Journal of Biogeography (J. Biogeogr.) (2008) 35, 1402–1414

ORIGINAL ARTICLE

Speciation and radiations track climate transitions since the Miocene Climatic Optimum: a case study of southern African chameleons Krystal A. Tolley1*, Brian M. Chase2 and Fe´lix Forest1,3 

1

South African National Biodiversity Institute, Kirstenbosch Research Centre, Cape Town, South Africa, 2Oxford University Centre for the Environment School of Geography, University of Oxford, Oxford, UK and 3Department of Botany, University of Cape Town, Cape Town, South Africa

ABSTRACT

Aim The high amount of species diversity concentrated in southern Africa has been attributed to palaeoclimatic factors, and the timing of radiations in some taxa corresponds to global palaeoclimatic trends. Using dwarf chameleons (Bradypodion: Chamaeleonidae) as a model system, we explored the relationship between palaeoclimatic fluctuations and cladogenesis with respect to both temporal and spatial patterns in an effort to understand the process of speciation in southern Africa. Location South Africa, with particular emphasis on the Cape Floristic Region and the Maputaland–Pondoland–Albany hotspot. Methods Mitochondrial sequence data (ND2 and 16S) were used to estimate the timing of major radiations and to examine the number of lineages through time. A dated phylogeny was constructed using Bayesian phylogenetic reconstruction, and a Bayesian relaxed molecular clock was used to estimate divergence times. Spatial data and lineage-through-time plots were used to identify geographic regions that underwent diversification in connection with major climatic events. Both parsimony and likelihood optimizations of habitat type on the phylogeny were used to determine whether major habitat shifts have occurred. On a coarse scale (half-degree grid cells), phylogenetic diversity (sum of the branch lengths linking terminals) was compared with species richness (absolute number of species) to identify areas of conservation importance. Results The complete species phylogeny of dwarf chameleons shows that the timing and mode of diversification exhibit spatio-temporal patterns that link to phases in the evolution of southern Africa’s climate over the last 14 Myr. Optimizations of habitat on the phylogenetic tree show a progression from closed to open habitats since the Mid-Miocene, corresponding to the shift from C3 to C4 environments, and later with the development of south-western Africa’s winter-rainfall regime. These shifts are not simultaneous across the region, with different geographic centres of diversity generated during different time periods.

*Correspondence: Krystal A. Tolley, Molecular Ecology and Evolution Program, South African National Biodiversity Institute, Private Bag X7, Claremont 7735, South Africa. E-mail: [email protected]  Present address: Jodrell Laboratory, Royal Botanic Gardens, Kew, Richmond TW9 3DS, UK.

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Main conclusions Regions that are prominent centres of chameleon diversification are encompassed by the current biodiversity hotspots as shown by chameleon species richness and phylogenetic diversity. Diversity within the Cape Floristic Region appears to be the result of a Late Pliocene radiation, whereas the diversity encompassed within the Maputaland– Pondoland–Albany hotspot is an aggregate of asynchronous radiation events, probably influenced by lineage losses. Overall, dwarf chameleons have experienced a shift in habitat types, with recent radiations occupying open habitats, and older lineages persisting in relictual forested habitats,

www.blackwellpublishing.com/jbi doi:10.1111/j.1365-2699.2008.01889.x

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Chameleon speciation tracks climate transitions

corresponding to the continental shift of vegetation types since the Miocene Climatic Optimum. Keywords Global cooling, hotspots, lizards, Miocene, palaeoclimate, Pliocene, reptiles, species radiation, southern Africa.

