus contains two genetically
distinct groups distributed between eastern and western
parts of Madagascar (Pastorini et al., 2003). This east–
west split has been found in other taxonomic groups as
well, including other geckos, tree boas and frogs (eg.
Nussbaum & Raxworthy, 1998; Andreone et al., 2002;
Orozco-ter Wengel et al., 2008 – but see the supplementary material of Vences et al., 2009 for a full review of
speciation studies). The south ⁄ north-west divergence is
less common, but has been highlighted in other studies.
For example, there is a genetically distinct southern
group and a widely dispersed north-western group
within the Malagasy plated lizard Zonosaurus laticaudatus
(Raselimanana et al., 2009).
Morphometric support for cryptic species
Canonical variates analyses of both males and females
found morphological differences among the three clades,
with CV1 accounting for 87.6% of the variation among
clades for males and 77.0% among females. CV1 likely
relates to changes in the landmarks associated with the
head casque (see Table 2 and Fig. S1), with eastern
individuals having a much lower head casque that
extends posteriorly, almost flattening into the dorsal
ridge of the body. The head casque on the southern clade
individuals are the most elevated, with north-west clade
individuals being slightly lower. Higher casque height has
been correlated with stronger bite force in lizards (Herrel
et al., 2001) because this development results in the
enlargement of the medialis and profundus portions of
the external jaw adductor (Rieppel, 1981). Interestingly,
in the field we noted differences in aggressive behaviour
exhibited between these three groups, with southern
individuals showing the greatest tendency to bite when
handled or placed with conspecifics in collecting bags.
This suggests that the southern F. lateralis group may
have more aggressive behaviour that may explain a
larger head casque and greater bite force. In contrast,
eastern individuals (with flattened head casques) were
extremely docile, displaying little aggression (Florio, pers.
obs.). Head casque differences between clades could also
be attributed to differential dietary preferences. One
study found that exaggerated head morphology and
absolute bite force are correlated with prey size in
chameleon species found in open habitats (Measey et al.,
2011). Little is known about dietary preference in Furcifer
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A . M. FL O R I O E T A L .
chameleons, but it is interesting to note that individuals
in the southern clade (with the highest head casques) are
generally distributed in open spiny desert habitat (Florio,
pers. obs.).
We found both similarities and differences between
our morphological results and previous studies. While
Hillenius (1959) noted a prevalence of white lines
under the tail in the F. lateralis complex distributed in
southern Madagascar, we found this to be more common in the eastern individuals (data not shown).
However, our results support Hillenius’ view that
axillary pits are absent in lizards of the southern region
of the island. Brygoo (1971) noted larger individuals in
the south-west that are less rich in colour, similar to our
findings that southern individuals are often pale green
and large in size (see Proposed Taxonomic Revision
section).
ENMs for species delimitation and phylogeographic
studies
Ecological niche models have the potential to help
delimit cryptic species when combined with other data,
such as phylogenetic and morphological analyses, by
providing evidence of (1) improved niche descriptions in
split versus lumped taxonomies, (2) niche divergence
and (3) geographic isolation between lineages (Raxworthy et al., 2007; Rissler & Apodaca, 2007; Leaché et al.,
2009). Separate ENMs for each of the three groups were
statistically significant and provided a better fit to the
data than the model treating F. lateralis as a single species,
and niches in geographic space are unique to each
species (although partially overlapping). These results
agree with the morphological and genetic divergences
found among these three potential cryptic species.
In addition to aiding in species delimitation, our ENMs
also provide insights into the processes driving divergence in the F. lateralis complex. On the basis of the
largely nonoverlapping distribution of the ENMs for the
eastern vs north-west ⁄ southern clades, initial divergence
may have been driven by adaptation across the western
to eastern climate gradient of Madagascar. This provides
support for the Ecogeographic Constraint model of
diversification (Yoder and Heckman, 2004) that proposes
that the distinction between the climate and resulting
vegetation of western and eastern Madagascar was the
driver of initial divergence between widely distributed
animal groups. With regarad to the humid eastern
distribution of F. lateralis, this ecological divergence is
especially significant in that its sister species F. labordi
occupies the more arid southern and western regions of
Madagascar, as do the other closely related species (e.g.
