Daniel T. Ksepka

Best Practices for Justifying Fossil Calibrations

Our ability to correlate biological evolution with climate change, geological evolution, and other historical patterns is essential to understanding the processes that shape biodiversity. Combining data from the fossil record with molecular phylogenetics represents an exciting synthetic approach to this challenge. The first molecular divergence dating analysis (Zuckerkandl and Pauling 1962) was based on a measure of the amino acid differences in the hemoglobin molecule, with replacement rates established (calibrated) using paleontological age estimates from textbooks (e.g., Dodson 1960). Since that time, the amount of molecular sequence data has increased dramatically, affording ever-greater opportunities to apply molecular divergence approaches to fundamental problems in evolutionary biology. To capitalize on these opportunities, increasingly sophisticated divergence dating methods have been, and continue to be, developed. In contrast, comparatively, little attention has been devoted to critically assessing the paleontological and associated geological data used in divergence dating analyses. The lack of rigorous protocols for assigning calibrations based on fossils raises serious questions about the credibility of divergence dating results (e.g., Shaul and Graur 2002; Brochu et al. 2004; Graur and Martin 2004; Hedges and Kumar 2004; Reisz and Muller 2004a, 2004b; Theodor 2004; van Tuinen and Hadly 2004a, 2004b; van Tuinen et al. 2004; Benton and Donoghue 2007; Donoghue and Benton 2007; Parham and Irmis 2008; Ksepka 2009; Benton et al. 2009; Heads 2011). The assertion that incorrect calibrations will negatively influence divergence dating studies is not controversial. Attempts to identify incorrect calibrations through the use of a posteriori methods are available (e.g., Near and Sanderson 2004; Near et al. 2005; Rutschmann et al. 2007; Marshall 2008; Pyron 2010; Dornburg et al. 2011). We do not deny that a posteriori methods are a useful means of evaluating calibrations, but there can be no substitute for a priori assessment of the veracity of paleontological data. Incorrect calibrations, those based upon fossils that are phylogenetically misplaced or assigned incorrect ages, clearly introduce error into an analysis. Consequently, thorough and explicit justification of both phylogenetic and chronologic age assessments is necessary for all fossils used for calibration. Such explicit justifications will help to ensure that divergence dating studies are based on the best available data. Unfortunately, the majority of previously published calibrations lack explicit explanations and justifications of the age and phylogenetic position of the key fossils. In the absence of explicit justifications, it is difficult to distinguish between correct and incorrect calibrations, and it becomes difficult to reevaluate previous claims in light of new data. Paleontology is a dynamic science, with new data and perspectives constantly emerging as a result of new discoveries (see Kimura 2010 for a recent case where the age of the earliest known record of a clade was more than doubled). Calibrations based upon the best available evidence at a given time can become inappropriate as the discovery of new specimens, new phylogenetic analyses, and ongoing stratigraphic and geochronologic revisions refine our understanding of the fossil record. Our primary goals in this paper are to establish the best practices for justifying fossils used for the temporal calibration of molecular phylogenies. Our examples derive mainly, but not exclusively, from the vertebrate fossil record. We hope that our recommendations will lead to more credible calibrations and, as a result, more reliable divergence dates throughout the tree of life. A secondary goal is to help the community (researchers, editors, and reviewers) who might be unfamiliar with fossils to understand and overcome the challenges associated with using paleontological data. In order to accomplish these goals, we present a specimen-based protocol for selecting and documenting relevant fossils and discuss future directions for evaluating and utilizing phylogenetic and temporal data from the fossil record. We likewise encourage biologists relying on nonfossil calibrations for molecular divergence estimates (e.g., ages of island or mountain range formations, continental drift, and biomarkers) to develop their own set of rigorous guidelines so that their calibrations may also be evaluated in a systematic way.

Fossil Evidence for Evolution of the Shape and Color of Penguin Feathers

Feather of the Penguin Penguins are highly adapted for their cold, aquatic environment. Changes in their wings and feathers have allowed rapid swimming and protection from the near-freezing water. Clarke et al. (p. 954, published online 30 September; see the cover) describe an early penguin, dating to about 35 million years ago, that includes well-preserved feathers. The melanosomes in the feathers, which influence their strength, as well as their color, are like those of many other aquatic birds and unlike those of present-day penguins, even though the morphology of the wings and feathers had already been modified. Thus, in penguins, the shape and form of the feather evolved before microstructural changes occurred. The melanosome arrangement also suggests that the penguin was mostly gray-brown. A fossil penguin shows that the wing and feather form evolved before distinctive microstructural changes in the feathers. Penguin feathers are highly modified in form and function, but there have been no fossils to inform their evolution. A giant penguin with feathers was recovered from the late Eocene (~36 million years ago) of Peru. The fossil reveals that key feathering features, including undifferentiated primary wing feathers and broad body contour feather shafts, evolved early in the penguin lineage. Analyses of fossilized color-imparting melanosomes reveal that their dimensions were similar to those of non-penguin avian taxa and that the feathering may have been predominantly gray and reddish-brown. In contrast, the dark black-brown color of extant penguin feathers is generated by large, ellipsoidal melanosomes previously unknown for birds. The nanostructure of penguin feathers was thus modified after earlier macrostructural modifications of feather shape linked to aquatic flight.

