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MicroRNAs (miRNAs) are important gene expression regulators implicated in many biological processes, but we lack a global understanding of how miRNA genes evolve and contribute to developmental canalization and phenotypic diversification. Whole-genome duplication events likely provide a substrate for species divergence and phenotypic change by increasing gene numbers and relaxing evolutionary pressures. To understand the consequences of genome duplication on miRNA evolution, we studied miRNA genes following the teleost genome duplication (TGD). Analysis of miRNA genes in four teleosts and in spotted gar, whose lineage diverged before the TGD, revealed that miRNA genes were retained in ohnologous pairs more frequently than protein-coding genes, and that gene losses occurred rapidly after the TGD. Genomic context influenced retention rates, with clustered miRNA genes retained more often than nonclustered miRNA genes and intergenic miRNA genes retained more frequently than intragenic miRNA genes, which often shared the evolutionary fate of their protein-coding host. Expression analyses revealed both conserved and divergent expression patterns across species in line with miRNA functions in phenotypic canalization and diversification, respectively. Finally, major strands of miRNA genes experienced stronger purifying selection, especially in their seeds and 3′-complementary regions, compared with minor strands, which nonetheless also displayed evolutionary features compatible with constrained function. This study provides the first genome-wide, multispecies analysis of the mechanisms influencing metazoan miRNA evolution after whole-genome duplication.

We evaluated the accuracy and precision of age estimates from ground sagittal otoliths, sectioned pectoral fin rays, and whole branchiostegal rays of Longnose Gar Lepisosteus osseus and Spotted Gar Lepisosteus oculatus using fish marked with oxytetracycline (OTC). The presence of OTC time stamps in these calcified structures and the ability to correctly identify post time-stamp annuli varied greatly. We identified time stamps in 66.7% and 91.7% of the otoliths and 43.8% and 61.4% of the pectoral fin rays for Longnose Gar and Spotted Gar, respectively; OTC marks were not observed in branchiostegal rays. Annual increment periodicity was validated in ground sagittal otoliths through age 10 for both species. For fish > age 10, accuracy declined to about 60% with most errors underestimating the number of post time-stamp annuli by one year. Ages derived from pectoral fin rays consistently underestimated the number of post time-stamp annuli because OTC marks were generally associated with the outer edge ...

The spotted gar (Lepisosteus oculatus) shows a disjunct natural distribution, with a core population extending from the central Mississippi River Basin to the U.S. gulf coast and a peripheral population in the southern Great Lakes Basin. Despite significant conservation concerns for this species in the Great Lakes watersheds where it occurs, few genetic examinations and comparisons of these populations have been performed. We investigated inter- and intrapopulational variation in several mitochondrial genetic markers (cytochrome oxidase subunit I, COI; cytochrome oxidase subunit II, COII; and 16S rRNA, 16S) from spotted gars taken from core and peripheral populations. Genetic diversity was highest in the Mississippi River Basin and lowest in the Great Lakes Basin, while the Nueces River Basin (Texas) population showed the greatest level of divergence from other populations. Average genetic distance among core and peripheral populations was over an order of magnitude less than that seen between L. oculatus and its sister species, the Florida gar (L. platyrhincus), although a significant correlation was found between genetic and geographical distance in L. oculatus. Genetic divergence in spotted gars is likely to be related to a combination of geographic isolation and founder effects associated with recent colonization following glacial retreat. Despite its apparent lack of significant genetic differentiation or haplotype diversity, the Great Lakes population of spotted gars has previously been shown to be a unique component of the species, and additional studies are needed to determine the genetic mechanisms underlying regional adaptations as well as potential morphological differentiation among spotted gar populations.

Whole genome duplications (WGD) are important evolutionary events. Our understanding of underlying mechanisms, including the evolution of duplicated genes after WGD, however remains incomplete. Teleost fish experienced a common WGD (teleost-specific genome duplication, or TGD) followed by a dramatic adaptive radiation leading to more than half of all vertebrate species. The analysis of gene expression patterns following TGD at the genome level has been limited by the lack of suitable genomic resources. The recent concomitant release of the genome sequence of spotted gar (a representative of holosteans, the closest lineage of teleosts that lacks the TGD) and the tissue-specific gene expression repertoires of over 20 holostean and teleostean fish species, including spotted gar, zebrafish and medaka (the PhyloFish project), offered a unique opportunity to study the evolution of gene expression following TGD in teleosts. We show that most TGD duplicates gained their current status (loss of one duplicate gene or retention of both duplicates) relatively rapidly after TGD (i.e. prior to the divergence of medaka and zebrafish lineages). The loss of one duplicate is the most common fate after TGD with a probability of approximately 80%. In addition, the fate of duplicate genes after TGD, including subfunctionalization, neofunctionalization, or retention of two ‘similar’ copies occurred not only before, but also after the radiation of species tested, in consistency with a role of the TGD in speciation and/or evolution of gene function. Finally, we report novel cases of TGD ohnolog subfunctionalization and neofunctionalization that further illustrate the importance of these processes.

Dorsoventral pigment patterning, characterized by a light ventrum and a dark dorsum, is one of the most widespread chromatic adaptations in vertebrate body coloration. In mammals, this countershading depends on differential expression of agouti-signaling protein (ASIP), which drives a switch of synthesis of one type of melanin to another within melanocytes. Teleost fish share countershading, but the pattern results from a differential distribution of multiple types of chromatophores, with black-brown melanophores most abundant in the dorsal body and reflective iridophores most abundant in the ventral body. We previously showed that Asip1 (a fish ortholog of mammalian ASIP) plays a role in patterning melanophores. This observation leads to the surprising hypothesis that agouti may control an evolutionarily conserved pigment pattern by regulating different mechanisms in mammals and fish. To test this hypothesis, we compared two ray-finned fishes: the teleost zebrafish and the nonteleost spotted gar (Lepisosteus oculatus). By examining the endogenous pattern of asip1 expression in gar, we demonstrate a dorsoventral-graded distribution of asip1 expression that is highest ventrally, similar to teleosts. Additionally, in the first reported experiments to generate zebrafish transgenic lines carrying a bacterial artificial chromosome (BAC) from spotted gar, we show that both transgenic zebrafish lines embryos replicate the endogenous asip1 expression pattern in adult zebrafish, showing that BAC transgenes from both species contain all of the regulatory elements required for regular asip1 expression within adult ray-finned fishes. These experiments provide evidence that the mechanism leading to an environmentally important pigment pattern was likely in place before the origin of teleosts.

