Integration drives rapid phenotypic evolution in flatfishes.

Evolutionary innovations are scattered throughout the tree of life, and have allowed the organisms that possess them to occupy novel adaptive zones. While the impacts of these innovations are well documented, much less is known about how these innovations arise in the first place. Patterns of covariation among traits across macroevolutionary time can offer insights into the generation of innovation. However, to date, there is no consensus on the role that trait covariation plays in this process. The evolution of cranial asymmetry in flatfishes (Pleuronectiformes) from within Carangaria was a rapid evolutionary innovation that preceded the colonization of benthic aquatic habitats by this clade, and resulted in one of the most bizarre body plans observed among extant vertebrates. Here, we use three-dimensional geometric morphometrics and a phylogenetic comparative toolkit to reconstruct the evolution of skull shape in carangarians, and quantify patterns of integration and modularity across the skull. We find that the evolution of asymmetry in flatfishes was a rapid process, resulting in the colonization of novel trait space, that was aided by strong integration that coordinated shape changes across the skull. Our findings suggest that integration plays a major role in the evolution of innovation by synchronizing responses to selective pressures across the organism.

Enormous gill chambers of deep-sea coffinfishes (Lophiiformes: Chaunacidae) support unique ventilatory specialisations such as breath holding and extreme inflation.

Deep sea habitats tend to favor species with low energetic demands, and therefore we predict that deep sea fishes will have behavioral and morphological specializations of the gill ventilatory system to reduce the energetic cost of pumping water across the gills. However, it is difficult to study functional morphology of deep sea fishes due the lack of ability to conduct laboratory experiments with living fishes. For this study, we combined analysis of publicly available video recorded by remote-operated vehicles (ROV) with detailed anatomical study of museum specimens to document the functional morphology of the massive gill chambers that are observed in coffinfishes (Lophiiformes: Chaunacidae). Chaunacids, like other lophiiforms, exhibit highly specialised ventilatory anatomy such as an enlarged branchiostegal apparatus and restricted gill openings, but videos show them using this anatomy in a new and unusual way. We observed eight individuals ventilating extremely slowly at rates of 0.03-0.004 Hz, during which the gill chambers were full yet we saw no inhalation or exhalation for periods of 26 to 245 s. This holding breath behaviour has not been observed in any other fishes and is probably highly energetically efficient. This inflation of the gill chambers also increases body volume by up to 30%, making them more globose and difficult to be taken as prey, much like stomach inflation in pufferfishes (Tetraodontidae). We also used micro computed-tomography (CT) scans to document the enormous branchiostegal rays and associated muscles that support this unique behaviour.

Effects of organism and substrate size on burial mechanics of English sole, Parophrys vetulus.

Flatfishes use cyclic body undulations to force water into the sediment and fluidize substrate particles, displacing them into the water column. When water velocity decreases, suspended particles settle back onto the fish, hiding it from view. Burial may become more challenging as flatfishes grow because the area to be covered increases exponentially with the second power of length. In addition, particle size is not uniform in naturally occurring substrates, and larger particles require higher water velocities for fluidization. We quantified the effects of organism and particle-size scaling on burial behavior of English sole, Parophrys vetulus We recorded burial events from a size range of individuals (5-32 cm total length, TL), while maintaining constant substrate grain size. Larger fish used lower cycle frequencies and took longer to bury, but overall burial performance was maintained (∼100% coverage). To test the effect of particle size on burial performance, individuals of similar lengths (5.7-8.1 cm TL) were presented with different substrate sizes (0.125-0.710 mm). Particle size did not affect cycle frequency or time to burial, but fish did not achieve 100% coverage with the largest particles because they could not fluidize this substrate. Taken together, these results suggest that both body size and substrate grain size can potentially limit the ability of flatfishes to bury: a very large fish (>150 cm) may move too slowly to fluidize all but the smallest substrate particles and some particles are simply too large for smaller individuals to fluidize.

Aquatic burst locomotion by hydroplaning and paddling in common eiders (Somateria mollissima).

