Of teeth and trees: A fossil tip‐dating approach to infer divergence times of extinct and extant squaliform sharks

Fossil tip‐dating allows for the inclusion of morphological data in divergence time estimates based on both extant and extinct taxa. Neoselachii have a cartilaginous skeleton, which is less prone to fossilization compared to skeletons of Osteichthyans. Therefore, the majority of the neoselachian fossil record is comprised of single teeth, which fossilize more easily. Neoselachian teeth can be found in large numbers as they are continuously replaced. Tooth morphologies are of major importance on multiple taxonomic levels for identification of shark and ray taxa. Here, we review dental morphological characters of squalomorph sharks and test these for their phylogenetic signal. Subsequently, we combine DNA sequence data (concatenated exon sequences) with dental morphological characters from 85 fossil and extant taxa to simultaneously infer the phylogeny and re‐estimate divergence times using information of 61 fossil tip‐dates as well as eight node age calibrations of squalomorph sharks. Our findings show that the phylogenetic placement of fossil taxa is mostly in accordance with their previous taxonomic allocation. An exception is the phylogenetic placement of the extinct genus ?Protospinax , which remains unclear. We conclude that the high number of fossil taxa as well as the comprehensive DNA sequence data for extant taxa may compensate for the limited number of morphological characters identifiable on teeth, serving as a backbone for reliably estimating the phylogeny of both extinct and extant taxa. In general, tip‐dating mostly estimates older node ages compared to previous studies based on calibrated molecular clocks.     

Bayesian phylogenetic estimation of fossil ages

Recent advances have allowed for both morphological fossil evidence and molecular sequences to be integrated into a single combined inference of divergence dates under the rule of Bayesian probability. In particular, the fossilized birth–death tree prior and the Lewis-Mk model of discrete morphological evolution allow for the estimation of both divergence times and phylogenetic relationships between fossil and extant taxa. We exploit this statistical framework to investigate the internal consistency of these models by producing phylogenetic estimates of the age of each fossil in turn, within two rich and well-characterized datasets of fossil and extant species (penguins and canids). We find that the estimation accuracy of fossil ages is generally high with credible intervals seldom excluding the true age and median relative error in the two datasets of 5.7% and 13.2%, respectively. The median relative standard error (RSD) was 9.2% and 7.2%, respectively, suggesting good precision, although with some outliers. In fact, in the two datasets we analyse, the phylogenetic estimate of fossil age is on average less than 2 Myr from the mid-point age of the geological strata from which it was excavated. The high level of internal consistency found in our analyses suggests that the Bayesian statistical model employed is an adequate fit for both the geological and morphological data, and provides evidence from real data that the framework used can accurately model the evolution of discrete morphological traits coded from fossil and extant taxa. We anticipate that this approach will have diverse applications beyond divergence time dating, including dating fossils that are temporally unconstrained, testing of the ‘morphological clock'', and for uncovering potential model misspecification and/or data errors when controversial phylogenetic hypotheses are obtained based on combined divergence dating analyses.This article is part of the themed issue ‘Dating species divergences using rocks and clocks’.     

Phylogeny of extant and fossil Juglandaceae inferred from the integration of molecular and morphological data sets

