Placental mammal diversification and the Cretaceous–Tertiary boundary

Analyses with the Complete Data Set, Different Priors, and Different Outgroups.

Fig. 2 depicts a time scale for placental mammal evolution based on the 16,397-bp data set with the opossum outgroup and the 105 mya prior for the base of the placental tree. Results of a second analysis, which used different starting states for the Markov chain, were almost identical and always within one million years (see supporting information). Similarly, switching to the diprotodontian marsupial outgroup had minimal impact: divergence estimates were always within one million years of those shown in Fig. 2 (see supporting information). Employing a mean value of 65 or 146 mya for the prior distribution of the root of the placental tree had minimal impact; divergence dates shown in Fig. 2 changed by no more than two million years (Table 1; see supporting information for complete results).

The deepest split among placentals is between Afrotheria and other taxa at 107 mya (Fig. 2). The split between Euarchontoglires and Laurasiatheria was estimated at 94 mya. Interordinal splits within Euarchontoglires are in the range of 82–87 mya; those within Laurasiatheria are in the range of 77–85 mya. Among interordinal splits, only divergences within Paenungulata occurred after the K/T boundary, and only when constrained with a maximum of 65 mya. Within orders, subsequent basal splits are approximately at the K/T boundary for Afrosoricida, Chiroptera, and Cetartiodactyla. These are followed by basal splits within Carnivora, Lagomorpha, and Perissodactyla at ≈10–15 mya after the K/T boundary. Eulipotyphla, Primates, Rodentia, and Xenarthra show basal splits that are 5–12 million years before the K/T boundary. Of these, the oldest split is within Primates at 77 mya. The earliest divergence within Rodentia was estimated at 74 mya, and the rat-mouse split was dated at 16 mya.

Analyses with the Complete Data Set and a Myomorph Root.

Select results of an analysis that used a myomorph root for the placental tree are depicted in Table 1. Dates were older for the root of the placental tree (131 mya) and several clades with more basal positions on the myomorph root tree including rat to mouse (35 mya), hystricid to caviomorph (71 mya), Primates (95 mya), and Lagomorpha (71 mya). Similarly, dates were younger for some clades with more derived positions on the tree, such as Afrosoricida (58 mya) and Xenarthra (66 mya).

Analyses with Reduced Constraints.

When constraints on basal cladogenesis in Paenungulata were relaxed, the point estimate for this node changed from ≈63 mya to 72 mya. Estimated dates for some of the other nodes also increased, including 112 mya for the base of Placentalia, 98 mya for Euarchontoglires to Laurasiatheria, and 17 mya for rat to mouse. Omitting the minimum constraint of 63 mya for shrew–hedgehog had minimal impact on estimates of dates in Eulipotyphla; hedgehog–shrew remained at 68 mya and the split between these two and mole remained at 76 mya. When the basal carnivore and basal perissodactyl constraints were relaxed, these divergence events were still estimated in the early Cenozoic (carnivores at 55 mya; perissodactyls at 59 mya). Basal cladogenesis for Chiroptera shifted from 65 to 70 mya when constraints on the megadermatid–pteropodid split were omitted. Omitting the minimum for hippo to Cetacea and the maximum for the base of Cetartiodactyla resulted in younger estimates for both splits (55 mya for base of Cetartiodactyla; 40 mya for hippo-Cetacea). Detailed results are given in the supporting information.

Analyses with Nuclear and Mitochondrial Data Partitions.

Select results of analyses based on individual data partitions are given in Table 2. Complete results and confidence intervals for all analyses are given in the supporting information. The analysis with the nuclear genes data set (14,750 bp) yielded dates that were always within two million years of those illustrated in Fig. 2 for the complete data set. Among different partitions of the nuclear data, older dates were consistently estimated with exons than with UTRs. Similarly, first and second codon positions recovered younger estimates than third codon positions of exons. All interordinal splits were estimated in the Cretaceous with all of the data partitions except for Paenungulata, which was constrained, and the Scandentia–Dermoptera split, which was estimated at 58 mya with the mitochondrial RNA data partition. Estimates for intraordinal cladogenesis remained in the Late Cretaceous for Eulipotyphla, Primates, Rodentia, and Xenarthra with both the nuclear and mitochondrial partitions. However, some estimates were only slightly older than the K/T boundary. For example, analysis of just first and second codon positions produced an estimate of 67 mya for the base of Xenarthra. Analyses based on the mitochondrial RNA data partition recovered a placental root at 97 mya, whereas the majority of the additional estimates did not depart considerably from results of the other partitions.

