- Research article
- Open Access
Influence of Tertiary paleoenvironmental changes on the diversification of South American mammals: a relaxed molecular clock study within xenarthrans
BMC Evolutionary Biologyvolume 4, Article number: 11 (2004)
Comparative genomic data among organisms allow the reconstruction of their phylogenies and evolutionary time scales. Molecular timings have been recently used to suggest that environmental global change have shaped the evolutionary history of diverse terrestrial organisms. Living xenarthrans (armadillos, anteaters and sloths) constitute an ideal model for studying the influence of past environmental changes on species diversification. Indeed, extant xenarthran species are relicts from an evolutionary radiation enhanced by their isolation in South America during the Tertiary era, a period for which major climate variations and tectonic events are relatively well documented.
We applied a Bayesian approach to three nuclear genes in order to relax the molecular clock assumption while accounting for differences in evolutionary dynamics among genes and incorporating paleontological uncertainties. We obtained a molecular time scale for the evolution of extant xenarthrans and other placental mammals. Divergence time estimates provide substantial evidence for contemporaneous diversification events among independent xenarthran lineages. This correlated pattern of diversification might possibly relate to major environmental changes that occurred in South America during the Cenozoic.
The observed synchronicity between planetary and biological events suggests that global change played a crucial role in shaping the evolutionary history of extant xenarthrans. Our findings open ways to test this hypothesis further in other South American mammalian endemics like hystricognath rodents, platyrrhine primates, and didelphid marsupials.
Paleobiological studies aim at assessing the influence of past environmental changes on the evolutionary history of organisms. To be reliable these macroevolutionary studies must consider groups possessing a high quality fossil record coupled with a well documented history of past environmental changes encompassing geological, ecological, climatic and biogeographical events . Inferring the impact of these historical events on the evolution of faunas is particularly difficult in cases where dispersal occurred repeatedly between biogeographic areas. For this reason, islands have for long attracted particular interest by acting as natural laboratories for the study of evolution . Owing to its "splendid isolation" from other continental land masses for a great part of the Tertiary era , South America offers a special case of continental island-like evolution with its very peculiar mammalian fauna composed of an initial nucleus of autochthonous taxa subsequently enriched by few successive waves of successful immigrants.
Numerous studies have attempted to relate the evolutionary history of mammalian fossil faunas in South America with well documented environmental changes [4–6]. However, these studies primarily rely on the quality of the paleontological record which is by essence incomplete. Since the seminal paper of Patterson and Pascual  several advances have been made on the understanding of the fossil record from this Southern hemisphere continent . This includes the discovery of additional fossil sites from the previously poorly sampled Tropical zone and new faunal horizons from key ages filling gaps in the stratigraphic sequence. These advances coupled with progress from multidisciplinary studies encompassing tectonic , isotopic  and radiochemical dating  evidences have shed new light on the biotic and environmental history of South America.
Armadillos, anteaters, and sloths belong to Xenarthra and represent the only placental group with living representatives from the initial South American mammalian stock . Owing to their longstanding evolutionary history on this island continent, xenarthrans constitute a promising model for studying the influence of past environmental changes on living organisms. Indeed, the earliest records of Xenarthra in the Paleocene, about 58 million years ago (Mya) [12, 13] already show the morphological features proper to the group (homodonty with simple hypsodont teeth lacking enamel) and possess armadillo-like carapace scutes, suggesting that the group likely originated earlier during the Paleocene or even possibly during the Late Cretaceous . Xenarthra subsequently underwent an impressive Tertiary radiation which led to a great diversity of fossil forms . This diversification was promoted by the South America's isolation from other continental masses at that time. Indeed, South America separated completely from Africa in the Late Cretaceous (80–65 Mya) although it maintained some connections with Antarctica until ca. 36 Mya . Its late Tertiary reconnection with North America via the Isthmus of Panama (3.0–2.5 Mya) led to the Great American Biotic Interchange . During this period of isolation, xenarthrans were one of the dominant groups of placental mammals in the successive South American faunas. They occupied a wide range of ecological niches with fossorial, terrestrial and semiarboreal taxa, gigantic forms such as armoured glyptodonts and ground sloths , and sloths even extending their adaptations to the aquatic environment . The late arrival of North American immigrants seems to have only slightly affected xenarthrans and giant terrestrial forms noteworthy became successful southern invaders of Central and North America . Nevertheless, a great part of the xenarthran diversity disappeared with the dramatic extinctions that took place at the end of the Pleistocene  and essentially affected the largest forms . This massive and global extinction event left only 30 living xenarthran species whose paleontological origins and phylogenetic relationships are very difficult to decipher from the available fossil record , although progresses have recently been made for understanding the origin of some groups of living armadillos .
Molecular phylogenetics is now providing a new tool to complement paleontological and geological studies by allowing to reconstruct both the phylogenetic relationships of living species and the timing of their diversification . Indeed, it is likely that global environmental changes left their footprints in the DNA of living organisms from which the evolutionary history can be reliably inferred . Furthermore, there is at present accumulating molecular evidence that past environmental changes have shaped the evolution of diverse terrestrial organisms such as frogs , squirrels , eulipotyphlan mammals  and elephant-shrews . A robust molecular picture of living xenarthran phylogenetic relationships have been recently established [27, 28] and estimates of their divergence dates have already been used to test biogeographic hypotheses . However, the recent development of improved molecular dating methods offers new possibilities in estimating divergence times and allows to go one step forward .