It is widely acknowledged that integrating fossils into data sets of extant taxa is imperative for proper placement of fossils, resolution of relationships, and a better understanding of character evolution. The importance of this process has been further magnified because of the crucial role of fossils in dating divergence times. Outstanding issues remain, including appropriate methods to place fossils in phylogenetic trees, the importance of molecules versus morphology in these analyses, as well as the impact of potentially large amounts of missing data for fossil taxa. In this study we used the angiosperm clade Juglandaceae as a model for investigating methods of integrating fossils into a phylogenetic framework of extant taxa. The clade has a rich fossil record relative to low extant diversity, as well as a robust molecular phylogeny and morphological database for extant taxa. After combining fossil organ genera into composite and terminal taxa, our objectives were to (1) compare multiple methods for the integration of the fossils and extant taxa (including total evidence, molecular scaffolds, and molecular matrix representation with parsimony [MRP]); (2) explore the impact of missing data (incomplete taxa and characters) and the evidence for placing fossils on the topology; (3) simulate the phylogenetic effect of missing data by creating "artificial fossils"; and (4) place fossils and compare the impact of single and multiple fossil constraints in estimating the age of clades. Despite large and variable amounts of missing data, each of the methods provided reasonable placement of both fossils and simulated "artificial fossils" in the phylogeny previously inferred only from extant taxa. Our results clearly show that the amount of missing data in any given taxon is not by itself an operational guideline for excluding fossils from analysis. Three fossil taxa (Cruciptera simsonii, Paleoplatycarya wingii, and Platycarya americana) were placed within crown clades containing living taxa for which relationships previously had been suggested based on morphology, whereas Polyptera manningii, a mosaic taxon with equivocal affinities, was placed firmly as sister to two modern crown clades. The position of Paleooreomunnea stoneana was ambiguous with total evidence but conclusive with DNA scaffolds and MRP. There was less disturbance of relationships among extant taxa using a total evidence approach, and the DNA scaffold approach did not provide improved resolution or internal support for clades compared to total evidence, whereas weighted MRP retained comparable levels of support but lost crown clade resolution. Multiple internal minimum age constraints generally provided reasonable age estimates, but the use of single constraints provided by extinct genera tended to underestimate clade ages.     

Fossil‐based comparative analyses reveal ancient marine ancestry erased by extinction in ray‐finned fishes

The marine‐freshwater boundary is a major biodiversity gradient and few groups have colonised both systems successfully. Fishes have transitioned between habitats repeatedly, diversifying in rivers, lakes and oceans over evolutionary time. However, their history of habitat colonisation and diversification is unclear based on available fossil and phylogenetic data. We estimate ancestral habitats and diversification and transition rates using a large‐scale phylogeny of extant fish taxa and one containing a massive number of extinct species. Extant‐only phylogenetic analyses indicate freshwater ancestry, but inclusion of fossils reveal strong evidence of marine ancestry in lineages now restricted to freshwaters. Diversification and colonisation dynamics vary asymmetrically between habitats, as marine lineages colonise and flourish in rivers more frequently than the reverse. Our study highlights the importance of including fossils in comparative analyses, showing that freshwaters have played a role as refuges for ancient fish lineages, a signal erased by extinction in extant‐only phylogenies.     

Assessment of available anatomical characters for linking living mammals to fossil taxa in phylogenetic analyses

Analyses of living and fossil taxa are crucial for understanding biodiversity through time. The total evidence method allows living and fossil taxa to be combined in phylogenies, using molecular data for living taxa and morphological data for living and fossil taxa. With this method, substantial overlap of coded anatomical characters among living and fossil taxa is vital for accurately inferring topology. However, although molecular data for living species are widely available, scientists generating morphological data mainly focus on fossils. Therefore, there are fewer coded anatomical characters in living taxa, even in well-studied groups such as mammals. We investigated the number of coded anatomical characters available in phylogenetic matrices for living mammals and how these were phylogenetically distributed across orders. Eleven of 28 mammalian orders have less than 25% species with available characters; this has implications for the accurate placement of fossils, although the issue is less pronounced at higher taxonomic levels. In most orders, species with available characters are randomly distributed across the phylogeny, which may reduce the impact of the problem. We suggest that increased morphological data collection efforts for living taxa are needed to produce accurate total evidence phylogenies.     