INTRODUCTION Changes in the global climate system are considered to have been crucial in igniting radiation events in a range of taxa across Africa (deMenocal, 1995; Potts, 1996). New light is being shed on the origins of floral diversity (Richardson et al., 2001; Linder, 2003, 2005; Jacobs, 2004; Hughes et al., 2005; Forest et al., 2007), and on the radiation of birds and mammals in connection with major climatic shifts (Kimbel, 1995; Behrensmeyer et al., 1997; Bobe et al., 2002; Bobe & Behrensmeyer, 2004; Bowie et al., 2004, 2006; deMenocal, 2004). In southern Africa, lineage diversification in a number of vertebrates and invertebrates is hypothesized to have tracked climatic shifts, especially in the Pliocene (e.g. Matthee & Flemming, 2002; Prendini et al., 2003; Daniels et al., 2004, 2006; Bauer & Lamb, 2005; Price et al., 2007; Smit et al., 2007; Lee-Thorp et al., 2007). However, this body of work has revealed just the tip of the iceberg, and the effects of these climatic scenarios on biogeographical patterns, especially in southern Africa, are only now coming to light. Whereas some taxonomic groups on the subcontinent are depauperate (for example, South Africa has 249 terrestrial mammals with only 14% endemics; Friedmann & Daly, 2004), other faunal assemblages are strikingly diverse. Notably, southern African lizard endemicity reaches 61%, and South Africa has the third richest lizard fauna on Earth, with 267 species of which 53% are endemic. Testudines reach their peak of diversity in southern Africa, with 33% of the world’s total number of land tortoises. Why should a temperate to subtropical region have such high reptile diversity in comparison to other regions, such as tropical rain forests, better known for their overall diversity? Much consideration has been given to the wet tropics as both ‘cradles’ of diversity and ‘museums’ retaining that diversity (Jablonski, 1993; Fjeldsa˚, 1994; Fjeldsa˚ & Lovett, 1997; Allen et al., 2002; Hillebrand, 2004; McKenna & Farrell, 2006). Although the mode of elevated speciation and lack of extinction in the tropics is not yet fully understood (Chown & Gaston, 2000), a number of hypotheses have been evoked, although a synergism of effects seems most plausible (Clarke & Gaston, 2006). Regardless of whether proposed factors such as total area, ecoclimatic zone, niche specialization, energy input, heterogeneity in altitude, or orbital forcing are responsible (Jablonski, 1993; Fjeldsa˚, 1994; Fjeldsa˚ & Lovett, 1997; Allen et al., 2002; Jansson & Dynesius, 2002; Hillebrand, 2004; McKenna & Farrell, 2006), an important consideration is that present-day environments do not necessarily have a

direct link back to the past processes that generated that diversity. To understand the patterns observed today, evolutionary history and palaeoenvironments must also be taken into account. In contrast to the tropics, Southern Hemisphere mesic environments with rich faunal assemblages represent a novel system for investigating palaeoclimatic trends in connection with diversification; they are a departure from environments traditionally considered cradles of diversity because they lack environmental features usually recognized as contributing to diversity. Furthermore, these areas did not experience the episodes of severe glaciations that prevailed in the Northern Hemisphere throughout the Pleistocene, which are known to have virtually erased genetic signatures in the high latitudes (Hewitt, 2000), so their evolutionary histories should be intact and readable through molecular markers. In this regard, the dwarf chameleons (Bradypodion: Chamaeleonidae) are of particular interest. The genus is endemic to southern Africa and comprises 15 described species representing almost 11% of the region’s endemic lizards. Recent phylogenetic studies have revealed nine additional terminal lineages that will be described as new species (Tolley et al., 2004, 2006; Tolley & Burger, 2007), meaning that species richness is higher than previously recognized. Although some clades seem to be the result of recent radiations (multiple terminal lineages with short branches), others appear to be much older relicts (few terminal lineages with long branches) (see Tolley et al., 2004, 2006). Using dwarf chameleons as a model, we investigated the hypothesis that climatic shifts and resulting habitat changes could have profound effects on speciation and extinction in southern Africa. A complete species-level phylogeny was constructed using DNA sequence data and calibrated using a Bayesian relaxed molecular clock to investigate the correspondence between speciation events and the effects of global and local shifts in climate on habitats. This phylogeny was also used to reconstruct ancestral habitat states, and to investigate lineage accumulation. We also assessed two measures of present-day diversity: species richness (SR), and phylogenetic diversity (PD, based on the calibrated ultrametric tree). These measures have been previously used to identify priority areas for conservation (Faith, 1992; Rodrigues & Gaston, 2002), although PD has untapped potential as a tool for understanding biogeographical and diversification patterns through the identification of rich evolutionary histories (Isaac et al., 2007).