Recent methodological advances in molecular dating have aimed at relaxing the assumption of the global molecular clock  by modelling the variation of the evolutionary rate along the phylogenetic tree [31–35]. Among these newly developed methods, the Bayesian relaxed molecular clock approach of Thorne et al.  appears particularly promising. Indeed, it allows the calculation of divergence time estimates in the presence of rate variation among lineages coupled with the possibility of incorporating multiple paleontological constraints used as priors . This method has already been successfully used to address long standing questions such as the age of the ancestor of the HIV-1 pandemic strain  and the diversification of placental mammals in relation to the Cretaceous/Tertiary (K/T) boundary [38–40]. By providing new molecular time scales, the application of the Bayesian relaxed clock has also revealed unsuspected biogeographic patterns in frogs  and unravelled the origins of the Malagasy mammalian fauna . These method are nonetheless still in their infancy and robustness of their assumptions to potential sources of errors has to be tested further [34, 42]. However, a significant advance has recently been made with the implementation of the Bayesian method to allow the analysis of genomic data sets by accounting for differences in evolutionary dynamics among genes . Here, we showed that divergence dates within xenarthrans can be accurately inferred by this method and used to unveil the influence of Tertiary environmental changes on the diversification of these South American endemics.
Results and discussion
Evolutionary rate variations and molecular dating methods
The molecular clock hypothesis is rejected for the combined DNA data set by a likelihood ratio test under the F84 + Γ8 model : δ = 2 x (lnL UNCONSTRAINED - lnL CLOCK) = 2 x [-87,895.39 - (-89,379.09)] = 2967.40; d.f. = 48; P < 0.0001. This reveals that extensive rate variation occurs among lineages, with fast evolving taxa like Tonatia (Laurasiatheria), murid rodents (Euarchontoglires) and Procavia (Afrotheria), and slow evolving ones like xenarthrans (Xenarthra), perissodactyls (Laurasiatheria), and Dugong and Elephas (Afrotheria) distributed all over the tree. The existence of such an extensive rate variation among lineages precludes the application of the linearized tree method  whose estimates can be highly biased when the molecular clock does not hold . Thus, the use of a relaxed molecular clock approach designed to accommodate rate variation is preferable for estimating divergence ages with this data set.
The molecular clock assumption can be relaxed in some ways by defining sub-groups in the tree that evolve locally clocklike and estimating divergence dates within a likelihood framework [47, 48]. Despite a definitive improvement in estimating divergence dates in the case of rate heterogeneity, these methods present some practical limitations pertaining to the arbitrary set up of local clocks and the use of a single calibration point [46, 49]. The management of simultaneous calibration points recently introduced in the local clock method appears nevertheless promising . Another class of methods has attained at modelling the evolutionary rate variation along the tree. The semi-parametric rate smoothing method implements a stochastic model of rate change based on the hypothesis of rate autocorrelation between adjacent branches [31, 35] whereas others attempt to model rate variation across the tree in a Bayesian framework [32–34, 36, 43]. Here, we used a Bayesian relaxed molecular clock method first introduced by Thorne et al.  that explicitly accounts for (i) paleontological uncertainty on calibration points by estimating the posterior distribution of rates and divergence times from prior distributions on rates and paleontological constraints , and (ii) contrasted patterns of evolutionary rate among genomic loci  as this has been shown to be the case for our data set .
Basal placental mammal divergences and the K/T boundary
Our dataset comprising 47 placental mammals allowed us to estimate divergence dates for the major placental clades in relation to the Cretaceous/Tertiary boundary (K/T). Indeed, there has been considerable debate [51, 52] on the impact of this massive extinction event on early placental mammal lineages . A recent molecular study by Springer et al.  using the same Bayesian dating method and calibration points – but for a longer data set – found support for the Long Fuse model of Archibald and Deutschman  by placing almost all interordinal divergences in the Late Cretaceous. The results we obtained in estimating the age of the Most Recent Common Ancestors (MRCAs) of seven basal placental clades using different data partitions of our nuclear data set are recapitulated in Table 1. It is noteworthy than the partitions dominated by 1st and 2nd codon positions (ABV 1+2 and A2BV2) generally yielded slightly more recent dates than the complete data set, a behaviour also noticed by Springer et al.  on nuclear exons. Nevertheless, irrespective of the data partition, all estimates obtained for the MRCAs of basal placental clades fell within the Late Cretaceous (Table 1). As there is only slight differences between the results obtained from the three different partition schemes, we will here focus on the results obtained from the longest dataset.
The chronogram obtained on the complete placental data set is presented in Figure 1. Based on this data set, the mean posterior age for the placental crown group, corresponding to the early emergence of Afrotheria, is estimated to be in the Early Cretaceous at 107 ± 6 Mya (95% Credibility Interval: 97–119 Mya). This early split was quickly followed by the divergence between Xenarthra and the remaining placentals at the end of the Early Cretaceous 103 ± 5 Mya (94–113). All four major placental clades have their last common ancestors in the Late Cretaceous (Figure 1) at respectively: 82 ± 6 Mya for Afrotheria (71–95), 65 ± 5 Mya for Xenarthra (55–75), 89 ± 4 Mya for Euarchontoglires (81–100), and 83 ± 3 Mya for Laurasiatheria (78–90). The divergence between Euarchontoglires and Laurasiatheria is estimated to have occurred at 94 ± 4 Mya (87–103). On the whole, our mean posterior estimates of basal placental divergence dates are fully compatible with those obtained by Springer et al.  on a similar set of characters (i.e. nuclear exons) and using the same priors on calibration points. The main placental divergences appear to have occurred in the Cretaceous (Figure 1) as previously proposed by other studies [55, 56] based on the results from the quartet dating approach  or by using the Bayesian relaxed molecular clock approach on complete mitochondrial genomes [38, 39] and concatenated nuclear DNA sequences [25, 40]. The good agreement between these studies in obtaining Cretaceous dates for basal placental divergences seems to validate the occurrence of a massive survival of modern placental lineages across the K/T boundary . However, more paleontological data on the degree of morphological and ecological specialization of early placental lineages are required to distinguish further between alternative models of diversification . In that respect, the recent discovery of the very well conserved eutherian fossil Eomaia in the cretaceous of China (125 Mya) is encouraging .
