Our study represents the first comprehensive sampling of mtDNAs for recent bears, including all living and two recently extinct bear species. The cave bear and the American giant short-faced bear are the third and fourth Pleistocene species for which mtDNAs have been determined. Moreover, the cave bear genome is the first determined from a Pleistocene sample obtained from a non-permafrost environment. Compared to the extinct moas from which complete mtDNAs have previously been determined from non-permafrost specimens , the cave bear genome extends the time frame by an order of magnitude, showing that complete mtDNA analysis can be performed using a wide range of samples. As is common in large scale ancient DNA analyses [7, 9, 24], we found a number of consistent differences between independent primary PCRs, all of which were either C to T or G to A substitutions (see Additional File 1). This confirms previous reports that deamination of cytosine is one of the most common, and probably the only type of miscoding lesion in ancient DNA [13, 24–26]. Moreover, the high number of consistent substitutions (81) observed in the cave bear genome sequences shows that each sequence position needs to be replicated when performing such large scale analyses.
This analysis has allowed the phylogenetic topology of the bear family to be resolved with high support values. Interestingly, it places the sloth bear basal to all other ursine bear species and the sun bear in a sister group related to the two black bear species. The latter observation coincides with paleontological information  and previous mtDNA studies [4, 17, 21]. An earlier study analysing six mtDNA fragments, also placed the sloth bear basal to all other members of the ursine bears . However, this study found weak support for the sun bear as being basal to the brown bear – polar bear clade rather than to the two black bear species.
The phylogenetic reconstruction also reveals the reasons for previous problems in resolving the relationships among ursine bears, as most of the internal branches for their phylogenetic tree are very short. Such a short internal branch structure (Figure 1) makes it likely that individual nuclear genes (or short sequences) may exhibit different tree topologies, as shown for nuclear loci from humans, chimpanzees and gorillas . Furthermore it was previously shown that despite being a non-recombining single genetic locus, individual genes on the mtDNA might produce different tree topologies [6, 8, 9, 18].
The mitogenomic data also has implications for bear taxonomy. Six ursine bears and the sloth bear are monophyletic with absolute support, which agrees with Hall and Nowak's inclusion (Table 1) of the Asian black bear, American black bear, sun bear, polar bear and brown bear within the genus Ursus [28, 29] and confirms the mitogenomic study by Yu et al . Given the short divergence time of the six ursine bears and the sloth bear we suggest, following Hall 1981, Nowak 1991 and Yu et al 2007 [4, 28, 29], that the sloth bear is grouped together with the other ursine bears in the genus Ursus and that the other genus names previously suggested for members of this radiation are discarded (Table 1).
Using this data set and multiple fossil calibration points, we have dated the various mtDNA divergence events during bear evolution with reasonable confidence. Strikingly, the divergence of the giant panda is estimated at about 19 Ma (95% HPD: 14.4–24.8 Ma, HPD: highest posterior density). This estimate is much earlier than previously reported for the divergence of the panda lineage from the Ursavus lineage based on teeth morphology of Agriarctos fossils (12–15 Ma) . The latter divergence date has been used in several studies as a calibration point for dating bear radiations [2, 4, 35]. We decided not to use this date as a calibration point, since the oldest known panda fossil, Ailuropoda microta, is less than 2.4 million years old , and therefore allows no inference about the date of divergence of this lineage. Moreover, the fossil record for both Ailuropoda and its potential ancestral species from the genus Agriarctos is sparse, making an early Miocene divergence date for the giant panda's lineage plausible. Interestingly, the next divergence event is not until 13 Ma (spectacled and American giant short-faced bear) followed by a gap until 6 Ma when a rapid radiation occurs. The American giant short-faced and spectacled bears diverged around 5.7 Ma, and the five ursine lineages diverged between 5.4 and 4.1 Ma (posterior mean age estimates) (Figures 1 and 2).
