Integrating biomechanics in evolutionary studies, with examples from the amphidromous goby model system.

The functional capacities of animals are a primary factor determining survival in nature. In this context, understanding the biomechanical performance of animals can provide insight into diverse aspects of their biology, ranging from ecological distributions across habitat gradients to the evolutionary diversification of lineages. To survive and reproduce in the face of environmental pressures, animals must perform a wide range of tasks, some of which entail tradeoffs between competing demands. Moreover, the demands encountered by animals can change through ontogeny as they grow, sexually mature or migrate across environmental gradients. To understand how mechanisms that underlie functional performance contribute to survival and diversification across challenging and variable habitats, we have pursued diverse studies of the comparative biomechanics of amphidromous goby fishes across functional requirements ranging from prey capture and fast-start swimming to adhesion and waterfall climbing. The pan-tropical distribution of these fishes has provided opportunities for repeated testing of evolutionary hypotheses. By synthesizing data from the lab and field, across approaches spanning high-speed kinematics, selection trials, suction pressure recordings, mechanical property testing, muscle fiber-type measurements and physical modeling of bioinspired designs, we have clarified how multiple axes of variation in biomechanical performance associate with the ecological and evolutionary diversity of these fishes. Our studies of how these fishes meet both common and extreme functional demands add new, complementary perspectives to frameworks developed from other systems, and illustrate how integrating knowledge of the mechanical underpinnings of diverse aspects of performance can give critical insights into ecological and evolutionary questions.

Locomotor Challenges of Waterfall-Climbing Gobies during Transitions between Media.

An amphidromous goby, Sicyopterus japonicus, migrates from the ocean to upstream regions of many streams and rivers in the Pacific coasts of Japan and Taiwan. Using its mouth and fused pelvic fins (pelvic sucker), this gobiid species exhibits a rock-climbing behavior and surmounts sizable waterfalls, which block the upstream movement of many of its competitors and predators. When gobies emerge from the water to commence their climbing behavior, the change in effective density (i.e., lack of buoyancy) that occurs in this transition substantially increases the force required for adhesion. Consequently, these fish must exert adhesive suction strong enough to support their body weight against gravity during climbing on the rock surface. Suction performance for adhesion and modulatory capacity of S. japonicus were evaluated with two different sets of experimental conditions: climbing on the vertical surface with no water flow versus climbing on a 60°-inclined surface with 2 L/min flow. Individuals of S. japonicus showed 50.7% greater mean safety factor (suction force for adhesion/gravitational force) and 56.6% shorter time to reach maximum pressure differential during climbing on the 60°-inclined surface with water rushing over their bodies than during climbing on the vertical surface with no water flow. These results indicate that when climbing with drag force from flowing water, greater functional demands are imposed and therefore, S. japonicus is required to increase neuromuscular stimulation of the pelvic muscles to elevate suction performance. In addition, S. japonicus individuals at different ontogenetic stages modulate their climbing behaviors and strategies to accommodate changing functional demands as they make transitions between different inclines, as well as media, while ascending waterfalls.

Adhesive force and endurance of the pelvic sucker across different modes of waterfall-climbing in gobiid fishes: Contrasting climbing mechanisms share aspects of ontogenetic change.

Waterfall-climbing gobiids from oceanic islands use a suction-based adhesive mechanism formed by fused pelvic fins (pelvic sucker) and exhibit rock-climbing behavior during upstream migration. Although adhesion is a common feature of locomotion in these fishes, two distinct climbing styles - powerburst climbing and inching - have evolved. We compared the performance of the pelvic sucker during climbing across a range of body sizes between two species that use these different styles, collecting new data from the powerburst climber Lentipes concolor, and comparing these to published data for the inching climber Sicyopterus japonicus. Suction force for adhesion generated during continuous climbing did not differ between the species, with similar mean safety factors of 2.5-3.0. However, L. concolor engaged its pelvic sucker for a significantly longer duration of time (approximately 34 % longer per climbing cycle) than S. japonicus during continuous climbing. During sustained adhesion, both species exhibited non-linear scaling of fatigue time, with intermediate-sized individuals (e.g., large juveniles to small adults) showing the greatest endurance. However, the two species exhibited strikingly different maxima and variability in the endurance of their pelvic suckers. Maximum time to fatigue in L. concolor was less than half that of S. japonicus, but L. concolor showed more than double the variability of S. japonicus in time to fatigue. Our comparisons of these species reveal that despite differences in several aspects of their adhesive performance, some features of sucker function remain similar across climbing styles, including several related to how performance changes through ontogeny.