Common eiders (Somateria mollissima) are heavy sea-ducks that spend a large portion of their time swimming at the water surface. Surface swimming generates a bow and hull wave that can constructively interfere and produce wave drag. The speed at which the wavelengths of these waves equal the waterline length of the swimming animal is the hull speed. To increase surface swimming speed beyond the hull speed, an animal must overtake the bow wave. This study found two distinct behaviors that eider ducks used to exceed the hull speed: (1) 'steaming', which involved rapid oaring with the wings to propel the duck along the surface of the water, and (2) 'paddle-assisted flying', during which the ducks lifted their bodies out of the water and used their feet to paddle against the surface while flapping their wings in the air. An average hull speed (0.732±0.046 m s(-1)) was calculated for S. mollissima by measuring maximum waterline length from museum specimens. On average, steaming ducks swam 5.5 times faster and paddle-assisted flying ducks moved 6.8 times faster than the hull speed. During steaming, ducks exceeded the hull speed by increasing their body angle and generating dynamic lift to overcome wave drag and hydroplane along the water surface. During paddle-assisted flying, ducks kept their bodies out of the water, thereby avoiding the limitations of wave drag altogether. Both behaviors provided alternatives to flight for these ducks by allowing them to exceed the hull speed while staying at or near the water surface.

Effect of parabranchial position on ventilatory pressures in the Pacific spiny dogfish (Squalus suckleyi).

The mechanics of ventilation in elasmobranchs have been described as a two-pump system which is dependent on the generation of differential pressures between the orobranchial and parabranchial cavities. However, this general model does not take into account sources of variation in parabranchial form and function. For example, the relative pressures that drive flow in each parabranchial chamber during ventilation remain largely unexplored. To address this gap, parabranchial pressures were collected from the Pacific spiny dogfish (Squalus suckleyi, n = 12) during routine ventilation using transducers inserted into parabranchial chambers 2, 3, and 5, numbered anteriorly to posteriorly. Pressure amplitudes collected from the three chambers displayed an attenuation of pressure amplitudes posteriorly, as well as differential, modular use of parabranchial chamber five These observations have implications for the functioning of the ventilatory pump and indicate distinct ventilatory modes, leading us to propose a new model to describe ventilation in Squalus suckleyi.

"How Do We Do This at a Distance?!" A Descriptive Study of Remote Undergraduate Research Programs during COVID-19.

The COVID-19 pandemic shut down undergraduate research programs across the United States. A group of 23 colleges, universities, and research institutes hosted remote undergraduate research programs in the life sciences during Summer 2020. Given the unprecedented offering of remote programs, we carried out a study to describe and evaluate them. Using structured templates, we documented how programs were designed and implemented, including who participated. Through focus groups and surveys, we identified programmatic strengths and shortcomings as well as recommendations for improvements from students' perspectives. Strengths included the quality of mentorship, opportunities for learning and professional development, and a feeling of connection with a larger community. Weaknesses included limited cohort building, challenges with insufficient structure, and issues with technology. Although all programs had one or more activities related to diversity, equity, inclusion, and justice, these topics were largely absent from student reports even though programs coincided with a peak in national consciousness about racial inequities and structural racism. Our results provide evidence for designing remote Research Experiences for Undergraduates (REUs) that are experienced favorably by students. Our results also indicate that remote REUs are sufficiently positive to further investigate their affordances and constraints, including the potential to scale up offerings, with minimal concern about disenfranchising students.

Linking ecomechanical models and functional traits to understand phenotypic diversity.

Physical principles and laws determine the set of possible organismal phenotypes. Constraints arising from development, the environment, and evolutionary history then yield workable, integrated phenotypes. We propose a theoretical and practical framework that considers the role of changing environments. This 'ecomechanical approach' integrates functional organismal traits with the ecological variables. This approach informs our ability to predict species shifts in survival and distribution and provides critical insights into phenotypic diversity. We outline how to use the ecomechanical paradigm using drag-induced bending in trees as an example. Our approach can be incorporated into existing research and help build interdisciplinary bridges. Finally, we identify key factors needed for mass data collection, analysis, and the dissemination of models relevant to this framework.

Pharyngeal Jaws Converge by Similar Means, Not to Similar Ends, When Minnows (Cypriniformes: Leuciscidae) Adapt to New Dietary Niches.

