* Centre for Evolutionary Biology and Biodiversity, School of Earth and Environmental Sciences, University of Adelaide, and Natural Sciences Division, The South Australian Museum, Adelaide, Australia
Correspondence: E-mail: Andrew.Hugall{at}adelaide.edu.au.
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Abstract |
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Key Words: molecular clock rate-smoothing ultrametric trees Agamidae Wallace line Gondwanan biogeography
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Introduction |
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The 64 or more species of agamid lizards ("dragons") constitute a sizeable component (13.5%) of the Australian lizard fauna and traditionally have been interpreted to have arrived by dispersal from the north (e.g., Cogger and Heatwole 1981; Schwaner et al. 1985; Greer 1989). This interpretation, however, has been challenged by a recent analysis of mtDNA data that inferred deep (150 MYA) divergence dates between Australasian agamids and their sister group (the SE Asian mainland Physignathus cocincinus) that could only be consistent with an ancient vicariance during the early breakup of Gondwana (Schulte, Melville, and Larson 2003; see also Moody [1980] and Macey et al. [2000]).
Schulte, Melville, and Larson (2003) used an approximately 1,600-bp segment of mtDNA to construct the phylogeny of Australasian and a sparser sampling of other agamids. Branch lengths were reconstructed using a parameter-rich maximum-likelihood (ML) model and then, because of significant rate variation, further modified using nonparametric rate smoothing (NPRS) (Sanderson 1997) to obtain an ultrametric tree. These reconstructed branch lengths were translated into absolute time using a calibration rate of 0.65% change per Myr along each lineage, calculated using uncorrected differences in Laudakia agamids (Macey et al. 1998), and also consistent with other ectothermic vertebrates (Weisrock et al. 2001). The divergence between the Australasian radiation and P. cocincinus was thus dated at around 150 MYA. The problem here is that a tree with highly "stretched" branches constructed under one model has been assigned absolute dates using a calibration rate inferred from (and applicable to) a tree with much shorter branches reconstructed under a very different model.
Here, we reanalyze the mtDNA data of Schulte, Melville, and Larson (2003) using more appropriate methods and taxon sampling. The results all suggest divergence dates around four to five times shallower (30 MYA) than proposed, with associated errors that are much greater than proposed. This more recent divergence is corroborated by analysis of new nuclear gene (c-mos) sequences.
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Mitochondrial Data |
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The results of Schulte, Melville, and Larson (2003) are erroneous because an ML+NPRS tree with greatly lengthened, reconstructed branch lengths has been related to absolute time using a divergence rate inferred in other studies using uncorrected sequence divergence. The obvious way (Rambaut and Bromham 1998; Benton and Ayala 2003) to avoid this error is to employ internal calibration(s), so that branch lengths (and inferred rates) subtending the calibration node(s) are modified in exactly the same fashion as branch lengths (and inferred rates) throughout the rest of the tree. Macey et al. (2000) analyzed agamid phylogeny using the same region as Schulte, Melville, and Larson (2003). The data set of Macey et al. (2000) is more appropriate to testing the questions posed in Schulte, Melville, and Larson (2003) than the data set in the latter study for two reasons: (1) taxon sampling is much more balanced, with many more non-Australasian agamids included, and (2) there are multiple geologically dated internal calibration points within Laudakia that are all highly consistent with each other and broadly consistent with other studies (Macey et al. 1998; Schulte, Melville, and Larson 2003). The divergence used to calibrate the present tree is the isolation of the Iranian plateau Laudakia (L. microlepis, L. erythrogastra, and L. caucasia) from their nearest northern relatives (identified as the L. himalayana clade by Macey et al. [2000]), which Macey et al. (1998) suggested was associated with a tectonic event of 10 MYA (the Pamir-TienShan uplift). Similarly, use of all the possible Laudakia calibration points simultaneously (e.g., using penalized-likelihood rate smoothing [Sanderson 2002]) would also give virtually identical results, given they are located on the same region of the tree and highly consistent with each other.
Using Macey et al.'s (2000) alignment (http://Systbiol url, matrix macey-99-59) and tree (their figure 8), with the GTRgi model, we employ the same procedureunconstrained GTRgi tree made ultrametric by (1) MLK and (2) NPRS (fig. 2). Analyses with MrBayes, and with other models such as HKYg, yield very similar topologies and branch lengths. For instance, using the most complex available model (GTRgi) increases the P. cocincinus branch length over HKYg by a factor of about 1.08 and over JC by about 2.4 (much the same as with the mtDNA analysis above).
