The bowfin genome illuminates the developmental evolution of ray-finned fishes.

The bowfin (Amia calva) is a ray-finned fish that possesses a unique suite of ancestral and derived phenotypes, which are key to understanding vertebrate evolution. The phylogenetic position of bowfin as a representative of neopterygian fishes, its archetypical body plan and its unduplicated and slowly evolving genome make bowfin a central species for the genomic exploration of ray-finned fishes. Here we present a chromosome-level genome assembly for bowfin that enables gene-order analyses, settling long-debated neopterygian phylogenetic relationships. We examine chromatin accessibility and gene expression through bowfin development to investigate the evolution of immune, scale, respiratory and fin skeletal systems and identify hundreds of gene-regulatory loci conserved across vertebrates. These resources connect developmental evolution among bony fishes, further highlighting the bowfin's importance for illuminating vertebrate biology and diversity in the genomic era.

Does the bowfin gas bladder represent an intermediate stage during the lung-to-gas bladder evolutionary transition?

Whether phenotypic evolution occurs gradually through time has prompted the search for intermediate forms between the ancestral and derived states of morphological features, especially when there appears to be a discontinuous origin. The gas bladder, a derived character of the Actinopteri, is a modification of lungs, which characterize the common ancestor of bony vertebrates. While gas bladders and lungs are similar in many ways, the key morphological difference between these organs is the direction of budding from the foregut during development; essentially, the gas bladder buds dorsally and the lungs bud ventrally from the foregut. Did the shift from ventral lungs to dorsal gas bladder transition through a lateral-budding stage? To answer this question, the precise location of budding during gas bladder development in bowfin, representing the sister lineage to teleosts, has been debated. In the early 20th-century, it was suggested that the bowfin gas bladder buds laterally from the right wall of the foregut. We used nano-CT scanning to visualize the early development of the bowfin gas bladder to verify the historical studies of gas bladder developmental morphology and determine whether the direction of gas bladder budding in bowfin could be intermediate between ventrally budding lungs and dorsally budding gas bladders. We found that the bowfin gas bladder buds dorsally from the anterior foregut; however, during early development, the posterior gas bladder twists right. As development progresses, the posterior, right-hand twist becomes shallower, and the gas bladder itself shifts toward a mid-dorsal position. The budding site is definitively dorsal, despite the temporary lateral twist of the posterior gas bladder.

Dorsoventral inversion of the air-filled organ (lungs, gas bladder) in vertebrates: RNAsequencing of laser capture microdissected embryonic tissue.

How modification of gene expression generates novel traits is key to understanding the evolutionary process. We investigated the genetic basis for the origin of the piscine gas bladder from lungs of ancestral bony vertebrates. Distinguishing these homologous organs is the direction of budding from the foregut during development; lungs bud ventrally and the gas bladder buds dorsally.

Unraveling hierarchical genetic structure in a marine metapopulation: A comparison of three high-throughput genotyping approaches.

Marine metapopulations often exhibit subtle population structure that can be difficult to detect. Given recent advances in high-throughput sequencing, an emerging question is whether various genotyping approaches, in concert with improved sampling designs, will substantially improve our understanding of genetic structure in the sea. To address this question, we explored hierarchical patterns of structure in the coral reef fish Elacatinus lori using a high-resolution approach with respect to both genetic and geographic sampling. Previously, we identified three putative E. lori populations within Belize using traditional genetic markers and sparse geographic sampling: barrier reef and Turneffe Atoll; Glover's Atoll; and Lighthouse Atoll. Here, we systematically sampled individuals at ~10 km intervals throughout these reefs (1,129 individuals from 35 sites) and sequenced all individuals at three sets of markers: 2,418 SNPs; 89 microsatellites; and 57 nonrepetitive nuclear loci. At broad spatial scales, the markers were consistent with each other and with previous findings. At finer spatial scales, there was new evidence of genetic substructure, but our three marker sets differed slightly in their ability to detect these patterns. Specifically, we found subtle structure between the barrier reef and Turneffe Atoll, with SNPs resolving this pattern most effectively. We also documented isolation by distance within the barrier reef. Sensitivity analyses revealed that the number of loci (and alleles) had a strong effect on the detection of structure for all three marker sets, particularly at small spatial scales. Taken together, these results illustrate empirically that high-throughput genotyping data can elucidate subtle genetic structure at previously-undetected scales in a dispersive marine fish.