F. oustaleti, F. verrucosus, and F. antimena) within the
complex. If the Ecogeographic Constraint model is
correct, this predicts that future speciation within the
eastern clade will be constrained to humid eastern
habitats. This may also include future range expansion
into the north-east, much of which has not yet been
occupied by this species. By contrast, the ENMs for the
sister southern and north-west clades are overlapping,
and the distributions of these clades meet at (or close to)
the Mangoky River, suggesting that this river (which is
one of the biggest in Madagascar) may have acted as a
barrier to gene flow. Martin (1972) first proposed a
riverine barrier model of diversification for Madagascar
in lemurs, and more recently large rivers have been
shown to drive divergence in several lemur groups (see
Goodman and Ganzhorn, 2003; Pastorini et al., 2003).
Recent fieldwork resulted in the discovery of individuals
belonging to the north-west clade distributed just south
of the Mangoky River. This could be due to humanmediated dispersal or could indicate that the Mangoky
River is no longer an isolating barrier between clades
(although it may have caused initial divergence). Further
analysis of this potential contact zone is needed to better
test this hypothesis. Although preliminary, current evidence suggests that at least two different speciation
processes may have driven diversification within the
F. lateralis complex.
The ENMs also provide interesting information on the
potential distribution of F. lateralis. The southern clade of
the F. lateralis complex has a disjunct area of overprediction in north-eastern Madagascar (Sambava
region) where southern F. lateralis are not known to occur.
This region may represent an area of potential endemism
for other Furcifer species (Raxworthy et al., 2002).
Proposed taxonomic revision
Given the results of this study, we conclude that the
F. lateralis complex represents three species corresponding to the three clades recovered from phylogenetic
analysis. These clades are not only well supported, but
individuals between clades also display differences in
head casque morphology and have diverged in environmental niche space. Although we are not the first to
propose or suspect that F. lateralis is a species complex
(Angel, 1921; Brygoo, 1971), this is the first study to
employ a combination of methods, and to find broad
support for three species within the group. Because
there are three available names for the species within
the complex – F. lateralis (Gray, 1845), F. lambertoni
(Angel, 1921) and F. lateralis major (Brygoo, 1971) – it is
important to assign their types to the appropriate species
identified in our study. The adult male syntype of
F. lateralis falls within the eastern CVA group of
F. lateralis and was not statistically different from the
other eastern individuals included in the analysis using
permutation tests of the Mahalanobis and Procrustes
distances (although the Procrustes distances was also
not statistically different to the north-west group).
Although this syntype has no precise locality within
Madagascar, based on the CVA results, we are confident
that Gray’s (1845) description refers to the eastern
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Cryptic species in a Malagasy chameleon
group recovered in this study, and other morphological
characters also diagnose the F. lateralis syntypes as
eastern individuals (see below).
The holotype of F. lambertoni MHNP 1921.269 collected
from Antananarivo (Central High Pleateau) is a juvenile
(SVL = 57 mm, with an open mouth) and therefore
could not be included in the geometric morphometric
analyses. Upon our examination of the F. lambertoni
holotype, we found a weak gular crest present on the
anterior part of the chin, which is a typical condition in
smaller juvenile F. lateralis. All other diagnostic characters (see below) also show no differences between the
F. lambertoni holotype and F. lateralis (from eastern and
central Madagascar). We also note that we have collected
typical F. lateralis from the F. lambertoni type locality,
Antananarivo. We therefore agree with Hillenius (1959)
that F. lambertoni is a junior synonym of F. lateralis.
Brygoo’s (1971) description of F. lateralis major from
Tanandava, south-western Madagascar, closely corresponds with the morphology of the southern group
recovered in our study. These individuals have higher
head casques and have a larger SVL. Unfortunately, we
were unable to examine the holotype (designated by
implication, see Klaver & Böhme, 1997), because this
specimen (457 ⁄ C, Brygoo’s chameleon collection) could
not be located during the time of this study and has not
been catalogued at the Muséum national d’Histoire
naturelle (Paris). However, based on the excellent
description and illustration of the holotype, we are
confident that the southern group should be assigned
to this taxon. We thus formally elevate here F. lateralis
major to the rank of full species: Furcifer major.
