Department of Biology, Institute of Molecular Evolutionary Genetics, and Astrobiology Research Center, Pennsylvania State University
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Abstract |
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Introduction |
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Avian molecular clocks have used internal (Cooper and Penny 1997
; Waddell et al. 1999
) and external (Hedges et al. 1996
; Härlid, Janke, and Arnason 1997, 1998
; Kumar and Hedges 1998
; Härlid and Arnason 1999
) calibrations. An advantage to the use of an internal calibration is that the time estimated is closer to the calibration time and therefore requires less extrapolation and associated error. One disadvantage is that the fossil record of the group in question (e.g., birds) may be poorer than that of another group (e.g., mammals) and therefore more likely to yield a significant underestimate of divergence time unless the fossil calibrations are chosen carefully. Another disadvantage is that an internal calibration could be considered nonindependent or paralogical. For example, if the question being posed involves the origin of avian orders, and the fossil record of avian orders is used to calibrate the clock, then the two are not independent. External calibrations have the advantage that they are independent and do not rely on the fossil record of the group in question. On the other hand, the larger extrapolation involved may lead to greater statistical error in the time estimate. An additional problem with external calibrations is that genes appropriate for resolving relationships within a group may be too fast-evolving for comparisons outside of the group.
A possible solution is to use the advantages of both internal and external calibrations without the disadvantages. This can be accomplished with a sequential calibration method. First, the divergence time is estimated for a basal divergence (anchor point) within the ingroup using an external calibration. Because it is a basal (early) divergence, the extrapolation error is reduced. Afterward, other genes and molecular data sets more appropriate for the ingroup can be calibrated with this anchor point. Here, we used this sequential calibration method to refine the avian molecular timescale. In this case, we chose the divergence between the order comprising the ducks (Anseriformes) and the order comprising the game fowl (Galliformes) as an anchor point because of its basal position and because those two groups are best represented in the sequence databases. Most avian orders are represented by only one or two protein sequences, whereas hundreds to thousands of sequences, representing dozens of genes, are available for galliforms and anseriforms. Several studies have supported a close relationship between those two orders and a close relationship between that clade (Galloanserae) and other neognathous birds (Neoaves; Cracraft 1988
; Sibley and Ahlquist 1990
; Caspers et al. 1997
; Groth and Barrowclough 1999
; van Tuinen, Sibley, and Hedges 2000
; fig. 1
). Alternative arrangements obtained with some mitochondrial sequences (Mindell et al. 1997, 1999
; Härlid and Arnason 1999
) may be the result of taxon-sampling biases (van Tuinen, Sibley, and Hedges 2000
).
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Materials and Methods |
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To investigate the extent of rate constancy among the mtDNA sequences, a branch length test was performed, after which the taxa showing significantly different branch lengths at the 1% level were excluded (Takezaki, Rzhetsky, and Nei 1995
). A second branch length test was performed on the pruned data set to determine if other taxa now exhibited heterogeneous branch lengths because of a possible change in the mean. Additional branch length tests were performed until all remaining sequences (n = 26) exhibited rate constancy (mean BLroot-tip = 0.013). Of the pruned sequences, several showed branch lengths smaller than the final mean. Rate constancy among the rate-constant taxa was confirmed by performing a two-cluster test (Takezaki, Rzhetsky, and Nei 1995
). Branch lengths and tree topologies in both tests were produced from a Kimura two-parameter distance including transversions only and the neighbor-joining tree building method. A linearized tree (Takezaki, Rzhetsky, and Nei 1995
) was produced from this pruned data set, and nodes were timed using the galliform-anseriform calibration. This molecular rate was then applied to the linearized tree of all taxa, and additional nodes were timed with a lineage-specific method (t = l1/rA; Takezaki, Rzhetsky, and Nei 1995
; Kumar and Hedges 1998
; Schubart, Diesel, and Hedges 1998
). To account for the difference in branch lengths between the calibration taxon and these slower- or faster-evolving taxa, branch length ratios were estimated for each of these taxa to the calibration taxon, and time estimates were multiplied by this ratio to obtain final weighted estimates.
