DiceCT for fishes: recommendations for pairing iodine contrast agents with µCT to visualize soft tissues in fishes.

Computed tomography (CT) scanning and other high-throughput three-dimensional (3D) visualization tools are transforming the ways we study morphology, ecology and evolutionary biology research beyond generating vast digital repositories of anatomical data. Contrast-enhanced chemical staining methods, which render soft tissues radio-opaque when coupled with CT scanning, encompass several approaches that are growing in popularity and versatility. Of these, the various diceCT techniques that use an iodine-based solution like Lugol's have provided access to an array of morphological data sets spanning extant vertebrate lineages. This contribution outlines straightforward means for applying diceCT techniques to preserved museum specimens of cartilaginous and bony fishes, collectively representing half of vertebrate species diversity. This study contrasts the benefits of using either aqueous or ethylic Lugol's solutions and reports few differences between these methods with respect to the time required to achieve optimal tissue contrast. It also explores differences in minimum stain duration required for different body sizes and shapes and provides recommendations for staining specimens individually or in small batches. As reported by earlier studies, the authors note a decrease in pH during staining with either aqueous or ethylic Lugol's. Nonetheless, they could not replicate the drastic declines in pH reported elsewhere. They provide recommendations for researchers and collections staff on how to incorporate diceCT into existing curatorial practices, while offsetting risk to specimens. Finally, they outline how diceCT with Lugol's can aid ichthyologists of all kinds in visualizing anatomical structures of interest: from brains and gizzards to gas bladders and pharyngeal jaw muscles.

The oldest Devonian circumpolar ray-finned fish?

Actinopterygians (ray-finned fishes) are the most diverse group of living fishes, but have a sparse Devonian fossil record restricted to low palaeolatitudes. Here we report a new actinopterygian from the Paraná Basin of Brazil, which occupied a circumpolar position in the Palaeozoic. Available geological evidence supports a Middle Devonian or older age for this taxon, which shares features of the mandibular symphysis with the latest Devonian Tegeolepis. A phylogenetic analysis resolves these two as sister taxa. This new record expands the palaeogeographic distribution of Devonian ray-fins and suggests that gaps in their fossil record might be filled by exploring poorly sampled high-latitude localities within the Malvinokaffric Realm.

The origin and early phylogenetic history of jawed vertebrates.

Fossils of early gnathostomes (or jawed vertebrates) have been the focus of study for nearly two centuries. They yield key clues about the evolutionary assembly of the group's common body plan, as well the divergence of the two living gnathostome lineages: the cartilaginous and bony vertebrates. A series of remarkable new palaeontological discoveries, analytical advances and innovative reinterpretations of existing fossil archives have fundamentally altered a decades-old consensus on the relationships of extinct gnathostomes, delivering a new evolutionary framework for exploring major questions that remain unanswered, including the origin of jaws.

Patterns of mammalian jaw ecomorphological disparity during the Mesozoic/Cenozoic transition.

The radiation of mammals after the Cretaceous/Palaeogene (K/Pg) boundary was a major event in the evolution of terrestrial ecosystems. Multiple studies point to increases in maximum body size and body size disparity, but patterns of disparity for other traits are less clear owing to a focus on different indices and subclades. We conducted an inclusive comparison of jaw functional disparity from the Early Jurassic-latest Eocene, using six mechanically relevant mandibular ratios for 256 species representing all major groups. Jaw functional disparity across all mammals was low throughout much of the Mesozoic and remained low across the K/Pg boundary. Nevertheless, the K/Pg boundary was characterized by a pronounced pattern of turnover and replacement, entailing a substantial reduction of non-therian and stem-therian disparity, alongside a marked increase in that of therians. Total mammal disparity exceeded its Mesozoic maximum for the first time during the Eocene, when therian mammals began exploring previously unoccupied regions of function space. This delay in the rise of jaw functional disparity until the Eocene probably reflects the duration of evolutionary recovery after the K/Pg mass extinction event. This contrasts with the more rapid expansion of maximum body size, which occurred in the Palaeocene.

Little evidence for enhanced phenotypic evolution in early teleosts relative to their living fossil sister group.