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K. A. Tolley, B. M. Chase and F. Forest MATERIALS AND METHODS Estimating the timing of diversification Phylogenetic analysis was performed using Bayesian inference on a 1400-bp mitochondrial sequence data set for two markers (ND2 and 16S) for the genus Bradypodion. Despite the limitations of a single marker system for phylogenetic reconstruction and dating (Bromham & Penny, 2003; Kumar, 2005), we relied on mitochondrial genes because nuclear genes (e.g. RAG) do not increase resolution or support of terminal nodes at the species level in this genus (K.A.T., unpublished data). Dwarf chameleons have been previously investigated for phylogenetic and phylogeographic patterns, as well as for causes of incomplete lineage sorting at some terminal tips, by Tolley et al. (2004, 2006). However, each of these studies focused on different aspects of the Bradypodion phylogeny from that addressed here. The phylogenetic tree used in this study includes previously published sequence data from 36 individuals, plus data from 12 new individuals to ensure complete taxon sampling for the genus (see Table S1 in the Supplementary Material). At least one representative of four other chameleon genera was also included, as well as a representative from the Agamidae, Calotes versicolor, defined as the outgroup. Laboratory protocols for the generation of new sequences follow Tolley et al. (2004). Once the initial phylogeny had been re-assessed, we used one representative of each lineage in all additional phylogenetic-based analyses [Bayesian dating, lineage-through-time (LTT) plots, habitat optimization, and diversity measures]. All phylogenetic analyses were performed using Bayesian inference as implemented by the software MrBayes 3.0b4 (Huelsenbeck & Ronquist, 2001). ModelTest (Posada & Crandall, 1998) indicated that the GTR+G+I model was the best model of DNA substitution for both genes, and thus six rate categories were used in MrBayes, including invariable sites, and the alpha shape parameter for the gamma distribution to account for among-site rate heterogeneity (Yang, 1997). Four data partitions (ND2 1st codon, ND2 2nd codon, ND2 3rd codon, and 16S) were allowed to run independently to estimate the model parameters. The Markov chain Monte Carlo (MCMC) was run three times, each with two parallel runs, for 10 · 106 generations with trees sampled every 1000 generations. Although log-likelihood scores for the MCMC reached a plateau at 10,000 generations for all runs, the average standard deviation of the split frequencies did not stabilize in fewer than 5 · 106 generations for any of the runs, so the first 5000 trees were excluded (‘burn-in’) from posterior probabilities (PP) and branch-length compilations for all runs. To infer divergence time estimates and associated credibility intervals, we used a Bayesian relaxed clock (Thorne et al., 1998; Thorne & Kishino, 2002) and followed the procedure outlined in Rutschmann (2004). This procedure relies on the use of three programs: Baseml (paml package; Yang, 1997), Estbranches (Thorne et al., 1998), and Multidivtime (Kishino et al., 2001; Thorne & Kishino, 2002). First, the model 1404

parameters (base frequencies, transistion/transversion ratio, alpha shape parameter of the gamma distribution accounting for among-site rate heterogeneity) were estimated from the data for both gene regions separately using the most complex substitution model implemented in Baseml, i.e. F84 + G (Felsenstein, 1993). Maximum likelihood estimates of the branch lengths and their variance–covariance matrix were then estimated by the program Estbranches for each data set. The following settings for the prior distributions were used in Multidivtime, which determines the posterior distributions of substitution rates and divergence times using a MCMC procedure: rttm set at 65 Ma and rttmsd set at 32.5 Ma, rtrate and rtratesd set at 0.0538, and brownmean and brownsd set at 0.15 (defined as 1/rttm, as suggested by J. Thorne). The bigtime value was set at 90 Ma, and all other parameters were left at their default values (see Multidivtime readme file; http:// www.statgen.ncsu.edu/thorne/multidivtime.html). The Markov chain was run for 106 generations and was sampled every 100 generations following an initial burn-in of 100,000 generations (not sampled). Two calibration points were included in the dating analysis, each with upper and lower limits. The oldest known chameleon fossil is estimated to be c. 26 Ma (Moody & Rocˇek, 1980), and, based on a Bayesian molecular clock, the split between most chameleon genera has been dated at c. 25–45 Ma (Matthee et al., 2004). Given the fossil record and the best available molecular dating, the upper and lower limits for divergence between chameleon genera were set at 25 and 45 Ma. The second calibration point, for the split between Bradypodion and all other chameleons, was assumed to have a lower limit of 6 Ma based on the presence of 5.2-Ma-old fragments of fossil Bradypodion skulls in the Langebaan fossil bed, South Africa (P. Haarhoff, West Coast Fossil Park, personal communication, 2007), with an upper limit of 45 Ma in the absence of any other information. Mode of diversification To examine the timing of radiation events with palaeoenvironmental shifts, the cumulative number of lineages in the phylogeny was plotted against the date of each lineage diversification event. Credibility intervals for each age estimate in the phylogeny were included (the 95% probability that the estimated date lies within the interval; Ellison, 2004). Furthermore, the cumulative number of lineages was compared with climate-change trends, as identified in d18O and d13C signals in marine cores based on data from Zachos et al. (2001). A lineage-through-time plot (LTT) was generated for the complete phylogeny of Bradypodion using genie ver. 3.0 (Pybus & Rambaut, 2002), and the chronogram was obtained using a relaxed Bayesian clock. To detect whether the rate of diversification deviated from a constant rate of speciation (Yule process), 1000 random trees were simulated in mesquite ver. 1.04 (Maddison & Maddison, 2006). LTT plots were generated for these 1000 trees in genie, with means and 95% confidence intervals estimated for each node. The 95%