Changes in Nkx2.1, Sox2, Bmp4, and Bmp16 expression underlying the lung-to-gas bladder evolutionary transition in ray-finned fishes.

The key to understanding the evolutionary origin and modification of phenotypic traits is revealing the responsible underlying developmental genetic mechanisms. An important organismal trait of ray-finned fishes is the gas bladder, an air-filled organ that, in most fishes, functions for buoyancy control, and is homologous to the lungs of lobe-finned fishes. The critical morphological difference between lungs and gas bladders, which otherwise share many characteristics, is the general direction of budding during development. Lungs bud ventrally and the gas bladder buds dorsally from the anterior foregut. We investigated the genetic underpinnings of this ventral-to-dorsal shift in budding direction by studying the expression patterns of known lung genes (Nkx2.1, Sox2, and Bmp4) during the development of lungs or gas bladder in three fishes: bichir, bowfin, and zebrafish. Nkx2.1 and Sox2 show reciprocal dorsoventral expression patterns during tetrapod lung development and are important regulators of lung budding; their expression during bichir lung development is conserved. Surprisingly, we find during gas bladder development, Nkx2.1 and Sox2 expression are inconsistent with the hypothesis that they regulate the direction of gas bladder budding. Bmp4 is expressed ventrally during lung development in bichir, akin to the pattern during mouse lung development. During gas bladder development, Bmp4 is not expressed. However, Bmp16, a paralogue of Bmp4, is expressed dorsally in the developing gas bladder of bowfin. Bmp16 is present in the known genomes of Actinopteri (ray-finned fishes excluding bichir) but absent from mammalian genomes. We hypothesize that Bmp16 was recruited to regulate gas bladder development in the Actinopteri in place of Bmp4.

Differences in Cell Proliferation and Craniofacial Phenotype of Closely Related Species in the Pupfish Genus Cyprinodon.

Understanding the genetic basis for phenotypic differences is fundamental to the study of macroevolutionary patterns of biological diversity. While technological advances in DNA sequencing have made researching genetic variation in wild taxa routine, fully understanding how these variants affect phenotype requires taking the next step to investigate how genetic changes alter cell and tissue interactions that ultimately produce phenotypes. In this article, we investigate a role for cell proliferation as a developmental source of craniofacial diversity in a radiation of 3 species of Cyprinodon from San Salvador Island, Bahamas. Patterns of cell proliferation in the heads of hatching-age fish differ among species of Cyprinodon, and correlate with differences in allometric growth rate among the jaws of 3 distinct species. Regional patterns of cell proliferation in the head are complex, resulting in an unintuitive result in which lower levels of cell proliferation in the posterior head region are associated with the development of relatively larger jaws in one species. We combine these data with previously published morphological and genomic data to show how studying the mechanisms generating phenotype at the cellular and tissue levels of biological organization can help mechanistically link genomic studies with classic morphological studies.

Co-opting evo-devo concepts for new insights into mechanisms of behavioural diversity.

We propose that insights from the field of evolutionary developmental biology (or 'evo-devo') provide a framework for an integrated understanding of the origins of behavioural diversity and its underlying mechanisms. Towards that goal, in this Commentary, we frame key questions in behavioural evolution in terms of molecular, cellular and network-level properties with a focus on the nervous system. In this way, we highlight how mechanistic properties central to evo-devo analyses - such as weak linkage, versatility, exploratory mechanisms, criticality, degeneracy, redundancy and modularity - affect neural circuit function and hence the range of behavioural variation that can be filtered by selection. We outline why comparative studies of molecular and neural systems throughout ontogeny will provide novel insights into diversity in neural circuits and behaviour.

An embryonic staging series up to hatching for Cyprinodon variegatus: An emerging fish model for developmental, evolutionary, and ecological research.