Adhesive force and endurance during waterfall climbing in an amphidromous gobiid, Sicyopterus japonicus (Teleostei: Gobiidae): Ontogenetic scaling of novel locomotor performance.

An amphidromous sicydiine goby, Sicyopterus japonicus, exhibits rock-climbing behavior during upstream migration along rivers and streams. Using a pelvic sucker formed by fused pelvic fins, S. japonicus generates suction adhesion on the climbing surface. By measuring performance variables that correlate with successful rock-climbing capability, we evaluated scaling relationships of adhesive suction force generated by the pelvic sucker and fatigue during climbing in S. japonicus during ontogeny. In continuous climbing on the experimental 60°-inclined surface, the pelvic sucker of S. japonicus exhibited strong positive allometry in generating suction force for adhesion during ontogeny. In contrast, fatigue time of the pelvic sucker muscles for sustained adhesion scaled non-linearly with body mass during ontogeny. In addition, fatigue time and body mass showed the best fit to a quadratic regression, which predicted intermediate-sized individuals (large juveniles to small adults) to have better performance in adhesive endurance than smaller or larger individuals. Our experimental results indicate that different sizes of waterfall-climbing gobies have different performance capacities for rock climbing perhaps because of physiological differences in their pelvic muscles. In addition, our data from S. japonicus indicates that selection pressures on the locomotor capacities of waterfall-climbing gobiids vary during ontogeny.

Comparative lever analysis and ontogenetic scaling in esocid fishes: Functional demands and constraints in feeding biomechanics.

When animals grow, the functional demands that they experience often change as a consequence of their increasing body size. In this study, we examined the feeding biomechanics in esocid species that represent different size classes (small, Esox americanus; intermediate, Esox niger; large, Esox lucius), and how their bite forces and associated functional variables change as they grow. In order to evaluate bite performance through ontogeny, we dissected and measured dimensions of the feeding apparatus and the adductor mandibulae muscle complex with its segmentum facialis subdivisions such as the ricto-malaris, stegalis and endoricto-malaris across a wide range of body sizes. The collected morphological data was used as input variables for a published anatomical model to simulate jaw function in these fish species. Maximum bite forces for both anterior bite and posterior bite increased in isometry in E. americanus and E. niger. The posterior bite of E. lucius also increases in isometry, however, the anterior bite increases in positive allometry. Intraspecific comparison within E. lucius indicated the increase of bite forces in more developed individuals accelerated after the fish grew out of fingerling stage. In addition, our analysis indicated functional differentiation between subdivisions of the adductor mandibulae segmentum facialis, as well as interspecific differences in the pattern of contribution to the bite performance by these subdivisions. Our study provides insights into not only the musculoskeletal basis of the jaw function of esocid species, but also the feeding capacity of this species in relation to the functional demands it faces as one of the top predators in lake and river systems. J. Morphol. 277:1447-1458, 2016. © 2016 Wiley Periodicals, Inc.

Musculoskeletal determinants of pelvic sucker function in Hawaiian stream gobiid fishes: interspecific comparisons and allometric scaling.

