The effects of salinity on swimming performance of two estuarine fishes,Fundulus heteroclitus and Fundulus majalis

Prolonged and high‐speed swimming performance measurements were used to explore the swimming abilities of two species of estuarine fishes, the mummichog Fundulus heteroclitus and the striped killifish Fundulus majalis, under different salinities. Critical swimming performance was significantly higher for F. majalis in high salinity than in low salinity, but no difference was observed in brief constant acceleration swimming trials in this species; however, the swimming performance of F. heteroclitus was not significantly affected by salinity changes, indicating that this species is well adapted to regular estuarine salinity oscillations. Fundulus majalis displayed higher swimming speeds than F. heteroclitus in both high and low salinities, and while this cannot be explained by their respective salinity preferences, the specific habitat preferences of F. majalis for sandy subtidal habitats and F. heteroclitus for vegetated marshes could explain the better swimming performance of F. majalis.     

Fundulus as the premier teleost model in environmental biology: opportunities for new insights using genomics

A strong foundation of basic and applied research documents that the estuarine fish Fundulus heteroclitus and related species are unique laboratory and field models for understanding how individuals and populations interact with their environment. In this paper we summarize an extensive body of work examining the adaptive responses of Fundulus species to environmental conditions, and describe how this research has contributed importantly to our understanding of physiology, gene regulation, toxicology, and ecological and evolutionary genetics of teleosts and other vertebrates. These explorations have reached a critical juncture at which advancement is hindered by the lack of genomic resources for these species. We suggest that a more complete genomics toolbox for F. heteroclitus and related species will permit researchers to exploit the power of this model organism to rapidly advance our understanding of fundamental biological and pathological mechanisms among vertebrates, as well as ecological strategies and evolutionary processes common to all living organisms.     

Phenotypic plasticity in gene expression contributes to divergence of locally adapted populations of Fundulus heteroclitus

We examine the interaction between phenotypic plasticity and evolutionary adaptation using muscle gene expression levels among populations of the fish Fundulus heteroclitus acclimated to three temperatures. Our analysis reveals shared patterns of phenotypic plasticity due to thermal acclimation as well as non‐neutral patterns of variation among populations adapted to different thermal environments. For the majority of significant differences in gene expression levels, phenotypic plasticity and adaptation operate on different suites of genes. The subset of genes that demonstrate both adaptive differences and phenotypic plasticity, however, exhibit countergradient variation of expression. Thus, expression differences among populations counteract environmental effects, reducing the phenotypic differentiation between populations. Finally, gene‐by‐environment interactions among genes with non‐neutral patterns of expression suggest that the penetrance of adaptive variation depends on the environmental conditions experienced by the individual.     

Competition and Coexistence Among Four Estuarine Species of Fundulus

Four sympatric species of Fundulus (F. heteroclitus, F. majalis,F. diaphanus, and F. luciae) are distributed along tidal heightand salinity gradients such that F. heteroclitus co-occurs witheach species, while the remaining species rarely occur together.Feeding habitats of all the species are similar, and food hasbeen shown to limit population size of F. heteroclitus. Thispaper examines whether competition is an important structuringforce within this guild by addressing two questions: 1) is thespatial separation exhibited by three of the species due tophysiological barriers or due to competitive exlusion? and 2)when F. heteroclitus occurs with other Fundulus species doescompetition for resources take place? Laboratory studies indicate that all four species are tolerantof a wide range of environmental conditions; available evidencesuggests that physiological barriers are an unlikely explanationfor spatial segregation among some members of this guild. Competitiveexclusion seems a likely alternate explanation, but is supportedonly by studies of diet overlap. Similarly, only inference fromdiet overlap is available to answer the second question. A fieldexperiment is presented here in which F. heteroclitus and F.majalis were placed in enclosures separately and together. Competitionbetween these species appears to be important, and similar fieldexperiments are recommended to investigate competitive interactionsamong other species pairs within the genus     

Heterogeneity and concordance in locus‐specific differentiation and introgression between species of towhees