Paleogene equatorial penguins challenge the proposed relationship between biogeography, diversity, and Cenozoic climate change

New penguin fossils from the Eocene of Peru force a reevaluation of previous hypotheses regarding the causal role of climate change in penguin evolution. Repeatedly it has been proposed that penguins originated in high southern latitudes and arrived at equatorial regions relatively recently (e.g., 4–8 million years ago), well after the onset of latest Eocene/Oligocene global cooling and increases in polar ice volume. By contrast, new discoveries from the middle and late Eocene of Peru reveal that penguins invaded low latitudes >30 million years earlier than prior data suggested, during one of the warmest intervals of the Cenozoic. A diverse fauna includes two new species, here reported from two of the best exemplars of Paleogene penguins yet recovered. The most comprehensive phylogenetic analysis of Sphenisciformes to date, combining morphological and molecular data, places the new species outside the extant penguin radiation (crown clade: Spheniscidae) and supports two separate dispersals to equatorial (paleolatitude ≈14°S) regions during greenhouse earth conditions. One new species, Perudyptes devriesi, is among the deepest divergences within Sphenisciformes. The second, Icadyptes salasi, is the most complete giant (>1.5 m standing height) penguin yet described. Both species provide critical information on early penguin cranial osteology, trends in penguin body size, and the evolution of the penguin flipper.

The phylogeny of the living and fossil Sphenisciformes (penguins)

We present the first phylogenetic analysis of the Sphenisciformes that extensively samples fossil taxa. Combined analysis of 181 morphological characters and sequence fragments from mitochondrial and nuclear genes (12S, 16S, COI, cytochrome b, RAG‐1) yields a largely resolved tree. Two species of the New Zealand Waimanu form a trichotomy with all other penguins in our result. The much discussed giant penguins Anthropornis and Pachydyptes are placed in two clades near the base of the tree. Stratigraphic and phylogenetic evidence suggest that some lineages of penguins attained very large body size rapidly and early in the clades evolutionary history. The only fossil taxa that fall inside the crown clade Spheniscidae are fossil species assigned to the genus Spheniscus. Thus, extant penguin diversity is more accurately viewed as the product of a successful radiation of derived taxa than as an assemblage of survivors belonging to numerous lineages. The success of the Spheniscidae may be due to novel feeding adaptations and a more derived flipper apparatus. We offer a biogeographical scenario for penguins that incorporates fossil distributions and paleogeographic reconstructions of the Southern continents positions. Our results do not support an expansion of the Spheniscidae from a cooling Continental Antarctica, but instead suggest those species that currently breed in that area are the descendants of colonizers from the Subantarctic. Many important divergence events in the clade Spheniscidae can instead be explained by dispersal along the paths of major ocean currents and the emergence of new islands due to tectonic events.

The Basal Penguin (Aves: Sphenisciformes) Perudyptes devriesi and a Phylogenetic Evaluation of the Penguin Fossil Record