The resolution of the gar genome affords an opportunity to examine the diversification and functional specialization of immune effector molecules at a distant and potentially informative point in phylogenetic development. Although innate immunity is effected by a particularly large number of different families of molecules, the focus here is to provide detailed characterization of several families of innate receptors that are encoded in large multigene families, for which orthologous forms can be identified in other species of bony fish but not in other vertebrate groups as well as those for which orthologs are present in other vertebrate species. The results indicate that although teleost fish and the gar, as a holostean reference species, share gene families thought previously to be restricted to the teleost fish, the manner in which the members of the multigene families of innate immune receptors have undergone diversification is different in these two major phylogenetic radiations. It appears that both the total genome duplication and different patterns of genetic selection have influenced the derivation and stabilization of innate immune genes in a substantial manner during the course of vertebrate evolution.

An ancient genome duplication (TGD or 3R) occurred in teleost fish after divergence from the lineage leading to gar. This genome duplication is shared by the three extant teleost lineages: Osteoglossomorpha (bony-tongues), Elopomorpha (eels and tarpons), and Clupeocephala (a large clade including salmon, carp, medaka, zebrafish, cichlids, pufferfish, stickleback, and ∼26,000 other species). After TGD, different clupeocephalan species retained different gene duplicates; this is seen clearly in Hox gene clusters but extends to all genes. Since divergent resolution of TGD paralogs is a potential driving force for speciation, it is possible this contributed to diversification of this clade. The extent to which divergent resolution of TGD paralogs occurred within Osteoglossomorpha has not been investigated in detail, and Hox cluster organization has been reported for just two species: Pantodon buchholzi (Pantodontidae) and Scleropages formosus (Osteoglossidae). We applied survey-scale genome sequencing and de novo assembly to three further osteoglossomorph taxa: Osteoglossum bicirrhosum (Osteoglossidae), Chitala ornata (Notopteridae), and Gnathonemus petersii (Mormyridae). We find that each retained more Hox genes than clupeocephalan taxa (excluding those that underwent additional genome duplication), but fewer than eels. Several Hox genes are missing in all teleosts, including duplicates of two Hox genes present in the slow evolving pre-TGD genome of the spotted gar. We find divergent resolution through individual gene losses, and whole cluster losses have been rampant across osteoglossomorphs, despite their extant species paucity. We suggest that reciprocal gene loss following TGD was probably insufficient to drive the exceptional diversification of teleosts.

Macroderoides luki n. sp. (Trematoda: Macroderoididae) is described from the intestine of spotted gar (Lepisosteus oculatus) collected from multiple locales on the Trinity River in eastern Texas, U.S.A. Members of the new species are most similar to Macroderoides trilobatus in that they possess an ovary composed of 3 lobes rather than an undivided ovary as in the other 6 species in North America. In the new species, the distance between the cirrus sac and ovary is far greater, the eggs are almost twice the size, and the body is longer than in M. trilobatus. This is the eighth species of Macroderoides named in North America and the largest specimens known from this group.

Genomic GC content can vary locally, and GC-rich regions are usually associated with increased DNA thermostability in thermophilic prokaryotes and warm-blooded eukaryotes. Among vertebrates, fish and amphibians appeared to possess a distinctly less heterogeneous AT/GC organization in their genomes, whereas cytogenetically detectable GC heterogeneity has so far only been documented in mammals and birds. The subject of our study is the gar, an ancient "living fossil" of a basal ray-finned fish lineage, known from the Cretaceous period. We carried out cytogenomic analysis in two gar genera (Atractosteus and Lepisosteus) uncovering a GC chromosomal pattern uncharacteristic for fish. Bioinformatic analysis of the spotted gar (Lepisosteus oculatus) confirmed a GC compartmentalization on GC profiles of linkage groups. This indicates a rather mammalian mode of compositional organization on gar chromosomes. Gars are thus the only analyzed extant ray-finned fishes with a GC compartmentalized genome. Since gars are cold-blooded anamniotes, our results contradict the generally accepted hypothesis that the phylogenomic onset of GC compartmentalization occurred near the origin of amniotes. Ecophysiological findings of other authors indicate a metabolic similarity of gars with mammals. We hypothesize that gars might have undergone convergent evolution with the tetrapod lineages leading to mammals on both metabolic and genomic levels. Their metabolic adaptations might have left footprints in their compositional genome evolution, as proposed by the metabolic rate hypothesis. The genome organization described here in gars sheds new light on the compositional genome evolution in vertebrates generally and contributes to better understanding of the complexities of the mechanisms involved in this process.

In this study, we characterize the retina of the spotted gar, Lepisosteus oculatus, a ray-finned fish. Gar did not undergo the whole genome duplication event that occurred at the base of the teleost fish lineage, which includes the model species zebrafish and medaka. The divergence of gars from the teleost lineage and the availability of a high-quality genome sequence make it a uniquely useful species to understand how genome duplication sculpted features of the teleost visual system, including photoreceptor diversity. We developed reagents to characterize the cellular organization of the spotted gar retina, including representative markers for all major classes of retinal neurons and Müller glia. We report that the gar has a preponderance of predicted short-wavelength shifted (SWS) opsin genes, including a duplicated set of SWS1 (ultraviolet) sensitive opsin encoding genes, a SWS2 (blue) opsin encoding gene, and two rod opsin encoding genes, all of which were expressed in retinal photoreceptors. We also report that gar SWS1 cones lack the geometric organization of photoreceptors observed in teleost fish species, consistent with the crystalline photoreceptor mosaic being a teleost innovation. Of note the spotted gar expresses both exo-rhodopsin (RH1-1) and rhodopsin (RH1-2) in rods. Exo-rhodopsin is an opsin that is not expressed in the retina of zebrafish and other teleosts, but rather is expressed in regions of the brain. This study suggests that exo-rhodopsin is an ancestral actinopterygian (ray finned fish) retinal opsin, and in teleosts its expression has possibly been subfunctionalized to the pineal gland.

