Evan T Saitta

Tyrannosaurs are among the most intensively studied and best-known dinosaurs. Despite this, their relationships and systematics are highly controversial. An ongoing debate concerns the validity of Nanotyrannus lancensis, interpreted either as a distinct genus of small-bodied tyrannosaur or a juvenile of Tyrannosaurus rex. We examine multiple lines of evidence and show that the evidence strongly supports recognition of Nanotyrannus as a distinct species for the following reasons: 1. High diversity of tyrannosaurs and predatory dinosaurs supports the idea that multiple tyrannosaurids inhabited the late Maastrichtian of Laramidia; 2. Nanotyrannus lacks characters supporting referral to Tyrannosaurus or Tyrannosaurinae but differs from T. rex in >150 morphological characters, while intermediate forms combining the features of Nanotyrannus and T. rex are unknown; 3. Histology shows specimens of Nanotyrannus showing (i) skeletal fusions, (ii) mature skull bone textures, (iii) slow growth rates relative to T. rex, (iv) decelerating growth in their final years of life, and (v) growth curves predicting adult masses of ~1500 kg or less, showing these animals are subadults and young adults, not juvenile Tyrannosaurus; 4. growth series of other tyrannosaurids, including Tarbosaurus and Gorgosaurus, do not show morphological changes proposed for a Nanotyrannus–Tyrannosaurus growth series, and deriving Tyrannosaurus from Nanotyrannus requires several changes inconsistent with known patterns of dinosaur development; 5. Juvenile T. rex exist, showing diagnostic features of Tyrannosaurus; 6. Phylogenetic analysis suggests that Nanotyrannus may lie outside Tyrannosauridae. Tyrannosaur diversity before the K-Pg extinction is higher than previously appreciated. The challenges inherent in diagnosing species based on fossils mean paleontologists may be systematically underestimating the diversity of ancient ecosystems.

Proteins are the most stable of the macromolecules that carry genetic information over long periods of time. Closed systems are more likely to retain endogenous proteins or their degradation products. Amino acid racemisation data in experimental and subfossil material suggests that mollusc shell and avian eggshell calcite crystals can demonstrate closed system behaviour, retaining endogenous amino acids. Here, Late Cretaceous (Campanian–Maastrichtian) Argentine titanosaurian sauropod eggshells show dark, organic stains under light microscopy/photography and fluorescence imaging. Raman spectroscopy can yield bands consistent with various organic molecules, possibly including N-bearing molecules or geopolymers. Pyrolysis-gas chromatography-mass spectrometry reveals pyrolysates consistent with amino acids as well as aliphatic hydrocarbon homologues that are not present in modern eggshell, consistent with kerogen formation deriving from eggshell lipids. High-performance liquid chromatography reveals that their intra-crystalline fraction can be enriched in some of the most stable amino acids (Glx, Gly, Ala, and possibly Val) and are fully racemic (despite being some of the slowest racemising amino acids), indicating ancient origin. This preservation varies across localities, but similar ancient amino acid profiles were also observed in Late Cretaceous Spanish titanosaurians from several localities and Chinese putative hadrosaurid eggshell. These amino acid results are consistent with previous studies on degradation trends deduced from modern, thermally matured, sub-fossil, and ∼3.8–6.5 Ma avian eggshell, as well as ∼30 Ma calcitic mollusc opercula. Selective preservation of certain fully racemic amino acids, which do not racemise in-chain, and the concentration of free amino acids suggests likely complete hydrolysis of original peptides. Liquid chromatography-tandem mass spectrometry supports this hypothesis by failing to detect any non-contamination peptide sequences from the Mesozoic eggshell. These closed-system amino acids are possibly the most thoroughly supported non-avian dinosaur endogenous protein-derived constituents, at least those that have not undergone oxidative condensation with other classes of biomolecules. Biocrystal matrices can help preserve mobile organic molecules by trapping them (perhaps with the assistance of resistant organic polymers), but trapped organics are nevertheless prone to diagenetic degradation, even if such reactions might be slowed in exceptional circumstances. Future work should survey fossil biocalcite to determine variability in amino acid preservation.