Gobiid fishes possess a distinctive ventral sucker, formed from fusion of the pelvic fins. This sucker is used to adhere to a wide range of substrates including, in some species, the vertical cliffs of waterfalls that are climbed during upstream migrations. Previous studies of waterfall-climbing goby species have found that pressure differentials and adhesive forces generated by the sucker increase with positive allometry as fish grow in size, despite isometry or negative allometry of sucker area. To produce such scaling patterns for pressure differential and adhesive force, waterfall-climbing gobies might exhibit allometry for other muscular or skeletal components of the pelvic sucker that contribute to its adhesive function. In this study, we used anatomical dissections and modeling to evaluate the potential for allometric growth in the cross-sectional area, effective mechanical advantage (EMA), and force generating capacity of major protractor and retractor muscles of the pelvic sucker (m. protractor ischii and m. retractor ischii) that help to expand the sealed volume of the sucker to produce pressure differentials and adhesive force. We compared patterns for three Hawaiian gobiid species: a nonclimber (Stenogobius hawaiiensis), an ontogenetically limited climber (Awaous guamensis), and a proficient climber (Sicyopterus stimpsoni). Scaling patterns were relatively similar for all three species, typically exhibiting isometric or negatively allometric scaling for the muscles and lever systems examined. Although these scaling patterns do not help to explain the positive allometry of pressure differentials and adhesive force as climbing gobies grow, the best climber among the species we compared, S. stimpsoni, does exhibit the highest calculated estimates of EMA, muscular input force, and output force for pelvic sucker retraction at any body size, potentially facilitating its adhesive ability.

Evolutionary novelty versus exaptation: oral kinematics in feeding versus climbing in the waterfall-climbing Hawaiian Goby Sicyopterus stimpsoni.

Species exposed to extreme environments often exhibit distinctive traits that help meet the demands of such habitats. Such traits could evolve independently, but under intense selective pressures of extreme environments some existing structures or behaviors might be coopted to meet specialized demands, evolving via the process of exaptation. We evaluated the potential for exaptation to have operated in the evolution of novel behaviors of the waterfall-climbing gobiid fish genus Sicyopterus. These fish use an "inching" behavior to climb waterfalls, in which an oral sucker is cyclically protruded and attached to the climbing surface. They also exhibit a distinctive feeding behavior, in which the premaxilla is cyclically protruded to scrape diatoms from the substrate. Given the similarity of these patterns, we hypothesized that one might have been coopted from the other. To evaluate this, we filmed climbing and feeding in Sicyopterus stimpsoni from Hawai'i, and measured oral kinematics for two comparisons. First, we compared feeding kinematics of S. stimpsoni with those for two suction feeding gobiids (Awaous guamensis and Lentipes concolor), assessing what novel jaw movements were required for algal grazing. Second, we quantified the similarity of oral kinematics between feeding and climbing in S. stimpsoni, evaluating the potential for either to represent an exaptation from the other. Premaxillary movements showed the greatest differences between scraping and suction feeding taxa. Between feeding and climbing, overall profiles of oral kinematics matched closely for most variables in S. stimpsoni, with only a few showing significant differences in maximum values. Although current data cannot resolve whether oral movements for climbing were coopted from feeding, or feeding movements coopted from climbing, similarities between feeding and climbing kinematics in S. stimpsoni are consistent with evidence of exaptation, with modifications, between these behaviors. Such comparisons can provide insight into the evolutionary mechanisms facilitating exploitation of extreme habitats.

Stairway to heaven: evaluating levels of biological organization correlated with the successful ascent of natural waterfalls in the Hawaiian stream goby Sicyopterus stimpsoni.

Selective pressures generated by locomotor challenges act at the level of the individual. However, phenotypic variation among individuals that might convey a selective advantage may occur across any of multiple levels of biological organization. In this study, we test for differences in external morphology, muscle mechanical advantage, muscle fiber type and protein expression among individuals of the waterfall climbing Hawaiian fish Sicyopterus stimpsoni collected from sequential pools increasing in elevation within a single freshwater stream. Despite predictions from previous laboratory studies of morphological selection, few directional morphometric changes in body shape were observed at successively higher elevations. Similarly, lever arm ratios associated with the main pelvic sucker, central to climbing ability in this species, did not differ between elevations. However, among climbing muscles, the adductor pelvicus complex (largely responsible for generating pelvic suction during climbing) contained a significantly greater red muscle fiber content at upstream sites. A proteomic analysis of the adductor pelvicus revealed two-fold increases in expression levels for two respiratory chain proteins (NADH:ubiquinone reductase and cytochrome b) that are essential for aerobic respiration among individuals from successively higher elevations. Assessed collectively, these evaluations reveal phenotypic differences at some, but not all levels of biological organization that are likely the result of selective pressures experienced during climbing.