Using multiple taxa to research development is necessary for making general conclusions about developmental patterns and mechanisms. We present a staging series for Cyprinodon variegatus as a basis for further study of the developmental biology of fishes in the genus Cyprinodon and for comparative work on teleost fishes beyond the standard models. Cyprinodon are small, euryhaline fishes, widely distributed in fresh, brackish, and hypersaline waters of southern and eastern North America. Cyprinodontids are closely related to fundulids, providing a comparative reference point to the embryological model, Fundulus heteroclitus. Ecologists and evolutionary biologists commonly study Cyprinodon, and we have been using Cyprinodon to study skull variation and its genetic basis among closely related species. We divided embryonic development of C. variegatus into 34 morphologically identifiable stages. We reference our staging series to that already defined for a related model species, Oryzias latipes (medaka) that is studied by a large community of researchers. We provide a description of the early chondrogenesis and ossification of skull and caudal fin bones during the latter stages of embryonic development. We show that Cyprinodon are tractable for studying development. Eggs can be obtained easily from breeding pairs and our study provides a staging system to facilitate future developmental studies.

The Cyprinodon variegatus genome reveals gene expression changes underlying differences in skull morphology among closely related species.

BACKGROUND: Understanding the genetic and developmental origins of phenotypic novelty is central to the study of biological diversity. In this study we identify modifications to the expression of genes at four developmental stages that may underlie jaw morphological differences among three closely related species of pupfish (genus Cyprinodon) from San Salvador Island, Bahamas. Pupfishes on San Salvador Island are trophically differentiated and include two endemic species that have evolved jaw morphologies unlike that of any other species in the genus Cyprinodon. RESULTS: We find that gene expression differs significantly across recently diverged species of pupfish. Genes such as Bmp4 and calmodulin, previously implicated in jaw diversification in African cichlid fishes and Galapagos finches, were not found to be differentially expressed among species of pupfish. Instead we find multiple growth factors and cytokine/chemokine genes to be differentially expressed among these pupfish taxa. These include both genes and pathways known to affect craniofacial development, such as Wnt signaling, as well as novel genes and pathways not previously implicated in craniofacial development. These data highlight both shared and potentially unique sources of jaw diversity in pupfish and those identified in other evolutionary model systems such as Galapagos finches and African cichlids. CONCLUSIONS: We identify modifications to the expression of genes involved in Wnt signaling, Igf signaling, and the inflammation response as promising avenues for future research. Our project provides insight into the magnitude of gene expression changes contributing to the evolution of morphological novelties, such as jaw structure, in recently diverged pupfish species.

Changes in growth rates of oral jaw elements produce evolutionary novelty in bahamian pupfish.

To understand the origins of novelty and the evolution of biological diversity, it is important to investigate the processes that generate phenotypic variation from genotypic variation. A number of path-breaking studies have revealed the genetic basis for phenotypic differences between distantly related taxa, but how qualitative change is produced during the early stages of divergence is largely unexplored. Here, we focus on striking differences in jaw morphology exhibited by three closely related sympatric pupfish species (genus Cyprinodon) from San Salvador Island, Bahamas as a basis for investigating the genetic sources of morphological variation in recently diverged species. San Salvador Island pupfish are trophically diverse and display derived jaw morphologies distinct from any other species in the genus. We illustrate these qualitative morphological differences between species with 3D-reconstructed CT-images and camera lucida drawings of the skulls of wild-caught fish. Quantitative data representing the size of individual bony skull elements in wild fish show how qualitatively novel morphologies arise as a consequence of changes to the size and shape of individual skull elements, particularly the dentary, premaxilla, and maxilla bones associated with the oral jaws. Consistent with these comparative data is that the growth rate of individual bony skull elements, measured on a developmental time series of lab-reared fish, differs between species. Our data provide a critical foundation for future studies developing San Salvador Cyprinodon pupfishes as a model system to understand the evolution and development of novel morphologies at the species level. J. Morphol. 277:935-947, 2016. © 2016 Wiley Periodicals, Inc.

Homology of lungs and gas bladders: insights from arterial vasculature.

