Hagfish from the Cretaceous Tethys Sea and a reconciliation of the morphological-molecular conflict in early vertebrate phylogeny.

Hagfish depart so much from other fishes anatomically that they were sometimes considered not fully vertebrate. They may represent: (i) an anatomically primitive outgroup of vertebrates (the morphology-based craniate hypothesis); or (ii) an anatomically degenerate vertebrate lineage sister to lampreys (the molecular-based cyclostome hypothesis). This systematic conundrum has become a prominent case of conflict between morphology- and molecular-based phylogenies. To date, the fossil record has offered few insights to this long-branch problem or the evolutionary history of hagfish in general, because unequivocal fossil members of the group are unknown. Here, we report an unequivocal fossil hagfish from the early Late Cretaceous of Lebanon. The soft tissue anatomy includes key attributes of living hagfish: cartilages of barbels, postcranial position of branchial apparatus, and chemical traces of slime glands. This indicates that the suite of characters unique to living hagfish appeared well before Cretaceous times. This new hagfish prompted a reevaluation of morphological characters for interrelationships among jawless vertebrates. By addressing nonindependence of characters, our phylogenetic analyses recovered hagfish and lampreys in a clade of cyclostomes (congruent with the cyclostome hypothesis) using only morphological data. This new phylogeny places the fossil taxon within the hagfish crown group, and resolved other putative fossil cyclostomes to the stem of either hagfish or lamprey crown groups. These results potentially resolve the morphological-molecular conflict at the base of the Vertebrata. Thus, assessment of character nonindependence may help reconcile morphological and molecular inferences for other major discords in animal phylogeny.

nkx3.2 mutant zebrafish accommodate jaw joint loss through a phenocopy of the head shapes of Paleozoic jawless fish.

The vertebrate jaw is a versatile feeding apparatus. To function, it requires a joint between the upper and lower jaws, so jaw joint defects are often highly disruptive and difficult to study. To describe the consequences of jaw joint dysfunction, we engineered two independent null alleles of a single jaw joint marker gene, nkx3.2, in zebrafish. These mutations caused zebrafish to become functionally jawless via fusion of the upper and lower jaw cartilages (ankylosis). Despite lacking jaw joints, nkx3.2 mutants survived to adulthood and accommodated this defect by: (a) having a remodeled skull with a fixed open gape, reduced snout and enlarged branchial region; and (b) performing ram feeding in the absence of jaw-generated suction. The late onset and broad extent of phenotypic changes in the mutants suggest that modifications to the skull are induced by functional agnathia, secondarily to nkx3.2 loss of function. Interestingly, nkx3.2 mutants superficially resemble ancient jawless vertebrates (anaspids and furcacaudiid thelodonts) in overall head shape. Because no homology exists in individual skull elements between these taxa, the adult nkx3.2 phenotype is not a reversal but rather a convergence due to similar functional requirements of feeding without moveable jaws. This remarkable analogy strongly suggests that jaw movements themselves dramatically influence the development of jawed vertebrate skulls. Thus, these mutants provide a unique model with which to: (a) investigate adaptive responses to perturbation in skeletal development; (b) re-evaluate evolutionarily inspired interpretations of phenocopies generated by gene knockdowns and knockouts; and (c) gain insight into feeding mechanics of the extinct agnathans.

Evolutionary transformation of mouthparts from particle-feeding to piercing carnivory in Viper copepods: Review and 3D analyses of a key innovation using advanced imaging techniques.