Another insight from our results is the fact that the paleontological constraint put on the paenungulate MRCA (Figure 1) appears to represent a large underestimate of the actual divergence time for this group (Table 1). In fact, the mean posterior estimate for this node a priori constrained at a minimum of 54 Mya was 69 ± 6 Mya (59–81). This suggests that the earliest proboscidean fossil  is far from representing the earliest Paenungulate and that older fossils belonging to this group remain to be found. This situation might be extended to the African clade (Afrotheria) as a whole since its fossil record remains poorly known despite the recent discovery of a spectacular Oligocene fauna . The assessment of the relative reliability of independent paleontological constraints represents a point that certainly needs more scrutiny in molecular clock analyses since it might have a strong impact on the results . The use of the more precise and reliable calibration constraints is indeed advocated to be of outmost importance in the application of Bayesian molecular clock methods . By allowing to reciprocally estimate the posterior age of different nodes for which paleontological constraint are available, the Bayesian molecular clock method might help to identify unreliable calibration points and groups for which older fossils are likely to be found.
A molecular timescale for xenarthran evolution
The age of the xenarthran crown group estimated by the Bayesian method in the present study (65 ± 5 Mya; 55–75) is consistent with previous results obtained from a local molecular clock approach  on the VWF alone suggesting a 59 to 76 million years (Myr) interval . Xenarthra thus appears to be the major placental clade with the youngest MRCA, but also with the longest ancestral lineage (Figure 1). The first appearance of xenarthrans in the fossil record is materialized by the occurrence of the earliest armadillo scutes in the late Paleocene (ca. 58 Mya) of Brazil [13, 15]. Therefore, our results are compatible with the age assumed for the xenarthran diversification, but suggest the existence of a xenarthran "ghost" lineage that left no fossil traces for almost 50 Myr since their purported origin estimated here around 105 Mya. This observation concurs with the fact that the origin of xenarthrans still constitutes a paleontological and biogeographic enigma [16, 60]. Only the discovery of new fossils – presumably of Cretaceous age according to our estimates – might help to solve this mystery.
The results obtained for the timing of the xenarthran diversification by the relaxed molecular clock approach are recapitulated in Table 1. Following the early split between Cingulata (armadillos) and Pilosa around 65 Mya at the end of the Cretaceous, anteaters and sloths separated at the transition between Paleocene and Eocene some 55 Mya (46–65). Within anteaters the lineage leading to the pygmy anteater (Cyclopes) emerged in the Middle Eocene around 40 Mya (32–49), whereas the Tamandua and Myrmecophaga lineages diverged 30 Myr later in the Late Miocene at 10 Mya (7–14). The oldest undoubted fossil of anteaters comes from the Colhuehuapian (ca. 20 Mya) South American Land Mammal Age (SALMA) and they are well known since the Santacrucian SALMA (ca. 16 Mya). Our estimates, matching those obtained in Delsuc et al. , suggest a long evolution of anteaters prior to the Colhuehuapian SALMA, and confirm the incompleteness of the early fossil record of anteaters. The molecular estimates also emphasize the relative antiquity of the pygmy anteater lineage whose only living representative (Cyclopes didactylus) is morphologically very divergent from the other two genera . The considerable evolutionary divergence of the pygmy anteater might be more efficiently reflected in the taxonomy by placing it in its own family as advocated by Barros et al. .
The Bayesian method estimates the separation between the two modern sloth lineages at 21 Mya (15–28). Unknown as fossils , the extant genera of two-toed (Choloepus) and three-toed sloths (Bradypus) were once placed into two distinct families (respectively Megalonychidae and Bradypodidae) on the basis of their numerous morphological differences and a presumably diphyletic origin . Recent cladistic analyses based on craniodental evidence, including the living forms and the three traditionally recognized families of fossil sloths (Megatheriidae, Megalonychidae and Mylodontidae), placed Bradypus as the sister-taxon of all remaining sloths and Choloepus with the extinct Megalonychidae . The oldest known sloths come from the Eocene of Patagonia and Antarctica but they cannot be precisely assigned to any of the recognized lineages . Our fairly ancient estimation confirms the considerable divergence between the two modern sloth genera bringing support for a taxonomic distinction at a high rank.
The almost complete generic sampling among armadillos (all living genera but the fairy armadillos Chlamyphorus) allows for the first time an adequate estimation of the divergence dates within this poorly known group of placental mammals. The early emergence of Dasypodinae – including the living long-nosed armadillos (Dasypodini) and the fossil Stegotheriini [13, 66] – within armadillos is estimated to have occurred during the Middle Eocene around 40 Mya (31–49). If the plesiomorphic Astegotheriini are removed from the Dasypodinae (see ), this estimation is consistent with the paleontological evidence that suggests an early divergence of this group from other lineages containing living representatives . The two divergent living species of long-nosed armadillos studied (Dasypus novemcinctus and D. kappleri) would have separated at 7 Mya (5–11) in the Late Miocene. This is an interesting result that obliges paleontologists to review the fossil evidence and could also be significant in the taxonomy of the living species. The earliest and most primitive Dasypodini (Anadasypus hondanus) comes from the middle Miocene Laventan SALMA (ca. 13 Myr) in Colombia . That means that the origin and radiation of the Dasypus genus must have occurred very soon after the emergence of the group and paleontologists should expect to find Dasypus remains at any stratigraphic level younger than the Late Miocene. However, the hard evidence of Pre-Pleistocene record is very weak. The oldest undoubted fossil record of the genus comes from the Late Pleistocene, around 0.8 Mya . This fossil evidence would suggest that the separation between D. novemcincus and D. kappleri should be much younger than proposed here, during the Pleistocene or even the Holocene. However, this can be very easily a matter of the paleontological record. Although the early existence of Dasypodinae is recorded in Patagonia, the origin and evolution of the Dasypodini seems to have happened mostly in Northern South America , where the paleontological exposures of appropriate age are not as spectacular as in Southern South America.