We studied the molecular responses to different water oxygen levels in gills and swim bladder of spotted gar (Lepisosteus oculatus), a bimodal breather. Fish at swim-up stage were exposed for 71 days to normoxic, hypoxic, and hyperoxic water conditions. Then, all aquaria were switched to normoxic conditions for recovery until the end of the experiment (120 days). Fish were sampled at the beginning of the experiment, and then at 71 days of exposure and at 8 days of recovery. We first cloned three hypoxia-related genes, hypoxia-inducible factor 2α (HIF-2α), Na(+) /H(+) exchanger 1 (NHE-1), and NHE-3, and uploaded their cDNA sequences in the GeneBank database. We then used One Step Taqman® real-time PCR to quantify the mRNA copies of target genes in gills and swim bladder of fish exposed to different water O2 levels. We also determined the protein expression of HIF-2α and neuronal nitric oxide synthase (nNOS) in the swim bladder by using confocal immunofluorescence. Hypoxic stress for 71 days significantly increased the mRNA copies of HIF-2α and NHE-1 in gills and swim bladder, whereas normoxic recovery for 8 days decreased the HIF-2α mRNA copies to control values in both tissues. We did not found significant changes in mRNA copies of the NHE-3 gene in either gills or swim bladder in response to hypoxia and hyperoxia. Unlike in normoxic swim bladder, double immunohistochemical staining in hypoxic and hyperoxic swim bladder using nNOS/HIF-2α showed extensive bundles of HIF-2α-positive nerve fibers in the trabecular musculature associated with a few varicose nNOS immunoreactive nerve terminals.

In order to compare its characteristics with those of jaw tooth collar enamel, normally developing and experimentally regenerating ganoine from ganoid scales of Lepisosteus oculatus (spotted gar), an actinopterygian fish species, was examined by Western blotting and immunohistochemistry. Amelogenin, a major enamel matrix protein (EMP), is widely found from sarcopterygian fish to mammals. Therefore, we used antimammalian amelogenin antibodies and antisera: an antibody against bovine amelogenin; antiserum against porcine amelogenin; and region-specific antibodies or antiserum against the C-terminus, middle region, or N-terminus of porcine amelogenin in this study. Positive immunoreactivity with the antibody against bovine amelogenin, antiserum against porcine amelogenin, and the middle and C-terminal region-specific antibodies was detected in both normally developing and regenerating ganoine matrix, as well as in granules found within inner ganoine epithelial cells. These immunohistochemical analyses indicated that the Lepisosteus ganoine matrix contains EMP-like proteins with epitopes similar to mammalian amelogenins. In Western blotting analyses of regenerating ganoid scales with the antibovine amelogenin antibody, two protein bands with molecular weights of approximately 78 and 65 kDa were detected, which were similar to those found in Lepisosteus tooth enamel. Our study suggests that in Lepisosteus, EMP-like proteins in the ganoine matrix corresponded to those in tooth enamel. However, it was revealed that the 78 and 65 kDa EMP-like proteins were different from 27 kDa bovine amelogenin.

Peripheral populations occupy the edge of a species' range and may exhibit adaptations to potentially “harsher” marginal environments compared with core populations. The peripheral population of Spotted Gar Lepisosteus oculatus in the Great Lakes basin represents the northern edge of the species' range and is completely disjunct from the core Mississippi River basin population. Age-0 Spotted Gars from the peripheral population experience a growing season approximately half that of the core population but reach similar sizes by winter, suggesting potential for countergradient variation in growth, i.e. an evolutionary response to an environmental gradient such as latitude to compensate for the usual phenotypic effect of that gradient. In this study we used two common garden experiments to investigate potential countergradient variation in growth of young-of-year Spotted Gars from peripheral populations in comparison with those from core populations. Our first experiment showed that in a common envir...

Understanding the spatial context of genetic variation for species at risk is important for effective management and long-term survival of the species. We use multilocus microsatellite data to investigate the population genetic structure of the spotted gar (Lepisosteus oculatus) across its northern range edge in Canada. We then compare these northern individuals with samples taken from the southern core of the species range. For the northern samples, significant genetic differentiation among groups of individuals forming two major genetically distinct populations, and as many as 7-9 smaller subpopulations, was recovered using hierarchical Bayesian assignment methods and non-equilibrial discriminant function analyses. Spatial genetic variation is present, particularly at higher hierarchical groupings; however, some population admixture at sites is evident and is indicative of dispersal and gene flow among some locations or shared ancestry. Gene flow estimates among populations and subpopulations is very low, ranging from essentially complete isolation to as high as 5 %-suggesting that mechanisms in addition to geographic isolation are operating to create genetic structure. In Lake Erie, the physical isolation of Point Pelee appears to confer distinct genetic differentiation for those populations and provide a source of genetic variation for Lake Erie proper when breaches to the barrier beach occur. Results indicate that the northern edge populations are distinct from southern populations and should be conserved to maintain the overall genetic diversity of this species. Additionally, the asymmetrical genetic connectivity among the Point Pelee and Rondeau Bay sites highlights the sensitivity of Point Pelee to environmental perturbation and habitat degradation.

Recreational angling for gars Lepisosteidae has become more popular in recent years; however, the fundamental understanding of population dynamics needed for effective management and conservation is lacking. Age data are essential for describing population dynamic rate functions, but few studies have addressed selection of ideal calcified structures for use in estimating ages in gars. We collected Spotted Gars Lepisosteus oculatus, Shortnose Gars L. platostomus, and Longnose Gars L. osseus from twelve Illinois waterbodies to assess the relative bias and precision of age estimates among branchiostegal rays, pectoral fin rays, cleithra, and sagittal otoliths. Age assignments differed among calcified structures for all three species. Branchiostegal rays underestimated the age of young fish and overestimated the age of old fish relative to all other structures. Pectoral fin rays consistently underestimated age relative to other structures and produced the lowest mean and maximum age estimates. Low rel...

Elucidating the genetic mechanisms of organismal adaptation to the Tibetan Plateau at a genomic scale can provide insights into the process of adaptive evolution. Many highland species have been investigated and various candidate genes that may be responsible for highland adaptation have been identified. However, we know little about the genomic basis of adaptation to Tibet in fishes. Here, we performed transcriptome sequencing of a schizothoracine fish (Gymnodiptychus pachycheilus) and used it to identify potential genetic mechanisms of highland adaptation. We obtained totally 66,105 assembled unigenes, of which 7,232 were assigned as putative one-to-one orthologs in zebrafish. Comparative gene annotations from several species indicated that at least 350 genes lost and 41 gained since the divergence between G. pachycheilus and zebrafish. An analysis of 6,324 orthologs among zebrafish, fugu, medaka, and spotted gar identified consistent evidence for genome-wide accelerated evolution in G. pachycheilus and only the terminal branch of G. pachycheilus had an elevated Ka/Ks ratio than the ancestral branch. Many functional categories related to hypoxia and energy metabolism exhibited rapid evolution in G. pachycheilus relative to zebrafish. Genes showing signature of rapid evolution and positive selection in the G. pachycheilus lineage were also enriched in functions associated with energy metabolism and hypoxia. The first genomic resources for fish in the Tibetan Plateau and evolutionary analyses provided some novel insights into highland adaptation in fishes and served as a foundation for future studies aiming to identify candidate genes underlying the genetic bases of adaptation to Tibet in fishes.