The maintenance or breakdown of reproductive isolation is an observable outcome of secondary contact between species. In cases where hybrids beyond the F1 are formed, the representation of each species' ancestry can vary dramatically among genomic regions. This genomic heterogeneity in ancestry and introgression can offer insight into evolutionary processes, particularly if introgression is compared in multiple hybrid zones. Similarly, considerable heterogeneity exists across the genome in the extent to which populations and species have diverged, reflecting the combined effects of different evolutionary processes on genetic variation. We studied hybridization across two hybrid zones of two phenotypically well‐differentiated bird species in Mexico (Pipilo maculatus and P. ocai), to investigate genomic heterogeneity in differentiation and introgression. Using genotyping‐by‐sequencing (GBS) and hierarchical Bayesian models, we genotyped 460 birds at over 41 000 single nucleotide polymorphism (SNP) loci. We identified loci exhibiting extreme introgression relative to the genome‐wide expectation using a Bayesian genomic cline model. We also estimated locus‐specific F_ST and identified loci with exceptionally high genetic divergence between the parental species. We found some concordance of locus‐specific introgression in the two independent hybrid zones (6–20% of extreme loci shared across zones), reflecting areas of the genome that experience similar gene flow when the species interact. Additionally, heterogeneity in introgression and divergence across the genome revealed another subset of loci under the influence of locally specific factors. These results are consistent with a history in which reproductive isolation has been influenced by a common set of loci in both hybrid zones, but where local environmental and stochastic factors also lead to genomic differentiation.     

Fundulus heteroclitus: ovarian reproductive physiology and the impact of environmental contaminants

Fundulus heteroclitus, the mummichog or Atlantic killifish, is the dominant small-bodied fish species of the east coast estuaries and salt marshes of Canada and the USA, where it is present as two subspecies, the northern F. h. macrolepidotus and the southern F. h. heteroclitus. Recently identified as the premier teleost model in environmental biology, the species has long been of value in understanding evolved tolerance to toxicants and more lately in adding to our knowledge about reproductive effects of environmental endocrine disruptors. The body of literature on F. heteroclitus ovarian physiology and reproduction, from both field and laboratory studies, provides the foundation for present work focused on understanding the reproductive effects and modes of action of environmental toxicants. In this paper, we review the environmental and endocrine factors controlling ovarian and reproductive cycling in F. heteroclitus, noting specifics related to field and laboratory studies on the two subspecies as well as key research gaps compared to other fish species. We also summarize recent development of methodologies to study the effects of environmental contaminants on endocrine signalling and egg production in F. heteroclitus. Continued efforts to progress both our fundamental understanding of reproductive physiology in mummichog, coupled with studies focused on the modes of action of environmental contaminants, have high potential to further develop this teleost model. While the model may presently lag behind those based on other species of fish, the unique biochemical and physiological adaptations which allow F. heteroclitus to adapt to changing environmental and toxic conditions provide a valuable experimental system for comparative physiologists, ecotoxicologists and evolutionary biologists.     

Divergent selection and drift shape the genomes of two avian sister species spanning a saline–freshwater ecotone

The role of species divergence due to ecologically based divergent selection—or ecological speciation—in generating and maintaining biodiversity is a central question in evolutionary biology. Comparison of the genomes of phylogenetically related taxa spanning a selective habitat gradient enables discovery of divergent signatures of selection and thereby provides valuable insight into the role of divergent ecological selection in speciation. Tidal marsh ecosystems provide tractable opportunities for studying organisms' adaptations to selective pressures that underlie ecological divergence. Sharp environmental gradients across the saline–freshwater ecotone within tidal marshes present extreme adaptive challenges to terrestrial vertebrates. Here, we sequence 20 whole genomes of two avian sister species endemic to tidal marshes—the saltmarsh sparrow (Ammospiza caudacutus) and Nelson's sparrow (A. nelsoni)—to evaluate the influence of selective and demographic processes in shaping genome‐wide patterns of divergence. Genome‐wide divergence between these two recently diverged sister species was notably high (genome‐wide F_ST = 0.32). Against a background of high genome‐wide divergence, regions of elevated divergence were widespread throughout the genome, as opposed to focused within islands of differentiation. These patterns may be the result of genetic drift resulting from past tidal march colonization events in conjunction with divergent selection to different environments. We identified several candidate genes that exhibited elevated divergence between saltmarsh and Nelson's sparrows, including genes linked to osmotic regulation, circadian rhythm, and plumage melanism—all putative candidates linked to adaptation to tidal marsh environments. These findings provide new insights into the roles of divergent selection and genetic drift in generating and maintaining biodiversity.     