Abstract We present the first detailed description of Perudyptes devriesi, a basal penguin from the middle Eocene (~42 Ma) Paracas Formation of Peru, and a new analysis of all published extinct penguin species as well as controversial fragmentary specimens. The Perudyptes devriesi holotype includes key regions of the skull and significant postcranial material, thus helping to fill a major phylogenetic and stratigraphic (~20 million year) gap between the earliest fossil penguins (Waimanu manneringi and Waimanu tuatahi, ~58–61.6 Ma) and the next oldest partial skeletons. Perudyptes devriesi is diagnosable by five autapomorphies: (1) an anteroventrally directed postorbital process, (2) marked anterior expansion of the parasphenoid rostrum, (3) posterior trochlear ridge of the humerus projecting distal to the middle trochlear ridge and conformed as a large, broadly curved surface, (4) convex articular surface for the antitrochanter of the femur, and (5) extremely weak anterior projection of the lateral condyle of the tibiotarsus. The skull of Perudyptes is characterized by deep temporal fossae and an elongate, narrow beak that differs from other reported stem penguins in its short mandibular symphysis. The wing skeleton of Perudyptes preserves a combination of plesiomorphic features also observed in the basal penguin Waimanu and derived features shared with more crownward penguins. Features of the wing optimized as primitive for Sphenisciformes include retention of a discrete dorsal supracondylar tubercle on the humerus and presence of a modestly projected pisiform process on the carpometacarpus. Derived features present in Perudyptes and all more crownward penguins, but absent in Waimanu, include a more flattened humerus, development of a trochlea for the tendon of m. scapulotriceps at the distal end of the humerus, and bowing of the anterior face of the carpometacarpus. A combined molecular and morphological dataset for Spheniciformes was expanded by adding 25 osteological and soft tissue characters as well as 11 taxa. In agreement with previous results, Perudyptes devriesi is identified as one of the most basal members of Sphenisciformes. This analysis also confirms the placement of the middle/late Miocene (~11–13 Ma) fossil Spheniscus muizoni as a member of the Spheniscus clade and places the late Miocene (~10 Ma) Madrynornis mirandus as sister taxon to extant Eudyptes. These two species, known from relatively complete partial skeletons, are the oldest crown clade penguin fossils and represent well-corroborated temporal calibration points for the Spheniscus-Eudyptula divergence and Megadyptes-Eudyptes divergence, respectively. Our results reaffirm that the Miocene penguin taxon Palaeospheniscus, recently proposed to represent a member of the crown radiation, belongs outside of the crown clade Spheniscidae. The phylogenetic positions of small Eocene Antarctic penguin taxa (Delphinornis, Marambiornis, and Mesetaornis) recently proposed as possible direct ancestors to crown Spheniscidae were further evaluated using alternate coding strategies for incorporating scorings from isolated elements that preserve critical morphologies and are thought to represent these taxa, although they cannot yet be reliably assigned to individual species. Under all scoring regimes, Delphinornis, Marambiornis, and Mesetaornis were recovered as distantly related to Spheniscidae. Using synapomorphies identified in the primary analysis, we evaluated the phylogenetic position of fragmentary specimens, including the holotypes of valid but poorly known species, specimens currently unassignable to the species level, and morphologically distinct specimens that have not yet been named. All pre-Miocene specimens can be excluded from Spheniscidae based on presence of plesiomorphies lost in all crown penguins, consistent with a recent radiation for the penguin crown clade. This study provides additional support for a scenario of penguin evolution characterized by an origin of flightlessness near the K-T boundary, dispersal throughout the Southern Hemisphere during the early Paleogene, and a late Cenozoic origin for the crown clade Spheniscidae. Stratigraphic distribution and phylogenetic relationships of fossil penguins are consistent with distinct radiations during the Eocene, Oligocene, and Miocene. While the Eocene and Oligocene penguin faunas are similar in many respects, the Miocene fauna is characterized by smaller average size and novel cranial morphologies, suggesting that an ecological shift in diet occurred close to the origin of crown Spheniscidae.

Bayesian Total-Evidence Dating Reveals the Recent Crown Radiation of Penguins

Abstract The total‐evidence approach to divergence time dating uses molecular and morphological data from extant and fossil species to infer phylogenetic relationships, species divergence times, and macroevolutionary parameters in a single coherent framework. Current model‐based implementations of this approach lack an appropriate model for the tree describing the diversification and fossilization process and can produce estimates that lead to erroneous conclusions. We address this shortcoming by providing a total‐evidence method implemented in a Bayesian framework. This approach uses a mechanistic tree prior to describe the underlying diversification process that generated the tree of extant and fossil taxa. Previous attempts to apply the total‐evidence approach have used tree priors that do not account for the possibility that fossil samples may be direct ancestors of other samples, that is, ancestors of fossil or extant species or of clades. The fossilized birth‐death (FBD) process explicitly models the diversification, fossilization, and sampling processes and naturally allows for sampled ancestors. This model was recently applied to estimate divergence times based on molecular data and fossil occurrence dates. We incorporate the FBD model and a model of morphological trait evolution into a Bayesian total‐evidence approach to dating species phylogenies. We apply this method to extant and fossil penguins and show that the modern penguins radiated much more recently than has been previously estimated, with the basal divergence in the crown clade occurring at ∼12.7 Ma and most splits leading to extant species occurring in the last 2 myr. Our results demonstrate that including stem‐fossil diversity can greatly improve the estimates of the divergence times of crown taxa. The method is available in BEAST2 (version 2.4) software www.beast2.org with packages SA (version at least 1.1.4) and morph‐models (version at least 1.0.4) installed.