We address the identity of putative ovarian follicles in Early Cretaceous bird fossils from the Jehol Biota (China), whose identification has previously been challenged. For the first time, we present a link to the botanical fossil record, showing that the “follicles” of some enantiornithine fossils resemble plant propagules from the Jehol Biota, which belong to Carpolithes multiseminalis. The botanical affinities of this “form-taxon” are currently unresolved, but we note that C. multiseminalis propagules resemble propagules associated with cone-like organs described as Strobilites taxusoides, which in turn are possibly associated with sterile foliage allocated to Liaoningcladus. Laser-Stimulated Fluorescence imaging furthermore reveals different intensities of fluorescence of “follicles” associated with a skeleton of the confuciusornithid Eoconfuciusornis zhengi, with a non-fluorescent circular micro-pattern indicating carbonaceous (or originally carbonaceous) matter. This is inconsistent with the interpretation of these structures as ovarian follicles. We therefore reaffirm that the “follicles” represent ingested food items, and even though the exact nature of the Eoconfuciusornis stomach contents remains elusive, at least some enantiornithines ingested plant propagules.

Despite reports of sexual dimorphism in extinct taxa, such claims in non-avian dinosaurs have been rare over the last decade and have often been criticized. Since dimorphism is widespread in sexually reproducing organisms today, under-reporting in the literature might suggest either methodological shortcomings or that this diverse group exhibited highly unusual reproductive biology. Univariate significance testing, especially for bimodality, is ineffective and prone to false negatives. Species recognition and mutual sexual selection hypotheses, therefore, may not be required to explain supposed absence of sexual dimorphism across the grade (a type II error). Instead, multiple lines of evidence support sexual selection and variation of structures consistent with secondary sexual characteristics, strongly suggesting sexual dimorphism in non-avian dinosaurs. We propose a framework for studying sexual dimorphism in fossils, focusing on likely secondary sexual traits and testing against all alternate hypotheses for variation in them using multiple lines of evidence. We use effect size statistics appropriate for low sample sizes, rather than significance testing, to analyse potential divergence of growth curves in traits and constrain estimates for dimorphism magnitude. In many cases, estimates of sexual variation can be reasonably accurate, and further developments in methods to improve sex assignments and account for intrasexual variation (e.g. mixture modelling) will improve accuracy. It is better to compare estimates for the magnitude of and support for dimorphism between datasets than to dichotomously reject or fail to reject monomorphism in a single species, enabling the study of sexual selection across phylogenies and time. We defend our approach with simulated and empirical data, including dinosaur data, showing that even simple approaches can yield fairly accurate estimates of sexual variation in many cases, allowing for comparison of species with high and low support for sexual variation.

Preserved melanin pigments have been discovered in fossilised integumentary appendages of several amniote lineages (fishes, frogs, snakes, marine reptiles, non-avialan dinosaurs, birds, and mammals) excavated from lagerstätten across the globe. Melanisation is a leading factor in organic integument preservation in these fossils. Melanin in extant vertebrates is typically stored in rod-to sphere-shaped, lysosome-derived, membrane-bound vesicles called melanosomes. Black, dark brown, and grey colours are produced by eumelanin, and reddish-brown colours are produced by phaeomelanin. Specific morphotypes and nanostructural arrangements of melanosomes and their relation to the keratin matrix in integumentary appendages create the so-called 'structural colours'. Reconstruction of colour patterns in ancient animals has opened an exciting new avenue for studying their life, behaviour and ecology. Modern relationships between the shape, arrangement, and size of avian melanosomes, melanin chemistry, and feather colour have been applied to reconstruct the hues and colour patterns of isolated feathers and plumages of the dinosaurs Anchiornis, Sinosauropteryx, and Microraptor in seminal papers that initiated the field of palaeocolour reconstruction. Since then, further research has identified countershading camouflage patterns, and informed subsequent predictions on the ecology and behaviour of these extinct animals. However, palaeocolour reconstruction remains a nascent field, and current approaches have considerable potential for further refinement, standardisation, and expansion. This includes detailed study of non-melanic pigments that might be preserved in fossilised integuments. A common issue among existing palaeocolour studies is the lack of contextualisation of different lines of evidence and the wide variety of techniques currently employed. To that end, this review focused on fossil amniotes: (i) produces an overarching framework that appropriately reconstructs palaeocolour by accounting for the chemical signatures of various pigments, morphology and local arrangement of pigment-bearing vesicles, pigment concentration, macroscopic colour patterns, and taphonomy; (ii) provides background context for the evolution of colour-producing mechanisms; and (iii) encourages future efforts in palaeocolour reconstructions particularly of less-studied groups such as non-dinosaur archosaurs and non-archosaur amniotes.