BACKGROUND: Novel feeding adaptations often facilitate adaptive radiation and diversification. But the evolutionary origins of such feeding adaptations can be puzzling if they require concordant change in multiple component parts. Pelagic, heterorhabdid copepods (Calanoida) exhibit diverse feeding behaviors that range from simple particle feeding to a highly specialized form of carnivory involving piercing mouthparts that likely inject venom. We review the evolutionary history of heterorhabdid copepods and add new high-resolution, 3D anatomical analyses of the muscular system, glands and gland openings associated with this remarkable evolutionary transformation. RESULTS: We examined four heterorhabdid copepods with different feeding modes: one primitive particle-feeder (Disseta palumbii), one derived and specialized carnivore (Heterorhabdus subspinifrons), and two intermediate taxa (Mesorhabdus gracilis and Heterostylites longicornis). We used two advanced, high-resolution microscopic techniques - serial block-face scanning electron microscopy and two-photon excitation microscopy - to visualize mouthpart form and internal anatomy at unprecedented nanometer resolution. Interactive 3D graphical visualizations allowed putative homologues of muscles and gland cells to be identified with confidence and traced across the evolutionary transformation from particle feeding to piercing carnivory. Notable changes included: a) addition of new gland cells, b) enlargement of some (venom producing?) glands, c) repositioning of gland openings associated with hollow piercing fangs on the mandibles, d) repurposing of some mandibular-muscle function to include gland-squeezing, and e) addition of new muscles that may aid venom injection exclusively in the most specialized piercing species. In addition, live video recording of all four species revealed mandibular blade movements coupled to cyclic contraction of some muscles connected to the esophagus. These behavioral and 3D morphological observations revealed a novel injection system in H. subspinifrons associated with piercing (envenomating?) carnivory. CONCLUSIONS: Collectively, these results suggest that subtle changes in mandibular tooth form, and muscle and gland form and location, facilitated the evolution of a novel, piercing mode of feeding that accelerated diversification of the genus Heterorhabdus. They also highlight the value of interactive 3D animations for understanding evolutionary transformations of complex, multicomponent morphological systems.

Connecting pattern to process: Growth of spiral shell sculpture in the gastropod Nucella ostrina (Muricidae: Ocenebrinae).

Shell morphology is a well-suited and underused system to examine the development of novel forms. The three-dimensional structure produced (the shell) is separate from the largely two-dimensional tissue that secretes it (the mantle), allowing us to disentangle the pattern from the process. Despite knowing a great deal about the mechanics of shell secretion (process), and the variety of shell shapes that exist (pattern), no effort has been made to understand how the mantle changes to produce different shell shapes. We investigated this question in the dimorphic snail Nucella ostrina, which exhibits both smooth and ribbed shells to determine how ribs are formed by the mantle. Rib thickenings are produced only in the outer calcitic shell layer and secreted by the distal Outer Mantle Epithelium (OME) with increased acid phosphatase activity. The evenly thick inner aragonitic layers are secreted by the proximal OME which expresses acid phosphatase. Here we show that locally thicker ribs in N. ostrina are produced by changing the dimensions of the distal OME: elongation in the direction of growth and increased cell height. This should increase the amount of shell material secreted, producing locally thicker shell (ribs). Preliminary evidence suggests this mechanism may be widespread in gastropods.

Mesoscale morphology at nanoscale resolution: serial block-face scanning electron microscopy reveals fine 3D detail of a novel silk spinneret system in a tube-building tanaid crustacean.

BACKGROUND: The study of morphology is experiencing a renaissance due to rapid improvements in technologies for 3D visualization of complex internal and external structures. But 3D visualization of the internal structure of mesoscale objects - those in the 10-1000 µm range - remains problematic. They are too small for microCT, many lack suitable specific fluorescent markers for confocal microscopy, or they require labor-intensive stacking and smoothing of individual TEM images. Here we illustrate the first comprehensive morphological description of a complete mesoscale biological system at nanoscopic resolution using ultra-modern technology for 3D visualization - serial block-face scanning electron microscopy (SBF-SEM). The SBF-SEM machine combines an in-chamber ultramicrotome, which creates a serial array of exposed surfaces, with an SEM that images each surface as it is exposed. The serial images are then stacked automatically by 3D reconstruction software. We used SBF-SEM to study the spinneret (thread-producing) system of a small, tube-dwelling crustacean that weaves tubes of silk. Thread-producing ability is critical for the survival of many small-bodied animals but the basic morphology of these systems remains mysterious due to the limits of traditional microscopy. RESULTS: SBF-SEM allowed us to describe - in full 3D - well-resolved components (glands, ducts, pores, and associated nerves and muscles) of the spinneret system in the thoracic legs and body segments of Sinelobus sp. (Crustacea, Peracarida, Tanaidacea), a tube-building tanaid only 2 mm in body length. The 3D reconstruction by SBF-SEM revealed at nanoscale resolution a unique structure to the gland and duct systems: In each of three thread-producing thoracic segments, two separate ducts, derived from two separate glands located in the body, run through the entire leg and merge at the leg tip just before the spinneret pore opening. We also resolved nerves connecting to individual setae, spines and pores on the walking legs, and individual muscles within each leg segment. CONCLUSIONS: Our results significantly expand our understanding of the diversity of spinneret systems in the Crustacea by providing the first well-resolved view of spinneret components in the peracarid crustacean order, Tanaidacea. More significantly, our results reveal the great power of SBF-SEM technology for comprehensive studies of the morphology of microscopic animals.