Divergence time estimates for the DNA hybridization data set were based on the mean T50 values reported in figs. 353369 in Sibley and Ahlquist (1990)
. For the immunological transferrin protein data, distance values were obtained from neighbor-joining branch lengths calculated from a complete data matrix of published reciprocal values (Ho et al. 1976
; Prager and Wilson 1976
; Prager et al. 1976
) under the assumption of rate homogeneity. Divergence times within neornithine birds were estimated from the three molecular data sets only for well-established groupings (see Olson 1985
; Cracraft 1988
; Sibley and Ahlquist 1990
; Feduccia 1996
) or for groupings consistent between all three molecular data sets (table 3
).
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Results |
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The galliform-anseriform divergence time obtained from the mean of the rate-constant genes ranged between 90 and 100 MYA, with two of the three calibrations yielding times close to 90 MYA (table 2
). Similar times were obtained from a concatenation of the rate-constant genes (92.0 MYA using RM calibration), in combination with a mean gamma parameter of 1.4 (among genes), when including the outliers or when averaging over all genes regardless of rate constancy. A slightly older time estimate (112 ± 11.7 MYA, n = 5 genes) for the galliform-anseriform divergence was published earlier (Kumar and Hedges 1998
), but it was based on fewer genes and a larger gamma parameter (a = 2). For subsequent timing within modern birds, a galliform-anseriform divergence time of 90 MYA (89.8 ± 6.97 MYA) was used as the internal calibration point because it was based on more than one external calibration and involved the most conservative (recent) estimate of the rate-constant genes.
Divergence Times Among Birds
Multiple standard errors were involved in the calculation of the time estimates based on the mtDNA data, and these are shown as single propagated errors for each node in table 3
. As a result, the calibration errors alone yielded a propagated standard error of about 12% of the final time estimate. For the DNA-DNA hybridization data set, the galliform-anseriform calibration yielded a rate of 0.255 °C/MYR (= 3.92 Myr/
°C). A slightly slower rate of 0.213
°C/MYR (= 4.69 Myr/
°C) was proposed by the authors of a study (Sibley and Ahlquist 1990
) based on an ostrich-rhea vicariance calibration now considered to be both phylogenetically and temporally incorrect (van Tuinen, Sibley, and Hedges 1998
). For the transferrin data set, the galliform-anseriform calibration yielded a rate of 0.72 units of immunological distance per Myr; a slower rate than the 1.01.2 units/Myr estimated by the original authors (Prager et al. 1974
). The original transferrin rate was estimated from the fossil record under the assumption of 100 MYA for the mean divergence time between bird orders and using 70 MYA for the divergence between phasianoid galliform and megapodioid galliform birds (Prager et al. 1974
). However, the bird fossil record is too sparse to be useful for accurate internal calibration. Among the different molecular data sets, general agreement exists between divergence times for each avian group, as shown by small standard errors (table 3
). As described previously (van Tuinen, Sibley, and Hedges 2000
), we found a high correlation between mtDNA versus hybridization pairwise distances (r = 0.90), but also high correlations (r = 0.93) between the other data sets (transferrin vs. mtDNA and vs. hybridization distances). These results suggest clocklike behavior in all three data sets (see also Wilson, Carlson, and White 1977
; Sibley and Ahlquist 1990
).
The earliest divergences within modern birds, between Palaeognathae and Neognathae and between Galloanserae and Neoaves, are estimated to have occurred in the mid-Cretaceous (100120 MYA; table 3 ). Considering the three molecular data sets simultaneously, the deepest splits within Palaeognathae, Galloanserae, and Neoaves, as well as some intraordinal divergences, also are estimated to have occurred in the Cretaceous (7590 MYA, fig. 3 ).
