The role of heterotopy and heterochrony during morphological diversification of otocephalan epibranchial organs.

Epibranchial organs (EBOs), found in at least five of the eight otomorphan families, are used to aggregate small prey inside the buccopharyngeal cavity and range in morphological complexity from a singular, small slit on the pharyngeal roof to several, elongated soft tissue tubes. Despite broad phylogenetic representation, little is known about the origin, development, or evolution of EBOs. We hypothesize that both heterochronic and heterotopic changes throughout the evolution of EBOs are at the root of their morphological diversity. Heterochrony is a foundational explanation in developmental studies, however, heterotopy, a developmental change in spatial or topographical relationships, can have even more profound effects on a given structure but has received relatively little attention. Here, we investigate how developmental mechanisms may drive morphological diversity of EBOs within otomorphan fishes. We compare early pharyngeal development in three species, Anchoa mitchilli (Engraulidae) which has the most basic EBO, B. tyrannus (Clupeidae) which has a more complex EBO, and Hypophthalmichthys molitrix (Cyprinidae) which has the most complex EBO yet described. Using branchial arch growth rates and morphological analyses, we illustrate how both heterochronic and heterotopic mechanisms are responsible for some of the phenotypic diversity seen in otomorphan EBOs. Importantly, we also identify conserved developmental patterns that further our understanding of how EBOs may have first originated and evolved across actinopterygian fishes.

Sticky, stickier and stickiest - a comparison of adhesive performance in clingfish, lumpsuckers and snailfish.

The coastal waters of the North Pacific are home to the northern clingfish (Gobiesox maeandricus), Pacific spiny lumpsucker (Eumicrotremus orbis) and marbled snailfish (Liparis dennyi) - three fishes that have evolved ventral adhesive discs. Clingfish adhesive performance has been studied extensively, but relatively little is known about the performance of other sticky fishes. Here, we compared the peak adhesive forces and work to detachment of clingfish, lumpsuckers and snailfish on surfaces of varying roughness and over ontogeny. We also investigated the morphology of their adhesive discs through micro-computed tomography scanning and scanning electron microscopy. We found evidence that adhesive performance is tied to the intensity and variability of flow regimes in the fishes' habitats. The northern clingfish generates the highest adhesive forces and lives in the rocky intertidal zone where it must resist exposure to crashing waves. Lumpsuckers and snailfish both generate only a fraction of the clingfish's adhesive force, but live more subtidal where currents are slower and less variable. However, lumpsuckers generate more adhesive force relative to their body weight than snailfish, which we attribute to their higher-drag body shape and frequent bouts into the intertidal zone. Even so, the performance and morphology data suggest that snailfish adhesive discs are stiffer and built more efficiently than lumpsucker discs. Future studies should focus on sampling additional diversity and designing more ecologically relevant experiments when investigating differences in adhesive performance.

Not your father's homodonty-stress, tooth shape, and the functional homodont.

Teeth tell the tale of interactions between predator and prey. If a dental battery is made up of teeth that look similar, they are morphologically homodont, but if there is an unspecified amount of regional specialization in size or shape, they are morphologically heterodont. These are vague terms with no useful functional implication because morphological homodonty does not necessarily equal functional homodonty. Teeth that look the same may not function the same. Conical teeth are prevalent in fishes, superficially tasked with the simple job of puncture. There is a great deal of variation in the shape and placement of conical teeth. Anterior teeth may be larger than posterior ones, larger teeth may be surrounded by small ones, and patches of teeth may all have the same size and shape. Such variations suggest that conical dentitions might represent a single morphological solution for different functional problems. We are interested in the concept of homodonty and using the conical tooth as a model to differentiate between tooth shape and performance. We consider the stress that a tooth can exert on prey as stress is what causes damage. To create a statistical measure of functional homodonty, stress was calculated from measurements of surface area, position, and applied force. Functional homodonty is then defined as the degree to which teeth along the jaw all bear/exert similar stresses despite changes in shape. We find that morphologically heterodont teeth are often functionally homodont and that position is a better predictor of performance than shape. Furthermore, the arrangement of teeth affects their function, such that there is a functional advantage to having several smaller teeth surrounding a singular large tooth. We demonstrate that this arrangement of teeth is useful to grab, rather than tear, prey upon puncture, with the smaller teeth dissipating large stress forces around the larger tooth. We show that measurements of how shape affects stress distribution in response to loading give us a clearer picture of the evolution of conically shaped teeth.