Performance and scaling of a novel locomotor structure: adhesive capacity of climbing gobiid fishes.

Many species of gobiid fishes adhere to surfaces using a sucker formed from fusion of the pelvic fins. Juveniles of many amphidromous species use this pelvic sucker to scale waterfalls during migrations to upstream habitats after an oceanic larval phase. However, adults may still use suckers to re-scale waterfalls if displaced. If attachment force is proportional to sucker area and if growth of the sucker is isometric, then increases in the forces that climbing fish must resist might outpace adhesive capacity, causing climbing performance to decline through ontogeny. To test for such trends, we measured pressure differentials and adhesive suction forces generated by the pelvic sucker across wide size ranges in six goby species, including climbing and non-climbing taxa. Suction was achieved via two distinct growth strategies: (1) small suckers with isometric (or negatively allometric) scaling among climbing gobies and (2) large suckers with positively allometric growth in non-climbing gobies. Species using the first strategy show a high baseline of adhesive capacity that may aid climbing performance throughout ontogeny, with pressure differentials and suction forces much greater than expected if adhesion were a passive function of sucker area. In contrast, large suckers possessed by non-climbing species may help compensate for reduced pressure differentials, thereby producing suction sufficient to support body weight. Climbing Sicyopterus species also use oral suckers during climbing waterfalls, and these exhibited scaling patterns similar to those for pelvic suckers. However, oral suction force was considerably lower than that for pelvic suckers, reducing the ability for these fish to attach to substrates by the oral sucker alone.

Jaw muscle fiber type distribution in Hawaiian gobioid stream fishes: histochemical correlations with feeding ecology and behavior.

Differences in fiber type distribution in the axial muscles of Hawaiian gobioid stream fishes have previously been linked to differences in locomotor performance, behavior, and diet across species. Using ATPase assays, we examined fiber types of the jaw opening sternohyoideus muscle across five species, as well as fiber types of three jaw closing muscles (adductor mandibulae A1, A2, and A3). The jaw muscles of some species of Hawaiian stream gobies contained substantial red fiber components. Some jaw muscles always had greater proportions of white muscle fibers than other jaw muscles, independent of species. In addition, comparing across species, the dietary generalists (Awaous guamensis and Stenogobius hawaiiensis) had a lower proportion of white muscle fibers in all jaw muscles than the dietary specialists (Lentipes concolor, Sicyopterus stimpsoni, and Eleotris sandwicensis). Among Hawaiian stream gobies, generalist diets may favor a wider range of muscle performance, provided by a mix of white and red muscle fibers, than is typical of dietary specialists, which may have a higher proportion of fast-twitch white fibers in jaw muscles to help meet the demands of rapid predatory strikes or feeding in fast-flowing habitats.

Femoral loading mechanics in the Virginia opossum, Didelphis virginiana: torsion and mediolateral bending in mammalian locomotion.

Studies of limb bone loading in terrestrial mammals have typically found anteroposterior bending to be the primary loading regime, with torsion contributing minimally. However, previous studies have focused on large, cursorial eutherian species in which the limbs are held essentially upright. Recent in vivo strain data from the Virginia opossum (Didelphis virginiana), a marsupial that uses a crouched rather than an upright limb posture, have indicated that its femur experiences appreciable torsion during locomotion as well as strong mediolateral bending. The elevated femoral torsion and strong mediolateral bending observed in D. virginiana might result from external forces such as a medial inclination of the ground reaction force (GRF), internal forces deriving from a crouched limb posture, or a combination of these factors. To evaluate the mechanism underlying the loading regime of opossum femora, we filmed D. virginiana running over a force platform, allowing us to measure the magnitude of the GRF and its three-dimensional orientation relative to the limb, facilitating estimates of limb bone stresses. This three-dimensional analysis also allows evaluations of muscular forces, particularly those of hip adductor muscles, in the appropriate anatomical plane to a greater degree than previous two-dimensional analyses. At peak GRF and stress magnitudes, the GRF is oriented nearly vertically, inducing a strong abductor moment at the hip that is countered by adductor muscles on the medial aspect of the femur that place this surface in compression and induce mediolateral bending, corroborating and explaining loading patterns that were identified in strain analyses. The crouched orientation of the femur during stance in opossums also contributes to levels of femoral torsion as high as those seen in many reptilian taxa. Femoral safety factors were as high as those of non-avian reptiles and greater than those of upright, cursorial mammals, primarily because the load magnitudes experienced by opossums are lower than those of most mammals. Thus, the evolutionary transition from crouched to upright posture in mammalian ancestors may have been accompanied by an increase in limb bone load magnitudes.