The split between the two remaining armadillo sub-families Tolypeutinae (Tolypeutes, Cabassous, and Priodontes) and Euphractinae (Euphractus, Chaetophractus, and Zaedyus) took place about 33 Mya (25–42). The respective diversifications of these two subfamilies appear to have happened in relatively short time periods between 20–22 Mya for Tolypeutinae and 6–7 Mya for Euphractinae (Table 1; Figures 2,3). The evolution of the Tolypeutinae seems to parallel that of the Dasypodini . This is particularly evident for the Tolypeutini, including the earliest known Pedrolypeutes praecursor from the Middle Miocene Laventan SALMA in Colombia, and the genus Tolypeutes, known since the Pleistocene of the pampean region. The fossil record of the giant armadillo (Priodontes) and naked-tailed armadillos (Cabassous) is virtually unknown, but the present distribution clearly suggests a Northern South American history as for the Tolypeutini and Dasypodini. On the other hand, the evolution of the euphractines is more related to Southern South America. The early history of Euphractini is recorded in Patagonia since the Deseadan SALMA (30–25 Mya) with Prozaedyus a fossil form that clearly resembles the living members of the group. However, Carlini and Scillato-Yané  proposed that the Late Miocene-Pliocene euphractines represent an independent radiation in temperate to warm environments of central and northern Argentina from that of the living genera.
These results confirm the fact that living Dasypodidae contains quite divergent taxa grouped into subfamilies of fairly ancient origins . This suggests that the still unclear paleontological roots of these groups have to be searched deeper than previously thought . The relatively younger dates obtained within armadillos in Delsuc et al.  might be explained by an effect of reduced taxon sampling since only three genera (Dasypus, Cabassous and Chaetophractus) were previously considered. The ancient dates obtained for the origin of each of the three armadillo subfamilies are coherent with their distinctive morphologies  and marked differences observed in the structure of their spermatozoa .
Xenarthran diversification and paleoenvironnemental changes
In the light of these results, it is striking to note that some diversification events within Xenarthra seem to be synchronous. This synchronicity among independent xenarthran lineages is illustrated by the correspondence between the distributions of posterior divergence time estimates sampled during Markov Chain Monte Carlo (MCMC) runs presented in Figure 2. Indeed, the separation of the Cyclopes lineage from other anteaters is perfectly correlated with the separation of the Dasypus lineage from other armadillos around 40 Mya in the Middle Eocene. The quasi perfect correspondence between these two speciation events is well illustrated by looking at the posterior distribution of the differences between the ages estimated for these nodes which is centred around 0 (Figure 2A). Similarly, the diversification of the armadillo subfamily Tolypeutinae (Priodontes, Cabassous and Tolypeutes) relates to the separation between the two modern sloths genera around 21–22 Mya in the Early Miocene (Figure 2B). And finally, the recent diversification of the subfamily Euphractinae (Chaetophractus, Euphractus and Zaedyus) also matches well with the separation between the two living species of the genus Dasypus here included at 6–7 Mya in the late Miocene (Figure 2C). Such a correlated history between independent lineages is unlikely to have occurred by chance alone and suggests a role for external factors in shaping the evolutionary history of living xenarthrans.
The influence of environmental changes that occurred during the Cenozoic of South America on the evolution of its endemic fauna has long been studied from the paleobiogeographical point of view . Indeed, this era was characterized by drastic climatic variations associated with changes in sea levels , major biotic modifications  and tectonic phases of Andean uplift . Moreover, recent spectacular advances have greatly improved the understanding of the fossil record of South America . These advances from multidisciplinary studies on tectonics , stable isotopes  and biostratigraphy and geochronology  have clarified the biotic and environmental history of South America during the Cenozoic. The making up of this integrated framework has rendered possible to retrace and identify general evolutionary patterns for terrestrial communities such as mammalian herbivores . Until recently, there has been only limited efforts to incorporate the results of molecular studies into this multidisciplinary canvas . However, some recent surveys have provided substantial evidence that past environmental changes may have significantly influenced the evolutionary history of mammalian groups at a large scale [24–26].
As the only South American endemic group of placental mammals whose evolutionary history encompasses all the Cenozoic, Xenarthra represents a good candidate to test the hypothesis that global environmental changes have influenced the diversification of the endemic fauna. Interpreting our results in the context of relatively well documented environmental variables like Andean tectonic crises, changes in ocean level, and temperature variations sheds light on the peculiar diversification pattern revealed by our molecular dating analyses. Indeed, each of the three correlated diversification events identified among independent xenarthran lineages appears to follow periods of major environmental changes (Figure 3). More specifically, these events seem to be slightly posterior to the major phases of Andean uplift defined as "tectonic crises" by Marshall and Sempere . First, the codiversification of Myrmecophagidae and Dasypodidae in the Middle Eocene occurred just after the large Incaic uplift episode (Figure 3). The estimated date of ca. 40 Mya for these synchronous diversification events matches up with a well dated pulse of this deformation episode in the Andes of Peru estimated at ca. 43 Mya . Second, the simultaneous diversification of modern sloth lineages and the proposed radiation of Tolypeutinae in the middle of the Early Miocene correlates with the end of the first major Bolivian crisis (Figure 3). This diastrophic event that occurred during the Late Oligocene-Early Miocene interval was an intense deformational and magmatic episode widespread along the Andes . The intensity of this crisis is seen as a turning point in Andean tectonics because the Andes became at that time the principal relief of the West coast of South America creating a rain shadow that significantly influenced South American climates . This period is also notably marked by a thermal optimum followed by a brief but deep glacial maximum  and sharp marine regressions . This leads to a major shift in South American mammalian fossil communities with the beginning of the Miocene radiation of ground sloths for example . Third, both the radiation of living euphractine armadillos and the separation between long-nosed armadillos correspond with the latest phases of the second major Bolivian tectonic crisis marked by a significant increase in Andes height (Figure 3). This period marks a stage of general cooling with the formation of the Antarctic ice sheet  resulting in a global drying of the Patagonian region and the formation of the Argentinean pampas in the Late Miocene . This major shift in vegetation with the beginning of the predominance of savannas and grasslands was recorded in isotopic studies in the form of an elevation of the C4/C3 ratio . The fact that euphractine armadillos represent a zoogeographically well defined group that occupies the pampas and savannas of Southern South America  adds credit to the idea that this major environmental change created ecological opportunities that triggered the diversification of this subfamily. The evolutionary history of extant xenarthrans also appears to have been influenced by the major environmental changes that marked the transition between the Eocene and Oligocene epochs with the split at this time between Tolypeutine and Euphractine armadillos (Figure 3). This period corresponds to a glacial optimum with the first formation of a concrete Antarctic ice sheet and the creation of the circum-Antarctic oceanic current that changed the atmospheric circulation and induced a drastic general cooling . This marked the passage from warm, humid, tropical-temperate forest environments to much more arid and dryer habitats dominated by savannas and grasslands  prompting the diversification of mammalian herbivore communities .