Many fields of biology--including vertebrate Evo-Devo research--are facing an explosion of genomic and transcriptomic sequence information and a multitude of fish species are now swimming in this "genomic tsunami." Here, we first give an overview of recent developments in sequencing fish genomes and transcriptomes that identify properties of fish genomes requiring particular attention and propose strategies to overcome common challenges in fish genomics. We suggest that the generation of chromosome-level genome assemblies--for which we introduce the term "chromonome"--should be a key component of genomic investigations in fish because they enable large-scale conserved synteny analyses that inform orthology detection, a process critical for connectivity of genomes. Orthology calls in vertebrates, especially in teleost fish, are complicated by divergent evolution of gene repertoires and functions following two rounds of genome duplication in the ancestor of vertebrates and a third round at the base of teleost fish. Second, using examples of spotted gar, basal teleosts, zebrafish-related cyprinids, cavefish, livebearers, icefish, and lobefin fish, we illustrate how next generation sequencing technologies liberate emerging fish systems from genomic ignorance and transform them into a new model army to answer longstanding questions on the genomic and developmental basis of their biodiversity. Finally, we discuss recent progress in the genetic toolbox for the major fish models for functional analysis, zebrafish, and medaka, that can be transferred to many other fish species to study in vivo the functional effect of evolutionary genomic change as Evo-Devo research enters the postgenomic era.

Novel molecular data from both mitochondrial (cytochrome c oxidase subunit 1) and ribosomal regions (18S, ITS1-5.8S, ITS2, and 28S) are provided for Sebekia mississippiensis Overstreet, Se...

Connection events between rivers and their adjacent floodplains can alter availability of resources. Riverine fishes opportunistically exploit seasonally available resources during periods of high water. Gars are known to utilize floodplain habitats throughout different life stages for spawning and feeding. Food habits of gars have been the focus of many studies; however, less is known regarding the partitioning of food resources between syntopic species overlapping in body size. Further, little information exists on food resources of shortnose gar, Lepisosteus platostomus. We report results of diet analysis of spotted, Lepisosteus oculatus, and shortnose gar from the Fourche LaFave River in central Arkansas. Stomachs were examined from 74 adult spotted gar (46–81 cm TL) and 91 adult shortnose gar (49–76 cm TL) collected between May and July 2007 during flooding. Forty-seven (64%) spotted and 54 (59%) shortnose gar contained identifiable prey items. Spotted and shortnose gars had low diet overlap (33.0%), indicating these two species partition food resources. Considering percent composition by weight (%Cw), spotted and shortnose gar appear to partition available food resources during summer flooding. Percent composition by weight of fishes and crustaceans was significantly greater in the diet of spotted gar than shortnose gar (Table 2). Shortnose gar had significantly greater %Cw of amphibians and terrestrial invertebrates compared to spotted gar. It is likely that the seasonal and consistent annual availability of terrestrial subsidies to the system holds a unique importance for the shortnose gar population.

Teleost fish have been shown to contain many superfamilies of transposable elements (TEs) that are absent from most tetrapod genomes. Since theories predict an increase in TE activity following polyploidization, such diversity might be linked to the 3R whole-genome duplication that occurred approximately 300 million years ago before the teleost radiation. To test this hypothesis, we have analyzed the genome of the spotted gar Lepisosteus oculatus, which diverged from the teleost lineage before the 3R duplication. Our results indicate that TE diversity and copy numbers are similar in gar and teleost genomes, suggesting that TE diversity was ancestral and not linked to the 3R whole-genome duplication. We propose that about 25 distinct superfamilies of TEs were present in the last ancestor of gars and teleost fish about 300 million years ago in the ray-finned fish lineage.

The Alligator Gar Atractosteus spatula has become a focal species of management and conservation due to declining populations and growing popularity among anglers. The Alligator Gar is sympatric with the Longnose Gar Lepisosteus osseus and Spotted Gar L. oculatus throughout much of its range, providing the potential for hybridization that can complicate conservation efforts. Hybridization between gars has been documented in captivity; however, natural (i.e., wild) hybridization has not been formally assessed. Population sampling and genetic analyses of Alligator Gars in Texas provided an opportunity to examine hybridization among gars and to assess the potential implications for Alligator Gar management. Specifically, we (1) developed markers to distinguish lepisosteid species and their hybrids, (2) characterized hybridization between species, and (3) assessed our ability to differentiate hybrids from parent species via field evaluation of snout morphology. Fin tissue samples from Alligator Gars and putative hybrids, along with a reference sample of Longnose Gars and Spotted Gars, were genotyped for a mitochondrial locus, two nuclear introns, two nuclear exons, and nine microsatellite loci. Natural hybridization was confirmed: 17 F1 Alligator Gar (female) × Longnose Gar (male) crosses and one F1 Longnose Gar × Spotted Gar were identified, as well as three additional hybrids of uncertain ancestry. Field identification of putative hybrids based on snout morphology was conservative: 21 of 26 (81%) were correctly classified as hybrid individuals, with no false negatives. Our data confirm that sympatric lepisosteid populations do hybridize and that general monitoring of hybridization rates can be accomplished afield. We recommend monitoring of hybridization between sympatric gar populations due to the risks associated with genetic introgression, particularly in systems with depressed Alligator Gar stocks. Received September 5, 2016; accepted November 21, 2016 Published online March 10, 2017