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Chameleon speciation tracks climate transitions confidence intervals did not deviate substantially from the mean (see Table S2). Habitat optimization under the maximum parsimony criterion was conducted using the software MacClade (Maddison & Maddison, 2000). Reconstructions based on the likelihood criterion were performed with the software mesquite using branch lengths representing absolute time (obtained from the Bayesian dating). We used the Mk1 model (Markov k-state one-parameter model; Lewis, 2001), in which the only parameter is the rate of change, and all changes are equally probable. Three character states were considered: (1) open habitat, (2) closed habitat, and (3) both habitats. Assignment of character state was made based on field observations and distribution records collected during the period 2001–2006. Open habitats include grassland, fynbos, strandveld, and savannah. Closed habitats include all thicket and forest types. Diversification rate changes in the phylogenetic tree were investigated with Symmetree ver. 1.1 (Chan & Moore, 2005). Given that taxon sampling was complete and the confidence in nodes was high, a single-tree analysis was conducted. Amonglineage diversification rate variation was estimated with two rate-shift statistics (MP and MS) and a tree imbalance statistic (B1), over the whole Bradypodion tree (Moore et al., 2004), with an a priori hypothesis that a shift in habitat resulted in an overall change in the rate of diversification. Diversity measures Point localities were obtained from 1002 museum records and distribution records corresponding to tissue samples lodged at the South African National Biodiversity Institute. Each terminal lineage was coded as present or absent from every halfdegree grid cell (c. 2700 km2) for generating measures of richness and diversity. Regional species richness (SR) was estimated based on the absolute number of described species present within each grid cell. Phylogenetic diversity (PD) measures for each grid cell represent the summation of the branch lengths linking all terminals found in a given grid cell, including the root. PD calculations were performed in r 2.5.1 (R Development Core Team, 2005) using the ape package (Paradis et al., 2004) and a script written by R. Grenyer (personal communication). Both measures were scaled to their respective largest value, for non-parametric comparisons of correlation (Spearman’s rho) and significant differences between the matrices (Wilcoxon’s signed ranks test). RESULTS The complete phylogenetic analysis shows that each lineage is well supported (‡0.95 posterior probabilities; see Fig. S1). Three well-supported clades correspond to distinct geographical areas in South Africa: clade A from the south-west coast, clade B from south inland to the south-east coast, and clade C from the north-east (Fig. 1a). Divergence times and their associated credibility intervals indicate that diversification in

the genus began at the end of the Mid-Miocene Climatic Optimum at c. 14.8 Ma and was followed by four broad phases of cladogenesis (11.0–9.0 Ma, 6.3–4.3 Ma, 2.9–1.6 Ma and < 0.5 Ma) (Fig. 1a; see also Tables S2 & S3). Radiations of extant lineages were mapped according to their present-day distributions at periods of marked climate change as identified in marine d18O and d13C signals in marine cores (Zachos et al., 2001; Fig. 2). These maps show links between cladogenesis events across the phylogeny and the past climatic episodes that could have triggered these radiations. We recognize that there may be a limitation to this method, given that modern distributions would have shifted over time, but an overall signature of regional cladogenesis should still be evident. The lineage accumulation plot shows sharp increases in the number of lineages across three main stages from the Late Miocene to Late Pliocene (Fig. 3). Although credibility intervals associated with the dates of diversification are broad, the dates still fit within the general trend for major climatic shifts. The observed LTT plot also demonstrates several diversification phases starting in the Mid to Late Miocene (Fig. 1b). These phases are separated by periods of stasis where no lineage accumulation is observed. At the start of each stage, the number of lineages observed matches that expected under a simple probability model of uniform speciation (Brown, 1994) but is followed by an increase in the number of lineages. Among-lineage rate variation can be detected by three topological shift statistics, MP, MS and B1, although the 97.5% frequentiles for P = 0.01–0.17, 0.01–0.22 and 0.01–0.51, respectively. Rate shifts along the branches are not detected, but this situation could result if diversification rates vary across the tree rather than being concentrated along any one branch (Moore et al., 2004). Strong shifts in the rate of lineage accumulation are not obvious within the phylogeny, suggesting that the pattern in the LTT is not caused by topological imbalances. The optimization of habitat types shows that closed habitat (i.e. forest) is the most likely ancestral habitat state for dwarf chameleons (Fig. 1a). Most members of clades A and C retain this state, although there are several independent transitions to more open habitat within these clades. In contrast, the ancestral node of clade B shows a shift to open habitat during the Late Miocene, just prior to the Pliocene radiation of this clade. The two scaled diversity measures used (SR, PD) indicate two areas of present-day dwarf chameleon diversity (Fig. 4). One occurs in the south and corresponds to the Cape Floristic Region (CFR) hotspot, and the other occurs in the east and corresponds to the Maputaland–Pondoland–Albany (MPA) hotspot. Non-parametric tests show that the two scaled measures are correlated (Spearman’s rho; P < 0.01), and show the same general geographic pattern, as has been observed in other taxonomic groups (Polasky et al., 2001; Rodrigues & Gaston, 2002). However, scaled values within certain individual half-degree squares (HDS) are significantly higher for scaled PD when compared with scaled SR (Wilcoxon’s signed ranks test; P < 0.001).