Analyses with the “K/T Body Size” Taxon Set.

The “K/T body size” taxon set was used to determine if analyses that only included smaller taxa resulted in younger divergence estimates. Some divergence estimates were slightly younger with this pruned taxon set relative to the full-taxon data set; however, the overall results were similar. The placental root was estimated at 100–104 mya. The split between Euarchontoglires and Laurasiatheria was estimated at 89–91 mya. Although comparisons were limited, all interordinal divergences were estimated in the Cretaceous, including 84–86 mya for Afrosoricida to Macroscelidea, 81–82 mya for the base of Laurasiatheria, and 83–85 mya for the base of Euarchontoglires. As with the full-taxon data set, results of analyses that used mean priors of 65, 105, and 146 million years for the base of Placentalia were in excellent agreement with each other. Point estimates using the 65 and 146 mya priors differed by no more than three million years from estimates using the 105 prior (see supporting information).

Molecular and Paleontological Support for the Long Fuse Model.

The fossil record has suggested to some paleontologists that the ordinal radiation and diversification of crown-group Placentalia is entirely or mostly post-Cretaceous, i.e., the Explosive Model (Fig. 1). This hypothesis regards most or all known species of Cretaceous eutherian mammals as stem eutherians. In contrast, McKenna and Bell (26) recognize 22 genera from the Late Cretaceous and one genus from the Early Cretaceous as crown-group Placentalia. Cladistic analyses that incorporate a diverse assemblage of Cretaceous eutherian fossils, as well as representatives of all extant orders, have not been reported. Lacking a robust phylogeny that integrates Cretaceous and early Cenozoic eutherian fossils into the emerging molecular tree (13, 14, 38–40), it is difficult to evaluate how compatible the fossil record is with Cretaceous interordinal divergences. Archibald et al.'s (8) cladistic study provides some support for the Long Fuse Model of placental diversification, but even this study samples only a few crown-group placentals.

Our results are generally compatible with the Long Fuse Model of placental diversification. Interordinal splits are concentrated in the Cretaceous, whereas basal cladogenesis within most orders is in the Cenozoic (Table 1; Fig. 2). Four orders show basal cladogenesis extending back into the Crtetaceous, but point estimates are only 1–16 million years before the K/T boundary. The suggestion that four orders have intraordinal cladogenesis in the Cretaceous exceeds Archibald and Deutschman's (1) depiction of a single order. However, we find no evidence for deep intraordinal splits in excess of 100 mya, as predicted by the Short Fuse Model.

These results are robust, having been obtained with three different priors (65, 105, and 146 mya) for the placental root, two different marsupial outgroups, different data partitions, and with a “K/T body size” taxon set (Table 1). Our estimates for a placental root at 97–112 mya, even with an allowance for rate variation among lineages and a mean prior that was set at 65 mya, demonstrate the difficulty of fitting molecular data to the Explosive Model, as do analyses with “K/T body size” taxa. These analyses resulted in slightly younger estimates of divergence times for most nodes, but all interordinal divergences were still placed in the Cretaceous. Therefore, the proposed negative correlation between body size and rate of molecular evolution (17) does not account for the apparent discrepancy between molecular dates and the Explosive Model.

To examine the cost of fitting these molecular data (14) to a strict interpretation of the Explosive Model, we added constraints for the base of Placentalia (minimum = 65 mya; maximum = 66 mya) and compared resulting interordinal and intraordinal rates of nucleotide substitution over the entire placental tree. The lowest interordinal rate was 72 times higher than the lowest intraordinal rate; the highest interordinal rate was 3.4 times higher than the highest intraordinal rate; the highest interordinal rate was 669 times higher than the lowest interordinal rate; and the median interordinal rate was 7 times higher than the median intraordinal rate (see supporting information). Additional results from this analysis further demonstrate the difficulties of fitting the Murphy et al. data to the Explosive Model. For example, the rate on the branch leading to Boreoeutheria was 19 times higher than the branch leading to rat and 30 times higher than the rate on the branch leading to mouse, even though murids have among the fastest rates among placental taxa (41). Indeed, the median rate on interordinal branches was 242 times higher than the median rate on terminal branches under the Explosive Model.