Background In the duplication-degeneration-complementation (DDC) model, a duplicated gene has three possible fates: it may lose functionality through the accumulation of mutations (nonfunctionalization), acquire a new function (neofunctionalization), or each duplicate gene may retain a subset of functions of the ancestral gene (subfunctionalization). The role that promoter evolution plays in retention of duplicated genes in eukaryotic genomes is not well understood. Fatty acid-binding proteins (Fabp) belong to a multigene family that are highly conserved in sequence and function, but differ in their gene regulation, suggesting selective pressure is exerted via regulatory elements in the promoter. Results In this study, we describe the PPAR regulation of zebrafish fabp1a, fabp1b.1, and fabp1b.2 promoters and compare them to the PPAR regulation of the spotted gar fabp1 promoter, representative of the ancestral fabp1 gene. Evolution of the fabp1 promoter was inferred by sequence analysis, and differential PPAR-agonist activation of fabp1 promoter activity in zebrafish liver and intestine explant cells, and in HEK293A cells transiently transfected with wild-type and mutated fabp1promoter-reporter gene constructs. The promoter activity of spotted gar fabp1, representative of the ancestral fabp1, was induced by both PPARα- and PPARγ-specific agonists, but displayed a biphasic response to PPARα activation. Zebrafish fabp1a was PPARα-selective, fabp1b.1 was PPARγ-selective, and fabp1b.2 was not regulated by PPAR. Conclusions The zebrafish fabp1 promoters underwent two successive rounds of subfunctionalization with respect to PPAR regulation leading to retention of three zebrafish fabp1 genes with stimuli-specific regulation. Using a pharmacological approach, we demonstrated here the divergent regulation of the zebrafish fabp1a, fabp1b.1, and fabp1b.2 with regard to subfunctionalization of PPAR regulation following two rounds of gene duplication. Electronic supplementary material The online version of this article (doi:10.1186/s12862-016-0717-x) contains supplementary material, which is available to authorized users.

The spotted gar Lepisosteus oculatus is designated as threatened in Canada under the federal Species at Risk Act. Identification and protection of critical habitat is an important component of recovering species at risk. To understand the habitat utilization of the adult life stage of the spotted gar in Rondeau Bay, a shallow coastal wetland of Lake Erie, external radio transmitters were surgically attached to 37 specimens in May 2007. These individuals were tracked at 224 discrete locations throughout the spring and summer of 2007. Aquatic macrophytes were present at 201 (90%) of these sites. Habitat and water chemistry data were collected at all tracked locations occupied by spotted gars. On the basis of electivity indices, in spring spotted gars showed a strong preference for shallow ( 2.5-m) waters with pH values

Increased interest in the management and conservation of gars (family Lepisosteidae) has resulted in the need to identify individual fish in wild and hatchery stocks; however, tagging methods have not been evaluated for these species. We estimated tag retention for passive integrated transponder (PIT) tags, T-bar anchor tags (Floy), and coded wire tags in alligator gar Atractosteus spatula, longnose gar Lepisosteus osseus, and spotted gar L. oculatus. Tag retention up to 876 d was similar across species for all tag types and tagging locations. Retention exceeded 98% for PIT tags injected into the muscle at the base of the dorsal fin and did not decrease with days posttagging. Retention was 97% for coded wire tags injected into the muscle at the base of the pectoral and anal fins. Retention of T-bar anchor tags was ≥ 97% through 500 d posttagging; however, T-bar anchor tag retention declined after 500 d and tags became difficult to read. The high retention of the tags evaluated in this study provi...

Turbidity, and associated sedimentation, is increasing in aquatic ecosystems globally and is thought to be a major driver of aquatic biodiversity loss. In this study, hatching success of Spotted Gar (Lepisosteus oculatus), a Threatened species in Canada, is reported for eggs held under clear and turbid conditions. Spotted Gar embryos were held in either clear or mildly turbid water (∼5 NTU). Fertilized eggs held in turbid water exhibited a final 24 % reduction in hatching success by the end of the hatching period. Turbidity is identified as a potential threat for this species in Canada. The decrease in hatching success found here indicates that this early life history stage is particularly vulnerable to disturbance by turbidity and sedimentation.

Bowfishing is an understudied method of fishing that appears to be legal throughout the United States. Therefore, species composition and harvest rates were determined at six bowfishing tournaments held in Arkansas at the lower White River, the Arkansas River at Lake Dardanelle, the Arkansas River at Piney Bay, Lake DeGray, Bull Shoals Lake, and Lake Ouachita between July 1999 and May 2000. A total of 3,280 fish were harvested at the six tournaments; of this total, 2,751 fish representing 19 species were identified. Total harvest per tournament ranged from 179 to 1,674 fish and from 6 to 12 species. Mean (±SD) harvest rate for tournament participants was 3.8 ± 1.1 fish/h; among tournament winners, the harvest rate was 7.7 ± 2.8 fish/h, which appears high compared with other sport fisheries (range = 0.28–2.59 fish/h). Five species accounted for 84% of fish harvested: spotted gar Lepisosteus oculatus, common carp Cyprinus carpio, shortnose gar L. platostomus, spotted sucker Minytrema melanops, and ...

Spotted Gar (Lepisosteus oculatus), a species listed as Threatened under the Canadian Species at Risk Act (SARA) was collected during May and June, 2007 from several sites in Rondeau Bay, a shallow coastal wetland of Lake Erie. The first pectoral fin ray was removed from 78 individuals to age the fish and to determine individual growth characteristics. To assess the validity of using pectoral rays to age Spotted Gar, we compared techniques (otoliths, branchiostegal rays and pectoral rays) for ten individuals captured in southwestern Michigan. Agreement between readers and amongst the three structures was high; thus aging of Spotted Gar using sectioned pectoral rays is an effective method. Rondeau Bay specimens varied in age from 3 to 10 years and from 515 to 761 mm total length. Regression analysis of length vs. age data was calculated to be $$ {\hbox{y}} = {19}.{\hbox{217x}} + {491}.{19}\left( {{{\hbox{R}}^{{2}}} = 0.{22}} \right) $$ . The low R 2 value is attributed to having males and females, which differ in growth rates, combined. Growth rates of Rondeau Bay specimens were compared to a Louisiana population using ANCOVA. No significant difference was found in the rate of growth between these populations; however, condition was low as compared to a standard weight equation. This may lead to lower fecundity, contributing to the species’ rarity in Canada.