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Discussion |
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An alternative interpretation of the discordance between molecular and fossil divergence times is that molecular clocks have accelerated during rapid adaptive radiations (Benton 1999
). However, it is often overlooked that most molecular-clock studies have tested for such deviations from rate constancy (e.g., in comparisons of lineages inside and outside of groups undergoing radiation). Moreover, clock studies using large numbers of genes and diverse taxa thus far have not encountered biases in species-rich versus species-poor lineages (Kumar and Hedges 1998
; Hedges and Kumar 1999
). In addition, the dichotomy between molecular estimates and the fossil record does not appear to be a general one, but is most pronounced for vertebrate divergences in the late Cretaceous (Kumar and Hedges 1998
). An acceleration in molecular clocks during the radiation of avian and mammalian orders is not consistent with this restricted (Cretaceous) nature of the gap or with mechanisms of molecular evolution (Easteal 1999
; Hedges and Kumar 1999
). Finally, most knowledge on the origin and early evolution of birds has been gained only in the last 15 years, and therefore more discoveries should be expected (Chiappe 1995
; Padian and Chiappe 1998
). Nonetheless, the importance of this evolutionary question requires continued scrutiny of both molecular time estimates and the fossil record.
Times of divergence are of particular interest for one group of birds, the ratites. These are large, flightless birds currently distributed on the southern continents of South America (rheas), Africa (ostrich), Australia and New Guinea (emu and cassowary), and New Zealand (kiwi). Their closest relatives are the smaller, fowl-like tinamous of Central and South America. There is disagreement as to whether the current distribution and phylogeny of the ratites is the result of dispersal (Houde 1986
; Feduccia 1996
) or involves some continental breakup (Cracraft 1974
; van Tuinen, Sibley, and Hedges 1998
). Moreover, the specific timing of divergences will have a bearing on alternative scenarios within the context of continental breakup (van Tuinen, Sibley, and Hedges 1998
).
Two recently proposed scenarios for the origin of ratites assume a close relationship between the South American (rhea) and Australasian (emu, cassowary, and kiwi) taxa based on molecular evidence (van Tuinen, Sibley, and Hedges 1998
). Also, both scenarios suggest that the breakup of Antarctica and Australia (
65 MYA; Smith, Smith, and Funnell 1994
) was the mechanism for the divergence of those two clades. The two scenarios differ in the location of the earliest ratite: Africa versus South America. One suggests that a proto-palaeognath stock was distributed on the Africa-South America supercontinent in the early to middle Cretaceous, leading to ratites (Africa) and tinamous (South America) when the two continents split 100105 MYA. Under this scenario, there was subsequent dispersal of one African lineage back to South America via a late Cretaceous proto-Antillean land connection. Alternatively, the proto-palaeognaths may have been isolated in South America after that continent separated from Africa. Under this second scenario, the tinamou-ratite divergence took place within South America, and a subsequent dispersal of a proto-ratite northward to Laurasia, and then to Africa, led to the African ratites. The presence of fossil ratites in the Cenozoic of Laurasia (Houde 1986
; Martin 1992
) is compatible with both hypotheses (van Tuinen, Sibley, and Hedges 1998
).
The mean divergence time estimates for palaeognaths (table 2
and fig. 1
) support the second scenario. Specifically, the divergences between tinamous and ratites (83 MYA) and between ostrich and other ratites (75 MYA) are younger than predicted by the first scenario and therefore suggest that ratites arose in South America. The divergence time estimate for the rhea versus emu/kiwi (65 MYA; table 3
) is also in agreement with tectonic and biogeographic models for the New World origin of Australasian taxa such as marsupials and hylid frogs (Maxson, Sarich, and Wilson 1975
; Woodburne and Case 1996
). The early Cenozoic divergence time (50 MYA; table 3
) between kiwis and emus suggests that the ancestor of the kiwi lineage may have dispersed over water from Australia to New Zealand. However, in some of these cases, the 95% confidence intervals for the divergence times do not permit rejection of the alternative scenario (fig. 3
). In the future, additional nuclear genes, particularly from nongalloanserine taxa, should help to further refine these time estimates and yield smaller confidence intervals.
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Acknowledgements |
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Footnotes |
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1 Keywords: bird
divergence time
molecular clock
ratite
sequential calibration
2 Address for correspondence and reprints: S. Blair Hedges, Department of Biology, 208 Mueller Laboratory, Pennsylvania State University, University Park, Pennsylvania 16802. E-mail: sbh1{at}psu.edu
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