Factors affecting plasticity in whole-organism thermal tolerance in common killifish (<Emphasis Type="Italic">Fundulus heteroclitus</Emphasis>)

We characterized the degree of plasticity in thermal tolerance (assessed as critical thermal maxima; CTMax) and the relationship between thermal tolerance and underlying physiological and biochemical factors in two subspecies of a teleost fish, Fundulus heteroclitus. CTMax was not affected by repeated daily heat shock, but increased within a few days in response to warm acclimation. Loss of tolerance with acclimation to lowered temperatures occurred more slowly. Exposure to hypoxia decreased CTMax, and hyperoxia had no effect. CTMax showed a daily rhythm in both subspecies. Thermal acclimation changed the value of CTMax but did not affect the amplitude of the rhythm. Exposure to altered photoperiod had complex effects with a summer photoperiod producing a daily rhythm at higher CTMax than a spring photoperiod, and a winter photoperiod removing the rhythm. There was no daily rhythm in routine metabolic rate in either subspecies. There was no relationship between CTMax and the protein levels of the constitutive 70 and 90 kDa heat shock proteins (HSC70, HSP90β) in gill, or with mRNA levels of hsc70 in liver. There was a daily rhythm in the basal levels of the inducible hsp70-2 mRNA. Induction of hsp70-2 mRNA with mild heat shock occurred only in the evening and at night, and not during the day. These results demonstrate that there is substantial plasticity of thermal tolerance in killifish, and that this plasticity does not differ between subspecies. CTMax has a complex relationship with physiological and biochemical mechanisms that have been hypothesized to affect thermal tolerance.     

Reproductive isolating mechanisms contributing to asymmetric hybridization in Killifishes (Fundulus spp.)

When species hybridize, one F1 hybrid cross type often predominates. Such asymmetry can arise from differences in a variety of reproductive barriers, but the relative roles and concordance of pre-mating, post-mating prezygotic, and post-zygotic barriers in producing these biases in natural animal populations have not been widely investigated. Here, we study a population of predominantly F1 hybrids between two killifish species (Fundulus heteroclitus and F. diaphanus) in which >95% of F1 hybrids have F. diaphanus mothers and F. heteroclitus fathers (D♀ × H♂). To determine why F. heteroclitus × F. diaphanus F1 hybrids (H♀ × D♂) are so rare, we tested for asymmetry in pre-mating reproductive barriers (female preference and male aggression) at a common salinity (10 ppt) and post-mating, pre-zygotic (fertilization success) and post-zygotic (embryonic development time and hatching success) reproductive barriers at a range of ecologically relevant salinities (0, 5, 10, and 15 ppt). We found that F. heteroclitus females preferred conspecific males, whereas F. diaphanus females did not, matching the observed cross bias in the wild. Naturally rare H♀ × D♂ crosses also had lower fertilization success than all other cross types, and a lower hatching success than the prevalent D♀ × H♂ crosses at the salinity found in the hybrid zone centre (10 ppt). Furthermore, the naturally predominant D♀ × H♂ crosses had a higher hatching success than F. diaphanus crosses at 10 ppt, which may further increase their relative abundance. The present study suggests that a combination of incomplete mating, post-mating pre-zygotic and post-zygotic reproductive isolating mechanisms act in concert to produce hybrid asymmetry in this system.     

Early Responses of Fishes and Crustaceans to Restoration of a Tidally Restricted New England Salt Marsh

Nekton (fishes and decapod crustaceans) is an abundant and productive faunal component of salt marshes, yet nekton responses to tidal manipulations of New England salt marshes remain unclear. This study examined nekton use of a tidally restricted salt marsh in Narragansett, Rhode Island relative to an unrestricted marsh during summer. In addition, a before‐after‐control‐impact design was used to examine early responses of nekton to the reintroduction of natural tidal flushing. Species richness and densities of Cyprinodon variegatus, Lucania parva, Menidia beryllina, and Palaemonetes pugio were higher in the restricted marsh compared with the unrestricted marsh. The unrestricted marsh supported higher densities of Menidia menidia and Fundulus majalis. Mean lengths of Carcinus maenas and P. pugio were greater in the restricted marsh. Tidal restoration resulted in increased tidal flushing, salinity, and water depth in the restricted marsh. Densities of Fundulus heteroclitus, F. majalis, and Callinectes sapidus were higher after 2 years of restoration. Density of L. parva decreased after restoration, probably in response to a loss of macroalgal habitat. Species richness also decreased after 2 years, from 20.9 species when the marsh was restricted to 13.0 species. Total nekton density did not change with restoration, but shifts in community composition were evident. In this study restoration induced rapid changes in the composition, density, size, and distribution of nekton species, but additional monitoring is necessary to quantify longer‐term effects of salt marsh restoration on nekton.     