Fossils, molecules, divergence times, and the origin of lissamphibians

A review of the paleontological literature shows that the early dates of appearance of Lissamphibia recently inferred from molecular data do not favor an origin of extant amphibians from temnospondyls, contrary to recent claims. A supertree is assembled using new Mesquite modules that allow extinct taxa to be incorporated into a time-calibrated phylogeny with a user-defined geological time scale. The supertree incorporates 223 extinct species of lissamphibians and has a highly significant stratigraphic fit. Some divergences can even be dated with sufficient precision to serve as calibration points in molecular divergence date analyses. Fourteen combinations of minimal branch length settings and 10 random resolutions for each polytomy give much more recent minimal origination times of lissamphibian taxa than recent studies based on a phylogenetic analyses of molecular sequences. Attempts to replicate recent molecular date estimates show that these estimates depend strongly on the choice of calibration points, on the dating method, and on the chosen model of evolution; for instance, the estimate for the date of the origin of Lissamphibia can lie between 351 and 266 Mya. This range of values is generally compatible with our time-calibrated supertree and indicates that there is no unbridgeable gap between dates obtained using the fossil record and those using molecular evidence, contrary to previous suggestions.     

Systematics and evolution of the Pan‐Alcidae (Aves,Charadriiformes)

Puffins, auks and their allies in the wing‐propelled diving seabird clade Pan‐Alcidae (Charadriiformes) have been proposed to be key pelagic indicators of faunal shifts in Northern Hemisphere oceans. However, most previous phylogenetic analyses of the clade have focused only on the 23 extant alcid species. Here we undertake a combined phylogenetic analysis of all previously published molecular sequence data (～ 12 kb) and morphological data (n = 353 characters) with dense species level sampling that also includes 28 extinct taxa. We present a new estimate of the patterns of diversification in the clade based on divergence time estimates that include a previously vetted set of twelve fossil calibrations. The resultant time trees are also used in the evaluation of previously hypothesized paleoclimatic drivers of pan‐alcid evolution. Our divergence dating results estimate the split of Alcidae from its sister taxon Stercorariidae during the late Eocene (～ 35 Ma), an evolutionary hypothesis for clade origination that agrees with the fossil record and that does not require the inference of extensive ghost lineages. The extant dovekie Alle alle is identified as the sole extant member of a clade including four extinct Miocene species. Furthermore, whereas an Uria + Alle clade has been previously recovered from molecular analyses, the extinct diversity of closely related Miocepphus species yields morphological support for this clade. Our results suggest that extant alcid diversity is a function of Miocene diversification and differential extinction at the Pliocene–Pleistocene boundary. The relative timing of the Middle Miocene climatic optimum and the Pliocene–Pleistocene climatic transition and major diversification and extinction events in Pan‐Alcidae, respectively, are consistent with a potential link between major paleoclimatic events and pan‐alcid cladogenesis.     

The position of Cetacea within mammalia: phylogenetic analysis of morphological data from extinct and extant taxa

Knowledge of the phylogenetic position of the order Cetacea (whales, dolphins, and porpoises) within Mammalia is of central importance to evolutionary biologists studying the transformations of biological form and function that accompanied the shift from fully terrestrial to fully aquatic life in this clade. Phylogenies based on molecular data and those based on morphological data both place cetaceans among ungulates but are incongruent in other respects. Morphologists argue that cetaceans are most closely related to mesonychians, an extinct group of terrestrial ungulates. They have disagreed, however, as to whether Perissodactyla (odd-toed ungulates) or Artiodactyla (even-toed ungulates) is the extant clade most closely related to Cetacea, and have long maintained that each of these orders is monophyletic. The great majority of molecule-based phylogenies show, by contrast, not only that artiodactyls are the closest extant relatives of Cetacea, but also that Artiodactyla is paraphyletic unless cetaceans are nested within it, often as the sister group of hippopotamids. We tested morphological evidence for several hypotheses concerning the sister taxon relationships of Cetacea in a maximum parsimony analysis of 123 morphological characters from 10 extant and 30 extinct taxa. We advocate treating certain multistate characters as ordered because such a procedure incorporates information about hierarchical morphological transformation. In all most-parsimonious trees, whether multistate characters are ordered or unordered, Artiodactyla is the extant sister taxon of Cetacea. With certain multistate characters ordered, the extinct clade Mesonychia (Mesonychidae + Hapalodectidae) is the sister taxon of Cetacea, and Artiodactyla is monophyletic. When all fossils are removed from the analysis, Artiodactyla is paraphyletic with Cetacea nested inside, indicating that inclusion of mesonychians and other extinct stem taxa in a phylogenetic analysis of the ungulate clade is integral to the recovery of artiodactyl monophyly. Phylogenies derived from molecular data alone may risk recovering inconsistent branches because of an inability to sample extinct clades, which by a conservative estimate, amount to 89% of the ingroup. Addition of data from recently described astragali attributed to cetaceans does not overturn artiodactyl monophyly.     