Gas bladders of ray-finned fishes serve a variety of vital functions and are thus an important novelty of most living vertebrates. The gas bladder has long been regarded as an evolutionary modification of lungs. Critical evidence for this hypothesized homology is whether pulmonary arteries supply the gas bladder as well as the lungs. Pulmonary arteries, paired branches of the fourth efferent branchial arteries, deliver blood to the lungs in osteichthyans with functional lungs (lungfishes, tetrapods, and the ray-finned polypterid fishes). The fact that pulmonary arteries also supply the respiratory gas bladder of Amia calva (bowfin) has been used to support the homology of lungs and gas bladders, collectively termed air-filled organs (AO). However, the homology of pulmonary arteries in bowfin and lunged osteichthyans has been uncertain, given the apparent lack of pulmonary arteries in critical taxa. To re-evaluate the homology of pulmonary arteries in bowfin and lunged osteichthyans, we studied, using micro-CT technology, the arterial vasculature of Protopterus, Polypterus, Acipenser, Polyodon, Amia, and Lepisosteus, and analyzed these data using a phylogenetic approach. Our data reveal that Acipenser and Polyodon have paired posterior branches of the fourth efferent branchial arteries, which are thus similar in origin to pulmonary arteries. We hypothesize that these arteries are vestigial pulmonary arteries that have been coopted for new functions due to the dorsal shift of the AO and/or the loss of respiration in these taxa. Ancestral state reconstructions support pulmonary arteries as a synapomorphy of the Osteichthyes, provide the first concrete evidence for the retention of pulmonary arteries in Amia, and support thehomology of lungs and gas bladders due to a shared vascular supply. Finally, we use ancestral state reconstructions to show that arterial AO supplies from the celiacomesenteric artery or dorsal aorta appear to be convergent between teleosts and nonteleost actinopterygians.

Expression of a lung developmental cassette in the adult and developing zebrafish swimbladder.

The presence of an air-filled organ (AO), either lungs or a swimbladder, is a defining character of the Osteichthyes (bony vertebrates, including tetrapods). Despite the functional and structural diversity of AOs, it was not previously known whether the same group of developmental regulatory genes are involved in the early development of both lungs and swimbladders. This study demonstrates that a suite of genes (Nkx2.1, FoxA2, Wnt7b, GATA6), previously reported to be co-expressed only in the tetrapod lung, is also co-expressed in the zebrafish swimbladder. We document the expression pattern of these genes in the adult and developing zebrafish swimbladder and compare the expression patterns to those in the mouse lung. Early-acting genes involved in endoderm specification are expressed in the same relative location and stage of AO development in both taxa (FoxA2 and GATA6), but the order of onset and location of expression are not completely conserved for the later acting genes (Nkx2.1 and Wnt7b). Co-expression of this suite of genes in both tetrapod lungs and swimbladders of ray-finned fishes is more likely due to common ancestry than independent co-option, because these genes are not known to be co-expressed anywhere except in the AOs of Osteichthyes. Any conserved gene product interactions may comprise a character identity network (ChIN) for the osteichthyan AO.

Using genetic networks and homology to understand the evolution of phenotypic traits.

Homology can have different meanings for different kinds of biologists. A phylogenetic view holds that homology, defined by common ancestry, is rigorously identified through phylogenetic analysis. Such homologies are taxic homologies (=synapomorphies). A second interpretation, "biological homology" emphasizes common ancestry through the continuity of genetic information underlying phenotypic traits, and is favored by some developmental geneticists. A third kind of homology, deep homology, was recently defined as "the sharing of the genetic regulatory apparatus used to build morphologically and phylogenetically disparate features." Here we explain the commonality among these three versions of homology. We argue that biological homology, as evidenced by a conserved gene regulatory network giving a trait its "essential identity" (a Character Identity Network or "ChIN") must also be a taxic homology. In cases where a phenotypic trait has been modified over the course of evolution such that homology (taxic) is obscured (e.g. jaws are modified gill arches), a shared underlying ChIN provides evidence of this transformation. Deep homologies, where molecular and cellular components of a phenotypic trait precede the trait itself (are phylogenetically deep relative to the trait), are also taxic homologies, undisguised. Deep homologies inspire particular interest for understanding the evolutionary assembly of phenotypic traits. Mapping these deeply homologous building blocks on a phylogeny reveals the sequential steps leading to the origin of phenotypic novelties. Finally, we discuss how new genomic technologies will revolutionize the comparative genomic study of non-model organisms in a phylogenetic context, necessary to understand the evolution of phenotypic traits.