Paramacroderoides kinsellai n. sp. is described based on specimens collected from the spotted gar (Lepisosteus oculatus) from the Pascagoula River, Jackson County, Mississippi, and the Florida gar (Lepisosteus platyrhincus) from Cross Creek, Alachua County, Florida. The new species is the third recognized species of Paramacroderoides. Two Indian species of Paramacroderoides, originally described within Pseudoparamacroderoides, are transferred into Macroderoides. Paramacroderoides kinsellai n. sp. differs from Paramacroderoides echinus in having a much longer body, greater body width/length ratio, ovary situated at a significant distance from the cirrus sac rather than immediately posterior to it, and anterior margin of vitellaria at a significant distance posterior to ventral sucker. The new species differs from Paramacroderoides pseudoechinus in having a much longer body, greater body width/length ratio, ovary situated at a significant distance from the cirrus sac rather than immediately posterior to it, anterior margin of vitellaria at a significant distance anterior to ovary, vitellaria extending further posteriorly, and relative position of the intestinal bifurcation. The generic diagnosis of Paramacroderoides is amended to incorporate features found in the new species. This is the first record of a species of Paramacroderoides from the spotted gar and the first record from any host in Mississippi. Comparison of more than 2,300 base-pair sequences of nuclear rDNA (partial 18S, complete ITS region, and partial 28S) of 3 specimens from Florida and 2 specimens from Mississippi revealed only 1 base-pair difference between specimens collected in the 2 areas from the 2 host species. Sequence comparison between P. kinsellai n. sp. and previously published sequences of 3 Macroderoides species revealed that the levels of divergence between members of Paramacroderoides and Macroderoides are not much higher than levels of divergence among some species of Macroderoides.

Carbon dioxide (CO(2)) transport in the blood of fishes is aided by the proton-binding properties of hemoglobin (Hb) through either a high-intrinsic buffer value and small oxylabile proton binding (Haldane effect), or a low buffer value and large Haldane effect. Primitive species, such as elasmobranchs and sarcopterygians have been shown to rely on the former, while derived species, such as teleosts rely on the latter. Both strategies are effective in the transport of CO(2) in the blood. However, there is a paucity of information on the nature of the transition between these two strategies that appears to occur within the intermediate group of fishes, the basal actinopterygians. The objective of the present study was to simultaneously assess the intrinsic Hb buffer values and Haldane effects of species within the basal actinopterygian lineage to characterize the transition in Hb-proton-binding strategy seen among the fishes. Expressed in order of most basal to most derived, the species investigated included American paddlefish (Polyodon spathula), white sturgeon (Acipenser transmontanus), spotted gar (Lepisosteus oculatus), alligator gar (Atractosteus spatula), bowfin (Amia calva), and mooneye (Hiodon tergisus). Hemolysates from these species were prepared and Hb titrations (under oxygenated and deoxygenated conditions) were performed in both the presence and absence of saturating levels of organic phosphates (GTP). The findings suggest that the nature of the Hb-proton-binding transition may have been punctuated rather than gradual, with the Hb buffer value decreasing and the Haldane effect increasing significantly in bowfin from fairly steady ancestral levels in the four more basal species. This change is coupled with the initial appearance of the choroid rete, as well as an increase in the magnitude and onset pH of the Root effect in bowfin, suggesting that the change in Hb-proton-binding strategy may be associated with the evolution of enhanced O(2) delivery to the eye and an in vivo operational Root effect.

The middle Brazos River, located in east-central Texas, is a meandering lowland river with many oxbow lakes on its flood plain. Flood dynamics of the Brazos River are aseasonal, and faunal exchange during lateral connections of the main river channel and oxbows is pulse-like and occurs only during floods that may be months or years apart. Patterns of resource use among sympatric gar species (Lepisosteus oculatus, Lepisosteus osseus and Atractosteus spatula) associated with river-flood plain connectivity were studied for a period of 2 years (May 2003 to May 2005). The first year was relatively dry yielding few lateral connections, whereas the second year was relatively wet resulting in more frequent lateral connections. This study focused on habitat and diet partitioning among the three gar species in oxbow habitats with different connection frequencies and an active river channel site. Overall, 684 gars were collected with experimental gillnets: 19 A. spatula (alligator gar), 374 L. oculatus (spotted gar) and 291 L. osseus (longnose gar). There was strong partitioning of habitat between spotted and longnose gars, in which 98% of spotted gars were captured in oxbow habitats and 84% of longnose gars were captured in the river channel. Hydrology did not appear to affect habitat partitioning, although longnose gar abundance significantly increased in oxbows during the wet year. Diet overlap was high between spotted and longnose gars. Temporal variation in diet was significantly influenced by flood pulses that connected oxbows with the river channel, and which allowed predators and their prey to move between habitats.

Adult spotted gar Lepisosteus oculatus are potentially important components of food webs given their predatory habits and high relative abundance. However, few studies have examined their foraging behavior. Therefore, we examined the effects of vegetation density on the foraging success of adult spotted gars preying on bluegill Lepomis macrochirus and fathead minnow Pimephales promelas. Spotted gars use ambush foraging and were more successful capturing fathead minnow than bluegills at all stem densities. Predation success was negatively correlated with stem density for bluegill prey but not for fathead minnow. Although fathead minnow and bluegills shoaled less as stem density increased, bluegills modified their behavior in the presence of a predator by moving lower in the water column at high stem densities, whereas fathead minnow did not. Predatory behaviors were directed at prey within a short distance and near the surface of the water. Foraging of spotted gar, a lie-in-wait predator, was prim...

The spotted gar, Lepisosteus oculatus, population in the Lake Pontchartrain estuary in southeastern Louisiana was sampled monthly to characterize its reproduction, age, and growth from November 1998 through October 1999. Males ranged from 285 to 568 mm standard length (SL) and 0 to 8 years; females ranged from 395 to 606 mm SL and 0 to 10 years. Both sexes were mature before age 2. Spawning was from February to June, and fecundity was highest during October (mean number of eggs = 13,798 ± 7,654 SE) and lowest during June (mean number of eggs = 1,772 ± 392 SE). Ova diameters varied among months and among individuals within a month. Mean ova diameter during the spawning season was 3.02 mm ± 0.02 SE. Males grew significantly faster than females, but had a shorter lifespan. Condition was generally lowest during fall and varied by sex among seasons.

The structure and physiology of enteric system are very similar in all classes of vertebrates, although they have been investigated only occasionally in non-mammalian vertebrates. Very little is known about the distribution of the neurotransmitters in the gut of actinopterygian fishes. Anatomical and physiological studies of enteric nervous systems in the spotted gar (Lepisosteus oculatus) and airbreathing catfish (Clarias batrachus), a non-teleost and teleost actinopterygian, respectively, have not been undertaken. This study provides the first comprehensive characterization of the range of neurochemical coding in the enteric nervous system of these two species, including the chemical diversity of the mucosal endocrine cells in the pyloric stomach of Clarias. Autonomic innervation of the secretory glands is also described and reported herein for the first time for fishes. We also report splanchnic (spinal) innervation of the stomach, submucosal ganglia (that also colocalize with nNOS) and caudal intestine of Clarias. In both fish species, numerous 5HT, ChAT, nNOS and TH-positive nerve fibres have been observed. These discoveries demonstrate that much more physiological and pharmacological data are needed before a comprehensive model of enteric nervous system control in vertebrates can be developed.