Morphological selection and the evaluation of potential tradeoffs between escape from predators and the climbing of waterfalls in the Hawaiian stream goby Sicyopterus stimpsoni.

Environmental pressures may vary over the geographic range of a species, exposing subpopulations to divergent functional demands. How does exposure to competing demands shape the morphology of species and influence the divergence of populations? We explored these questions by performing selection experiments on juveniles of the Hawaiian goby Sicyopterus stimpsoni, an amphidromous fish that exhibits morphological differences across portions of its geographic range where different environmental pressures predominate. Juvenile S. stimpsoni face two primary and potentially opposing selective pressures on body shape as they return from the ocean to freshwater streams on islands: (1) avoiding predators in the lower reaches of a stream; and (2) climbing waterfalls to reach the habitats occupied by adults. These pressures differ in importance across the Hawaiian Islands. On the youngest island, Hawai'i, waterfalls are close to shore, thereby minimizing exposure to predators and placing a premium on climbing performance. In contrast, on the oldest major island, Kaua'i, waterfalls have eroded further inland, lengthening the exposure of juveniles to predators before migrating juveniles begin climbing. Both juvenile and adult fish show differences in body shape between these islands that would be predicted to improve evasion of predators by fish from Kaua'i (e.g., taller bodies that improve thrust) and climbing performance for fish from Hawai'i (e.g., narrower bodies that reduce drag), matching the prevailing environmental demand on each island. To evaluate how competing selection pressures and functional tradeoffs contribute to the divergence in body shape observed in S. stimpsoni, we compared selection imposed on juvenile body shape by (1) predation by the native fish Eleotris sandwicensis versus (2) climbing an artificial waterfall (∼100 body lengths). Some variables showed opposing patterns of selection that matched predictions: for example, survivors of predation had lower fineness ratios than did control fish (i.e., greater body depth for a given length), whereas successful climbers had higher fineness ratios (reducing drag) than did fish that failed. However, most morphological variables showed significant selection in only one treatment rather than opposing selection across both. Thus, functional tradeoffs between evasion of predators and minimizing drag during climbing might influence divergence in body shape across subpopulations, but even when selection is an important contributing mechanism, directly opposite patterns of selection across environmental demands are not required to generate morphological divergence.

Jaw lever analysis of Hawaiian gobioid stream fishes: a simulation study of morphological diversity and functional performance.

Differences in feeding behavior and performance among the five native Hawaiian gobioid stream fishes (Sicyopterus stimpsoni, Lentipes concolor, Awaous guamensis, Stenogobius hawaiiensis, and Eleotris sandwicensis) have been proposed based on the skeletal anatomy of their jaws and dietary specialization. However, performance of the feeding apparatus likely depends on the proportions and configurations of the jaw muscles and the arrangement of the jaw skeleton. We used a published mathematical model of muscle function to evaluate potential differences in jaw closing performance and their correlations with morphology among these species. For example, high output force calculated for the adductor mandibulae muscles (A2 and A3) of both A. guamensis and E. sandwicensis matched expectations based on the morphology of these species because these muscles are larger than in the other species. In contrast, Stenogobius hawaiiensis exhibited an alternative morphological strategy for achieving high relative output forces of both A2 and A3, in which the placement and configuration of the muscles conveyed high mechanical advantage despite only moderate cross-sectional areas. These differing anatomical pathways to similar functional performance suggest a pattern of many-to-one mapping of morphology to performance. In addition, a functional differentiation between A2 and A3 was evident for all species, in which A2 was better suited for producing forceful jaw closing and A3 for rapid jaw closing. Thus, the diversity of feeding performance of Hawaiian stream gobies seems to reflect a maintenance of functional breadth through the retention of some primitive traits in combination with novel functional capacities in several species.