Something Darwin didn't know about barnacles: spermcast mating in a common stalked species.

Most free-living barnacles are hermaphroditic, and eggs are presumed to be fertilized either by pseudo-copulation or self-fertilization. Although the common northeast Pacific intertidal gooseneck barnacle, Pollicipes polymerus, is believed only to cross-fertilize, some isolated individuals well outside penis range nonetheless bear fertilized eggs. They must therefore either self-fertilize or-contrary to all prior expectations about barnacle mating-obtain sperm from the water. To test these alternative hypotheses, we collected isolated individuals bearing egg masses, as well as isolated pairs where at least one parent carried egg masses. Using 16 single nucleotide polymorphism markers, we confirmed that a high percentage of eggs were fertilized with sperm captured from the water. Sperm capture occurred in 100 per cent of isolated individuals and, remarkably, even in 24 per cent of individuals that had an adjacent partner. Replicate subsamples of individual egg masses confirmed that eggs fertilized by captured sperm occurred throughout the egg mass. Sperm capture may therefore be a common supplement to pseudo-copulation in this species. These observations (i) overturn over a century of beliefs about what barnacles can (or cannot) do in terms of sperm transfer, (ii) raise doubts about prior claims of self-fertilization in barnacles, (iii) raise interesting questions about the capacity for sperm capture in other species (particularly those with short penises), and (iv) show, we believe for the first time, that spermcast mating can occur in an aquatic arthropod.

Intertidal sea stars (Pisaster ochraceus) alter body shape in response to wave action.

Sea stars are some of the largest mobile animals able to live in the harsh flow environment of wave-exposed, rocky intertidal shores. In addition, some species, such as the northeastern Pacific Pisaster ochraceus, are ecologically significant predators in a broad range of environments, from sheltered lagoons to the most wave-exposed shorelines. How they function and survive under such an extreme range of wave exposures remains a puzzle. Here we examine the ability of P. ochraceus to alter body form in response to variation in flow conditions. We found that sea stars in wave-exposed sites had narrower arms and were lighter per unit arm length than those from sheltered sites. Body form was tightly correlated with maximum velocity of breaking waves across four sites and also varied over time. In addition, field transplant experiments showed that these differences in shape were due primarily to phenotypic plasticity. Sea stars transplanted from a sheltered site to a more wave-exposed site became lighter per unit arm length, and developed narrower arms, after 3 months. The tight correlation between water flow and morphology suggests that wave force must be a significant selective factor acting on body shape. On exposed shores, narrower arms probably reduce both lift and drag in breaking waves. On protected shores, fatter arms may provide more thermal inertia to resist overheating, or more body volume for gametes. Such plastic changes in body shape represent a unique method by which sea stars adapt to spatial, seasonal and possibly short-term variation in flow conditions.

Developmental plasticity and the origin of novel forms: unveiling cryptic genetic variation via "use and disuse".

Natural selection eliminates phenotypic variation from populations, generation after generation-an observation that haunted Darwin. So, how does new phenotypic variation arise, and is it always random with respect to fitness? Repeated behavioral responses to a novel environment-particularly those that are learned-are typically advantageous. If those behaviors yield more extreme or novel morphological variants via developmental plasticity, then previously cryptic genetic variation may be exposed to natural selection. Significantly, because the mean phenotypic effect of "use and disuse" is also typically favorable, previously cryptic genetic variation can be transformed into phenotypic variation that is both visible to selection and biased in an adaptive direction. Therefore, use-induced developmental plasticity in a very real sense "creates" new phenotypic variation that is nonrandom with respect to fitness, in contrast to the random phenotypic effects of mutation, recombination, and "direct effects" of environment (stress, nutrition). I offer here (a) a brief review of the immense literature on the effects of "use and disuse" on morphology, (b) a simple yet general model illustrating how cryptic genetic variation may be exposed to selection by developmentally plastic responses that alter trait performance in response to "use and disuse," and (c) a more detailed model of a positive feedback loop between learning (handed behavior) and morphological plasticity (use-induced morphological asymmetry) that may rapidly generate novel phenotypic variation and facilitate the evolution of conspicuous morphological asymmetries. Evidence from several sources suggests that handed behaviors played an important role both in the origin of novel forms (asymmetries) and in their subsequent evolution.