Sucker with a fat lip: The soft tissues underlying the viscoelastic grip of remora adhesion.

Remoras are fishes that attach to a broad range of hosts using an adhesive disc on their head that is derived from dorsal fin elements. Research on the adhesive mechanism of remoras has focused primarily on the skeletal components of the disc and their contribution to generating suction and friction. However, the soft tissues of the disc, such as the soft lip surrounding the bony disc and the muscles that control the bony lamellae, have been largely ignored. To understand the sealing mechanism of the disc, it is imperative to understand the tissue morphology and material properties of the soft lip. Here, we show that the soft lip surrounding the remora disc is comprised of discrete multilayered collagen, fat, and elastic tissues which we hypothesize to have specific roles in the viscoelastic sealing mechanism of the remora disc. The central, heavily vascularized fat and collagen layer are infiltrated by strands of elastic tissue and surrounded by crossed-fiber collagen. A newly described jubilee muscle underneath the adhesive disc provides a mechanism for stopping venous return from the disc lip, thereby allowing it to become engorged and create a pressurized fit to the attachment substrate. Thus, the remora lip acts as a vascular hydrostat.

Developmental ecomorphology of the epibranchial organ of the silver carp, Hypophthalmichthys molitrix.

Silver carp regularly consume and digest particles of food as small as 5 µm. This ability drives their efficient consumption of phytoplankton and because they feed low on the food chain they have an important place in aquaculture worldwide. In North America, where they are considered invasive, silver carp deplete food resources for native species and in so doing occupy increased niche space. Here, we determine the ontogenetic stage and size at which silver carp are morphologically capable of primarily feeding on particles <10 µm. Ecological studies on this species have shown that there is an ontogenetic shift in diet as predominantly zooplanktivorous juveniles later switch to eating much smaller phytoplankton. The occupation of this new trophic niche presents both a metabolic and a mechanical challenge to these fish, since it is unclear how they can efficiently feed on such small particles. We hypothesize that the epibranchial organ (EBO) in silver carp is essential in aggregating these small particles of food, allowing the species to consume mass quantities of tiny particles, thus mitigating metabolic constraints. In this study, we investigate early ontogeny of the EBO in silver carp to determine when this structure achieves the requisite morphology to become functional. We find that at around 80 mm standard length (SL) the EBOs are consistently filled with food, demonstrating that this accumulating organ has become functional. This size corresponds with previous ecological data documenting important shifts in the type of food consumed. While the basic bauplan of the EBO is established very early in ontogeny (by 15 mm SL), multiple waves of histological maturation of muscle, cartilage, gill rakers and epithelium ultimately form the functional structure.

Tooth and consequences: Heterodonty and dental replacement in piranhas and pacus (Serrasalmidae).

Tooth replacement in piranhas is unusual: all teeth on one side of the head are lost as a unit, then replaced simultaneously. We used histology and microCT to examine tooth-replacement modes across carnivorous piranhas and their herbivorous pacu cousins (Serrasalmidae) and then mapped replacement patterns onto a molecular phylogeny. Pacu teeth develop and are replaced in a manner like piranhas. For serrasalmids, unilateral tooth replacement is not an "all or nothing" phenomenon; we demonstrate that both sides of the jaws have developing tooth rows within them, albeit with one side more mineralized than the other. All serrasalmids (except one) share unilateral tooth replacement, so this is not an adaptation for carnivory. All serrasalmids have interlocking teeth; piranhas interdigitate lateral tooth cusps with adjacent teeth, forming a singular saw-like blade, whereas lateral cusps in pacus clasp together. For serrasalmids to have an interlocking dentition, their teeth need to develop and erupt at the same time. We propose that interlocking mechanisms prevent tooth loss and ensure continued functionality of the feeding apparatus. Serrasalmid dentitions are ubiquitously heterodont, having incisiform and molariform dentitions reminiscent of mammals. Finally, we propose that simultaneous tooth replacement be considered as a synapomorphy for the family.