Recent speciation between sympatric Tanganyikan cichlid colour morphs.

Lake Tanganyika, Africa's oldest lake, harbours an impressive diversity of cichlid fishes. Although diversification in its radiating groups is thought to have been initially rapid, cichlids from Lake Tanganyika show little evidence for ongoing speciation. In contrast, examples of recent divergence among sympatric colour morphs are well known in haplochromine cichlids from Lakes Malawi and Victoria. Here, we report genetic evidence for recent divergence between two sympatric Tanganyikan cichlid colour morphs. These Petrochromis morphs share mitochondrial haplotypes, yet microsatellite loci reveal that their sympatric populations form distinct genetic groups. Nuclear divergence between the two morphs is equivalent to that which arises geographically within one of the morphs over short distances and is substantially smaller than that among other sympatric species in this genus. These patterns suggest that these morphs diverged only recently, yet that barriers to gene flow exist which prevent extensive admixture despite their sympatric distribution. The morphs studied here provide an unusual example of active diversification in Lake Tanganyika's generally ancient cichlid fauna and enable comparisons of speciation processes between Lake Tanganyika and other African lakes.

Reinventing species selection with molecular phylogenies.

Species selection as a potential driver of macroevolutionary trends has been relegated to a largely philosophical position in modern evolutionary biology. Fundamentally, species selection is the outcome of heritable differences in speciation and extinction rates among lineages when the causal basis of those rate differences can be decoupled from genotypic (within-population) fitnesses. Here, we discuss the rapidly growing literature on variation in species diversification rates as inferred from molecular phylogenies. We argue that modern studies of diversification rates demonstrate that species selection is an important process influencing both the evolution of biological diversity and distributions of phenotypic traits within higher taxa. Explicit recognition of multi-level selection refocuses our attention on the mechanisms by which traits influence speciation and extinction rates.

Contrasting patterns of spatial genetic structure in sympatric rock-dwelling cichlid fishes.

The cichlid fish of Lake Tanganyika in Eastern Africa are a celebrated example of both ecological and species diversification. Because population subdivision is likely to play an important role in the speciation process, understanding how habitat features interact with species' demographic, behavioral and ecological attributes to influence gene flow and population divergence may help explain the causes of high species richness in this and other systems. Here, we test the roles of isolation-by-habitat and isolation-by-distance in generating fine-scale population genetic structure in three sympatric species of habitat-restricted cichlids in Lake Tanganyika. Using multilocus microsatellite genotypes, we contrast patterns of population differentiation in these habitat specialists along a mosaic coastline of both favorable and unfavorable habitat. Despite their close phylogenetic relationship and shared habitat affinity, these species show striking differences in their pattern of genetic subdivision within the same geographical region, suggesting substantially different patterns of gene flow. In particular, two trophically specialized species exhibit much more restricted gene flow over sandy habitat than a trophically opportunistic species. This result suggests that ecological and behavioral traits have a strong influence on the scale and degree of population subdivision, a finding that has potentially important implications for understanding differential propensities for diversification among lineages and phylogenetic patterns of diversity.

Evolution and the latitudinal diversity gradient: speciation, extinction and biogeography.

A latitudinal gradient in biodiversity has existed since before the time of the dinosaurs, yet how and why this gradient arose remains unresolved. Here we review two major hypotheses for the origin of the latitudinal diversity gradient. The time and area hypothesis holds that tropical climates are older and historically larger, allowing more opportunity for diversification. This hypothesis is supported by observations that temperate taxa are often younger than, and nested within, tropical taxa, and that diversity is positively correlated with the age and area of geographical regions. The diversification rate hypothesis holds that tropical regions diversify faster due to higher rates of speciation (caused by increased opportunities for the evolution of reproductive isolation, or faster molecular evolution, or the increased importance of biotic interactions), or due to lower extinction rates. There is phylogenetic evidence for higher rates of diversification in tropical clades, and palaeontological data demonstrate higher rates of origination for tropical taxa, but mixed evidence for latitudinal differences in extinction rates. Studies of latitudinal variation in incipient speciation also suggest faster speciation in the tropics. Distinguishing the roles of history, speciation and extinction in the origin of the latitudinal gradient represents a major challenge to future research.