Because there is no available name for the north-west
species, we describe it as a new species: Furcifer viridis
new species (Fig. 5).
Holotype: AMNH 152603 (RAX 5989), a mature male,
collected 1 March 2003 at Ambinanitelo (14.22556S,
48.96297E), 1250- to 1300-m elevation, Tsaratanana
Massif, Mahajanga Province, Madagascar, by N. Rabibisoa, S. Mahaviasy & N. Rakotozafy. Left hind limb was
removed and preserved in ethanol for DNA extraction.
Paratopotypes: AMNH 152604 (RAX 5990) and AMNH
152606 (RAX 5995) – both mature females collected in
the same locality and on the same date as the holotype.
Other specimens: See Supporting information.
Diagnosis: A Furcifer chameleon from Madagascar with
a double row of scales along the dorsal body ridge, which
can be distinguished from all other species with this
character by the following: 14–18 tubercles on the
parietal crest (F. major 10–13, F. campani 7–8); axillary
pits always present or at least indicated (F. major absent);
a typical solid green adult colour in life, with or without a
single pale line on the flank (F. lateralis and F. campani
with complex pale and dark spotting, often on a dark
brown or reddish brown background, and for F. campani
2–3 pale lines on lateral body); maximum SVL of
120 mm for males and 98 mm for females (F. lateralis
1409
with maximum SVL of 98 mm for males, and 92 mm for
females; F. campani with maximum SVL of 70 mm for
males and females), head casque height ⁄ head height
> 0.5 (F. campani and F. lateralis < 0.5); and no regular
rows of enlarged round tubercles on flanks (F. campani,
regular rows of enlarged round tubercles on flanks).
Furcifer viridis is also diagnosable from other species based
on phylogenetic analysis of the mitochondrial and
nuclear loci (see results).
Description of holotype: Male, in good condition, but
missing the left hind limb, which was removed and
preserved in ethanol for DNA extraction; hemipenes
everted; SVL, 81 mm; tail, 89 mm; axilla–groin distance,
56 mm; eye horizontal diameter, 9 mm.
Head lacks a rostral appendage, the orbital crests do not
make contact anteriorly at the snout tip; orbital and
lateral crests (the latter of which are weakly defined)
form a dorsal helmet; helmet posteriorly comes to a blunt
point and is elevated above the dorsal ridge of the body;
parietal crest weakly developed, formed by a row of 18
tubercles; temporal crest not obvious behind eye; orbital
crest rounded in lateral view and formed by a single row
of scales; no occipital lobes or folds on each side of the
head; gular crest formed by a row of pointed tubercles,
which continues to the thorax. Head casque height,
8.5 mm; head height, 15 mm; ratio of head casque
height ⁄ head height, 0.57.
Dorsal ridge of body with a vertebral double row of
rounded tubercles that do not form a crest and flanked
below by two other rows of regularly arranged tubercles;
body laterally with homogeneous scalation that lack
enlarged tubercles; thorax with a ventral crest of short
pointed tubercles; body otherwise lacks a ventral crest of
pointed tubercles; axillary pits present; limbs with scattered slightly enlarged rounded tubercles, tail with a
vertebral double row of rounded tubercles that do not
form a crest, feet without tarsal spines. Hemipenes
quadriform with calyces on the truncus; apex smooth
with a pair of elongate pedunculi bearing 14–16 short
papillae; and a small denticulated auricula on the
external lateral side and a tuft of papillae on the sulcal
side at the base of each pedunculus.
In preservation, the coloration of head, body, limbs
and tail is black, with a single pale lateral line present
on the flank running from above the front limb to just
anterior, and above, the hind limb insertion point.
There are a few reddish brown blotches on the
posterior head and neck. Ventrally there is a prominent
white line that begins in the gular region and fades out
under the tail. Just behind the cloaca, there is a pair of
short white lines that extend 4 mm onto the ventral
tail base.