To determine sexual dimorphism in spotted gar (Lepisosteus oculatus), variation in standard length, mass, snout length, snout width and body depth was compared between sexes using analysis of covariance. Females were significantly longer and had longer snouts when effects of variation in mass, snout width, body depth and age were accounted for. Discriminant function analysis (DFA) discriminated between sexes with 78% accuracy from 159 field collected specimens using standard length and snout length. Using coefficients from the DFA, the sex of 65 museum specimens was predicted to 85% accuracy. Sexually dimorphic standard length is probably related to gonad size differences between males and females. Reasons for snout length dimorphism are not apparent. This study demonstrates the existence of sexual dimorphism in spotted gar.

During population dynamic and reproductive biology research on three species of Lepisosteidae (i.e., alligator gar Lepisosteus spatula (also known as Atractosteus spatula), longnose gar L. osseus, and spotted gar L. oculatus), we determined that misidentification of sex through gross examination of the gonads was probable. During gross examination of the gonads of all three species, postspawning and juvenile ovaries frequently resembled testes due to the envelopment of the ovaries by fatty tissue. We examined the gonads and gamete release pathways of 598 individuals and found that gamete release pathways (i.e., oviduct in females and vasa efferentia in males) were differentiable and can be used reliably in all seasons for all reproductive stages of the three species. Misidentification of sex can lead to inaccurate estimation of sex ratios and sex-dependent mortality and growth rates. Accurate estimates of Lepisosteidae population structure and vital rates are needed for successful conservation an...

We used radiotelemetry to determine diel and seasonal movements, habitat use, and home range behavior of 37 adult spotted gars Lepisosteus oculatus in the Atchafalaya River basin, Louisiana. The Atchafalaya River exhibits a distinct spring flood pulse each year, inundating a 3,640-km2 floodplain that includes a complex network of canals, bayous, and lakes. During nonflood months, diel locations of 27 fish recorded at 2-h intervals (median = 24 locations/fish) indicated that most spotted gars were shoreline oriented (P < 0.0001), preferred submerged branches as cover, and avoided areas of exposed bank (P < 0.0001). Median spotted gar movement rates were higher during summer (40.4 m/h) than fall–winter (15.1 m/h), and during both seasons, rate of movement and percentage of home range used were significantly greater at night than during dawn, day, or dusk periods (P < 0.03). Increased nocturnal activity appeared to be related to feeding periodicity, as a substantially greater proportion (70%) of spo...

Adult, juvenile and larval fishes were sampled in a managed isolated wetland in the Red Chute Bayou floodplain, LA from December 1994 to July 1995. Nineteen species were captured. Principal components analysis of adult fishes showed little difference between sites, but a gradient of habitats and seasons reflecting variable richness was observed. Bowfin (Amia calva), black bullhead (Ameiurus meals), spotted gar (Lepisosteus oculatus), bluegill (Lepomis macrochirus), warmouth (Lepomis gulosus), black crappie (Pomoxis nigromaculatus) and fliers (Centrarchus macropterus) were the dominant adult fishes. Juvenile and post-larval fishes had low catch-per-unit-effort and were dominated by fliers. Principal components analysis distinguished three habitat groups and significant seasonal variation in the juvenile/post-larval fish community. The ichthyofauna resembled that described for irregularly inundated isolated floodplain ponds of the lower Mississippi River valley. The relatively low diversity of this...

Many osteichthyan fishes obtain oxygen from both air, using a lung, and water, using gills. Although it is commonly thought that fishes air-breathe to survive hypoxic aquatic habitats, other reasons may be more important in many species. This study was undertaken to determine the significance of air-breathing in two fish species while exercising in oxygen-rich water. Oxygen consumption from air and water was measured during mild activity in bowfin (Amia calva) and spotted gar (Lepisosteus oculatus) by sealing a fish in an acrylic flume that contained an air-hole. At 19–23°C, the rate of oxygen consumption from air in both species was modest at rest. During low-level exercise, more than 50% of the oxygen consumed by both species was from the air (53.0±22.9% L. oculatus; 66.4±8.3% A. calva).

Functionality of the tetrameric hemoglobin molecule seems to be determined by a few amino acids located in key positions. Oxygen binding encompasses structural changes at the interfaces between the α1β2 and α2β1 dimers, but also subunit interactions are important for the oxygen binding affinity and stability. The latter packing contacts include the conserved Arg B12 interacting with Phe GH5, which is replaced by Leu and Tyr in the α A and α D chains, respectively, of birds and reptiles. Searching all known hemoglobins from a variety of gnathostome species (jawed vertebrates) revealed the almost invariant Arg B12 coded by the AGG triplet positioned at an exon-intron boundary. Rare substitutions of Arg B12 in the gnathostome β globins were found in pig, tree shrew and scaled reptiles. Phe GH5 is also highly conserved in the β globins, except for the Leu replacement in the β1 globin of five marine gadoid species, gilthead seabream and the Comoran coelacanth, while Cys and Ile were found in burbot and yellow croaker, respectively. Atlantic cod β1 globin showed a Leu/Met polymorphism at position GH5 dominated by the Met variant in northwest-Atlantic populations that was rarely found in northeast-Atlantic cod. Site-specific analyses identified six consensus codons under positive selection, including 122β(GH5), indicating that the amino acid changes identified at this position may offer an adaptive advantage. In fact, computational mutation analysis showed that the replacement of Phe GH5 with Leu or Cys decreased the number of van der Waals contacts essentially in the deoxy form that probably causes a slight increase in the oxygen binding affinity. The almost invariant Arg B12 and the AGG codon seem to be important for the packing contacts and pre-mRNA processing, respectively, but the rare mutations identified might be beneficial. The Leu122β1(GH5)Met and Met55β1(D6)Val polymorphisms in Atlantic cod hemoglobin modify the intradimer contacts B12-GH5 and H2-D6, while amino acid replacements at these positions in avian hemoglobin seem to be evolutionary adaptive in air-breathing vertebrates. The results support the theory that adaptive changes in hemoglobin functions are caused by a few substitutions at key positions.