Channeling vorticity: modeling the filter-feeding mechanism in silver carp using µCT and 3D PIV.

Invasive silver carp are thriving within eutrophic environments in the United States, in part because of their highly efficient filter-feeding mechanism. Silver carp utilize modified gill rakers to capture a specific range of food; however, their greatly modified filtering morphology allows them to feed on phytoplankton and zooplankton ranging in size from 4 to 85 µm. The filtering apparatus of silver carp comprises rigid filtering plates where the outer anatomy of these plates is characterized by long parallel channels that change in orientation along the length of the plate. Here, we investigate the underlying morphology and concomitant hydrodynamics that support the filtration mechanisms of silver and bighead carp. Bighead carp are also invasive filter feeders, but their filtering apparatus is morphologically distinct from that of silver carp. Using 3D particle image velocimetry, we determined how particles and fluid interact with the surface of the gill rakers/plates. Filtering plates in silver carp induce strong directed vortical flow, whereas the filtering apparatus of bighead carp resulted in a type of haphazard cross-flow filtration. The organized vortical flow established by silver carp likely increased the number of interactions that the particle-filled water had with the filtering membrane. This strong vortical organization is maintained only at 0.75 body lengths s-1, and vortical flow is poorly developed and maintained at slower and faster speeds. Moreover, we found that absolute vorticity magnitude in silver carp is an order of magnitude greater than in bighead carp.

Lip service: Histological phenotypes correlate with diet and feeding ecology in herbivorous pacus.

Complex prey processing requires the repositioning of food between the teeth, as modulated by a soft tissue appendage like a tongue or lips. In this study, we trace the evolution of lips and ligaments, which are used during prey capture and prey processing in an herbivorous group of fishes. Pacus (Serrasalmidae) are Neotropical freshwater fishes that feed on leaves, fruits, and seeds. These prey are hard or tough, require high forces to fracture, contain abrasive or caustic elements, or deform considerably before failure. Pacus are gape-limited and do not have the pharyngeal jaws many bony fishes use to dismantle and/or transport prey. Despite their gape limitation, pacus feed on prey larger than their mouths, relying on robust teeth and a hypertrophied lower lip for manipulation and breakdown of food. We used histology to compare the lip morphology across 14 species of pacus and piranhas to better understand this soft tissue. We found that frugivorous pacus have larger, more complex lips which are innervated and folded at their surface, while grazing species have callused, mucus-covered lips. Unlike mammalian lips or tongues, pacu lips lack any intrinsic skeletal or smooth muscle. This implies that pacu lips lack dexterity; however, we found a novel connection to the primordial ligament which suggests that the lips are actuated by the jaw adductors. We propose that pacus combine hydraulic repositioning of prey inside the buccal cavity with direct oral manipulation, the latter using a combination of a morphologically heterodont dentition and compliant lips for reorienting food.

Formation of a fringe: A look inside baleen morphology using a multimodal visual approach.

Filter-feeding has been present for hundreds of millions of years, independently evolving in aquatic vertebrates' numerous times. Mysticete whales are a group of gigantic, marine filter-feeders that are defined by their fringed baleen and are divided into two groups: balaenids and rorquals. Recent studies have shown that balaenids likely feed using a self-cleaning, cross-flow filtration mechanism where food particles are collected and then swept to the esophagus for swallowing. However, it is unclear how filtering is achieved in the rorquals (Balaenopteridae). Lunging rorqual whales engulf enormous masses of both prey and water; the prey is then separated from the water through baleen plates lining the length of their upper jaw and positioned perpendicular to flow. Rorqual baleen is composed of both major (larger) and minor (smaller) keratin plates containing embedded fringe that extends into the whale's mouth, forming a filtering fringe. We used a multimodal approach, including microcomputed tomography (µCT) and scanning electron microscopy (SEM), to visualize and describe the variability in baleen anatomy across five species of rorqual whales, spanning two orders of magnitude in body length. For most morphological measurements, larger whales exhibited hypoallometry relative to body length. µCT and SEM revealed that the major and minor plates break away from the mineralized fringes at variable distances from the gums. We proposed a model for estimating the effective pore size to determine whether flow scales with body length or prey size across species. We found that pore size is likely not a proxy for prey size but instead, may reflect changes in resistance through the filter that affect fluid flow.