Since Darwin, biologists have been struck by the extraordinary diversity of teleost fishes, particularly in contrast to their closest "living fossil" holostean relatives. Hypothesized drivers of teleost success include innovations in jaw mechanics, reproductive biology and, particularly at present, genomic architecture, yet all scenarios presuppose enhanced phenotypic diversification in teleosts. We test this key assumption by quantifying evolutionary rate and capacity for innovation in size and shape for the first 160 million y (Permian-Early Cretaceous) of evolution in neopterygian fishes (the more extensive clade containing teleosts and holosteans). We find that early teleosts do not show enhanced phenotypic evolution relative to holosteans. Instead, holostean rates and innovation often match or can even exceed those of stem-, crown-, and total-group teleosts, belying the living fossil reputation of their extant representatives. In addition, we find some evidence for heterogeneity within the teleost lineage. Although stem teleosts excel at discovering new body shapes, early crown-group taxa commonly display higher rates of shape evolution. However, the latter reflects low rates of shape evolution in stem teleosts relative to all other neopterygian taxa, rather than an exceptional feature of early crown teleosts. These results complement those emerging from studies of both extant teleosts as a whole and their sublineages, which generally fail to detect an association between genome duplication and significant shifts in rates of lineage diversification.

Two pulses of morphological diversification in Pacific pelagic fishes following the Cretaceous-Palaeogene mass extinction.

Molecular phylogenies suggest some major radiations of open-ocean fish clades occurred roughly coincident with the Cretaceous-Palaeogene (K/Pg) boundary, however the timing and nature of this diversification is poorly constrained. Here, we investigate evolutionary patterns in ray-finned fishes across the K/Pg mass extinction 66 million years ago (Ma), using microfossils (isolated teeth) preserved in a South Pacific sediment core spanning 72-43 Ma. Our record does not show significant turnover of fish tooth morphotypes at the K/Pg boundary: only two of 48 Cretaceous tooth morphotypes disappear at the event in the South Pacific, a rate no different from background extinction. Capture-mark-recapture analysis finds two pulses of origination in fish tooth morphotypes following the mass extinction. The first pulse, at approximately 64 Ma, included short-lived teeth, as well as forms that contribute to an expansion into novel morphospace. A second pulse, centred at approximately 58 Ma, produced morphotype novelty in a different region of morphospace from the first pulse, and contributed significantly to Eocene tooth morphospace occupation. There was no significant increase in origination rates or expansion into novel morphospace during the early or middle Eocene, despite a near 10-fold increase in tooth abundance during that interval. Our results suggest that while the K/Pg event had a minor impact on fish diversity in terms of extinction, the removal of the few dominant Cretaceous morphotypes triggered a sequence of origination events allowing fishes to rapidly diversify morphologically, setting the stage for exceptional levels of ray-finned fish diversity in the Cenozoic.

Initial radiation of jaws demonstrated stability despite faunal and environmental change.

More than 99 per cent of the roughly 58,000 living vertebrate species have jaws. This major clade, whose members are collectively known as gnathostomes ('jawed mouths'), made its earliest definitive appearance in the Silurian period, 444-416 million years (Myr) ago, with both the origin of the modern (crown-group) radiation and the presumptive invasion of land occurring by the end of the Devonian period (359 Myr ago). These events coincided with a major faunal shift that remains apparent today: the transition from Silurian ecosystems dominated by jawless fishes (agnathans) to younger assemblages composed almost exclusively of gnathostomes. This pattern has inspired several qualitative descriptions of the trophic radiation and ecological ascendance of the earliest jawed vertebrates. Here we present a quantitative analysis of functional variation in early gnathostome mandibular elements, placing constraints on our understanding of evolutionary patterns during this critical interval. We document an initial increase in functional disparity in the Silurian that stabilized by the first stage of the Devonian, before the occurrence of an Emsian (∼400 Myr ago) oxygenation event implicated in the trophic radiation of vertebrates. Subsequent taxonomic diversification during the Devonian did not result in increased functional variation; instead, new taxa revisited and elaborated on established mandibular designs. Devonian functional space is dominated by lobe-finned fishes and 'placoderms'; high disparity within the latter implies considerable trophic innovation among jaw-bearing stem gnathostomes. By contrast, the major groups of living vertebrates--ray-finned fishes and tetrapods--show surprisingly conservative mandibular morphologies with little indication of functional diversification or innovation. Devonian gnathostomes reached a point where they ceased to accrue further mandibular functional disparity before becoming taxonomic dominants relative to 'ostracoderm'-grade jawless fishes, providing a new perspective on classic adaptive hypotheses concerning this fundamental shift in vertebrate biodiversity.