A Total Evidence Phylogeny of the Arctoidea (Carnivora: Mammalia): Relationships Among Basal Taxa

A total evidence phylogenetic analysis was performed for 14 extant and 18 fossil caniform genera using a data matrix of 5.6 kbp of concatenated sequence data from six independent loci and 80 morphological characters from the cranium and dentition. Maximum parsimony analysis recovered a single most parsimonious cladogram (MPC). The topology of the extant taxa in the MPC agreed with previous molecular phylogenies. Phylogenetic positions for fossil taxa indicate that several taxa previously described as early members of extant families (e.g., Bathygale and Plesictis) are likely stem taxa at the base of the Arctoidea. Taxa in the “Paleomustelidae” were found to be paraphyletic, but a monophyletic Oligobuninae was recovered within this set of taxa. This clade was closely related to the extant genera Gulo and Martes, therefore, nested within the extant radiation of the family Mustelidae. This analysis provides a resolution to several discrepancies between phylogenies considering either fossil taxa or extant taxa separately, and provides a framework for incorporating fossil and extant taxa into comprehensive combined evidence analyses.     

Partially incorrect fossil data augment analyses of discrete trait evolution in living species

Ancestral state reconstruction of discrete character traits is often vital when attempting to understand the origins and homology of traits in living species. The addition of fossils has been shown to alter our understanding of trait evolution in extant taxa, but researchers may avoid using fossils alongside extant species if only few are known, or if the designation of the trait of interest is uncertain. Here, I investigate the impacts of fossils and incorrectly coded fossils in the ancestral state reconstruction of discrete morphological characters under a likelihood model. Under simulated phylogenies and data, likelihood-based models are generally accurate when estimating ancestral node values. Analyses with combined fossil and extant data always outperform analyses with extant species alone, even when around one quarter of the fossil information is incorrect. These results are especially pronounced when model assumptions are violated, such as when there is a trend away from the root value. Fossil data are of particular importance when attempting to estimate the root node character state. Attempts should be made to include fossils in analysis of discrete traits under likelihood, even if there is uncertainty in the fossil trait data.     

The past and the present through phylogenetic analysis: the rove beetle tribe Othiini now and 99 Ma

In order to classify and taxonomically describe the first two fossil Othiini (Coleoptera: Staphylinidae: Staphylininae) species from three well‐preserved specimens in Cretaceous Burmese amber, a phylogenetic analysis was conducted, combining extant and extinct taxa. A dataset of 76 morphological characters scored for 33 recent species across the subfamilies Staphylininae and Paederinae was analysed using maximum parsimony and Bayesian inference methods. The many differing phylogenetic hypotheses for higher‐level relationships in the large rove beetle subfamilies Staphylininae and Paederinae were summarized and their hitherto known fossil record was reviewed. Based on the analyses, the new extinct genus Vetatrecus gen.n. is described with two new species: V. adelfiae sp.n. and V. secretum sp.n. Both species share character states that easily distinguish them from all recent Othiini and demonstrate a missing morphological link between subfamilies Staphylininae and Paederinae. This is the first morphology‐based evidence for the paraphyly of Staphylininae with respect to Paederinae, suggested earlier by two independent molecular‐based phylogenies of recent taxa. Our newly discovered stem lineage of Othiini stresses the importance of fossils in phylogenetic analyses conducted with the aim of improving the natural classification of extant species. It also suggests that the definitions of Staphylininae and Paederinae, long‐established family‐group taxa, may have to be reconsidered. This published work has been registered in ZooBank, http://zoobank.org/urn:lsid:zoobank.org:pub:817F39C4-F36B-4FD9-96CD-5F8FB064C39E .     