Variation: See Table 3 for summary data of the
examined material (listed in Supporting information).
Adult males vary greatly in size (SVL, 65–120 mm) but
females never exceed 98 mm (SVL, 64–98 mm).
Females and juveniles lack the swollen tail base of
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A . M. FL O R I O E T A L .
(a)
(b)
(c)
(d)
(e)
(f)
Fig. 5 Live adults of the Furcifer lateralis complex: (a) male F. lateralis (Kianjavato), (b) female F. lateralis (Mandalahy Forest), (c) male Furcifer
major (Andoharano), (d) female F. major (Andranohinaly Village), (e) male Furcifer viridis (Anoalakely), (f) female F. viridis (Makay Massif).
adult males. The posterior head casque in females is
lower than that in equivalent sized males. The parietal
crest tubercle count varies from 14 to 18. Specimens
always have axillary pits or they are at least indicated,
with specimens from the most southern populations
tend to have more poorly developed axillary pits. The
pair of white ventral tail base lines may be weakly
marked in some specimens.
In life, the unstressed coloration of the body, head,
limbs and tail in adult males is vivid green, with a white
lateral line on the body flank and a white labial line. The
casque, eye turret and flanks may be marked with
scattered small pale blue flecks. Adult females have a
similar coloration except that the ground colour may be
pale green, pale brown, or greenish brown, with sometimes a pale yellowish brown vertebral line, and without
blue flecking. The white lateral body line and labial line
may also be weakly displayed in females. Juveniles have
a more uniform pale brown or green body coloration.
Preserved specimens are often dark, with a weak pale
lateral line on the flanks and a bright white mid-ventral
line on the throat and body. Some specimens have pale
gular skin between the scales that gives a striated
appearance on the throat.
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Table 3 Morphological variation in all Furcifer species with a
vertebral double row of tubercles on the body. All measurements in
mm. Coloration is based on live animals at rest.
1411
often also show the presence of three dark circles on the
lateral body, which are intercepted by a single pale lateral
line.
Furcifer species
Character
viridis
major
lateralis
campani
Maximum male SVL
Maximum female SVL
Tubercles on parietal crest
Head casque height ⁄ head
height
Axillary pits
Enlarged round tubercules
on flanks
Body mostly solid green
or brown
Body with pale and dark
spotting
Number of pale lines on
lateral body
120
98
14–18
> 0.5
117
112
10–13
> 0.5
98
92
14–20
< 0.5
70
70
7–8
< 0.5
+
–
–
–
+
–
+
+
+
+
–
–
–
–
+
+
1
1
1
2–3
SVL, snout-vent length.
Distribution: This species has a large distribution
throughout western and northern Madagascar, ranging
from Ambodiampana (13.7S, 49.6E) in the northeast, to the Makay Massif in the south (21.6S, 45.1E)
and extending into the interior of the island as far east
as Mandoto (19.6S, 46.3E – see localities on Fig. 1).
At the type locality, it has been found at a maximum
elevation of 1250–1300 m, but the species also occupies
lower coastal areas. In western Madagascar, F. viridis
occupies dry deciduous forest, scrub and grasslands;
and on the western high plateau and in northern
Madagascar, it occupies more humid and transitional
forests, grasslands and scrub. Like F. lateralis and
F. major, F. viridis individuals are tolerant of habitat
degradation and are often found near rice fields,
streams and rivers.
Remarks: Hillenius (1959) reported geographic variation in F. lateralis concerning the development of
axillary pits and the ventral white tail line. The western
and north-western populations that he examined represent populations of this new species, and he noted a
general trend for these populations to have betterdeveloped axillary pits and a more obvious ventral
white tail line.
Etymology: This species is named to recognize the
predominantly green body.
Identification key for the Furcifer lateralis group in
Madagascar
The species in this group are readily recognized by the
double row of scales on the body dorsal ridge, the lack of
rows of enlarged rounded tubercles on the lateral body
and an obvious white ventral line. As live adults, they
1a.
1b.
2a.
2b.
10–13 parietal crest tubercles on the head casque;
no axillary pits.
14–18 parietal crest tubercles on the head casque;
axillary pits present.