'Exceptional fossils' of dinosaurs preserving feathers have radically changed the way we view their paleobiology and the evolution of birds. Understanding how such soft tissues preserve is imperative to accurately interpreting the morphology of fossil feathers. Experimental taphonomy has been integral to such investigations. One such experiment used a printing press to mimic compaction, done subaerially and without sediment burial, and concluded that the leaking of bodily fluid could lead to the clumping of feathers by causing barbs to stick together such that they superficially resemble simpler, less derived, filamentous structures. Here we use a novel, custom-built experimental setup to more accurately mimic subaque-ous burial and compaction under low-energy, fine-grain depositional environments applicable to the taphonomic settings most plumage-preserving 'exceptional fossils' are found in. We find that when submerged and subsequently buried and compacted, feathers do not clump together and they maintain their original arrangement. Submersion in fluid in and of itself does not lead to clumping of barbs; this would only occur upon pulling feathers out from water into air. Furthermore, sediment encases the feathers, fixing them in place during compaction. Thus, feather clumping that leads to erroneously plesiomorphic morphological interpretations may not be a taphonomic factor of concern when examining fossil feathers. Our current methodology is amenable to further improvements that will continue to more accurately mimic subaqueous burial and compaction, allowing for various hypothesis testing.

The evolution of integumentary structures, particularly in relation to feathers in dinosaurs, has become an area of intense research. Our understanding of the molecular evolution of keratin protein is greatly restricted by the fact that such information is lost during diagenesis and cannot be derived from fossils. In this study, decay and maturation experiments are used to determine if different keratin types or integumentary structures show different patterns of degradation early in their taphonomic histories and if such simulations might advance our understanding of different fossilization pathways. Although different distortion patterns were observed in different filamentous structures during moderate maturation and ultrastructural textures unique to certain types of scales persisted in moderate maturation, neither of these have been observed in fossils. It remains uncertain whether these degradation patterns would ever occur in natural sediment matrix, where microbial and chemical decay happens well before significant diagenesis. It takes some time for remains to be buried, meaning that keratin may not be left for moderate maturation to produce such patterns. Higher, more realistic maturation conditions produce a thick, and water soluble fluid that lacks all morphological and ultrastructural details of the original keratin, suggesting that such textural or distortion patterns are unlikely to be preserved in fossils. Although different degradation patterns among keratinous structures are intriguing, it is unlikely that such information could be recorded in the fossil record. Calcium phosphates and pigments are the surviving components of integumentary structures. Thus, the results here are likely of more relation to the archaeological record than fossil record. Other noteworthy results of these experiments are that melanin may not be the leading factor in determining the rate of microbial decay in feathers but may reduce the rate of degradation from maturation, that the existence of rachis filamentous subunits similar to plumulaceous barbules is supported, and that previously reported dinosaur ‘erythrocytes' may be taphonomic artifacts of degraded organic material.