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K. A. Tolley, B. M. Chase and F. Forest (a)

(b)

Figure 1 (a) Ultrametric tree (time scale in million years ago; Ma) showing lineage diversification for the genus Bradypodion. Clades are labelled (A)–(C) according to the text. The numbers on each node refer to Table S3 (divergence dates and 95% CIs). Habitat optimizations are represented by blue (closed habitat), red (open habitat) and black (mixed habitats), with likelihood optimization probabilities for the habitat types represented by pie charts at each node. Pie charts for nodes 10 and 11 are not shown but have open habitat frequencies > 0.99. Branches are colour-coded according to parsimony optimization. (b) Observed lineage-through-time plot (black dots) superimposed over a simulation using a Yule model (black line) representing the expected number of lineages through time (based on 1000 simulated trees).

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Chameleon speciation tracks climate transitions Figure 2 Spatio-temporal patterning of Bradypodion during periods of marked global climate change as identified in d18O and d13C records from marine cores. The number/intensity of diversification events (red through yellow grid cells) are mapped according to the present-day distribution of the associated extant lineages. The location and intensity of radiations for each timeslice are mapped according to the cumulative number of lineages observed in the phylogeny (Fig. 3).

DISCUSSION Present-day diversity in dwarf chameleons is concentrated in two main epicentres corresponding to known hotspots of biodiversity, namely the CFR and the MPA. The geographic distribution of contemporary diversity is demonstrated by both SR (species richness) and PD (phylogenetic diversity). Despite the correlation between the measures, PD provides an indication of which areas have the greatest diversity in evolutionary history, as some areas exhibit higher PD values compared with the associated SR. These discrepancies highlight the fact that SR alone provides limited information about the quantification of evolutionary diversity (Forest et al., 2007). Such inferences can be better made using PD, as areas with relatively higher PD values reflect either multiple, shallow, and closely related branching events (phylogenetic clustering), or several, deep, unrelated branching events in the phylogeny (phylogenetic over-dispersion). Thus, PD is a powerful tool for identifying areas where evolutionary processes should be examined in the light of ecological and palaeoclimatic data. In dwarf chameleons, PD is greatest in areas where the diversity was generated as a result of radiations over different time periods. PD is elevated within the MPA owing to radiations in the Late Miocene (clade C lineages with long branches) and Pleistocene (clade C lineages with short branches). PD patterns in Bradypodion do not seem to be the result of phylogenetic dispersion, as found in the eastern CFR for floral assemblages (Forest et al., 2007). Although modern centres of richness can be identified through diversity measures, it is important to understand their history in order to ensure that the full evolutionary potential of the species found within them is protected. Diversification in dwarf chameleons shows regional specificity in relation to major climatic and geological events. We suggest that the environmental mechanisms influencing these spatio-temporal patterns are related to the global trends of progressive cooling and atmospheric CO2 reductions, which have resulted in changes in rainfall seasonality and contributed to the development of increasingly arid climates with a shift to more open vegetation in the African sector of the Southern Hemisphere. Lineage accumulation There is no strong evidence for shifts in lineage diversification rate variation, but these methods do not take into consideration temporal aspects of the phylogeny. Certainly, the lineage Journal of Biogeography 35, 1402–1414 ª 2008 The Authors. Journal compilation ª 2008 Blackwell Publishing Ltd

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MPA CFR (a)

(b)

Figure 3 Comparison of the cumulative number of lineages in Bradypodion (top line; error bars represent 95% credibility intervals for the dates associated with the Bayesian dating) with d18O (centre line) and d13C (bottom line) records from marine cores (Zachos et al., 2001), which are proxies of global climate change.

accumulation and LTT plots reveal punctuated episodes of diversification in the latter half of the Late Miocene, during the Miocene–Pliocene transition, and in the very latest Pliocene. These changes can be linked to the progressive development of hypothermic conditions that prevailed following the MidMiocene Climatic Optimum. Whereas warmer and moister conditions prior to the Late Miocene maintained widespread forests in southern Africa (Coetzee, 1982, 1983, 1986; Scott et al., 1997), subsequent global cooling and the development of the Antarctic ice sheet initiated a shift from moist woodlands with predominantly summer rainfall to a range of more arid to semi-arid environments with increasingly seasonal rainfall regimes (Udeze & Oboh-Ikuenobe, 2005). A period of Earth low orbital eccentricity between 13.87 and 13.84 Ma (Holbourn et al., 2005) induced a marked expansion in the East Antarctic ice sheet, and the beginning of a permanent ice sheet presence on the continent (Zachos et al., 2001). The development of this ice sheet is believed to be related to the development of the Antarctic Circumpolar Current (Livermore et al., 2005), to the drawdown of atmospheric CO2 associated with Himalayan uplift (Raymo & Ruddiman, 1992), or to both (Barker & Thomas, 2004). Sharp d18O excursions between 13.4 1408