Furcifer
major
2
Low adult head casque, with the highest point of the
lateral crest lower than the highest point of the orbital
crest; ratio of head casque height : head height < 0.5
in males and females; adult male and female SVL,
60–98 mm; live colour typically heavily speckled with
red, blue, yellow, black blotches.
Raised adult head casque, with the highest point of the
lateral crest higher than the highest point of the orbital
crest; ratio of head casque : head height ‡ 0.5 in
males and usually females; adult male SVL,
65–120 mm, adult female SVL, 64–98 mm; live colour
typically solid green or brown (in some females) without
heavily speckled red, blue, yellow or black blotches.
Furcifer
lateralis
Furcifer
viridis
Conclusions
This study of the widespread carpet chameleon
(F. lateralis sensu lato) finds support for three clades
within the complex, which correspond with geography
and have diverged in both morphological and ecological
space. On the basis of these results, we recognize three
species within the F. lateralis complex. Initial ecological
divergence within the species complex, associated with
the speciation of the eastern F. lateralis, best fits the
Ecographic Constraint species diversification model (between western and eastern Madagascar). The second
speciation event best supports a riverine barrier model, as
the contact zone between F. major and F. viridis is in close
proximity to the Mangoky River. More generally, this
study also demonstrates (1) the utility of using a multiple
method approach for species delimitation within morphologically conservative species complexes, and (2) the
existence of cryptic speciation and regional endemism
within what was previously (and incorrectly) considered
a widespread habitat generalist species.
Acknowledgments
Research for this project was funded by the Richard
Gilder Graduate School (RGGS) at the American
Museum of Natural History (AMNH) and the U.S.
National Science Foundation (DEB 9984496, 0423286
and 0641023). Field studies in Madagascar were made
possible due through the agreement of the Ministries des
Eaux et Forêts, the Association Nationale pour la Gestion
des Aires Protégés (ANGAP) and the Université d’Antananarivo, Département de Biologie Animale (especially
D. Rakotondravony and H. Razafindraibe). We thank
ª 2012 THE AUTHORS. J. EVOL. BIOL. 25 (2012) 1399–1414
JOURNAL OF EVOLUTIONARY BIOLOGY ª 2012 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY
1412
A . M. FL O R I O E T A L .
A. Yoder and R.A. Nussbaum for generously providing
access to F. lateralis specimens, as well as Ivan Ineich at
The Muséum national d’Histoire naturelle (Paris, France)
and Tracy Heath at the Natural History Museum
(London, England) for providing access to the type
specimens used in this study. We also thank the many
people who have aided or contributed to this research
programme including A. Carnaval, R. DeSalle and S.
Perkins for useful feedback; J. Denton and S. Edwards for
advice with the morphometric analyses; and R. Pearson
for help with ecological niche modelling. Fieldwork was
accomplished with the help of many people, including R.
Andrianasolo, B. Falk, S. Mahaviasy, N. Rakotoarisoa, A.
Rakotondrazafy, J. Randrianirina, and many local guides,
reserve agents and volunteers too numerous to mention.
We are grateful to David Marjanović and an anonymous
reviewer whose comments and suggestions greatly
helped to improve this manuscript. J. Florio provided
helpful edits and comments on the final version of this
paper.
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Supporting information
Additional Supporting Information may be found in the
online version of this article:
Figure S1 Landmarks recorded on the left lateral view of
the Furcifer lateralis head.
Table S1 Catalog numbers, localities, coordinates, and
Genbank accession numbers for all samples in this study.
Table S2 Primer information for the genes utilized in this
study.
Table S3 All specimens examined for canonical variates
analysis, and associated locality and clade information.
Table S4 Identical haplotypes and their corresponding
‘TYPE’ name as found in Fig 2.
As a service to our authors and readers, this journal
provides supporting information supplied by the authors.
Such materials are peer-reviewed and may be reorganized for online delivery, but are not copy-edited
or typeset. Technical support issues arising from supporting information (other than missing files) should be
addressed to the authors.
Received 28 January 2012; revised 31 March 2012; accepted 3 April
2012
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