Recent studies have suggested the presence of keratin in fossils dating back to the Mesozoic. However, ultrastructural studies revealing exposed melanosomes in many fossil keratinous tissues suggest that keratin should rarely, if ever, be preserved. In this study, keratin's stability through diagenesis was tested using microbial decay and maturation experiments on various keratinous structures. The residues were analysed using pyrolysis-gas chromatography-mass spectrometry and compared to unpublished feather and hair fossils and published fresh and fossil melanin from squid ink. Results show that highly matured feathers (200–250°C/250 bars/24 h) become a volatile-rich, thick fluid with semi-distinct pyrolysis compounds from those observed in less degraded keratins (i.e. fresh, decayed, moderately matured, and decayed and moderately matured) suggesting hydrolysis of peptide bonds and potential degradation of free amino acids. Neither melanization nor keratin (secondary) structure (e.g. ⍺- vs β-keratin) produced different pyrograms; melanin pyrolysates are largely a subset of those from proteins, and proteins have characteristic pyrolysates. Analyses of fossil fur and feather found a lack of amides, succinimide and piperazines (present even in highly matured keratin) and showed pyrolysis compounds more similar to fossil and fresh melanin than to non-matured or matured keratin. Although the highly matured fluid was not water soluble at room temperature, it readily dissolved at elevated temperatures easily attained during diagenesis, meaning it could leach away from the fossil. Future interpretations of fossils must consider that calcium phosphate and pigments are the only components of keratinous structures known to survive fossilization in mature sediments.

Conclusive evidence for sexual dimorphism in non-avian dinosaurs has been elusive. Here it is shown that dimorphism in the shape of the dermal plates of Stegosaurus mjosi (Upper Jurassic, western USA) does not result from non-sex-related individual, interspecific, or ontogenetic variation and is most likely a sexually dimorphic feature. One morph possessed wide, oval plates 45% larger in surface area than the tall, narrow plates of the other morph. Intermediate morphologies are lacking as principal component analysis supports marked size- and shape-based dimorphism. In contrast, many non-sex-related individual variations are expected to show intermediate morphologies. Taphonomy of a new quarry in Montana (JRDI 5ES Quarry) shows that at least five individuals were buried in a single horizon and were not brought together by water or scavenger transportation. This new site demonstrates co-existence, and possibly suggests sociality, between two morphs that only show dimorphism in their plates. Without evidence for niche partitioning, it is unlikely that the two morphs represent different species. Histology of the new specimens in combination with studies on previous specimens indicates that both morphs occur in fully-grown individuals. Therefore, the dimorphism is not a result of ontogenetic change. Furthermore, the two morphs of plates do not simply come from different positions on the back of a single individual. Plates from all positions on the body can be classified as one of the two morphs, and previously discovered, isolated specimens possess only one morph of plates. Based on the seemingly display-oriented morphology of plates, female mate choice was likely the driving evolutionary mechanism rather than male-male competition. Dinosaur ornamentation possibly served similar functions to the ornamentation of modern species. Comparisons to ornamentation involved in sexual selection of extant species, such as the horns of bovids, may be appropriate in predicting the function of some dinosaur ornamentation.

Body size is undoubtedly one of the most useful measures of sexual dimorphism and, by proxy, sexual selection. Here, I examine large, published datasets of average sexual size dimorphism (SSD) in four clades of amniotes: birds, mammals, squamates, and turtles. Most sexual variation is of subtle magnitude; attempts to discretely categorize species as monomorphic may overlook genuine and common sexual variations of small magnitude (e.g., <10-20% difference). Mammals, squamates, and turtles have unimodal SSD distributions centered close to zero that vary in skew. Mammals skew towards a preponderance of taxa with larger males than females, and mammals with the most extreme SSD have larger males than females. Turtles, however, skew strongly towards a preponderance of taxa with larger females than males, and turtles with the most extreme SSD have larger females than males. Squamates are intermediate to these two clades. Birds are unique in that they 1) are noticeably deficient in taxa near monomorphism, 2) have a bimodal distribution with peaks closely and roughly equidistantly straddling either side of monomorphism, and 3) have a high preponderance of taxa with larger males than females. This suggests stronger disruptive selection or constraints against monomorphism in birds compared to other amniotes. Bird data from Dunning (2007) yields bimodality, while other datasets do not, possibly due to data artefacts/errors. Although Rensch's rule (RR) is difficult to apply to broad clades, scaling patterns were nevertheless examined here. While turtles and squamates show full adherence to RR, mammals show weaker adherence. Mammal scaling is comparatively less male-biased with increased size than scaling in squamates and turtles, and sex-role reversed mammals instead approach isometry between male and female size. Although bird taxa with larger males than females follow RR, sex-role reversed birds show the converse RR pattern. In birds, increasing size leads to increased dimorphism magnitude regardless of the direction of dimorphism, even though regression of the entire clade deceptively suggests they scale isometrically. This paradoxical scaling explains their unusual bimodal SSD distribution, as shown here through simulation. Equidistant bimodality from monomorphism might suggest disruptive selection where both mating systems have mirrored sexual selection dynamics of comparable effect. Scaling patterns between dimorphism magnitude and overall taxon size in non-reversed and reversed systems might not be readily apparent when examining the whole clade. Large mammals have disproportionately male-biased and more extreme SSD magnitudes. In comparison, large birds have relatively numerous sex-role reversed taxa as well as more extreme SSD magnitudes. These results deserve further testing with tighter phylogenetic controls and comparison of data sources. Additional ecological, physiological, and behavioral variables should also be examined in relation to SSD (e.g., altriciality vs. precociality, oviparity vs. viviparity, clutch size, neonate mass).