An exceptionally preserved Late Devonian actinopterygian provides a new model for primitive cranial anatomy in ray-finned fishes.

Actinopterygians (ray-finned fishes) are the most diverse living osteichthyan (bony vertebrate) group, with a rich fossil record. However, details of their earliest history during the middle Palaeozoic (Devonian) 'Age of Fishes' remains sketchy. This stems from an uneven understanding of anatomy in early actinopterygians, with a few well-known species dominating perceptions of primitive conditions. Here we present an exceptionally preserved ray-finned fish from the Late Devonian (Middle Frasnian, ca 373 Ma) of Pas-de-Calais, northern France. This new genus is represented by a single, three-dimensionally preserved skull. CT scanning reveals the presence of an almost complete braincase along with near-fully articulated mandibular, hyoid and gill arches. The neurocranium differs from the coeval Mimipiscis in displaying a short aortic canal with a distinct posterior notch, long grooves for the lateral dorsal aortae, large vestibular fontanelles and a broad postorbital process. Identification of similar but previously unrecognized features in other Devonian actinopterygians suggests that aspects of braincase anatomy in Mimipiscis are apomorphic, questioning its ubiquity as stand-in for generalized actinopterygian conditions. However, the gill skeleton of the new form broadly corresponds to that of Mimipiscis, and adds to an emerging picture of primitive branchial architecture in crown gnathostomes. The new genus is recovered in a polytomy with Mimiidae and a subset of Devonian and stratigraphically younger actinopterygians, with no support found for a monophyletic grouping of Moythomasia with Mimiidae.

Evolutionary innovation accelerates morphological diversification in pufferfishes and their relatives.

Evolutionary innovations have played an important role in shaping the diversity of life on Earth. However, how these innovations arise, and their downstream effects on patterns of morphological diversification remain poorly understood. Here, we examine the impact of evolutionary innovation on trait diversification in tetraodontiform fishes (pufferfishes, boxfishes, ocean sunfishes, and allies). This order provides an ideal model system for studying morphological diversification owing to their range of habitats and divergent morphologies, including the fusion of the teeth into a beak in several families. Using three-dimensional geometric morphometric data for 176 extant and fossil species, we examine the effect of skull integration and novel habitat association on the evolution of innovation. Strong integration may be a requirement for rapid trait evolution and facilitating the evolution of innovative structures, like the tetraodontiform beak. Our results show that the beak arose in the presence of highly conserved patterns of integration across the skull, suggesting that integration did not limit the range of available phenotypes to tetraodontiforms. Furthermore, we find that beaks have allowed tetraodontiforms to diversify into novel ecological niches, irrespective of habitat. Our results suggest that general rules pertaining to evolutionary innovation may be more nuanced than previously thought.

Bony-fish-like scales in a Silurian maxillate placoderm.

Major groups of jawed vertebrates exhibit contrasting conditions of dermal plates and scales. But the transition between these conditions remains unclear due to rare information on taxa occupying key phylogenetic positions. The 425-million-year-old fish Entelognathus combines an unusual mosaic of characters typically associated with jawed stem gnathostomes or crown gnathostomes. However, only the anterior part of the exoskeleton was previously known for this very crownward member of the gnathostome stem. Here, we report a near-complete post-thoracic exoskeleton of Entelognathus. Strikingly, its scales are large and some are rhomboid, bearing distinctive peg-and-socket articulations; this combination was previously only known in osteichthyans and considered a synapomorphy of that group. The presence in Entelognathus of an anal fin spine, previously only found in some stem chondrichthyans, further illustrates that many characters previously thought to be restricted to specific lineages within the gnathostome crown likely arose before the common ancestor of living jawed vertebrates.

Parallel evolutionary trajectories underlie the origin of giant suspension-feeding whales and bony fishes.

Giant suspension feeders such as mysticete whales, basking and whale sharks, and the extinct (indicated by '†') †pachycormiform teleosts are conspicuous members of modern and fossil marine vertebrate faunas. Whether convergent anatomical features common to these clades arose along similar evolutionary pathways has remained unclear because of a lack of information surrounding the origins of all groups of large-bodied suspension feeders apart from baleen whales. New investigation reveals that the enigmatic ray-finned fish †Ohmdenia, from the Lower Jurassic (Toarcian, 183.0-175.6 Ma) Posidonia Shale Lagerstätte, represents the immediate sister group of edentulous †pachycormiforms, the longest lived radiation of large vertebrate suspension feeders. †Ohmdenia bisects the long morphological branch leading to suspension-feeding †pachycormiforms, providing information on the sequence of anatomical transformations preceding this major ecological shift that can be compared to changes associated with the origin of modern mysticetes. Similarities include initial modifications to jaw geometry associated with the reduction of dentition, followed by the loss of teeth. The evolution of largest body sizes within both radiations occurs only after the apparent onset of microphagy. Comparing the fit of contrasting evolutionary models to functionally relevant morphological measurements for whales and †pachycormiform fishes reveals strong support for a common adaptive peak shared by suspension-feeding members of both clades.