Elevated CO2 affects shell dissolution rate but not calcification rate in a marine snail.

As CO(2) levels increase in the atmosphere, so too do they in the sea. Although direct effects of moderately elevated CO(2) in sea water may be of little consequence, indirect effects may be profound. For example, lowered pH and calcium carbonate saturation states may influence both deposition and dissolution rates of mineralized skeletons in many marine organisms. The relative impact of elevated CO(2) on deposition and dissolution rates are not known for many large-bodied organisms. We therefore tested the effects of increased CO(2) levels--those forecast to occur in roughly 100 and 200 years--on both shell deposition rate and shell dissolution rate in a rocky intertidal snail, Nucella lamellosa. Shell weight gain per day in live snails decreased linearly with increasing CO(2) levels. However, this trend was paralleled by shell weight loss per day in empty shells, suggesting that these declines in shell weight gain observed in live snails were due to increased dissolution of existing shell material, rather than reduced production of new shell material. Ocean acidification may therefore have a greater effect on shell dissolution than on shell deposition, at least in temperate marine molluscs.

Reproduction: widespread cloning in echinoderm larvae.

Asexual reproduction by free-living invertebrate larvae is a rare and enigmatic phenomenon and, although it is known to occur in sea stars and brittle stars, it has not been detected in other echinoderms despite more than a century of intensive study. Here we describe spontaneous larval cloning in three species from two more echinoderm classes: a sea cucumber (Holothuroidea), a sand dollar and a sea urchin (Echinoidea). Larval cloning may therefore be an ancient ability of echinoderms and possibly of deutero-stomes - the group that includes echinoderms, acorn worms, sea squirts and vertebrates.

Experimental evidence for the rapid evolution of behavioral canalization in natural populations.

Canalization-the evolutionary loss of the capacity of organisms to develop different phenotypes in different environments-is an evolutionary phenomenon suspected to occur widely, although examples in natural populations are elusive. Because behavior is typically a highly flexible component of an individual's phenotype, it provides fertile ground for studying the evolution of canalization. Here we report how snail populations exposed for different lengths of time to a predatory crab introduced from Europe to America exhibit different degrees of canalization of an adaptive antipredator behavior: soft tissue withdrawal, measured as angular retraction depth. Where crab-snail contact is shortest (60 years), snails showed the highest behavioral flexibility. Where crabs invaded 110 years ago, snails showed significantly less behavioral flexibility, and where the interaction is ancient (Europe), snails exhibited highly canalized behavior. Selection therefore appears to have acted rapidly to increase canalization in wild snail populations, leading ultimately to the hard-wired behavior seen in European conspecifics.

Remarkably loud snaps during mouth-fighting by a sponge-dwelling worm.

Many aquatic animals, including mammals, fishes, crustaceans and insects, produce loud sounds underwater [1-6]. Soft-bodied worms would seem unlikely to produce a loud snap or pop because such brief, intense sounds normally require extreme movements and sophisticated energy storage and release mechanisms [5]. Surprisingly, we discovered a segmented marine worm that makes loud popping sounds during a highly stereotyped intraspecific agonistic behavior we call 'mouth fighting'. These sounds - sound pressures up to 157 dB re 1 µPa at 1 m, with frequencies in the 1-100 kHz range and a strong signal at ∼6.9 kHz - are comparable to those made by snapping shrimps, which are among the most intense biological sounds that have been measured in the sea [6]. We suggest a novel mechanism for generating ultrafast movements and loud sounds in a soft-bodied animal: thick, muscular pharyngeal walls appear to allow energy storage and cocking; this permits extremely rapid expansion of the pharynx within the worm's body during the strike, which yields an intense popping sound (likely via cavitation) and a rapid influx of water. Clearly, even soft-bodied marine invertebrates can produce remarkably loud sounds underwater. How they do so remains an intriguing biomechanical puzzle that hints at a new type of extreme biology.

Precisely proportioned: intertidal barnacles alter penis form to suit coastal wave action.