Applicability of RAD‐tag genotyping for interfamilial comparisons: empirical data from two cetaceans

Restriction‐site‐associated DNA tag (RAD‐tag) sequencing has become a popular approach to generate thousands of SNPs used to address diverse questions in population genomics. Comparatively, the suitability of RAD‐tag genotyping to address evolutionary questions across divergent species has been the subject of only a few recent studies. Here, we evaluate the applicability of this approach to conduct genome‐wide scans for polymorphisms across two cetacean species belonging to distinct families: the short‐beaked common dolphin (Delphinus delphis; n = 5 individuals) and the harbour porpoise (Phocoena phocoena; n = 1 individual). Additionally, we explore the effects of varying two parameters in the Stacks analysis pipeline on the number of loci and level of divergence obtained. We observed a 34% drop in the total number of loci that were present in all individuals when analysing individuals from the distinct families compared with analyses restricted to intraspecific comparisons (i.e. within D. delphis). Despite relatively stringent quality filters, 3595 polymorphic loci were retrieved from our interfamilial comparison. Cetaceans have undergone rapid diversification, and the estimated divergence time between the two families is relatively recent (14–19 Ma). Thus, our results showed that, for this level of divergence, a large number of orthologous loci can still be genotyped using this approach, which is on par with two recent in silico studies. Our findings constitute one of the first empirical investigations using RAD‐tag sequencing at this level of divergence and highlights the great potential of this approach in comparative studies and to address evolutionary questions.     

Molecular cloning and brain distribution of three types of gonadotropin‐releasing hormone from mummichog Fundulus heteroclitus

Complementary DNAs encoding gonadotropin‐releasing hormone (GnRH) precursors were cloned from the mummichog Fundulus heteroclitus brain, showing that this species has three GnRH forms, i.e. medaka Oryzias latipes GnRH (mdGnRH), chicken GnRH‐II (cGnRH‐II) and Atlantic salmon Salmo salar GnRH (sGnRH). The F. heteroclitus prepro GnRHs have common structural architectures of vertebrate GnRHs, consisting of the signal peptide, 10 amino acids of mature peptide, GKR sequence and GnRH‐associated peptide (GAP). Phylogenetic analysis of fish prepro GnRHs showed that F. heteroclitus mdGnRH is a homologue of sbGnRHs and mdGnRHs of other acanthopterygian. Quantitative real‐time PCR revealed that mdGnRH was abundantly expressed in the olfactory bulb and in olfactory lobe areas and is expressed in the pituitary. The cGnRH‐II was mainly expressed in the midbrain and interbrain areas, and the sGnRH was expressed not only in the olfactory bulb but also in other regions of the brain. These results suggest that the mdGnRH is involved in the stimulation of gonadotrophs in the pituitary, whereas cGnRH‐II and sGnRH are involved in neurotransmission and neuromodulation.     

Evolutionary conservation,diversity and specificity of LTR‐retrotransposons in flowering plants: insights from genome‐wide analysis and multi‐specific comparison

The availability of complete or nearly complete genome sequences from several plant species permits detailed discovery and cross‐species comparison of transposable elements (TEs) at the whole genome level. We initially investigated 510 long terminal repeat‐retrotransposon (LTR‐RT) families comprising 32 370 elements in soybean (Glycine max (L.) Merr.). Approximately 87% of these elements were located in recombination‐suppressed pericentromeric regions, where the ratio (1.26) of solo LTRs to intact elements (S/I) is significantly lower than that of chromosome arms (1.62). Further analysis revealed a significant positive correlation between S/I and LTR sizes, indicating that larger LTRs facilitate solo LTR formation. Phylogenetic analysis revealed seven Copia and five Gypsy evolutionary lineages that were present before the divergence of eudicot and monocot species, but the scales and timeframes within which they proliferated vary dramatically across families, lineages and species, and notably, a Copia lineage has been lost in soybean. Analysis of the physical association of LTR‐RTs with centromere satellite repeats identified two putative centromere retrotransposon (CR) families of soybean, which were grouped into the CR (e.g. CRR and CRM) lineage found in grasses, indicating that the ‘functional specification’ of CR pre‐dates the bifurcation of eudicots and monocots. However, a number of families of the CR lineage are not concentrated in centromeres, suggesting that their CR roles may now be defunct. Our data also suggest that the envelope‐like genes in the putative Copia retrovirus‐like family are probably derived from the Gypsy retrovirus‐like lineage, and thus we propose the hypothesis of a single ancient origin of envelope‐like genes in flowering plants.     