Overall, our results are consistent with the hypothesis of a major influence of global environmental change on the evolution of xenarthrans. This process appears to have been apparently mainly driven by the consequences of Andean tectonics as suggested by Marshall and Sempere  even if the causal link between climate change and the rise of the Andes might be more complicated than previously though . These authors argued as a working hypothesis that geological and tectonic processes might have played a key role in restructuring the mammalian communities by drastically remodelling the South American environments and climates. Some groups have consequently been driven to extinction and new ecological opportunities have been provided for others to diversify. In particular, the finding that the two radiation events identified within armadillos  appear to follow two major tectonic crises is consistent with Marshall and Sempere's hypothesis and suggests that other living members of South American endemic groups might also have recorded these events in their genomes.
We have used a Bayesian relaxed molecular clock approach explicitly taking into account paleontological uncertainty and contrasted evolutionary dynamics between genomic loci to obtain divergence time estimates for living xenarthrans. We proposed a time scale for the diversification of this major placental clade with a sparse fossil record, based on the analysis of three nuclear genes for 50 mammals. This molecular timescale put in relation with well documented environmental changes that occurred during the Cenozoic of South America revealed the crucial influence that global change – possibly induced by Andean tectonic processes – might have played on the evolutionary history of these peculiar mammals. It is finally suggested that these major paleoenvironmental changes may also have left their footprints in the genomes of other South American endemic mammalian taxa. This hypothesis has yet to be tested in hystricognath rodents, platyrrhine monkeys or didelphid marsupials for example.
Data sets and topology
The original data set we use here is the one obtained by Delsuc et al.  consisting of 50 mammalian taxa for the three nuclear genes α2B adrenergic receptor (ADRA2B), breast cancer susceptibility exon 11 (BRCA1), and von Willebrand factor exon 28 (VWF). The complete alignment of these genes represents a total of 5130 nucleotide sites . Different partitions from this data set were used for calculating divergence times: ABV (1+2) = concatenation of first and second codon positions of the three genes (3421 sites); A2BV2 = concatenation of first and second codon positions of ADRA2B and VWF plus all codon positions of BRCA1 (4350 sites); and ABV (1+2+3) = concatenation of all codon positions of the three genes (5130 sites). As the relaxed molecular clock approach relies on a topology to infer divergence times, we used the ML topology previously identified on the complete data set  which conforms to the current views on placental mammals relationships based on the latest large scale analyses .
In the Bayesian relaxed molecular clock approach , it is important to use prior constraints on independent calibration points dispersed across the tree in order to reduce potential regional effects . To render things comparable among studies, we used the calibrations of Springer et al.  that are compatible with our taxon sampling. Thus, the six calibration points defining the following eight prior constraints were used (see Figure 1): (1) a minimum of 54 Mya for Paenungulata (PAE), (2) a minimum of 12 Mya for the divergence between the Muridae Mus and Rattus (MUR); (3) a minimum of 43 Mya and a maximum of 60 Mya for Megachiroptera (MEG); (4) a minimum of 54 Mya and maximum of 58 Mya for Perissodactyla (PER); (5) a conservative maximum of 65 Mya for Cetartiodactyla (ART); and (6) a minimum of 52 Mya for the first appearance of Cetacea (CET). We do not used prior constraint on the origin of Xenarthra because we wanted to make independent inferences about their divergence dates. We verified however that constraining the base of Xenarthra with a minimum of 60 Mya  does not significantly affect our molecular estimates (data not shown). For the same reasons of independence, we did not use intra Xenarthra calibrations, but also because of a lack thereof. Indeed, if the fossil record of xenarthrans is especially rich for extinct giant sloths for example, the paleontological origins of extant species are almost unknown . In all subsequent Bayesian analyses these eight prior constraints on calibration points were simultaneously used to derive posterior estimates of divergence ages .
Divergence time estimates
The Bayesian relaxed molecular clock approach was applied using the program package MULTIDIVTIME . The module ESTBRANCHES was first used to estimate branch lengths of the constrained topology and the corresponding variance-covariance matrices for each data partition using the three marsupials Macropus, Didelphis and Vombatus as outgroups for the 47 placental taxa. The F84+Γ8 model was used with maximum likelihood parameters previously estimated by PAML version 3.13 . Then, the module MULTIDIVTIME used the variance-covariance matrices produced by ESTBRANCHES to run a MCMC for estimating mean posterior divergence times on nodes with associated standard-deviation (SD) and 95% credibility interval (CredI). In all calculations, the MCMC was sampled 10,000 times every 100 cycles and the burn-in stage was set to 100,000 cycles in all subsequent analyses. The following priors were used for the mammalian data set: 110 Mya (SD = 55 Mya) for the expected number of time units between tip and root if there has been no constraint on node times, and 200 Mya for the highest possible number of time units between tip and root. Other priors for gamma distribution of the rate at root node and the Brownian motion constant describing the rate variation (i.e., the degree of rate autocorrelation along the descending branches of the tree), were derived from the median branch length for each data set as advised by Thorne et al. .
Janis CM: Tertiary mammal evolution in the context of changing climates, vegetation, and tectonic changes. Annu Rev Ecol Syst. 1993, 24: 467-500. 10.1146/annurev.es.24.110193.002343.
Darwin C: On the origin of species by means of natural selection. 1859, London: Murray
Simpson GG: Splendid isolation: The curious history of South American mammals. 1980, Yale University Press
Pascual R, Ortiz Jaureguizar E: Evolving climates and mammal faunas in Cenozoic South America. J Hum Evol. 1990, 19: 23-60.