Two classes of deleterious recessive alleles in a natural population of zebrafish, Danio rerio.

Natural populations carry deleterious recessive alleles which cause inbreeding depression. We compared mortality and growth of inbred and outbred zebrafish, Danio rerio, between 6 and 48 days of age. Grandparents of the studied fish were caught in the wild. Inbred fish were generated by brother-sister mating. Mortality was 9% in outbred fish, and 42% in inbred fish, which implies at least 3.6 lethal equivalents of deleterious recessive alleles per zygote. There was no significant inbreeding depression in the growth, perhaps because the surviving inbred fish lived under less crowded conditions. In contrast to alleles that cause embryonic and early larval mortality in the same population, alleles responsible for late larval and early juvenile mortality did not result in any gross morphological abnormalities. Thus, deleterious recessive alleles that segregate in a wild zebrafish population belong to two sharply distinct classes: early-acting, morphologically overt, unconditional lethals; and later-acting, morphologically cryptic, and presumably milder alleles.

Twenty ways to lose your bladder: common natural mutants in zebrafish and widespread convergence of swim bladder loss among teleost fishes.

Convergence is an important evolutionary phenomenon often attributed solely to natural selection acting in similar environments. The frequency of mutation and number of ways a phenotypic trait can be generated genetically, however, may also affect the probability of convergence. Here we report both a high frequency of loss of gas bladder (swim bladder) mutations in zebrafish and widespread convergent loss of gas bladders among teleost fishes. The phenotypes of 22 of 27 recessive lethal mutations, carried by a sample of 26 wild-caught zebrafish, involve loss or noninflation of the gas bladder. Nine of these bladderless mutations showed no other obvious phenotypic abnormalities other than the lack of an inflated gas bladder. At least 19 of the 22 bladderless mutations are genetically distinct, as shown by unique morphology or complementation. Although we were not able to obtain eggs for all 21 required crosses to demonstrate definitively that the remaining three mutations are different from all other bladderless mutations, all available evidence suggests that these mutants are also distinct. At least 79 of 425 families of extant teleosts include one or more species lacking a gas bladder as adults. Analysis of the trait's phylogenetic distribution shows that the gas bladder has been lost at least 30-32 times independently. Although adaptive explanations for gas bladder loss are convincing, a developmental bias toward bladderless phenotypes may also have contributed to the widespread convergence of this trait among teleosts. If gas bladder development in teleosts is as vulnerable to genetic perturbation as it is in zebrafish, then perhaps a supply of bladderless phenotypes has been readily available to natural selection under conditions for which it is advantageous not to have a gas bladder. In this way, developmental bias and selection can work together to produce widespread convergence.

Evidence for developmental linkage of pigment patterns with body size and shape in danios (Teleostei: Cyprinidae).

Variation in pigment patterns in fishes is known to be subject to natural and sexual selection, but the mechanisms by which that variation is generated are only beginning to be understood. Theoretical models of pigment pattern formation in animals suggest that the size and shape of the organism at the time of pattern determination as well as subsequent growth time are important determinants of pattern. However, few data document the empirical relationship of pigment patterning with size, shape, and growth. Here we document patterns of growth in relation to pigment pattern formation in the zebrafish (Danio rerio) and six close relatives. In all species examined, establishment of adult pigment pattern within a particular region of the body is associated with a period of substantial growth and shape change in that region of the body. Furthermore, forms with more horizontal stripes on the midbody as adults (Danio rerio and D. rerio "leo") are larger at the time pigment cells begin to assume their adult pattern. Finally, continued deepening of the body as the pigment pattern develops is associated with vertical distortions and reticulations in the patterns of D. malabaricus and D. browni. These results are consistent with the predictions of theoretical models that size, growth, and shape change are critical determinants of pigment patterning, and suggest that variation in pigment pattern may arise in part through differential allometric growth between species.