Figure 4 Half-degree grid maps showing two measures of diversity, namely (a) species richness (SR), and (b) phylogenetic diversity (PD), for Bradypodion in South Africa. Each shaded cell represents at least one species occurrence, with darker shades indicating higher values for each measure. Inset shows approximate locations of the Cape Floristic Region (CFR) and the Maputaland–Pondoland–Albany (MPA) hotspot.

and 9.4 Ma (the Mi 3–7 events; (Miller et al., 1991) are registered off the south-west African coast (Westerhold et al., 2005) and provide evidence for a coeval cooling of ocean temperatures (Kastanja et al., 2006). Furthermore, uplift of the Great Escarpment (Partridge & Maud, 1987) is thought to have isolated the interior plateau, contributing to aridification of the subcontinent (Sepulchre et al., 2006). This, combined with the prevailing cooler conditions and reduced atmospheric CO2, resulted in the spread of savanna and grasslands and a shift from C3- to more C4-dominant vegetation (Cerling et al., 1997; Bredenkamp et al., 2002; Lee-Thorp et al., 2007).

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Chameleon speciation tracks climate transitions Periods of lineage diversification interrupted by repeated static phases highlight the changes taking place in the palaeolandscape. Corresponding pollen evidence from the west coast of southern Africa (Scott, 1995; Udeze & ObohIkuenobe, 2005) indicates progression from a sub-tropical environment with palm and mixed montane forests to drought-tolerant taxa beginning in the Mid-Miocene. Along the west coast, the concurrent development of the Benguela upwelling system (Diester-Haass et al., 2004) would have reinforced this trend towards a semi-hyperarid climate regime, further stressing and thus restricting forests in the region. Commensurate with progressive aridification and increased seasonality, closed habitats would have shrunk into the few refugia in which they are found today along the south coast and in the east (Scott et al., 1997; Chase & Meadows, 2007). Forest lineages dominate clades A and C, and the dated phylogeny suggests that their origin dates to the Late Miocene. The long branches observed in these lineages and lack of extant sister species could be indicative of lineage extinctions, associated with the increasing aridification and fragmentation of forests, which isolated species in shrinking refuges. Although the presence alone of long branches is not in itself evidence for lineage extinctions, a complete sampling at the species level, as in the studied group, renders this assertion more plausible (see also below). Regional patterns and processes During the Late Miocene–Pliocene, the expansion of the ice sheet and the initiation of sea-ice were key factors in the development of South Africa’s winter-rainfall zone (WRZ) (Stuut et al., 2004). The present-day WRZ core lies along the south-western coast, and appears to have existed for at least the last c. 5 Myr (Franz-Odendaal et al., 2002). This environment is thought to have promoted the progressive development, expansion and establishment of the fynbos biome, an exceptionally rich floral assemblage that forms the heart of the CFR (Meadows & Sugden, 1993; Cowling & Lombard, 2002; Chase & Meadows, 2007). Several other taxonomic groups located in the CFR are thought to have diversified during this period, especially in the western CFR (e.g. Cowling & Lombard, 2002; Price et al., 2007; Smit et al., 2007). Although the evidence is still patchy for fauna, the data that exist suggest a major turnover of faunal lineages in the CFR during this time period. It has been proposed (Cockcroft et al., 1987) that during Pleistocene glacial periods the WRZ would have expanded to encompass much of southern Africa. Although still a subject for debate, a recent review of the available evidence broadly supports this hypothesis (Chase & Meadows, 2007). It is suggested here that, during the Pliocene, centres of chameleon diversification tracked what would have been a progressive west-to-east expansion of a zone of enhanced seasonality and increasingly significant winter rainfall influence (Fig. 2). The Pliocene development of the boundary conditions controlling these climatic conditions (e.g. Marlow et al., 2000; Udeze & Oboh-Ikuenobe, 2005) is coincident with significant changes