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Nuclear Data |
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There was little stretching of branches using the HKYg or more complex models over the observed differences, suggesting minimal problems with saturation. Limited taxon sampling may hide some saturation among the basal splits; however, the low amount of inferred multiple hits in complex models in the c-mos data suggests this may only amount to a small underestimate. This contrasts with the high inferred sequence divergence and great elongation of basal branches in the mtDNA analyses above, which indicate likely problems with saturation. The observed average pairwise difference across the Australasian-P. cocincinus is 3.5% (uncorrected p-distance), increasing to 4.2% with an HKYg-MLK model and to 6.6% with HKYg-NPRS (type 1) model; much less than for the mtDNA. The likelihood penalty of imposing a molecular clock assumption on the c-mos tree is slight (lnL = 15.7, chi-square test, P = 0.003 with df = 13) and recovers the same tree as the unconstrained search. Given the minor deviation from the molecular clock and low divergence levels, the MLK transformation could well be suitable and even preferable to NPRS (Sanderson 1997, 2002). For this reason, we focus on the MLK analysis, but NPRS dates are very similar (see below). Here, NPRS again increases branch lengths and lowers the ratio of deeper to shallower node distances. This pattern is also apparent in other studies (e.g., Barraclough and Vogler 2001; Martin et al. 2004) and so may be a general effect.
For four well-supported (>90% bootstrap) nodes, variance in branch lengths (and thus divergence dates) was assessed using the nonparametric bootstrap. This involves, for each bootstrap replicate, using the HKYg-MLK model to search for the optimal topology then calculating the depth of the most recent common ancestor of a given set of taxa (Baldwin and Sanderson 1998). Parametric bootstrapping of branch lengths (as employed by Schulte, Melville, and Larson [2003]) gives low variances because it measures only the inherent stochasticity in the chosen substitution model (Felsenstein 2004). In contrast, nonparametric bootstrapping of branch lengths captures errors caused by variation in character sampling and tree topology, which are additional and usually much larger sources of error. The curves showing bootstrap variance in branch lengths are shown in figure 3. Broadly, the 95% confidence interval usually includes 10% to 20% variation in divergence-time estimates, orders of magnitude larger than the standard errors indicated by Schulte, Melville, and Larson (2003) for the mitochondrial data. Even these variance estimates are overly conservative because they ignore such factors as errors in estimating divergence linearity ("stretching") in MLK or NPRS methods and calibration errors (which would uniformly stretch or compress the entire tree). The latter continues to be unjustifiably ignored in molecular clock studies (as emphasized by Graur and Martin [2004]; see also Hedges and Kumar [2004]). However, the magnitude of such fossil calibration errors is difficult to quantify, as it is itself an amalgam of several errors: (1) how closely the first known fossil of a group approaches the (necessarily earlier) true origin of the group, (2) uncertainties in the relative and absolute geochronological dating techniques used to assign an age to the stratum containing the fossil, and (3) uncertainties in the taxonomic assignment of the fossil (i.e., which node or clade the fossil is assumed to "date").
For the c-mos tree, if the Australasian-P. cocincinus split is assumed to be 150 MYA (fide Schulte, Melville, and Larson 2003), the split between Leiolepis and the clade containing the Australasian taxa gets pushed beyond 450 MYA, before the origin of reptiles and, in fact, of terrestrial vertebrates. In particular, the chameleon-agamid split is always at least three times the age of Australasian-P. cocincinus split. These findings are robust to lack of linearity and, hence, rule out any Gondwanan scenario.
There are no well-corroborated calibration points within the agamids for the c-mos tree, but there are two fossil dates to provide benchmarks for the most basal (acrodont-iguanid) split, with the caveat of assuming reasonable linearity. The earliest taxon that can be provisionally assigned to either side of this split is a likely stem acrodont from India, dated at around 190 MYA (Evans, Prasad, and Manhas 2002). However, this taxon is known only from jaw fragments, and the first unequivocal iguanians (priscagamids and Pristiguana) only occur much later (e.g., Evans 1993). The priscagamids have been rigorously shown to be stem acrodonts (Frost and Etheridge 1989) and first occur in Aptian-Albian deposits of Asia, around 110 MYA (Evans, Prasad, and Manhas 2002, and references therein). The phylogenetic relationships of other contemporaneous iguanians have not been properly analyzed, and they cannot yet be used for calibration. If the tentative, earlier date is used to calibrate the tree, the Australasian-P. cocincinus split is dated at 33 MYA (95% CI = 2441 MYA), whereas the Australasian-Leiolepis split is dated at 106 MYA (95% CI = 90126 MYA). If the later calibration date is used as an absolute minimum boundary, these divergences reduced to 19 MYA (95% CI = 1424 MYA) and 62 MYA (95% CI = 5273 MYA), respectively. For the c-mos tree with nonparametric rate smoothing (type 3), the two splits are dated at 50 MYA and 119 MYA (using the 190 MYA calibration) or at 29 MYA and 69 MYA (using the 110 MYA calibration). These dates are comparable with the revised mitochondrial DNA dates calculated above (MLK dates of 28 and 58 MYA, and NPRS type 3 dates of 27 and 52 MYA). Note that the Leiolepis split is approximately three times as old as the P. cocincinus split in the nuclear trees, compared with only twice as old in the mtDNA trees. The compression of the basal nodes in the mtDNA versus the c-mos trees suggests greater saturation effects in the mtDNA.