Ecomorphological selectivity among marine teleost fishes during the end-Cretaceous extinction.

Despite the attention focused on mass extinction events in the fossil record, patterns of extinction in the dominant group of marine vertebrates-fishes-remain largely unexplored. Here, I demonstrate ecomorphological selectivity among marine teleost fishes during the end-Cretaceous extinction, based on a genus-level dataset that accounts for lineages predicted on the basis of phylogeny but not yet sampled in the fossil record. Two ecologically relevant anatomical features are considered: body size and jaw-closing lever ratio. Extinction intensity is higher for taxa with large body sizes and jaws consistent with speed (rather than force) transmission; resampling tests indicate that victims represent a nonrandom subset of taxa present in the final stage of the Cretaceous. Logistic regressions of the raw data reveal that this nonrandom distribution stems primarily from the larger body sizes of victims relative to survivors. Jaw mechanics are also a significant factor for most dataset partitions but are always less important than body size. When data are corrected for phylogenetic nonindependence, jaw mechanics show a significant correlation with extinction risk, but body size does not. Many modern large-bodied, predatory taxa currently suffering from overexploitation, such billfishes and tunas, first occur in the Paleocene, when they appear to have filled the functional space vacated by some extinction victims.

A Permian fish reveals widespread distribution of neopterygian-like jaw suspension.

The actinopterygian crown group (comprising all living ray-finned fishes) originated by the end of the Carboniferous. However, most late Paleozoic taxa are stem actinopterygians, and broadly resemble stratigraphically older taxa. The early Permian †Brachydegma caelatum is notable for its three-dimensional preservation and past phylogenetic interpretations as a nested member of the neopterygian crown. Here, we use computed microtomography to redescribe †Brachydegma, uncovering an unanticipated combination of primitive (e.g., aortic canal; immobile maxilla) and derived (e.g., differentiated occipital ossifications; posterior stem of parasphenoid; two accessory hyoidean ossifications; double jaw joint) dermal and endoskeletal traits relative to most other Paleozoic actinopterygians. Some of these features were previously thought to be restricted to the neopterygian crown. The precise phylogenetic position of †Brachydegma is unclear, with placements either on the polypterid stem or as an early-diverging stem neopterygian. However, our analyses decisively reject previous placements of †Brachydegma in the neopterygian crown. Critically, we demonstrate that key endoskeletal components of the hyoid portion of the suspensorium of crown neopterygians appeared deeper in the tree than previously thought.

The rapid evolution of lungfish durophagy.

Innovations relating to the consumption of hard prey are implicated in ecological shifts in marine ecosystems as early as the mid-Paleozoic. Lungfishes represent the first and longest-ranging lineage of durophagous vertebrates, but how and when the various feeding specializations of this group arose remain unclear. Two exceptionally preserved fossils of the Early Devonian lobe-finned fish Youngolepis reveal the origin of the specialized lungfish feeding mechanism. Youngolepis has a radically restructured palate, reorienting jaw muscles for optimal force transition, coupled with radiating entopterygoid tooth rows like those of lungfish toothplates. This triturating surface occurs in conjunction with marginal dentition and blunt coronoid fangs, suggesting a role in crushing rather than piercing prey. Bayesian tip-dating analyses incorporating these morphological data indicate that the complete suite of lungfish feeding specializations may have arisen in as little as 7 million years, representing one of the most striking episodes of innovation during the initial evolutionary radiations of bony fishes.

Large-bodied sabre-toothed anchovies reveal unanticipated ecological diversity in early Palaeogene teleosts.