Convergent evolution is at the forefront of many form-function studies. There are many examples of multiple independent lineages evolving a similar morphology in response to similar functional demands, providing a framework for testing hypotheses of form-function evolution. However, there are numerous clades with underappreciated convergence, in which there is a perceived homogeneity in morphology. In these groups, it can be difficult to investigate causal relationships of form and function (e.g., diet influencing the evolution of jaw morphology) without the ability to disentangle phylogenetic signal from convergence. Leuciscids (Cypriniformes: Leuciscidae; formerly nested within Cyprinidae) are a species-rich clade of fishes that have diversified to occupy nearly every freshwater trophic niche, yet are considered to have relatively low morphological diversity relative to other large freshwater clades. Within the North American leuciscids, many genera contain at least one herbivore, insectivore, and larvaphage. We created 3D models from micro-computed tomography scans of 165 leuciscid species to measure functionally relevant traits within the pharyngeal jaws of these fishes. Using a published phylogeny, we tested these metrics for evolutionary integration, phylogenetic signal, and correlation with diet. Measurements of the pharyngeal jaws, muscle attachment areas, and teeth showed strong positive evolutionary correlation with each other and negative evolutionary correlation with measurements of the inter-ceratobranchial ligament (ICB ligament). Using diet data from published literature, we found extensive dietary convergence within Leuciscidae. The most common transitions we found were between herbivorous and invertivorous taxa and between insectivore types (aquatic vs. terrestrial). We document a trade-off in which herbivorous leuciscids have large teeth, short ICB ligaments, and large muscle attachment areas, whereas insectivorous leuciscids showed the opposite pattern. Inverse patterns of morphological integration between the ICB ligament the rest of the pharyngeal jaw correspond this dietary trade-off, which indicates that coordinated evolution of morphological traits contributes to functional diversity in this clade. However, these patterns only emerge in the context of phylogeny, meaning that the pharyngeal jaws of North American leuciscids converge by similar means (structural changes in response to dietary demands), but not necessarily to similar ends (absolute phenotype).

A walking behavior generates functional overland movements in the tidepool sculpin, Oligocottus maculosus.

Tidepool sculpins (Oligocottus maculosus) have been observed moving overland in the rocky intertidal, and we documented the terrestrial walking behavior that they use to accomplish this. We quantified the terrestrial movements of O. maculosus and compared them to (1) their aquatic locomotion, (2) terrestrial locomotion of closely-related subtidal species (Leptocottus armatus and Icelinus borealis), and (3) terrestrial movements of walking catfishes (Clarias spp.). We recorded sculpin movements (210 fps) on a terrestrial platform and in a water tank and tracked body landmarks for kinematic analysis. The axial-appendage-based terrestrial locomotion of O. maculosus is driven by cyclic lateral oscillations of the tail, synchronized with alternating rotations about the base of the pectoral fins, a behavior that appears similar to a military "army crawl." The pectoral fins do not provide propulsion, but act as stable points for the body to rotate around. In contrast, individuals of O. maculosus use primarily axial undulation during slow-speed swimming. The army crawl is a more effective terrestrial behavior (greater distance ratio) than the movements produced by L. armatus and I. borealis, which use rapid, cyclic oscillations of the tail, without coordinated pectoral fin movements. Relative to Clarias spp., O. maculosus rotated the body about the base of the pectoral fin, rather than the tip of the fin, which may cause O. maculosus to have a lower distance ratio. Since O. maculosus lack major morphological adaptations for terrestrial locomotion, instead relying on behavioral adaptations, we propose behavioral adaptations may evolutionarily predate morphological adaptations for terrestrial locomotion in vertebrates.

Evolution of skeletal and muscular morphology within the functionally integrated lower jaw adduction system of sculpins and relatives (Cottoidei).