Feeding kinematics and performance of Hawaiian stream gobies, Awaous guamensis and Lentipes concolor: linkage of functional morphology and ecology.

Distributions of Hawaiian stream fishes are typically interrupted by waterfalls that divide streams into multiple segments. Larvae hatch upstream, are flushed into the ocean, and must climb these waterfalls to reach adult habitats when returning back to freshwater as part of an amphidromous life cycle. Stream surveys and studies of climbing performance show that Lentipes concolor Gill can reach fast-flowing upper stream segments but that Awaous guamensis Valenciennes reaches only slower, lower stream segments. Gut content analyses for these two species indicate considerable overlap in diet, suggesting that feeding kinematics and performance of these two species might be comparable. Alternatively, feeding kinematics and performance of these species might be expected to differ in relation to the different flow regimes in their habitat (feeding in faster stream currents for L. concolor versus in slower currents for A. guamensis). To test these alternative hypotheses, we compared food capturing kinematics and performance during suction feeding behaviors of A. guamensis and L. concolor using morphological data and high-speed video. Lentipes concolor showed both a significantly larger gape angle and faster jaw opening than A. guamensis. Geometric models calculated that despite the inverse relationship of gape size and suction pressure generation, the fast jaw motions of L. concolor allow it to achieve higher pressure differentials than A. guamensis. Such elevated suction pressure would enhance the ability of L. concolor to successfully capture food in the fast stream reaches it typically inhabits. Differences in jaw morphology may contribute to these differences in performance, as the lever ratio for jaw opening is about 10% lower in L. concolor compared with A. guamensis, suiting the jaws of L. concolor better for fast opening.

Morphological selection in an extreme flow environment: body shape and waterfall-climbing success in the Hawaiian stream fish Sicyopterus stimpsoni.

Flow characteristics are a prominent factor determining body shapes in aquatic organisms, and correlations between body shape and ambient flow regimes have been established for many fish species. In this study, we investigated the potential for a brief period of extreme flow to exert selection on the body shape of juvenile climbing Hawaiian gobiid fishes. Because of an amphidromous life history, juvenile gobies that complete an oceanic larval phase return to freshwater habitats, where they become adults. Returning juveniles often must scale waterfalls (typically with the use of a ventral sucker) in order to reach the habitats they will use as adults, thereby exposing these animals to brief periods of extreme velocities of flow. Hydrodynamic theory predicts that bodies with larger suckers and with lower heights that reduce drag would have improved climbing success and, thus, be well suited to meet the demands of the flows in waterfalls. To test the potential for the flow environment of waterfalls to impose selection that could contribute to differences in body shape between islands, we subjected juvenile Sicyopterus stimpsoni to climbing trials up artificial waterfalls (∼100 body lengths) and measured differences in body shape between successful and unsuccessful climbers. Waterfalls appear to represent a significant selective barrier to these fishes, as nearly 30% failed our climbing test. However, the effects of selection on morphology were not straightforward, as significant differences in shape between successful and unsuccessful climbers did not always match hydrodynamic predictions. In both selection experiments and in adult fish collected from habitats with different prevailing conditions of flow (the islands of Hawai'i versus Kaua'i), lower head heights were associated with exposure to high-flow regimes, as predicted by hydrodynamic theory. Thus, a premium appears to be placed on the reduction of drag via head morphology throughout the ontogeny of this species. The congruence of phenotypic selection patterns observed in our experiments, with morphological character divergence documented among adult fish from Hawai'i and Kaua'i, suggests that differences in morphology between subpopulations of adult climbing gobies may result, at least in part, from the selective pressures of high-velocity flows encountered by migrating juveniles.