It Pays to Be Bumpy: Drag Reducing Armor in the Pacific Spiny Lumpsucker, Eumicrotremus orbis.

Armor is a multipurpose set of structures that has evolved independently at least 30 times in fishes. In addition to providing protection, armor can manipulate flow, increase camouflage, and be sexually dimorphic. There are potential tradeoffs in armor function: increased impact resistance may come at the cost of maneuvering ability; and ornate armor may offer visual or protective advantages, but could incur excess drag. Pacific spiny lumpsuckers (Eumicrotremus orbis) are covered in rows of odontic, cone-shaped armor whorls, protecting the fish from wave driven impacts and the threat of predation. We are interested in measuring the effects of lumpsucker armor on the hydrodynamic forces on the fish. Bigger lumpsuckers have larger and more complex armor, which may incur a greater hydrodynamic cost. In addition to their protective armor, lumpsuckers have evolved a ventral adhesive disc, allowing them to remain stationary in their environment. We hypothesize a tradeoff between the armor and adhesion: little fish prioritize suction, while big fish prioritize protection. Using micro-CT, we compared armor volume to disc area over lumpsucker development and built 3D models to measure changes in drag over ontogeny. We found that drag and drag coefficients decrease with greater armor coverage and vary consistently with orientation. Adhesive disc area is isometric but safety factor increases with size, allowing larger fish to remain attached in higher flows than smaller fish.

Internal vertebral morphology of bony fishes matches the mechanical demands of different environments.

Fishes have repeatedly evolved characteristic body shapes depending on how close they live to the substrate. Pelagic fishes live in open water and typically have narrow, streamlined body shapes; benthic and demersal fishes live close to the substrate; and demersal fishes often have deeper bodies. These shape differences are often associated with behavioral differences: pelagic fishes swim nearly constantly, demersal fishes tend to maneuver near the substrate, and benthic fishes often lie in wait on the substrate. We hypothesized that these morphological and behavioral differences would be reflected in the mechanical properties of the body, and specifically in vertebral column stiffness, because it is an attachment point for the locomotor musculature and a central axis for body bending. The vertebrae of bony fishes are composed of two cones connected by a foramen, which is filled by the notochord. Since the notochord is more flexible than bony vertebral centra, we predicted that pelagic fishes would have narrower foramina or shallower cones, leading to less notochordal material and a stiffer vertebral column which might support continuous swimming. In contrast, we predicted that benthic and demersal fishes would have more notochordal material, making the vertebral column more flexible for diverse behaviors in these species. We therefore examined vertebral morphology in 79 species using micro-computed tomography scans. Six vertebral features were measured including notochordal foramen diameter, centrum body length, and the cone angles and diameters for the anterior and posterior vertebral cones, along with body fineness. Using phylogenetic generalized least squares analyses, we found that benthic and pelagic species differed significantly, with larger foramina, shorter centra, and larger cones in benthic species. Thus, morphological differences in the internal shape of the vertebrae of fishes are consistent with a stiffer vertebral column in pelagic fishes and with a more flexible vertebral column in benthic species.

Pacific Spiny Lumpsucker armor-Development, damage, and defense in the intertidal.