Vertebrate lever mechanics are defined by the morphology of skeletal elements and the properties of their muscular actuators; these metrics characterize functional diversity. The components of lever systems work in coordination ("functional integration") and may show strong covariation across evolutionary history ("evolutionary integration"), both of which have been hypothesized to constrain phenotypic diversity. We quantified evolutionary integration in a functionally integrated system - the lower jaw of sculpins and relatives (Actinopterygii: Cottoidei). Sculpins primarily rely on suction feeding for prey capture, but there is considerable variation in evasiveness of their prey, resulting in variation in anatomy of the lower jaw-closing mechanism. We used functionally-relevant linear measurements to characterize skeletal and muscular components of this system among 25 cottoid species and two outgroup Hexagrammoidei (greenling) species. We quantified evolutionary covariation and correlation of jaw-closing mechanical advantage (i.e., skeletal leverage) and muscle architecture (i.e., gearing) by correlating phylogenetically independent contrasts and fitting phylogenetically corrected generalized least squares models. We found no evidence of evolutionary covariation in muscle architecture and skeletal leverage. While we found a positive evolutionary correlation between out-lever length and adductor muscle fiber length, there was no significant evolutionary correlation between in-lever length and adductor muscle fiber length. We also found a positive evolutionary correlation between in- and out-lever lengths. These results suggest that skeletal morphology and muscle morphology contribute independently to biomechanical diversity among closely related species, indicating the importance of considering both skeletal and muscular variation in studies of ecomorphological diversification.

Functional morphology of gill ventilation of the goosefish, Lophius americanus (Lophiiformes: Lophiidae).

The goosefish, Lophius americanus, is a dorso-ventrally compressed marine fish that spends most of its life sitting on the substrate waiting to ambush prey. Species in the genus Lophius have some of the slowest ventilatory cycles recorded in fishes, with a typical cycle lasting more than 90s. They have a large gill chamber, supported by long branchiostegal rays and ending in a siphon-like gill opening positioned underneath and behind the base of the pectoral fin. Our goals were to characterize the kinematics of gill ventilation in L. americanus relative to those of more typical ray-finned fishes, address previous assertions about ventilation in this genus, and describe the anatomy of the gill opening. We found that phase 1 of ventilation (during which both the buccal and gill chamber are expanding) is greatly increased in duration relative to that of typical ray-finned fishes (ranging from 62 to 127s), and during this phase, the branchiostegal rays are slowly expanding. This slow expansion is almost visually imperceptible, especially from a dorsal view. Despite this unusually long phase 1, the pattern of skeletal movements follows that of a typical actinopterygian, refuting previous assertions that Lophius does not use its jaws, suspensorium, and operculum during ventilation. When individuals were disturbed from the sediment, they tended to breathe more rapidly by decreasing the duration of phase 1 (to 18-30s). Dissections of the gill opening revealed a previously undocumented dorsal extension of the adductor hyohyoideus muscle, which passes from between the branchiostegal rays, through the ventro-medial wall of the gill opening, and to the dorsal midline of the body. This morphology of the adductor hyohyoideus shares similarities with that of many Tetraodontiformes, and we suggest that it may be a synapomorphy for Lophiiformes+Tetraodontiformes. The specialized anatomy and function of the gill chamber of Lophius represents extreme modifications that provide insight into the potential limits of the actinopterygian gill ventilatory system.

Look before you leap: Visual navigation and terrestrial locomotion of the intertidal killifish Fundulus heteroclitus.

Mummichogs (Fundulus heteroclitus; Cyprinodontiformes) are intertidal killifish that can breathe air and locomote on land. Our goals were to characterize the terrestrial locomotion of mummichogs and determine their method of navigation towards water in a terrestrial environment. We used high-speed video to record behavior during stranding experiments and found that mummichogs use a tail-flip jump to move overland, similarly to other Cyprinodontiformes. However, mummichogs also prop themselves upright into a prone position between each jump, a previously undescribed behavior. After becoming prone, mummichogs rotate about their vertical axis, directing the caudal fin towards the water. Then, they roll back onto their lateral aspect and use a tail-flip behavior to leap into a caudally-directed, ballistic flight path. We conducted experiments to determine the sensory stimulus used to locate a body of water by placing mummichogs on a square platform with one side adjacent to a sea table. Under artificial light, mummichogs moved towards the sea table with a higher frequency than towards the other sides. Under dark conditions, mummichogs did not show a preference for moving towards the sea table. When the surface of the water was covered with reflective foil, mummichogs moved towards it as if it were a body of water. These results suggest that mummichogs primarily use visual cues, specifically reflected light, to orient towards the water. The uprighting behavior that mummichogs perform between terrestrial jumps may provide an opportunity for these fish to receive visual information that allows them to safely return to the water. J. Exp. Zool. 325A:57-64, 2016. © 2015 Wiley Periodicals, Inc.