Marshall LG, Sempere T: Evolution of the Neotropical land mammal fauna in its geochronologic, stratigraphic and tectonic context. In: Biological relationships between Africa and South America. Edited by: Goldblatt P. 1993, New Heaven and London: Yale University Press, 329-392.
MacFadden BJ: Cenozoic mammalian herbivores from the Americas: reconstructing ancient diets and terrestrial communities. Annu Rev Ecol Syst. 2000, 31: 33-59. 10.1146/annurev.ecolsys.31.1.33.
Patterson B, Pascual R: The fossil mammal fauna of South-America. In: Evolution, mammals and southern continents. Edited by: Keast A, Erk FC, Glass BP. 1972, Albany: State University of New-York Press, 247-309.
Flynn JJ, Wyss AR: Recent advances in South American mammalian paleontology. Trends Ecol Evol. 1998, 13: 449-454. 10.1016/S0169-5347(98)01457-8.
Sempere T, Marshall LG, Rivano S, Godoy E: Late Oligocene-Early Miocene compresssional tectosedimentary episode and associated land-mammal faunas in the Andes of central Chile and adjacent Argentina (32°-37°S). Tectonophysics. 1994, 229: 251-264. 10.1016/0040-1951(94)90032-9.
Latorre C, Quade J, McIntosh WC: The expansion of C4 grasses and global change in the late Miocene: Stable isotope evidence from the Americas. Earth Planet Sci Lett. 1997, 146: 83-96. 10.1016/S0012-821X(96)00231-2.
Kay RF, Madden RH, Vucetich MG, Carlini AA, Mazzoni MM, Re GH, Heizler M, Sandeman H: Revised geochronology of the Casamayoran South American Land Mammal Age: Climatic and biotic implications. Proc Natl Acad Sci USA. 1999, 96: 13235-13240. 10.1073/pnas.96.23.13235.
Scillato-Yané GJ: Sobre un Dasypodidae (Mammalia, Xenarthra) de edad Riochiquense (Paleoceno superior) de Itaboraí, Brasil. An Acad Brasil Ciênc. 1976, 48: 527-530.
Vizcaíno SF: Sistemática y Anatomía de los Astegotheriini Ameghino, 1906 (nuevo rango) (Dasypodidae, Dasypodinae). Ameghiniana. 1994, 31: 3-13.
Oliveira E, Bergqvist L: A new Paleocene armadillo (Mammalia, Dasypodoidea) from the Itaboraí Basin, Brazil. Paleógeno de América del Sur y de la Península Antártica, Asociación Paleontológica Argentina, Publicación especial 5. 1998, 30: 35-40.
Vizcaíno SF, Reguero MA, Goin FJ, Pascual R: Antarctica as background for mammalian evolution. Paleógeno de América del Sur y de la Península Antártica, Asociación Paleontológica Argentina, Publicación especial 5. 1998, 30: 201-211.
Stehli FG, Webb SD: The Great American Biotic Interchange. 1985, New York: Plenum Press
Bargo MS: Biomechanics and palaeobiology of the Xenarthra: The state of the art (Mammalia, Xenarthra). Senckenbergiana Biologica.
McDonald HG, de Muizon C: The cranial anatomy of Thalassocnus (Xenarthra, Mammalia), a derived nothrothere from the Neogene of the Pisco Formation (Peru). Journal of Vertebrate Paleontology. 2002, 22: 349-365.
Lessa EP, Van Valkenburgh B, Fariña RA: Testing hypotheses of differential mammalian extinctions subsequent to the Great American Biotic Interchange. Palaeogeogr Palaeoclimatol Palaeoecol. 1997, 135: 157-162. 10.1016/S0031-0182(97)00042-4.
Carlini AA, Vizcaíno SF, Scillato-Yané GJ: Armoured xenarthrans: a unique taxonomic and ecologic assemblage. In: Vertebrate Paleontology of the Miocene Honda Group, Republic of Colombia. Edited by: Kay RF, Cifelli RL, Flynn JJ, Madden RH. 1997, Washington, Smithsonian Institution Press, 213-226.
Bromham L, Penny D: The modern molecular clock. Nat Rev Genet. 2003, 4: 216-224. 10.1038/nrg1020.
Benner SA, Caraco MD, Thomson JM, Gaucher EA: Planetary biology – paleontological, geological, and molecular histories of life. Science. 2002, 296: 864-868. 10.1126/science.1069863.
Bossuyt F, Milinkovitch MC: Amphibians as indicators of early Tertiary "out-of-India" dispersal of vertebrates. Science. 2001, 292: 93-95. 10.1126/science.1058875.
Mercer JM, Roth VL: The effects of Cenozoic global change on squirrel phylogeny. Science. 2003, 299: 1568-1572. 10.1126/science.1079705.
Douady CJ, Douzery EJP: Molecular estimation of eulipotyphlan divergence times and the evolution of "Insectivora". Mol Phylogenet Evol. 2003, 28: 285-296. 10.1016/S1055-7903(03)00119-2.
Douady CJ, Catzeflis F, Raman J, Springer MS, Stanhope MJ: The Sahara as a vicariant agent, and the role of Miocene climatic events, in the diversification of the mammalian order Macroscelidea (elephant shrews). Proc Natl Acad Sci USA. 2003, 100: 8325-8330. 10.1073/pnas.0832467100.
Delsuc F, Scally M, Madsen O, Stanhope MJ, de Jong WW, Catzeflis FM, Springer MS, Douzery EJP: Molecular phylogeny of living xenarthrans and the impact of character and taxon sampling on the placental tree rooting. Mol Biol Evol. 2002, 19: 1656-1671.
Delsuc F, Stanhope MJ, Douzery EJP: Molecular systematics of armadillos (Xenarthra, Dasypodidae): Contribution of maximum likelihood and Bayesian analyses of mitochondrial and nuclear genes. Mol Phylogenet Evol. 2003, 28: 261-275. 10.1016/S1055-7903(03)00111-8.