Many modern groups of marine fishes first appear in the fossil record during the early Palaeogene (66-40 Ma), including iconic predatory lineages of spiny-rayed fishes that appear to have originated in response to ecological roles left empty after the Cretaceous/Palaeogene extinction. The hypothesis of extinction-mediated ecological release likewise predicts that other fish groups have adopted novel predatory ecologies. Here, we report remarkable trophic innovation in early Palaeogene clupeiforms (herrings and allies), a group whose modern representatives are generally small-bodied planktivores. Two forms, the early Eocene (Ypresian) †Clupeopsis from Belgium and a new genus from the middle Eocene (Lutetian) of Pakistan, bear conspicuous features indicative of predatory ecology, including large size, long gapes and caniniform dentition. Most remarkable is the presence of a single, massive vomerine fang offset from the midline in both. Numerous features of the neurocranium, suspensorium and branchial skeleton place these taxa on the engraulid (anchovy) stem as the earliest known representatives of the clade. The identification of large-bodied, piscivorous anchovies contributes to an emerging picture of a phylogenetically diverse guild of predatory ray-finned fishes in early Palaeogene marine settings, which include completely extinct lineages alongside members of modern marine groups and taxa that are today restricted to freshwater or deep-sea environments.

The nearshore cradle of early vertebrate diversification.

Ancestral vertebrate habitats are subject to controversy and obscured by limited, often contradictory paleontological data. We assembled fossil vertebrate occurrence and habitat datasets spanning the middle Paleozoic (480 million to 360 million years ago) and found that early vertebrate clades, both jawed and jawless, originated in restricted, shallow intertidal-subtidal environments. Nearshore divergences gave rise to body plans with different dispersal abilities: Robust fishes shifted shoreward, whereas gracile groups moved seaward. Fresh waters were invaded repeatedly, but movement to deeper waters was contingent upon form and short-lived until the later Devonian. Our results contrast with the onshore-offshore trends, reef-centered diversification, and mid-shelf clustering observed for benthic invertebrates. Nearshore origins for vertebrates may be linked to the demands of their mobility and may have influenced the structure of their early fossil record and diversification.

A new stem sarcopterygian illuminates patterns of character evolution in early bony fishes.

Discoveries of putative stem sarcopterygians from the late Silurian and Early Devonian of South China have increased our knowledge of the initial diversification of osteichthyans while also highlighting incongruities in character evolution in this major jawed vertebrate group. Character-rich endocrania are incompletely preserved for early bony fishes, limiting a detailed understanding of complex internal morphology and evolutionary changes in the cranium. Here we report a new sarcopterygian (Ptyctolepis brachynotus gen. et sp. nov.) from the Pragian (Early Devonian) of South China, which preserves a unique example of a completely ossified otoccipital division of the braincase in a stem lobe-finned fish. The hyomandibular facets are paired but lie dorsal to the jugular canal, representing a hitherto unobserved combination of derived and primitive character states. This new taxon prompts a reassessment of early osteichthyan interrelationships, including the phylogenetic placement of psarolepids, which might branch from the osteichthyan-rather than sarcopterygian-stem.

A three-dimensional placoderm (stem-group gnathostome) pharyngeal skeleton and its implications for primitive gnathostome pharyngeal architecture.

The pharyngeal skeleton is a key vertebrate anatomical system in debates on the origin of jaws and gnathostome (jawed vertebrate) feeding. Furthermore, it offers considerable potential as a source of phylogenetic data. Well-preserved examples of pharyngeal skeletons from stem-group gnathostomes remain poorly known. Here, we describe an articulated, nearly complete pharyngeal skeleton in an Early Devonian placoderm fish, Paraplesiobatis heinrichsi Broili, from Hunsrück Slate of Germany. Using synchrotron light tomography, we resolve and reconstruct the three-dimensional gill arch architecture of Paraplesiobatis and compare it with other gnathostomes. The preserved pharyngeal skeleton comprises elements of the hyoid arch (probable ceratohyal) and a series of branchial arches. Limited resolution in the tomography scan causes some uncertainty in interpreting the exact number of arches preserved. However, at least four branchial arches are present. The final and penultimate arches are connected as in osteichthyans. A single median basihyal is present as in chondrichthyans. No dorsal (epibranchial or pharyngobranchial) elements are observed. The structure of the pharyngeal skeleton of Paraplesiobatis agrees well with Pseudopetalichthys from the same deposit, allowing an alternative interpretation of the latter taxon. The phylogenetic significance of Paraplesiobatis is considered. A median basihyal is likely an ancestral gnathostome character, probably with some connection to both the hyoid and the first branchial arch pair. Unpaired basibranchial bones may be independently derived in chondrichthyans and osteichthyans.