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.

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) .

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 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 ).

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.

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.

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 ).

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, 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 ).

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 Maxentv3.3.31 ( Phillips et al. , 2006 ). All occurrence localities and environmental variables were resampled to an oblique Mercator projection at 1 km 2 resolution ( Pearson et al. , 2007 ) using ArcMap ( 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 ArcMap by reclassifying the continuous data to create a binary prediction, and all values above the LPT were reclassified as suitable environment.

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.

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 ).

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.

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 phase v2.1 ( Stephens et al. , 2001 ; Stephens & Sheet, 2005 ) and implemented using DnaSP v5 ( Librado & Rozas, 2009 ). Runs consisted of 1000 main iterations with an initial 1000 iterations for burn‐in and a thinning interval of 1. splitstree 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 (D xy ) and nucleotide diversity (Pi) within groups ( Takahata & Nei, 1985 ) was assessed also using DnaSP v5, with individuals with ambiguity codes in their sequences excluded from analysis.

Phylogenetic analyses were conducted using maximum parsimony (MP) and maximum likelihood (ML). MP was carried out with tnt v1.1 ( Goloboff et al. , 2008 ) and Winclada 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 RAxML ( Stamatakis, 2006 ) with the RAxMLgui0.93 ( 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 + Γ but at significantly lower computational costs and memory consumption ( Stamatakis, 2006 ).

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 PCRμ96 Filter plates (Millipore, Billerica, MA, USA) and sequenced in both directions using BigDye v.3.0 (Applied Biosystems, Foster City, CA, USA) on an ABI 3730 automated DNA sequencer. Sequences were edited in geneious 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.

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, 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.

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 over‐prediction in north‐eastern Madagascar, where the species complex is known to be absent (see Fig. 1 ).

The ENMs are significant for the eastern (mean AUC: 0.9314, 0.894–0.9765; P < 0.05 for 6/6 trials), north‐west (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.

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.

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.

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) 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.

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 mitochondrial analysis are split into two groups, with little geographic pattern evident. Most notably, several RAG1 haplotypes from different mtDNA clades are identical.

(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.

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) north‐west (MP 100%, ML 100%) ( Fig. 2a ). However, the 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%).

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 phylo‐geographic 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 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 (D xy : 7.8% and 6.9%, respectively). Mitochondrial divergence between the north‐west and southern clades is D xy = 3.6% ( Table 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.

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 635‐bp 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 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 100%). The southern and north‐west clades are recovered as sister groups by both MP and ML analyses (MP 99%, ML 100%).

Discussion

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 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; Leachéet 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 human‐mediated 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 over‐prediction 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 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). Figure 5 Open in figure viewer PowerPoint 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). Holotype: AMNH 152603 (RAX 5989), a mature male, collected 1 March 2003 at Ambinanitelo (14.22556°S, 48.96297°E), 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 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 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. 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. Character Furcifer species viridis major lateralis campani Maximum male SVL 120 117 98 70 Maximum female SVL 98 112 92 70 Tubercles on parietal crest 14–18 10–13 14–20 7–8 Head casque height/head height > 0.5 > 0.5 < 0.5 < 0.5 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 1 1 1 2–3 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. Distribution: This species has a large distribution throughout western and northern Madagascar, ranging from Ambodiampana (13.7°S, 49.6°E) in the north‐east, to the Makay Massif in the south (21.6°S, 45.1°E) and extending into the interior of the island as far east as Mandoto (19.6°S, 46.3°E – 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 better‐developed axillary pits and a more obvious ventral white tail line. Etymology: This species is named to recognize the predominantly green body.