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Discussion |
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The more appropriate analyses of the agamid data here demonstrate that both the mitochondrial and nuclear sequence data imply a relatively recent divergence between Australasian agamids and the SE Asian P. cocincinus (between 14 and 41 MYA). These dates are sufficiently recent to exclude the Gondwanan vicariance hypothesis (Macey et al. 2000; Schulte, Melville, and Larson 2003), which suggested the split between Australasian agamids and P. cocincinus occurred before 120 MYA, when the last terrains split off from Gondwana and drifted north to Eurasia (Metcalfe 2001). Furthermore, for both the mtDNA and c-mos data sets, the proposed approximately 150-MYA split between P. cocincinus and Australasian agamids (Schulte, Melville, and Larson 2003; see also Macey et al. [2000]) implies implausibly ancient divergences in basal living agamids (300400 MYA). These inferred divergence dates predate the fossil record for lizards and are as old as, or older than, the earliest fossil record for reptiles (Lee 1999; Graur and Martin 2004). Similary, their inferred dates (>110 MYA) for splits within Varanus imply more basal splits happened implausibly early (e.g., 300 MYA for the Lanthanotus-Varanus split, again coincident with the earliest fossil reptiles). Any proposed dates for particular nodes should be strongly suspect when they imply such unlikely dates for other splits within the tree.
Contrary to Schulte, Melville, and Larson (2003), the recent discovery of a 190-Myr-old putative acrodont (Evans, Prasad, and Manhas 2002) does not provide much support for their proposed dates. Using this very early fossil to calibrate the iguanid tree (fig. 3, c-mos) still only yields a divergence date for P. cocincinus and the Australasian agamids of 33 MYA, much more recent than the predicted 150 MYA. Finding a stem agamid that is 700 Myr old would approach the latter result; however, despite the problem with quantifying calibration uncertainty (see above), it seems safe to say such a scenario is unlikely. The "deep divergence" hypothesis also implies that every major agamid clade arose before the formation of Pangea in the Late Permian (250 MYA) and that multiple lineages from each clade should have been present on most continents. It thus has difficulty explaining the total absence of fossil and living agamids in both North and South America and the absence of agamid fossils in all continents before 210 MYA, even those regions with an excellent Permo-Triassic record (e.g., southern Africa and Russia). Finally, one might attempt to recast the Gondwanan vicariance model by assuming that the combined P. cocincinus-Australasian clade (rather than the Australasian clade alone) arose by vicariance, with P. cocincinus secondarily colonizing Eurasia. However, even this more inclusive clade appears too young (65 to 112 MYA using iguanid calibration bounds) to have formed through the early breakup of Gondwana, which requires it to be over 120 Myr old (see Schulte, Melville, and Larson [2003]).
Both the mtDNA and nuclear data indicate that the extant Australasian agamid radiation is between 14 and 41 Myr old (i.e., Oligo-Miocene). This age is consistent with the time frames for dispersal between Asia and Australasia suggested by plate tectonics (Cogger and Heatwole 1981). The gradual northward drift of the Australasian plate does not provide absolute constraints on the timing of dispersals (Hutchinson and Donnellan 1993; contra Hall [1996]). All that can be assumed is that during the Tertiary, dispersal between Asia and Australasia became progressively more likely with time as the plates approached one another. The latest studies suggest that short-range, island-hopping dispersal between Asia and Australasia could have occurred at least as long ago as 30 Myr (Metcalfe 2001), and longer-range dispersal was possible much earlier. The arboreal and/or amphibious habits of many acrodonts (including P. cocincinus and many basal Australasian agamids) would make them reasonable candidates for early dispersal through rafting. Schulte, Melville, and Larson (2003) suggested that agamids were not adept over-water dispersers; however, the three clades of iguanians (chamaeleons, agamids, and iguanids) all appear to contain forms that readily cross water barriers. Chameleons have been inferred to have crossed repeatedly between Madagascar and Africa (Raxworthy, Forstner, and Nussbaum 2002), and agamids and iguanids are found on numerous islands that have always been separated from the mainland by large water gaps (e.g., Moody 1980).
The dates estimated here are also compatible with the presence of agamids in Africa, Asia, and Australasia and their absence from the Americas. This is consistent with the relatively recent origin of agamids in Eurasia (where they potentially have their earliest fossil record at 190 MYA [Evans, Prasad, and Manhas 2002]), followed by dispersal into the Gondwanan landmasses of Africa, India, and Australasia when they drifted northwards. Since the breakup of Pangea, South America has never been connected to Eurasia, and North America connected only at high latitudes. The dates inferred here for basal divergences in agamids are also broadly consistent with the fossil record, placing the agamid radiation within the past 200 Myr, matching paleontological estimates of the timing of the lizard radiation (Estes 1983; Evans 1993).
Within agamids, the mtDNA data show large divergences, evidence of saturation effects, and considerable sensitivity to details of substitution model and rate-smoothing methods. In contrast, the large portion of c-mos appears less affected, performs well at retrieving basal nodes and giving stable estimates for deeper branch lengths in iguanians and should be a useful locus for other similar studies.
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Acknowledgements |
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Footnotes |
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