The effect of acute warming and thermal acclimation on maximum heart rate of the common killifish Fundulus heteroclitus

Common killifish Fundulus heteroclitus were acclimated to ecologically relevant temperatures (5, 15 and 33°C) and their maximum heart rate (f_Hmax) was measured at each acclimation temperature during an acute warming protocol. Acclimation to 33°C increased peak f_Hmax by up to 32% and allowed the heart to beat rhythmically at a temperature 10°C higher when compared with acclimation to 5°C. Independent of acclimation temperature, peak f_Hmax occurred about 3°C cooler than the temperature that first produced cardiac arrhythmias. Thus, when compared with previously published values for the critical thermal maximum of F. heteroclitus, the temperature for peak f_Hmax was cooler and the temperature that first produced cardiac arrhythmias was similar to these critical thermal maxima. The considerable thermal plasticity of f_Hmax demonstrated in the present study is entirely consistent with eurythermal ecology of killifish, as shown previously for another eurythermal fish Gillichthys mirabilis.     

Salinity affects growth but not thermal preference of adult mummichogs Fundulus heteroclitus

The effects of an ecologically relevant range of salinities (2, 12, 22, 32) on thermal preferences and growth of adult mummichogs Fundulus heteroclitus were determined for fish from a southern Chesapeake Bay population. Salinity did not affect the mean temperature selected by F. heteroclitus in a thermal gradient, which was identified as 26.6°C based on observations of 240 individuals. Salinity and temperature had significant and interacting effects on growth rates of F. heteroclitus measured over 12 weeks. Growth rates were highest overall and remained high over a broader range of temperatures at moderate salinities (12 and 22), while high growth rates were shifted toward lower temperatures for fish grown at a salinity of 2 and higher temperatures at a salinity of 32. Significant reductions in growth relative to the optimal conditions (28.6°C, salinity of 22) were observed at the coolest (19.6°C) and warmest (33.6°C) temperature tested at all salinities, as well as temperatures ≥ 26.6°C at a salinity of 2, ≥ 28.6°C at a salinity of 12 and ≤ 26.6°C at a salinity of 32. Growth rates provide a long-term, organismal measure of performance and results of this study indicate that performance may be reduced under conditions that the highly euryhaline F. heteroclitus can otherwise easily tolerate. The combination of reduced salinity and increased temperature that is predicted for temperate estuaries as a result of climate change may have negative effects on growth of this ecologically important species.     

Phenotypic plasticity and divergence in gene expression

The extent to which phenotypic plasticity, or the ability of a single genotype to produce different phenotypes in different environments, impedes or promotes genetic divergence has been a matter of debate within evolutionary biology for many decades (see, for example, Ghalambor et al. 2007 ; Pfennig et al. 2010 ). Similarly, the role of evolution in shaping phenotypic plasticity remains poorly understood (Pigliucci 2005 ). In this issue of Molecular Ecology, Dayan et al. ( 2015 ) provide empirical data relevant to these questions by assessing the extent of plasticity and divergence in the expression levels of 2272 genes in muscle tissue from killifish (genus Fundulus) exposed to different temperatures. F. heteroclitus (Fig.  1 A) and F. grandis are minnows that inhabit estuarine marshes (Fig.  1 B) along the coasts of the Atlantic Ocean and Gulf of Mexico in North America. These habitats undergo large variations in temperature both daily and seasonally, and these fish are known to demonstrate substantial phenotypic plasticity in response to temperature change (e.g. Fangue et al. 2006 ). Furthermore, the range of F. heteroclitus spans a large latitudinal gradient of temperatures, such that northern populations experience temperatures that are on average ~10°C colder than do southern populations (Schulte 2007 ). By comparing gene expression patterns between populations of these fish from different thermal habitats held in the laboratory at three different temperatures, Dayan et al. ( 2015 ) address two important questions regarding the interacting effects of plasticity and evolution: (i) How does phenotypic plasticity affect adaptive divergence? and (ii) How does adaptive divergence affect plasticity?