For their size, barnacles possess the longest penis of any animal (up to eight times their body length). However, as one of few sessile animals to copulate, they face a trade-off between reaching more mates and controlling ever-longer penises in turbulent flow. We observed that penises of an intertidal barnacle (Balanus glandula) from wave-exposed shores were shorter than, stouter than, and more than twice as massive for their length as, those from nearby protected bays. In addition, penis shape variation was tightly correlated with maximum velocity of breaking waves, and, on all shores, larger barnacles had disproportionately stouter penises. Finally, field experiments confirmed that most of this variation was due to phenotypic plasticity: barnacles transplanted to a wave-exposed outer coast produced dramatically shorter and wider penises than counterparts moved to a protected harbour. Owing to the probable trade-off between penis length and ability to function in flow, and owing to the ever-changing wave conditions on rocky shores, intertidal barnacles appear to have acquired the capacity to change the size and shape of their penises to suit local hydrodynamic conditions. This dramatic plasticity in genital form is a valuable reminder that factors other than the usual drivers of genital diversification--female choice, sexual conflict and male-male competition--can influence genital form.

Lamellose Axial Shell Sculpture Reduces Gastropod Vulnerability to Sea Star Predation.

Marine gastropods exhibit a stunning diversity of shell sculpture, but the functional significance of many sculpture types remains unknown. Unfortunately, experimental tests of the functional significance of differences between species are complicated by other morphological differences, such as shell microstructure, aperture shape, and shell thickness, that may confound interpretation. The most robust experimental tests are therefore performed using different shell forms within a species. We took advantage of the extensive intraspecific shell variation in the common intertidal gastropod Nucella lamellosa to test the adaptive significance of axial lamellae, a type of shell sculpture found in numerous marine gastropod subfamilies. We offered three forms of N. lamellosa (lamellose, artificially smooth, and naturally smooth) to the predatory sea star Pisaster ochraceus under controlled laboratory conditions. Pisaster ochraceus consumed significantly fewer lamellose snails than either artificially or naturally smooth snails. We suggest that shell lamellae deter sea star predation by impairing their ability to capture or manipulate snail prey or by increasing prey effective size. These results suggest a credible hypothesis for the adaptive significance of lamellar sculpture in marine gastropods and provide a valuable missing piece to the story about adaptive phenotypic plasticity in N. lamellosa shell form.

Parallel Saltational Evolution of Ultrafast Movements in Snapping Shrimp Claws.

How do stunning functional innovations evolve from unspecialized progenitors? This puzzle is particularly acute for ultrafast movements of appendages in arthropods as diverse as shrimps [1], stomatopods [2], insects [3-6], and spiders [7]. For example, the spectacular snapping claws of alpheid shrimps close so fast (∼0.5 ms) that jetted water creates a cavitation bubble and an immensely powerful snap upon bubble collapse [1]. Such extreme movements depend on (1) an energy-storage mechanism (e.g., some kind of spring) and (2) a latching mechanism to release stored energy quickly [8]. Clearly, rapid claw closure must have evolved before the ability to snap, but its evolutionary origins are unknown. Unearthing the functional mechanics of transitional stages is therefore essential to understand how such radical novel abilities arise [9-11]. We reconstructed the evolutionary history of shrimp claw form and function by sampling 114 species from 19 families, including two unrelated families within which snapping evolved independently (Alpheidae and Palaemonidae) [12, 13]. Our comparative analyses, using micro-computed tomography (microCT) and confocal imaging, high-speed video, and kinematic experiments with select 3D-printed scale models, revealed a previously unrecognized "slip joint" in non-snapping shrimp claws. This slip joint facilitated the parallel evolution of a novel energy-storage and cocking mechanism-a torque-reversal joint-an apparent precondition for snapping. Remarkably, these key functional transitions between ancestral (simple pinching) and derived (snapping) claws were achieved by minute differences in joint structure. Therefore, subtle changes in form appear to have facilitated wholly novel functional change in a saltational manner. VIDEO ABSTRACT.

How reversible is development? Contrast between developmentally plastic gain and loss of segments in barnacle feeding legs.

Segmented organisms and structures have fascinated biologists since William Bateson first described homeotic transformation and recognized the fundamental evolutionary significance of segmental organization. On evolutionary time scales, segments may be lost or gained during major morphological transitions. But how segment loss compares to gain on developmental time scales remains mysterious. Here, we examine the ease of reverse development (opposite to normal growth) by comparing developmentally plastic leg segment loss versus gain in individual barnacles transplanted between different water flow conditions. Plastic segment addition occurred rapidly (one to two molts) and exclusively near the limb base. In contrast, developmentally plastic segment loss-the first observation in any arthropod-took much longer (>10 molts) and, remarkably, occurred throughout the leg (23% of losses occurred mid-limb). Segment loss was not a simple reversal of segment addition. Intersegmental membranes fused first, followed by elimination of duplicate tendons and gradual shortening (but not loss) of duplicate setae. Setal loss, in particular, may impose a severe developmental constraint on arthropod segment fusion. This asymmetric developmental potential (time lag of phenotypic response)-plastic segment addition (amplified normal development) is faster and more orderly than segment loss (reverse development)-adds a new dimension to models of developmental plasticity because the cost of making a developmental mistake in one direction will be greater than in the other.