in East Africa (Herna´ndez Ferna´ndez & Vrba, 2006) and eastern South Africa (Vrba, 1985; Lee-Thorp et al., 2007) that mark the development of drier conditions and the continued expansion of C4 grasslands that followed the closing of the Panama seaway and the intensification of the Northern Hemisphere glaciation (Lear et al., 2003; deMenocal, 2004). Together, these dramatic changes in global and regional climates and controls are considered to be likely mechanisms for the fragmentation of closed habitats and the radiation of chameleon lineages through the Pliocene. The MPA represents an interesting case whereby current diversity is an admixture of ancient and recent radiations, punctuated by extinctions. A diverse range of studies suggest that most northern MPA forests are refugia (Eeley et al., 1999; Mazus, 2000; Lawes et al., 2007), and contraction of MPA forests in the Pleistocene produced localized faunal extinctions (Lawes et al., 2007). Consistent with this pattern, several dwarf chameleon lineages (clade C) are presently known from these forests (Bradypodion nemorale, B. sp. 5 and B. sp. 6), but all have long branches and no closely related sister species, suggesting that fragmentation, isolation, and extinction filtering has characterized these chameleon lineages. Perhaps more strikingly, only two dwarf chameleon species presently occupy coastal lowland forests in the MPA (B. setaroi and B. caffer), and these are represented by terminal taxa with long branch lengths with no sister species. Coastal lowland forests expanded in the region after the Last Glacial Maximum (Finch, 2005; Mucina & Geldenhuys, 2006), and these surviving relict lineages of chameleons could have taken advantage of this emerging forest. Overall, the pattern suggests that, although the origins of these five MPA lineages date to the Late Miocene, the disappearance of forests in association with Pleistocene climate shifts have filtered out the more vulnerable sister taxa. Although the MPA is thought to have undergone a Pleistocene–Holocene vegetation shift with isolated Afromontane forest surviving during the Last Glacial Maximum (Eeley et al., 1999; Mucina & Geldenhuys, 2006), a subsequent increase in southern MPA forests appears to have occurred during the mid-Holocene (Finch, 2005; Mucina & Geldenhuys, 2006; Lawes et al., 2007). A renewed increase in forest habitat would have provided habitats for chameleon lineages that had survived the widespread forest loss up until that time. Indeed, four lineages within clade C have recently radiated in that region (B. thamnobates, B. melanocephalum, B. sp. 7 and B. sp. 8). Although MPA forests have since retreated again (Finch, 2005) and open habitats now dominate the region (Mucina & Geldenhuys, 2006), these lineages remain in surviving pockets of mistbelt and lowland forests. In addition, at least three of the four lineages appear to be making a transition to open habitats, as they occur either in a mixture of habitats (B. thamnobates, B. sp. 8) or in open grassland habitat (B. melanocephalum). Not surprisingly, genetic differences among these young lineages are low, and, as expected, lineage sorting is incomplete (see Fig. S1; Maddison & Knowles, 2006). However, they show strong morphological differences, and this could be a result of strong selection in response to habitat shifts. Chameleons, as well as a

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K. A. Tolley, B. M. Chase and F. Forest number of other lizards (e.g. anoles, geckos), are highly responsive to selective pressure on the phenotype (Losos et al., 1997, 1998; Lamb & Bauer, 2005; Measey et al., unpublished data), and both empirical studies and direct observations show that considerable morphological differences can arise despite a lack of lineage sorting. CONCLUSIONS Increasingly, it appears that southern Africa may truly be a ‘cradle of faunal diversity’, as the few taxa studied to date appear to have radiated since the Miocene and especially since the Pliocene (e.g. Matthee & Flemming, 2002; Price et al., 2007; Smit et al., 2007). For chameleons and perhaps for other taxonomic groups (Lawes et al., 2007), biogeographical patterns are coupled to lineage turnover: radiations occurred in lineages that were able to take advantage of increasing C4 habitats, but extinctions mark lineages that remained confined to shrinking C3 habitats. In this regard, if southern Africa is a ‘cradle of diversity’, it is probably not a ‘museum’ retaining that diversity, as are the tropics. As a result of global and local palaeoclimatic shifts, several episodes of regional cladogenesis are proposed for dwarf chameleons in the formation of current epicentres of diversity. Within the MPA the total diversity is an aggregate of asynchronous radiation events, whereas within the CFR the bulk of the diversity is a result of a Pliocene radiation. Individual radiation events were preceded by periods of stasis, which coincided broadly with periods of limited environmental change. Reductions in closed habitats would have occurred during the progressive evolution towards our present ‘icehouse’ Earth, leading to subsequent increases in numbers of lineages, suggesting that cladogenesis was initiated repeatedly through the generation of novel habitats. Closed habitats are ancestral for dwarf chameleons, with a shift to open habitats synchronized with vegetation shifts brought about by climatic changes. Most species presently occur within a single habitat type, although some extant lineages occur in both habitat types. In these lineages, different ecomorphs occupy each habitat type, suggesting strong selection pressure on the phenotype (Tolley & Burger, 2007; Measey et al., unpublished data), and underlining the importance of these vegetation shifts in the initiation of radiations (Streelman & Danley, 2003) in dwarf chameleons. Our results offer a model for the evolution of southern African environments by coupling patterns of species radiation and climatic change in this diverse group of lizards. Contemporary diversity is greatly influenced by environmental upheaval, and the fracturing of habitats and the strong link between past climates and biodiversity patterns exemplified in dwarf chameleons further highlights the tremendous influence that future climatic perturbations will have on all forms of life. ACKNOWLEDGEMENTS We thank Bayworld (Port Elizabeth Museum), the Iziko South African Museum, and the Transvaal Museum for access to 1410