Morphology, fossils, divergence timing, and the phylogenetic relationships of Gavialis

Although morphological data have historically favored a basal position for the Indian gharial (Gavialis gangeticus) within Crocodylia and a Mesozoic divergence between Gavialis and all other crocodylians, several recent molecular data sets have argued for a sister-group relationship between Gavialis and the Indonesian false gharial (Tomistoma schlegelii) and a divergence between them no earlier than the Late Tertiary. Fossils were added to a matrix of 164 discrete morphological characters and subjected to parsimony analysis. When morphology was analyzed alone, Gavialis was the sister taxon of all other extant crocodylians whether or not fossil ingroup taxa were included, and a sister-group relationship between Gavialis and Tomistoma was significantly less parsimonious. In combination with published sequence and restriction site fragment data, Gavialis was the sister taxon of all other living crocodylians, but the position of Tomistoma depended on the inclusion of fossil ingroup taxa; with or without fossils, preferred morphological and molecular topologies were not significantly different. Fossils closer to Gavialis than to Tomistoma can be recognized in the Late Cretaceous, and fossil relatives of Tomistoma are known from the basal Eocene, strongly indicating a divergence long before the Late Tertiary. Comparison of minimum divergence time from the fossil record with different measures of molecular distance indicates evolutionary rate heterogeneity within Crocodylia. Fossils strongly contradict a post-Oligocene divergence between Gavialis and any other living crocodylian, but the phylogenetic placement of Gavialis is best viewed as unresolved.     

Phylogenetic relationships of the Cretaceous frog Beelzebufo from Madagascar and the placement of fossil constraints based on temporal and phylogenetic evidence

The placement of fossil calibrations is ideally based on the phylogenetic analysis of extinct taxa. Another source of information is the temporal variance for a given clade implied by a particular constraint when combined with other, well-supported calibrations. For example, the frog Beelzebufo ampinga from the Cretaceous of Madagascar has been hypothesized to be a crown-group member of the New World subfamily Ceratophryinae, which would support a Late Cretaceous connection with South America. However, phylogenetic analyses and molecular divergence time estimates based on other fossils do not support this placement. We derive a metric, Δt, to quantify temporal divergence among chronograms and find that errors resulting from mis-specified calibrations are localized when additional nodes throughout the tree are properly calibrated. The use of temporal information from molecular data can further assist in testing phylogenetic hypotheses regarding the placement of extinct taxa.     

Are pollen fossils useful for calibrating relaxed molecular clock dating of phylogenies? A comparative study using Myrtaceae

The identification and application of reliable fossil calibrations represents a key component of many molecular studies of evolutionary timescales. In studies of plants, most paleontological calibrations are associated with macrofossils. However, the pollen record can also inform age calibrations if fossils matching extant pollen groups are found. Recent work has shown that pollen of the myrtle family, Myrtaceae, can be classified into a number of morphological groups that are synapomorphic with molecular groups. By assembling a data matrix of pollen morphological characters from extant and fossil Myrtaceae, we were able to measure the fit of 26 pollen fossils to a molecular phylogenetic tree using parsimony optimisation of characters. We identified eight Myrtaceidites fossils as appropriate for calibration based on the most parsimonious placements of these fossils on the tree. These fossils were used to inform age constraints in a Bayesian phylogenetic analysis of a sequence alignment comprising two sequences from the chloroplast genome (matK and ndhF) and one nuclear locus (ITS), sampled from 106 taxa representing 80 genera. Three additional analyses were calibrated by placing pollen fossils using geographic and morphological information (eight calibrations), macrofossils (five calibrations), and macrofossils and pollen fossils in combination (12 calibrations). The addition of new fossil pollen calibrations led to older crown ages than have previously been found for tribes such as Eucalypteae and Myrteae. Estimates of rate variation among lineages were affected by the choice of calibrations, suggesting that the use of multiple calibrations can improve estimates of rate heterogeneity among lineages. This study illustrates the potential of including pollen-based calibrations in molecular studies of divergence times.     