Modelling tooth-prey interactions in sharks: the importance of dynamic testing.

The shape of shark teeth varies among species, but traditional testing protocols have revealed no predictive relationship between shark tooth morphology and performance. We developed a dynamic testing device to quantify cutting performance of teeth. We mimicked head-shaking behaviour in feeding large sharks by attaching teeth to the blade of a reciprocating power saw fixed in a custom-built frame. We tested three tooth types at biologically relevant speeds and found differences in tooth cutting ability and wear. Teeth from the bluntnose sixgill (Hexanchus griseus) showed poor cutting ability compared with tiger (Galeocerdo cuvier), sandbar (Carcharhinus plumbeus) and silky (C. falciformis) sharks, but they also showed no wear with repeated use. Some shark teeth are very sharp at the expense of quickly dulling, while others are less sharp but dull more slowly. This demonstrates that dynamic testing is vital to understanding the performance of shark teeth.

Undulation frequency affects burial performance in living and model flatfishes.

Flatfishes bury themselves under a thin layer of sand to hide from predators or to ambush prey. We investigated the role of undulation frequency of the body in burial in five species of flatfishes (Isopsetta isolepis, Lepidopsetta bilineata, Hippoglossoides elassodon, Parophrys vetulus, and Psettichthys melanostictus). High-speed videos show that undulations begin cranially and pass caudally while burying, as in forward swimming in many other fishes. The flatfishes also flick the posterior edge of their dorsal and anal fins during burial, which may increase the total surface area covered by substrate. We built a simple physical model - a flexible, oval silicone plate with a motorized, variable-speed actuator - to isolate the effect of undulation frequency on burial. In both the model and actuated dead flatfish, increased undulation frequency resulted in an increase in the area of sand coverage. Complete coverage required an undulation frequency of no more than 10Hz for our models, and that was also sufficient for live flatfishes. The model shows that undulation is sufficient to bury the animal, but live flatfishes showed a superior ability to bury, which we attribute to the action of the median fins.

Evolution of the branchiostegal membrane and restricted gill openings in Actinopterygian fishes.

A phylogenetic survey is a powerful approach for investigating the evolutionary history of a morphological characteristic that has evolved numerous times without obvious functional implications. Restricted gill openings, an extreme modification of the branchiostegal membrane, are an example of such a characteristic. We examine the evolution of branchiostegal membrane morphology and highlight convergent evolution of restricted gill openings. We surveyed specimens from 433 families of actinopterygians for branchiostegal membrane morphology and measured head and body dimensions. We inferred a relaxed molecular clock phylogeny with branch length estimates based on nine nuclear genes sampled from 285 species that include all major lineages of Actinopterygii. We calculated marginal state reconstructions of four branchiostegal membrane conditions and found that restricted gill openings have evolved independently in at least 11 major actinopterygian clades, and the total number of independent origins of the trait is likely much higher. A principal component analysis revealed that fishes with restricted gill openings occupy a larger morphospace, as defined by our linear measurements, than do fishes with nonrestricted openings. We used a decision tree analysis of ecological data to determine if restricted gill openings are linked to certain environments. We found that fishes with restricted gill openings repeatedly occur under a variety of ecological conditions, although they are rare in open-ocean pelagic environments. We also tested seven ratios for their utility in distinguishing between fishes with and without restricted gill openings, and we propose a simple metric for quantifying restricted gill openings (RGO), defined as a ratio of the distance from the ventral midline to the gill opening relative to half the circumference of the head. Functional explanations for this specialized morphology likely differ within each clade, but its repeated evolution indicates a need for a better understanding of diversity of ventilatory morphology among fishes. J. Morphol. 276:681-694, 2015. © 2015 Wiley Periodicals, Inc.