Feathers are complex integumentary structures with high diversity across species and within plumage and have varied functions (e.g., thermoregulation, flight). Flight is lost in many crown lineages, and frequently occurs in island founding or semiaquatic context. Different extant lineages lost flight across at least three orders of magnitude of time (~79.58 Ma-15 Ka). Flight loss effect on sensory capacity, brain size, and skeletomusculature have been studied, but less work exists on relations between flightlessness and feathers. To understand how flight loss affects feather anatomy, we measured 11 feather metrics (e.g., barb length, barb angle) from primaries, tertials, rectrices, and contour feathers on skins of 30 flightless taxa and their phylogenetically closest volant taxa, supplemented with broader sampling of primaries across all orders of volant crown birds. Our sample includes 27 independent losses of flight; the sample contains nearly half the extant flightless species count and matches its ~3:2 terrestrial:semiaquatic ratio. Vane symmetry increases in flightless lineages, and these patterns are strongest in flight feathers and weakest in coverts. Greatest changes in feathers are in the oldest flightless lineages like penguins, which show robust filaments (rachis, barbs, and barbules) on small feathers, and ratites, which show high interspecific diversity with plumulaceous filaments and/or filament loss. Phylogenetic comparative methods show that some of these microscopic feather traits, such as barb/barbule length and rachis width, are not as dramatically modified upon flight loss as are body mass increase and relative wing and tail fan reduction, whereas the effect on vane symmetry is more easily detected. Upon relaxing selection for flight, feathers do not soon significantly modify many of their flight adaptations, although increased vane symmetry is likely the most detectable shift. Feathers of recently flightless lineages are in many ways like those of their volant relatives. Feather microstructure evolution is often subtle in flightless taxa, except when flight loss is ancient, perhaps because developmental constraints act upon feathers and/or selection for novel feather morphologies is not strong. Changes in skeletomusculature of the flight apparatus are likely more evident in recently flightless taxa and may be a more reliable way to detect flight loss in fossils, with increased vane symmetry as potentially a microscopic signal. Finally, we see an intriguing, reversed pattern in feather evolution after flight loss from the pattern proposed in popular developmental models of feathers, with the later stages of feather development (asymmetric displacement of barb loci) being lost more readily, while early stages of development (e.g., differentiated barb ridges on follicle collar) are only lost after many millions of years of flightlessness.