A phylogeny of fossil and living neocoleoid cephalopods

Coleoid cephalopod phylogeny is well studied via both molecular and morphological data, yet although some agreement has been reached (e.g. that extant Decapodiformes and Octopoda are monophyletic) many details remain poorly resolved. Fossil coleoids, for which much data exists, have hitherto not been incorporated into analyses. Their inclusion is highly desirable for the support of neontological phylogenies, to better reconstruct character‐state histories, and to investigate the placement of the fossil groups themselves. In this study we present and analyse a morphological data matrix including both extinct and extant taxa. Homology assumptions in our data are discussed. Our results are presented both with and without the constraint of a monophyletic Decapodiformes imposed. When analysed with this constraint our results are strikingly congruent with those from molecular phylogeny, for instance placing Idiosepius in a basal position within Decapodiformes, and recovering Oegopsida and Bathyteuthoidea (although as grades). Our results support an Octopodiformes clade (“vampire squid” Vampyroteuthis as sister to Octopoda) and an octopodiform interpretation for most fossil coleoids. They suggest the fossil sister taxon to the octopods to be Plesioteuthididae. Most fossil higher taxa are supported, although many genera, especially within suborder Teudopseina, appear para‐ or polyphyletic.     

When Earth started blooming: insights from the fossil record

Recent palaeobotanical studies have greatly increased the quantity and quality of information available about the structure and relationships of Cretaceous angiosperms. Discoveries of extremely well preserved Cretaceous flowers have been especially informative and, combined with results from phylogenetic analyses of extant angiosperms (based mainly on molecular sequence data), have greatly clarified important aspects of early angiosperm diversification. Nevertheless, many questions still persist. The phylogenetic origin of the group itself remains as enigmatic as ever and, in some cases, newly introduced techniques from molecular biology have given confusing results. In particular, relationships between the five groups of extant seed plants remain uncertain, and it has sometimes proved difficult to reconcile estimates of the time of divergence between extant lineages made using a 'molecular clock' with the fossil record. One result, however, is becoming increasingly clear: a great deal of angiosperm diversity is extinct. Some groups of angiosperms were evidently more diverse in the past than they are today. In other cases, fossils defy assignment to extant groups at the family level or below. This raises the possibility that evolutionary conclusions based solely upon extant taxa that are merely relics of groups that were once much more diverse might be misled by the effects of extinction. It also introduces the possibility that some early enigmatic fossils might represent lineages that diverged from the main line of angiosperm evolution below the most recent common ancestor of all extant taxa. These, and other questions, are among those that need to be addressed by future palaeobotanical research.     

Early fossils illuminate character evolution and interrelationships of Lampridiformes (Teleostei,Acanthomorpha)

Lampridiformes is a peculiar clade of pelagic marine acanthomorph (spiny‐rayed) teleosts. Its phylogenetic position remains ambiguous, and varies depending on the type of data (morphological or molecular) used to infer interrelationships. Because the extreme morphological specializations of lampridiforms may have overwritten the ancestral features of the group with a bearing on its relationships, the inclusion of fossils that exhibit primitive character state combinations for the group as a whole is vital in establishing its phylogenetic position. Therefore, we present an osteological data set of extant (ten taxa) and fossil (14 taxa) acanthomorphs, including early Late Cretaceous taxa for which a close relationship with extant Lampridiformes has been suggested: ?Aipichthyoidea, ?Pharmacichthyidae, and ?Pycnosteroididae. We find that all three taxa plus Lampridiformes form a clade that we call Lampridomorpha. Under this hypothesis, ?Aipichthyoidea is paraphyletic. The inclusion of fossils in the analysis changes the topology, highlighting their critical importance in phylogenetic studies of morphological characters. When fossils are included, Lampridomorpha is sister to Euacanthomorpha (all other extant acanthomorphs), concurring with most previous anatomical studies, but conflicting with most molecular results. Lampridomorpha as a whole was a major component of the earliest acanthomorph faunas, notably in the Cenomanian. © 2014 The Linnean Society of London