Thus, taking the confidence intervals for the molecular dating into account, seven lineages radiated between 3.7 and 7 Ma. Such rapid radiations are also observed in other mammals, such as the cats  and procyonids , as well as in bird families like the woodpeckers . Strikingly, the major radiation wave for these families also occurred at the end of the Miocene. In combination with the fossil record, the mtDNA divergence estimates suggest that the rapid radiation of the bear family around the Miocene-Pliocene boundary followed a major extinction of some of the main bear genera such as Ursavus, Indarctos, Agriotherium, and the Hemicyoninae (Figure 2). Similar species turnover events were also observed for other mammals over a limited time span near the Miocene-Pliocene boundary resulting in a massive extinction of more than 60–70% of all Eurasian genera and 70–80% of North American genera . The cause of this widespread species turnover during this time period remains unclear. Some studies suggest that the initial opening of the Bering Strait at the beginning of the Pliocene around 5.3 Ma caused a major separation of northern hemisphere habitats . Major climatic changes occurred during that time, such as the Messinian crisis during which the Mediterranean Sea lost its connection to the world ocean system and became desiccated . These changes resulted in forest cover decline and the spread of arid habitats in Northern America and Eurasia [38, 39] as well as a global increase in C4 biomass . During that time, open grassland habitats, which were exploited by an entirely new suite of mammals , replaced the earlier less seasonal woodland forest habitats. Thus, it is possible that the environmental changes associated with the Miocene-Pliocene boundary and the following emergence of new ecological niches such as open grasslands caused an adaptive radiation in Old and New World bears similar to a number of other species groups . This could explain the divergence of the Tremarctinae with the spectacled bear adapted to closed habitats and the American giant short-faced bears being predators dwelling in open habitats [12, 27]. The latter adaptation was also described in other predator species that evolved around the Miocene-Pliocene boundary and were built for hunting in open habitats such as the cats [32, 35]. Other events such as the opening of the Bering Strait could have additionally promoted allopatric speciation in black bears. Our divergence time estimates suggest that the American black bear could have spread to America before the Bering Strait opened around 5.3 Ma . An early migration of ursine bears into the Americas is also supported by the oldest known Ursus fossil in North America, Ursus abstrusus , which was dated at 4.3 Ma, suggesting that U. abstrusus may be ancestral to the American black bear lineage.
Obviously, the Miocene-Pliocene global changes had a major impact on the radiation of bears and other species, both between and within the Old and New Worlds. It is interesting to note that African apes experienced a similar species turnover at the end of the Miocene, including the divergence of the chimpanzee and human lineages . This latter event has been attributed to a magnified climatic variability starting at the end of the Miocene . More studies are necessary to address the relationships between global changes and species radiations at the beginning of the Pliocene. Our results strongly support the idea of a major wave of bear radiations during that time.
Our data also indicate a much earlier divergence for the cave bear and brown bear lineages than those previously assumed, with a mean estimate at 2.8 Ma. This date agrees with recent results suggesting the existence of representatives of the brown bear lineage in Europe as early as 1.5 Ma (G. Rabeder, personal observation). Nevertheless, it questions other studies suggesting a later divergence time for this species pair at around 1.2–1.4 Ma based on the fossil record  and molecular data . Loreille et al. , however, used Taberlet & Bouvet's estimated divergence date for the two European brown bear lineages (Western and Eastern) of 850 ka , which in turn was based on an application of Vigilant et al.'s  intraspecific human rate of 8.4 × 10-8 substitutions/site/year – Taberlet & Bouvet cautioned that their estimates could be prone to uncertainty as they imported a human evolutionary rate. Given recent reports of problems in estimated intraspecific divergence times based on interspecific calibrations and vice versa, the implicit use of indirectly extrapolated evolutionary rates is not recommended [51, 52].
Most of the youngest fossils for Ursus etruscus, the assumed ancestor of the cave and brown bear, have been dated to 2–2.7 Ma , suggesting that a late divergence for the two lineages around 1.2 Ma is rather unlikely. These dates also partially overlap with the divergence date we obtained (range of posterior means across methods: 2.4–3.1 Ma). A greater number of reliably dated fossils from early members of both the cave bear and brown bear lineages are necessary to date the divergence of U. spelaeus. However, around 2.8 Ma, the climate again changed dramatically with the onset of the first major cooling events and climatic oscillations at the end of the Pliocene that eventually led to the Pleistocene glaciations . Thus, if bear speciation events were influenced by climate change, cave bears and brown bears may indeed have separated as early as 2.8 Ma.