Tyrannosaurs, giant predatory dinosaurs from the end of the Cretaceous, are among the most intensively researched and best-known groups of dinosaurs. Despite this, their relationships and systematics are highly controversial, and the number of tyrannosaur species occurring in the latest Cretaceous in North America is debated. An ongoing debate concerns the status of Nanotyrannus lancensis, which has variously been interpreted as a distinct taxon of small-bodied tyrannosaur or a juvenile of the coeval Tyrannosaurus rex. Here, we review multiple lines of evidence and show that the totality of evidence strongly supports recognition of Nanotyrannus as a distinct species: 1. The high diversity of Late Cretaceous tyrannosaurs and predatory dinosaurs in general is consistent with the idea that more than one species lived in the late Maastrichtian of Western North America; 2. Nanotyrannus shows few if any diagnostic characters allowing referral specifically to Tyrannosaurus or even Tyrannosaurinae, but is differentiated from T. rex by at least 77 morphological characters, while intermediate forms, combining characteristics of Nanotyrannus and T. rex, remain unknown; 3. Histological evidence shows that individuals previously referred to Nanotyrannus lancensis show (i) skeletal fusions consistent with maturity, (ii) skull bone textures consistent with maturity, (iii) slow growth rates relative to T. rex, (iv) decelerating growth in their final years of life, and (v) growth curves predicting adult body sizes of ~1500 kg or less, implying that these animals are young adults, not juveniles of Tyrannosaurus; 4. Juveniles of other tyrannosaurids, including Gorgosaurus and Tarbosaurus, do not show the kinds of changes proposed for a Nanotyrannus-Tyrannosaurus growth series, suggesting the Nanotyrannus morphology cannot simply be explained as the result of immaturity; 5. Small T. rex exist, comparable in size to Nanotyrannus, which exhibit diagnostic features of Tyrannosaurus, and which differ from Nanotyrannus; 6. Phylogenetic analysis suggests that Nanotyrannus is not a tyrannosaurid. Taken together, the totality of the evidence rejects referral of Nanotyrannus to T. rex. Phylogenetic analysis tentatively supports placement of Nanotyrannus outside of Tyrannosauridae as a nontyrannosaurid member of Tyrannosauroidea. Tyrannosaur diversity appears to have been higher than previously appreciated in the latest Cretaceous before the K-Pg extinction. The difficulties in recognizing species based on fossils alone mean that paleontologists may be systematically biased towards underestimating the species diversity of ancient ecosystems.

The Society of Vertebrate Paleontology has urged scientific journals to reject studies that use data from privately owned fossil collections. Here, I argue that the Society’s perspective on reproducibility in science is overly simplistic. Their suggested publication policy, at best, slows the progress of science and, at worst, permits scientific misconduct through a form of data falsification and provides a potential mechanism to bully and censor researchers. The best way to ensure the long-term survival of fossil data is to collect and publish the data while the specimens are available.

Rates of peptide bond hydrolysis and other diagenetic reactions are not favourable for Mesozoic protein survival. Proteins hydrolyse into peptide fragments and free amino acids that, in open systems such as bone, can leach from the specimen and be further degraded. However, closed systems are more likely to retain degradation products derived from endogenous proteins. Amino acid racemisation data in experimental and subfossil material suggests that mollusc shell and avian eggshell calcite crystals can demonstrate closed system behaviour, retaining endogenous amino acids. Here, high-performance liquid chromatography reveals that the intra-crystalline fraction of Late Cretaceous (estimated ~80 Ma) titanosaur sauropod eggshell is enriched in some of the most stable amino acids (Glx, Gly, Ala, and possibly Val) and those that racemise are fully racemic, despite being some of the slowest racemising amino acids. These results are consistent with degradation trends deduced from modern, thermally matured, sub-fossil, and ~3.8 Ma avian eggshell, as well as ~30 Ma calcitic mollusc opercula. Selective preservation of certain fully racemic amino acids, which do not racemise in-chain, along with similar concentrations of free versus total hydrolysable amino acids, likely suggests complete hydrolysis of original peptides. Liquid chromatography-tandem mass spectrometry supports this hypothesis by failing to detect any non-contamination peptide sequences from the Mesozoic eggshell. Pyrolysis-gas chromatography-mass spectrometry reveals pyrolysates consistent with amino acids as well as aliphatic hydrocarbon homologues that are not present in modern eggshell, suggestive of kerogen formation deriving from eggshell lipids. Raman spectroscopy yields bands consistent with various organic molecules, possibly including N-bearing molecules or geopolymers. These closed-system amino acids are possibly the most thoroughly supported non-avian dinosaur endogenous protein-derived constituents, at least those that have not undergone oxidative condensation with other classes of biomolecules. Biocrystal matrices can help preserve mobile organic molecules by trapping them (perhaps with the assistance of resistant organic polymers), but trapped organics are nevertheless prone to diagenetic degradation even if such reactions might be slowed in exceptional circumstances. The evidence for complete hydrolysis and degradation of most amino acids in the eggshell raises concern about the validity of reported polypeptide sequences from open-system non-avian dinosaur bone and other Mesozoic fossils.