Background Although the vertebrate skeleton arose in the sea 500 million years ago, our understanding of the molecular fingerprints of chondrocytes and osteoblasts may be biased because it is informed mainly by research on land animals. In fact, the molecular fingerprint of teleost osteoblasts differs in key ways from that of tetrapods, but we do not know the origin of these novel gene functions. They either arose as neofunctionalization events after the teleost genome duplication (TGD), or they represent preserved ancestral functions that pre-date the TGD. Here, we provide evolutionary perspective to the molecular fingerprints of skeletal cells and assess the role of genome duplication in generating novel gene functions. We compared the molecular fingerprints of skeletogenic cells in two ray-finned fish: zebrafish (Danio rerio)--a teleost--and the spotted gar (Lepisosteus oculatus)--a "living fossil" representative of a lineage that diverged from the teleost lineage prior to the TGD (i.e., the teleost sister group). We analyzed developing embryos for expression of the structural collagen genes col1a2, col2a1, col10a1, and col11a2 in well-formed cartilage and bone, and studied expression of skeletal regulators, including the transcription factor genes sox9 and runx2, during mesenchymal condensation. Results Results provided no evidence for the evolution of novel functions among gene duplicates in zebrafish compared to the gar outgroup, but our findings shed light on the evolution of the osteoblast. Zebrafish and gar chondrocytes both expressed col10a1 as they matured, but both species' osteoblasts also expressed col10a1, which tetrapod osteoblasts do not express. This novel finding, along with sox9 and col2a1 expression in developing osteoblasts of both zebrafish and gar, demonstrates that osteoblasts of both a teleost and a basally diverging ray-fin fish express components of the supposed chondrocyte molecular fingerprint. Conclusions Our surprising finding that the "chondrogenic" transcription factor sox9 is expressed in developing osteoblasts of both zebrafish and gar can help explain the expression of chondrocyte genes in osteoblasts of ray-finned fish. More broadly, our data suggest that the molecular fingerprint of the osteoblast, which largely is constrained among land animals, was not fixed during early vertebrate evolution.

Prey capture in the spotted gar, Lepisosteus oculatus, was studied by high-speed cinematography synchronized with electromyographic recordings of cranial muscle activity. Muscle activity patterns were recorded during each of the three major phases of feeding: the initial strike at the prey, manipulation of the prey following capture, and swallowing. With one exception, the obliquus superioris, all muscles at the strike are active in a bilaterally symmetrical pattern. During the manipulation phase two distinct muscle activity patterns occur: one is characterized by symmetrical activity in the epaxial muscles and obliquus inferioris, the other by complete asymmetry between the right and left sternohyoideus, obliquus superioris, and epaxial muscles. Low-amplitude manipulatory movements are characterized by activity in one side of the sternohyoideus only, all other muscles being generally inactive. The adductor mandibulae and obliquus inferioris are always active symmetrically. Asymmetrical activity in the sternohyoideus, epaxial muscles, and obliquus superioris correlates with lateral head movements during feeding and acts to rotate prey into the preferred orientation for swallowing. The pattern of asymmetrical activity between right and left side muscles is discussed in relation to previous studies of feeding which utilized only unilateral muscle recordings.

During the summer of 1965, stomach contents of three species of gars from brackish water of the Mississippi Coast were examined. An attempt was made to correlate the types and amounts of food items eaten with ecological conditions which prevailed at the time of the capture. The alligator gar, Lepisosteus spatula, fed heavily on Galeichthys felis and other fishes which were periodically discarded around docks and piers as refuse. The longnose gar, L. osseus, which fed primarily on Brevoortia patronus at night, was a more active predator. In contrast, L. oculatus, the spotted gar, was found to prey extensively on Uca pugnax and Callinectes sapidus in the shallows during rising and high tides.

Fish populations in seven backwater lakes were sampled by rotenone poisoning. On the average, 397 pounds of fish per acre comprising 103 pounds of game fish and 169 pounds of commercial fish were recovered from the lakes. Recoveries from individual lakes ranged from 142 to 651 pounds of fish per acre. Non-predaceous fish made up 56 percent of the total population. Bluegill sunfish, warmouth sunfish, gizzard shad, fresh-water drum, largemouth bass, black crappie and spotted gar were the principal fishes in the lakes. An average of 78 pounds of available game fish (harvestable-size game fish) per acre was recovered from the lakes. Fairly large concentrations of fish occurred in relatively small areas in some of the backwater lakes. A total of 974 pounds of fish, of which 475 pounds were game fish, was recovered from a single 1 acre sampling area. On the average, there were 1.9 pounds of non-predaceous fish per pound of predaceous fish. There were considerably less available forage fish (by weight) ...

Several factors influencing the rate of aerial breathing and metabo- lism of young-of-year gars were studied. Without access to atmospheric oxygen, the lethal oxygen concentration was found to be approximately 2.45 mg/1 at 73F. The average rate of oxygen consumption of these fishes was 0.295 ml 0,/gm/hr. Pre- vention of aerial breathing produced no observable ill effects on the gars tested pro- vided the dissolved oxygen content of the water was above 3 mg/1. Undoubtedly, the gar's air bladder serves a hydrostatic function as well as a respiration function, and thus complicates the analysis of aerial breathing of these fishes. The temperature acclimation time of alligator gar, based on the rate of aerial breathing, requires ap- proximately 48 hours. INTRODUCTION. Biologists who first described the habits of Lep- isosteus believed that the swim bladder of this fish preformed a respi- ratory function. This belief was based on several observations: (1) the internal surface of the walls of the swim bladder are cellular and spongy; (2) a rich network of capillaries supplies blood to this organ; (3) a pneumatic duct connects the swim bladder with the pharynx and a glottis is present at this connection; and (4) gars often appear to gulp air. Investigations by Potter (1927) showed that gar could live 20 days in deoxygenated water if permitted to gulp air at the surface. Con- versely, he found that gar could live and apparently remain healthy for several days when confined below the surface, if they were main- tained in oxygenated water. He also demonstrated that there was an exchange of gases in the swim bladder resulting in a decrease in the percentage of oxygen and an increase in the percentage of carbon dioxide between inhalations. From this, he concluded that the swim bladder of Lepisosteus is an organ of supplementary respiration. Working with the longnose gar, L. osseus, and the spotted gar, L. oculatus, Saksena (1963) showed a significant increase in the rate of aerial breathing for both species with a rise in water temperature. He 45

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