Predation, combat, and the slings and arrows of an abrasive and high impact environment, represent just some of the biotic and abiotic stressors that fishes are armored against. The Pacific Spiny Lumpsucker (Eumicrotremus orbis) found in the subtidal of the Northern Pacific Ocean is a rotund fish covered with epidermal, cone-shaped, enamel odontodes. The Lumpsucker is a poor swimmer in the wave swept rocky intertidal, and this armor may be a lightweight solution to the problem of collisions with abiotic obstacles. We use micro-CT and scanning electron microscopy to reveal the morphology and ontogeny of the armor, and to quantify the amount of mineralization relative to the endoskeleton. The non-overlapping odontodes are organized into eight rows-six rows on the body, one row surrounding the eye, and one row underneath the chin. Odontodes start as a single, hooked cone; and they grow by the addition of cusps that accrete into a spiral. The mineral investment in armor compared to skeleton increases over ontogeny. Damage to the armor occurs both through passive abrasion and breakage from impact; and there is no evidence of replacement, or repair of damaged odontodes.

The Evolutionary Continuum of Functional Homodonty to Heterodonty in the Dentition of Halichoeres Wrasses.

Vertebrate dentitions are often collapsed into a few discrete categories, obscuring both potentially important functional differences between them and insight into their evolution. The terms homodonty and heterodonty typically conflate tooth morphology with tooth function, and require context-dependent subcategories to take on any specific meaning. Qualifiers like incipient, transient, or phylogenetic homodonty attempt to provide a more rigorous definition but instead highlight the difficulties in categorizing dentitions. To address these issues, we recently proposed a method for quantifying the function of dental batteries based on the estimated stress of each tooth (inferred using surface area) standardized for jaw out-lever (inferred using tooth position). This method reveals a homodonty-heterodonty functional continuum where small and large teeth work together to transmit forces to a prey item. Morphological homodonty or heterodonty refers to morphology, whereas functional homodonty or heterodonty refers to transmission of stress. In this study, we use Halichoeres wrasses to explore how functional continuum can be used in phylogenetic analyses by generating two continuous metrics from the functional homodonty-heterodonty continuum. Here we show that functionally heterodont teeth have evolved at least three times in Halichoeres wrasses. There are more functionally heterodont teeth on upper jaws than on lower jaws, but functionally heterodont teeth on the lower jaws bear significantly more stress. These nuances, which have functional consequences, would be missed by binning entire dentitions into discrete categories. This analysis points out areas worth taking a closer look at from a mechanical and developmental point of view with respect to the distribution and type of heterodonty seen in different jaws and different areas of jaws. These data, on a small group of wrasses, suggest continuous dental variables can be a rich source of insight into the evolution of fish feeding mechanisms across a wider variety of species.

Knowing when to stick: touch receptors found in the remora adhesive disc.

Remoras are fishes that piggyback onto larger marine fauna via an adhesive disc to increase locomotor efficiency, likelihood of finding mates and access to prey. Attaching rapidly to a large, fast-moving host is no easy task, and while research to date has focused on how the disc supports adhesion, no attention has been paid to how or if remoras are able to sense attachment. We identified push-rod-like mechanoreceptor complexes embedded in the soft lip of the remora adhesive disc that are known in other organisms to respond to touch and shear forces. This is, to our knowledge, the first time such mechanoreceptor complexes are described in fishes as they were only known previously in monotremes. The presence of push-rod-like mechanoreceptor complexes suggests not only that fishes may be able to sense their environment in ways not heretofore described but that specialized tactile mechanoreceptor complexes may be a more basal vertebrate feature than previously thought.

Grand Challenges in Comparative Tooth Biology.

Teeth are a model system for integrating developmental genomics, functional morphology, and evolution. We are at the cusp of being able to address many open issues in comparative tooth biology and we outline several of these newly tractable and exciting research directions. Like never before, technological advances and methodological approaches are allowing us to investigate the developmental machinery of vertebrates and discover both conserved and excitingly novel mechanisms of diversification. Additionally, studies of the great diversity of soft tissues, replacement teeth, and non-trophic functions of teeth are providing new insights into dental diversity. Finally, we highlight several emerging model groups of organisms that are at the forefront of increasing our appreciation of the mechanisms underlying tooth diversification.