Delsuc F, Catzeflis FM, Stanhope MJ, Douzery EJP: The evolution of armadillos, anteaters and sloths depicted by nuclear and mitochondrial phylogenies: implications for the status of the enigmatic fossil Eurotamandua. Proc R Soc Lond BS. 2001, 268: 1605-1615. 10.1098/rspb.2001.1702.
Zuckerkandl E, Pauling L: Evolutionary divergence and convergence in proteins. In: Evolving genes and proteins. Edited by: Bryson V, Vogel HJ. 1965, New York: Academic Press, 97-166.
Sanderson MJ: A nonparametric approach to estimating divergence times in the absence of rate constancy. Mol Biol Evol. 1997, 14: 1218-1231.
Thorne JL, Kishino H, Painter IS: Estimating the rate of evolution of the rate of molecular evolution. Mol Biol Evol. 1998, 15: 1647-1657.
Huelsenbeck JP, Larget B, Swofford DL: A compound poisson process for relaxing the molecular clock. Genetics. 2000, 154: 1879-1892.
Aris-Brosou S, Yang Z: Effects of models of rate evolution on estimation of divergence dates with special reference to the metazoan 18S ribosomal RNA phylogeny. Syst Biol. 2002, 51: 703-714. 10.1080/10635150290102375.
Sanderson MJ: Estimating absolute rates of molecular evolution and divergence times: A penalized likelihood approach. Mol Biol Evol. 2002, 19: 101-109.
Kishino H, Thorne JL, Bruno WJ: Performance of a divergence time estimation method under a probabilistic model of rate evolution. Mol Biol Evol. 2001, 18: 352-361.
Korber B, Muldoon M, Theiler J, Gao F, Gupta R, Lapedes A, Hahn BH, Wolinsky S, Bhattacharya T: Timing the ancestor of the HIV-1 pandemic strains. Science. 2000, 288: 1789-1796. 10.1126/science.288.5472.1789.
Cao Y, Fujiwara M, Nikaido M, Okada N, Hasegawa M: Interordinal relationships and timescale of eutherian evolution as inferred from mitochondrial genome data. Gene. 2000, 259: 149-158. 10.1016/S0378-1119(00)00427-3.
Hasegawa M, Thorne JL, Kishino H: Timescale of eutherian evolution estimated without assuming a constant rate of molecular evolution. Genes Genet Syst. 2003, 78: 267-283. 10.1266/ggs.78.267.
Springer MS, Murphy WJ, Eizirik E, O'Brien SJ: Placental mammal diversification and the Cretaceous-Tertiary boundary. Proc Natl Acad Sci USA. 2003, 100: 1056-1061. 10.1073/pnas.0334222100.
Yoder AD, Burns MM, Zehr S, Delefosse T, Veron G, Goodman SM, Flynn JJ: Single origin of Malagasy Carnivora from an African ancestor. Nature. 2003, 421: 734-737. 10.1038/nature01303.
Sanderson MJ, Doyle JA: Sources of error and confidence intervals in estimating the age of angiosperms from rbcL and 18S rDNA data. Am J Bot. 2001, 88: 1499-1516.
Thorne JL, Kishino H: Divergence time and evolutionary rate estimation with multilocus data. Syst Biol. 2002, 51: 689-702. 10.1080/10635150290102456.
Felsenstein J: Phylogenies from molecular sequences: Inference and reliability. Annu Rev Genet. 1988, 22: 521-565. 10.1146/annurev.ge.22.120188.002513.
Takezaki N, Rzhetsky A, Nei M: Phylogenetic test of the molecular clock and linearized trees. Mol Biol Evol. 1995, 12: 823-833.
Douzery EJP, Delsuc F, Stanhope MJ, Huchon D: Local molecular clocks in three nuclear genes: Divergence times for rodents and other mammals, and incompatibility among fossil calibrations. J Mol Evol. 2003, 57: S201-213. 10.1007/s00239-003-0028-x.
Rambaut A, Bromham L: Estimating divergence dates from molecular sequences. Mol Biol Evol. 1998, 15: 442-448.
Yoder AD, Yang Z: Estimation of primate speciation dates using local molecular clocks. Mol Biol Evol. 2000, 17: 1081-1090.
Huchon D, Catzeflis FM, Douzery EJP: Variance of molecular datings, evolution of rodents and the phylogenetic affinities between Ctenodactylidae and Hystricognathi. Proc R Soc Lond B. 2000, 267: 393-402. 10.1098/rspb.2000.1014.
Yang Z, Yoder AD: Comparison of likelihood and Bayesian methods for estimating divergence times using multiple gene loci and calibration points, with application to a radiation of cute-looking mouse lemur species. Syst Biol. 2003, 52: 705-716. 10.1080/10635150390235557.
Benton MJ: Early origins of modern birds and mammals: Molecules vs. morphology. Bioessays. 1999, 21: 1043-1051. 10.1002/(SICI)1521-1878(199912)22:1<1043::AID-BIES8>3.3.CO;2-2.
Easteal S: Molecular evidence for the early divergence of placental mammals. Bioessays. 1999, 21: 1052-1058. 10.1002/(SICI)1521-1878(199912)22:1<1052::AID-BIES9>3.0.CO;2-6. discussion 1059
Bromham L, Phillips MJ, Penny D: Growing up with dinosaurs: Molecular dates and the mammalian radiation. Trends Ecol Evol. 1999, 14: 113-118. 10.1016/S0169-5347(98)01507-9.
Archibald JD, Deutschman DH: Quantitative analysis of the timing of the origin and diversification of extant placental orders. J Mamm Evol. 2001, 8: 107-124. 10.1023/A:1011317930838.
Eizirik E, Murphy WJ, O'Brien SJ: Molecular dating and biogeography of the early placental mammal radiation. J Hered. 2001, 92: 212-219. 10.1093/jhered/92.2.212.
Huchon D, Madsen O, Sibbald MJ, Ament K, Stanhope MJ, Catzeflis F, de Jong WW, Douzery EJP: Rodent phylogeny and a timescale for the evolution of Glires: Evidence from an extensive taxon sampling using three nuclear genes. Mol Biol Evol. 2002, 19: 1053-1065.