distribution records, and J. Measey and T. Nowell for comments on the manuscript. South African National Biodiversity Institute data records were contributed by K.A.T., M. Burger, M. Cunningham, C. Henderson, K. Hopkins, D. Houniet, A. Moussalli, D. Stuart-Fox, C. Tilbury, and A. Turner. Funding was provided by the South African National Biodiversity Institute to K.A.T. and by the Smuts Memorial Botanical Fellowship (University of Cape Town) to F.F. REFERENCES Allen, A.P., Brown, J.K.M. & Gillooly, J.F. (2002) Global biodiversity, biochemical kinetics, and the energetic-equivalence rule. Science, 297, 1545–1548. Barker, P.F. & Thomas, E. (2004) Origin, signature and palaeoclimatic influence of the Antarctic Circumpolar Current. Earth-Science Reviews, 66, 143–162. Bauer, A.A. & Lamb, T. (2005) Phylogenetic relationships of the southern African geckos in the Pachydactylus group (Squamata: Gekkonidae). African Journal of Herpetology, 52, 105–129. Behrensmeyer, A.K., Todds, N.E., Potts, R. & McBrinn, G.E. (1997) Late Pliocene faunal turnover in the Turkana Basin, Kenya and Ethiopia. Science, 278, 1589–1594. Bobe, R. & Behrensmeyer, A.K. (2004) The expansion of grassland ecosystems in Africa in relation to mammalian evolution and the origin of the genus Homo. Evolution of grass-dominated ecosystems during the late Cenozoic Session at the North American Paleontological Convention, 2001. Palaeogeography, Palaeoclimatology, Palaeoecology, 207, 399–420. Bobe, R., Behrensmeyer, A.K. & Chapman, R.E. (2002) Faunal change, environmental variability and late Pliocene hominin evolution. Journal of Human Evolution, 42, 475–497. Bowie, R.C.K., Fjeldsa˚, J., Hackett, S.J. & Crowe, T.M. (2004) Molecular evolution in space and through time: mtDNA phylogeography of the Olive Sunbird (Nectarinia olivacea/ obscura) throughout continental Africa. Molecular Phylogenetics and Evolution, 33, 56–74. Bowie, R.C.K., Fjeldsa˚, J., Hackett, S.J., Bates, J.M. & Crowe, T.M. (2006) Coalescent models reveal the relative roles of ancestral polymorphism, vicariance, and dispersal in shaping phylogeographical structure of an African montane forest robin. Molecular Phylogenetics and Evolution, 38, 171– 188. Bredenkamp, G.J., Spada, F. & Kazmierczak, E. (2002) On the origin of Northern and Southern Hemisphere grasslands. Plant Ecology, 163, 209–229. Bromham, L. & Penny, D. (2003) The modern molecular clock. Nature Reviews Genetics, 4, 216–224. Brown, J.K.M. (1994) Probabilities of evolutionary trees. Systematic Biology, 43, 78–91. Cerling, T.E., Harris, J.M., MacFadden, B.J., Leakey, M.G., Quade, J., Eisenmann, V. & Ehleringer, J.R. (1997) Global vegetation change through the Miocene/Pliocene boundary. Nature, 389, 153–158.

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K. A. Tolley, B. M. Chase and F. Forest BIOSKETCHES Krystal Tolley leads the Molecular Ecology and Evolution Program at the South African National Biodiversity Institute. Her interests include historical biogeography and speciation processes in southern African reptiles, with particular emphasis on chameleons. Brian Chase is a palaeoclimatologist specializing in the environmental evolution of southern Africa. He is currently focusing on developing the range of palaeoenvironmental proxies contained within the region’s fossilized herbivore middens. Fe´lix Forest is head of Molecular Systematics at the Royal Botanic Gardens, Kew, and is mainly interested in speciation, biogeography and the effects of environmental pressures on biodiversity, with a particular focus on the flora of the Cape region of South Africa.

Editor: S¸ erban Proches¸

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