Intraordinal Divergences.

Of the four orders that consistently display intraordinal diversification in the Late Cretaceous, some interpretations of the fossil record suggest that lipotyphlan insectivores extend into the Cretaceous (1, 26). However, this issue remains contentious (4, 5, §). Given that molecular data suggest Lipotyphla is diphyletic (10, 14, 42), the phylogenetic affinities of Cretaceous insectivores are highly questionable and demand further scrutiny.

Rodentia, Primates, and Xenarthra first appear in the Paleocene (26). Our analyses suggest intraordinal diversification in the Late Cretaceous, but only 5–11 (rodents), 4–15 (primates), and 2–16 (xenarthrans) million years before the K/T boundary in different analyses and with different data sets. Further, 95% confidence intervals in some analyses extend into the Tertiary for each of these orders (e.g., 63 mya for rodents in “K/T body size” analysis). On the other hand, Tavaré et al. (43) estimated that living primates last shared a common ancestor 81.5 mya (95% confidence interval, 72.0–89.6 mya) based on a statistical analysis of the fossil record that takes into account fossil preservation rate.

Our sampling includes taxa that index basal or near basal divergences in 10 of 18 eutherian orders (Afrosoricida, Carnivora, Cetartiodactyla, Chiroptera, Eulipotyphla, Lagomorpha, Perissodactyla, Primates, Rodentia, Xenarthra). Taxa that represent basal crown-group cladogenesis were not indexed for the remaining orders: Tubulidentata, Hyracoidea, Proboscidea, Sirenia, Pholidota, Scandentia, Dermoptera, and Macroscelidea. However, available evidence suggests that none of these orders has crown-group cladogenesis as far back as the K/T boundary (supporting information).

Estimates Based on Different Data Partitions.

Dates based on different partitions were generally in good agreement, although dates based on UTRs and first and second codon positions were consistently younger than dates based on third codon positions. Whether the former two are underestimates or the latter are inflated deserves investigation. One potential problem with the latter is base-compositional heterogeneity. A χ² test indicates base-compositional heterogeneity for third codon positions, but not for UTRs or first and second codon positions. Huchon et al. (44) also found significant base-compositional heterogeneity for third codon positions, but not for first and second codon positions. Huchon et al. (44) obtained a median estimate of 101 mya for the base of Placentalia by using quartet dating and first and second codon positions for three nuclear genes. This finding agrees with our estimate of 103 mya based on first and second codon positions.

Deep Rodent Dates.

A potential stumbling block for the acceptance of molecular dates is deep cladogenesis within Rodentia, either as a monophyletic or paraphyletic group (11, 15). Our 70–77 mya estimates for basal cladogenesis within this order are younger than molecular dates in the range of 112–125 mya (9, 11, 45, 46) and agree with Adkins et al.'s (47) study of rodent genes, which estimates the basal rodent split at 75 mya. Huchon et al. (44) obtained even younger dates (median = 56 mya) for the base of Rodentia by using quartet dating. In addition, paleontological evidence supports the view that Mus and Rattus diverged at 12–14 mya (32), whereas many molecular estimates have departed significantly from fossil evidence, ranging as high as 41–46 mya (11, 47). Our analyses with the Murphy et al. (14) maximum likelihood topology suggest dates (14–24 mya) that are more concordant with the fossil record. Our results also suggest a possible explanation for the deep split between rat and mouse that has emerged from some molecular studies. Specifically, placement of rodents at or near the base of the eutherian tree (46). In our analysis that rooted Placentalia at the base of myomorph rodents, we obtained an estimate of 35 mya for the rat–mouse split. However, this requires a topology that contradicts the monophyly of Rodentia, Glires, Euarchontoglires, and Boreoeutheria, all of which are supported by Bayesian posterior probabilities of 1.00 and bootstrap support scores of 100% (14). Further, the monophyly of Euarchontoglires is supported by two independent deletions (48). Nevertheless, this analysis highlights the sensitivity of molecular dates to the underlying topology.