Ji Q, Luo ZX, Yuan CX, Wible JR, Zhang JP, Georgi JA: The earliest known eutherian mammal. Nature. 2002, 416: 816-822. 10.1038/416816a.
Gheerbrant E, Sudre J, Cappetta H: A Palaeocene proboscidean from Morocco. Nature. 1996, 383: 68-70. 10.1038/383068a0.
Kappelman J, Rasmussen DT, Sanders WJ, Feseha M, Bown T, Copeland P, Crabaugh J, Fleagle J, Glantz M, Gordon A, Jacobs B, Maga M, Muldoon K, Pan A, Pyne L, Richmond B, Ryan T, Seiffert ER, Sen S, Todd L, Wiemann MC, Winkler A: Oligocene mammals from Ethiopia and faunal exchange between Afro-Arabia and Eurasia. Nature. 2003, 426: 549-552. 10.1038/nature02102.
McKenna MC: Toward a phylogenetic classification of the Mammalia. In: Phylogeny of the Primates. Edited by: Luckett WP, Szalay FS. 1975, New York: Plenum Press, 21-46.
Gaudin TJ, Branham DG: The phylogeny of the Myrmecophagidae (Mammalia, Xenarthra, Vermilingua) and the relationship of Eurotamandua to the Vermilingua. J Mammal Evol. 1998, 5: 237-265. 10.1023/A:1020512529767.
Barros MC, Sampaio I, Schneider H: Phylogenetic analysis of 16S mitochondrial DNA data in sloths and anteaters. Genet Mol Biol. 2003, 26: 5-11.
Webb SD: The interrelationships of tree sloths and ground sloths. In: The Evolution and Ecology of Armadillos, Sloths and Vermilinguas. Edited by: Montgomery GG. 1985, Washington: Smithsonian Institution Press, 105-112.
Gaudin TJ: Phylogenetic relationships among sloths (Mammalia, Xenarthra, Tardigrada): the craniodental evidence. Zool J Linn Soc. 2004, 140: 225-305. 10.1111/j.1096-3642.2003.00100.x.
Vizcaíno SF, Scillato-Yané GJ: An Eocene Tardigrada (Mammalia, Xenarthra) from Seymour Island, Antarctica. Antarctic Sci. 1995, 7: 407-408.
Gaudin TJ, Wible JR: The phylogeny of living and extinct armadillos (Mammalia, Xenarthra, Cingulata): a craniodental analysis. In: Amniote Paleobiology: Phylogenetic and Functional Perspectives on the Evolution of Mammals, Birds and Reptiles. Edited by: Carrano MT, Gaudin TJ, Blob RW, Wible JR. Chicago: University of Chicago Press,
Vizcaíno SF, Perea D, Ubilla M: Presencia de Dasypus (Mammalia, Edentata, Dasypodidae) en la Formación Sopas (Pleistoceno tardío) de Uruguay. Distribución cronológica del género. Revista Chilena de Historia Natural. 1995, 68: 95-99.
Carlini AA, Scillato-Yané GJ: Un Euphractini (Mammalia, Dasypodidae) del Plioceno de Chapadmalal (Buenos Aires, Argentina). Consideraciones filogenéticas sobre los Euphractini. Revista del Museo de La Plata. 1996, 9: 225-238.
Wetzel RM: Taxonomy and distribution of armadillos, Dasypodidae. In: The Evolution and Ecology of Armadillos, Sloths and Vermilinguas. Edited by: Montgomery GG. 1985, Washington: Smithsonian Institution, 23-46.
Cetica PD, Solari AJ, Merani MS, De Rosas JC, Burgos MH: Evolutionary sperm morphology and morphometry in armadillos. J Submicroscop Cytol Pathol. 1998, 30: 309-314.
Haq BU, Hardenbol J, Vail PR: Chronology of fluctuating sea levels since the Triassic. Science. 1987, 235: 1156-1167.
Zachos J, Pagani M, Sloan L, Thomas E, Billups K: Trends, rhythms, and aberrations in global climate 65 Ma to present. Science. 2001, 292: 686-693. 10.1126/science.1059412.
Lamb S, Davis P: Cenozoic climate change as a possible cause for the rise of the Andes. Nature. 2003, 425: 792-797. 10.1038/nature02049.
Amrine-Madsen H, Koepfli KP, Wayne RK, Springer MS: A new phylogenetic marker, apolipoprotein B, provides compelling evidence for eutherian relationships. Mol Phylogenet Evol. 2003, 28: 225-240. 10.1016/S1055-7903(03)00118-0.
Yang Z: PAML: A program package for phylogenetic analysis by maximum likelihood. Comput Appl Biosci. 1997, 13: 555-556.
1999 Geological timescale. [http://www.geosociety.org/science/timescale/timescl.pdf]
The authors whish to thank two anonymous referees for helpful comments on the manuscript. This work has been supported by the TMR Network "Mammalian phylogeny" (contract FMRX – CT98 – 022) of the European Community, the "Genopole Montpellier Languedoc-Roussillon", the "Action Bioinformatique inter-EPST" of the CNRS, and by the IFR119 "Biodiversité Continentale Méditerranéenne et Tropicale" (Montpellier) and INFOBIOGEN (Evry, France) computing facilities. FD acknowledges the financial support of a MENRT Doctoral Grant (contract 99075) and a Lavoisier Postdoctoral Grant from the French Ministry of Foreign Affairs. This is publication EPML-002 of the Equipe-Projet multi-laboratoires CNRS-STIC "Méthodes informatiques pour la biologie moléculaire" and ISEM 2004-021 of the Institut des Sciences de l'Evolution de Montpellier (UMR 5554 – CNRS). It is a contribution to the projects UNL N336 and PICT 07-06348 (SFV).
FD and EJPD contributed equally to this work in initiating the study, assembling the data, and designing and running the calculations. FD drafted the manuscript and drew the figures. EJPD and SFV assisted with drafting, revising and editing more specifically the molecular and paleontological parts of the manuscript, respectively. All authors read and approved the final manuscript.