New fossil penguins (Aves, Sphenisciformes) from the Oligocene of New Zealand reveal the skeletal plan of stem penguins

ABSTRACT Three skeletons collected from the late Oligocene Kokoamu Greensand of New Zealand are among the most complete Paleogene penguins known. These specimens, described here as Kairuku waitaki, gen. et sp. nov., and Kairuku grebneffi, sp. nov., reveal new details of key elements of the stem penguin skeleton associated with underwater flight, including the sternum, flipper, and pygostyle. Relative proportions of the trunk, flippers, and hind limbs can now be determined from a single individual for the first time, offering insight into the body plan of stem penguins and improved constraints on size estimates for ‘giant’ taxa. Kairuku is characterized by an elongate, narrow sternum, a short and flared coracoid, an elongate narrow flipper, and a robust hind limb. The pygostyle of Kairuku lacks the derived triangular cross-section seen in extant penguins, suggesting that the rectrices attached in a more typical avian pattern and the tail may have lacked the propping function utilized by living penguins. New materials described here, along with re-study of previously described specimens, resolve several long-standing phylogenetic, biogeographic, and taxonomic issues stemming from the inadequate comparative material of several of the first-named fossil penguin species. An array of partial associated skeletons from the Eocene—Oligocene of New Zealand historically referred to Palaeeudyptes antarcticus or Palaeeudyptes sp. are recognized as at least five distinct species: Palaeeudyptes antarcticus, Palaeeudyptes marplesi, Kairuku waitaki, Kairuku grebneffi, and an unnamed Burnside Formation species.

Broken gears in the avian molecular clock: new phylogenetic analyses support stem galliform status for Gallinuloides wyomingensis and rallid affinities for Amitabha urbsinterdictensis

Galliformes (landfowl) have been the focus of numerous divergence dating studies that seek a refined understanding of the early radiation of living birds. The Eocene fossil birds Amitabha urbsinterdictensis (Bridger Formation) and Gallinuloides wyomingensis (Green River Formation) have been used extensively in studies dealing with the timing of evolution in crown Galliformes. Divergence estimates from studies incorporating these fossils as calibration points suggest that multiple galliform lineages radiated in the Cretaceous and survived the Cretaceous–Tertiary mass extinction. However, the phylogenetic position of both fossils has been disputed, particularly with regard to crown or stem status. In order to resolve this debate, a new study of A. urbsinterdictensis and G. wyomingensis was undertaken. Further preparation and re‐examination of the A. urbsinterdictensis holotype indicates this fossil falls outside both crown and stem Galliformes, and reveals evidence for a relationship with Rallidae (rails). In order to reassess the status of G. wyomingensis, a matrix of 120 morphological characters was constructed by revising and expanding on previous studies. Phylogenetic analyses using this matrix place G. wyomingensis basal to all crown Galliformes. Stem placement of G. wyomingensis is retained and resolution is improved in combined analyses incorporating sequence data from cytochrome b, NADH dehydrogenase subunit 2, mitochondrial control region, 12S rDNA, and nuclear ovomucoid intron G. All evidence indicates that A. urbsinterdictensis and G. wyomingensis are inappropriate internal calibration points for Galliformes and may have contributed to overestimation of divergence event ages. Though stem galliforms existed in the Cretaceous, the divergence of crown lineages in the Cretaceous remains inconclusively demonstrated. Because few galliform fossils have been evaluated phylogenetically, further investigations into the tempo of galliform evolution must await identification of proper fossil calibration points.

Synthesizing and databasing fossil calibrations: Divergence dating and beyond

Divergence dating studies, which combine temporal data from the fossil record with branch length data from molecular phylogenetic trees, represent a rapidly expanding approach to understanding the history of life. National Evolutionary Synthesis Center hosted the first Fossil Calibrations Working Group (3–6 March, 2011, Durham, NC, USA), bringing together palaeontologists, molecular evolutionists and bioinformatics experts to present perspectives from disciplines that generate, model and use fossil calibration data. Presentations and discussions focused on channels for interdisciplinary collaboration, best practices for justifying, reporting and using fossil calibrations and roadblocks to synthesis of palaeontological and molecular data. Bioinformatics solutions were proposed, with the primary objective being a new database for vetted fossil calibrations with linkages to existing resources, targeted for a 2012 launch.

Redescription and Phylogenetic Position of the Early Miocene Penguin Paraptenodytes Antarcticus from Patagonia

Abstract Paraptenodytes antarcticus is one of the best-known and most complete fossil penguins. This taxon is so distinctive that it has traditionally been classified in its own subfamily (Sphenisciformes: Paraptenodytinae) separate from all living penguins (Spheniscinae). The well-preserved partial skull of P. antarcticus is one of our richest sources of data on early penguin cranial morphology. We provide an updated description of the skull of P. antarcticus in a comparative context and use this information to explore the phylogenetic relationships of this taxon. Three cladistic analyses using an osteology dataset, a larger morphological dataset (including osteological, soft tissue, behavior, and oological characters) and a combined (morphological + molecular) dataset all recover Paraptenodytes as the sister taxon to a clade including all extant penguins. The placement of Paraptenodytes outside the crown clade of extant penguins reveals the order in which many spheniscid synapomorphies were acquired and lends support to the hypothesis that modern penguins had Subantarctic ancestors.