Morphological phylogeny of alpheid shrimps: parallel preadaptation and the origin of a key morphological innovation, the snapping claw.

The Alpheidae-possibly the most diverse family of recent decapod crustaceans-offers attractive opportunities to study the evolution of many intriguing phenomena, including key morphological innovations like spectacular snapping claws, highly specialized body forms, facultative and obligate symbioses with many animal groups, and sophisticated behaviors like eusociality. However, studies of these remarkable adaptations remain hampered by insufficient phylogenetic information. We present the first phylogenetic hypothesis of relationships among 36 extant genera of alpheid shrimps, based on a cladistic analysis of 122 morphological characters from 56 species, and we use this hypothesis to explore evolutionary trends in morphology and species diversity. Our results strongly supported a monophyletic Alpheidae that included two hitherto difficult-to-place genera (Yagerocaris and Pterocaris). Of 35+ nodes among genera, all were supported by at least one morphological character (24 were supported by two or more) and 17 received greater than 50% jackknife support. Unfortunately, many basal nodes were only weakly supported. Six genera appeared nonmonophyletic, including the dominant genus Alpheus (paraphyletic due to inclusion of one clade with three minor genera). Evolutionary trends in alpheid claw form shed some revealing light on how key innovations evolve. First, several functionally significant features of the cheliped (claw bearing leg) evolved independently multiple times, including: asymmetry, folding, inverted orientation, sexual dimorphism, adhesive plaques that enhance claw cocking, and tooth-cavity systems on opposing claw fingers, a preadaptation for snapping. Many conspicuous features of alpheid claw form therefore appear prone to parallel evolution. Second, although tooth-cavity systems evolved multiple times, a functional snapping claw, which likely facilitated an explosive radiation of over 550 species, evolved only once (in Synalpheus + [Alpheus + satellite genera]). Third, adhesive plaques (claw cocking aids) also evolved multiple times, and within snapping alpheids are associated with the most diverse clade (Alpheus + derivative genera). This pattern of parallel preadaptation-multiple independent evolutionary origins of precursors (preadaptations) to what ultimately became a key innovation (adaptation)-suggests alpheid shrimp claws are predisposed to develop features like tooth-cavity and adhesive plaque systems for functional or developmental reasons. Such functional/developmental predisposition may facilitate the origin of key innovations. Finally, moderate orbital hoods-anterior projections of the carapace partly or completely covering the eyes-occur in many higher Alpheidae and likely evolved before snapping claws. They are unique among decapod crustaceans, and their elaboration in snapping alpheids suggests they may protect the eyes from the stress of explosive snaps. Thus one key innovation (orbital hoods) may have facilitated evolution of a second (snapping claws).

What determines direction of asymmetry: genes, environment or chance?

Conspicuous asymmetries seen in many animals and plants offer diverse opportunities to test how the development of a similar morphological feature has evolved in wildly different types of organisms. One key question is: do common rules govern how direction of asymmetry is determined (symmetry is broken) during ontogeny to yield an asymmetrical individual? Examples from numerous organisms illustrate how diverse this process is. These examples also provide some surprising answers to related questions. Is direction of asymmetry in an individual determined by genes, environment or chance? Is direction of asymmetry determined locally (structure by structure) or globally (at the level of the whole body)? Does direction of asymmetry persist when an asymmetrical structure regenerates following autotomy? The answers vary greatly for asymmetries as diverse as gastropod coiling direction, flatfish eye side, crossbill finch bill crossing, asymmetrical claws in shrimp, lobsters and crabs, katydid sound-producing structures, earwig penises and various plant asymmetries. Several examples also reveal how stochastic asymmetry in mollusc and crustacean early cleavage, in Drosophila oogenesis, and in Caenorhabditis elegans epidermal blast cell movement, is a normal component of deterministic development. Collectively, these examples shed light on the role of genes as leaders or followers in evolution.This article is part of the themed issue 'Provocative questions in left-right asymmetry'.