Paralog switching facilitates diadromy: ontogenetic, microevolutionary and macroevolutionary evidence.

Identifying how the demands of migration are met at the level of gene expression is critical for understanding migratory physiology and can potentially reveal how migratory forms evolve from nonmigratory forms and vice versa. Among fishes, migration between freshwater and seawater (diadromy) requires considerable osmoregulatory adjustments, powered by the ion pump Na+, K+-ATPase (NKA) in the gills. Paralogs of the catalytic α-subunit of the pump (NKA α1a and α1b) are reciprocally upregulated in fresh- and seawater, a response known as paralog-switching, in gills of some diadromous species. We tested ontogenetic changes in NKA α-subunit paralog expression patterns, comparing pre-migrant and migrant alewife (Alosa pseudoharengus) sampled in their natal freshwater environment and after 24 h in seawater. In comparison to pre-migrants, juvenile out-migrants exhibited stronger paralog switching via greater downregulation of NKA α1a in seawater. We also tested microevolutionary changes in the response, exposing juvenile diadromous and landlocked alewife to freshwater (0 ppt) and seawater (30 ppt) for 2, 5, and 15 days. Diadromous and landlocked alewife exhibited salinity-dependent paralog switching, but levels of NKA α1b transcription were higher and the decrease in NKA α1a was greater after seawater exposure in diadromous alewife. Finally, we placed alewife α-subunit NKA paralogs in a macroevolutionary context. Molecular phylogenies show alewife paralogs originated independently of paralogs in salmonids and other teleosts. This study demonstrated that NKA paralog switching is tied to halohabitat profile and that duplications of the NKA gene provided the substrate for multiple, independent molecular solutions that support a diadromous life history.

Forecasting hydrologic controls on juvenile anadromous fish out-migration with process-based modeling and machine learning.

River herring (Alosa sp.) are ecologically and economically foundational species in freshwater streams, estuaries, and oceanic ecosystems. The migration between fresh and saltwater is a key life stage of river herring, where the timing and magnitude of out-migration by juveniles can be limited when streams dry and hydrologic connectivity is lost. Operational decisions by water managers (e.g., restricting community water use) can impact out-migration success; however, these decisions are largely made without reliable predictions of outmigration potential across the migration season. This research presents a model to generate short-term forecasts of the probability of herring out-migration loss. We monitored streamflow and herring out-migration for 2 years at three critical runs along Long Island Sound (CT, USA) to develop empirical understandings of the hydrologic controls on out-migration. We used calibrated Soil and Water Assessment Tool hydrologic models of each site to generate 10,000 years of daily synthetic meteorological and streamflow records. These synthetic meteorological and streamflow data were used to train random forest models to provide rapid within-season forecasts of out-migration loss from two simple predictors: current spawning reservoir depth and the previous 30-day precipitation total. The resulting models were approximately 60%-80% accurate with a 1.5-month lead time and 70-90% accurate within 2 weeks. We anticipate that this tool will support regional decisions on spawning reservoir operations and community water withdrawals. The architecture of this tool provides a framework to facilitate broader predictions of the ecological consequences of streamflow connectivity loss in human-impacted watersheds.

Direct and size-mediated effects of temperature and ration-dependent growth rates on energy reserves in juvenile anadromous alewives (Alosa pseudoharengus).

Growth rate and energy reserves are important determinants of fitness and are governed by endogenous and exogenous factors. Thus, examining the influence of individual and multiple stressors on growth and energy reserves can help estimate population health under current and future conditions. In young anadromous fishes, freshwater habitat quality determines physiological state and fitness of juveniles emigrating to marine habitats. In this study, the authors tested how temperature and food availability affect survival, growth and energy reserves in juvenile anadromous alewives (Alosa pseudoharengus), a forage fish distributed along the eastern North American continent. Field-collected juvenile anadromous A. pseudoharengus were exposed for 21 days to one of two temperatures (21°C and 25°C) and one of two levels of food rations (1% or 2% tank biomass daily) and compared for differences in final size, fat mass-at-length, lean mass-at-length and energy density. Increased temperature and reduced ration both led to lower growth rates, and the effect of reduced ration was greater at higher temperature. Fat mass-at-length decreased with dry mass, and energy density increased with total length, suggesting size-based endogenous influences on energy reserves. Lower ration also directly decreased fat mass-at-length, lean mass-at-length and energy density. Given the fitness implications of size and energy reserves, temperature and food availability should be considered important indicators of nursery habitat quality and incorporated in A. pseudoharengus life-history models to improve forecasting of population health under climate change.

Transcriptomic imprints of adaptation to fresh water: parallel evolution of osmoregulatory gene expression in the Alewife.

Comparative approaches in physiological genomics offer an opportunity to understand the functional importance of genes involved in niche exploitation. We used populations of Alewife (Alosa pseudoharengus) to explore the transcriptional mechanisms that underlie adaptation to fresh water. Ancestrally anadromous Alewives have recently formed multiple, independently derived, landlocked populations, which exhibit reduced tolerance of saltwater and enhanced tolerance of fresh water. Using RNA-seq, we compared transcriptional responses of an anadromous Alewife population to two landlocked populations after acclimation to fresh (0 ppt) and saltwater (35 ppt). Our results suggest that the gill transcriptome has evolved in primarily discordant ways between independent landlocked populations and their anadromous ancestor. By contrast, evolved shifts in the transcription of a small suite of well-characterized osmoregulatory genes exhibited a strong degree of parallelism. In particular, transcription of genes that regulate gill ion exchange has diverged in accordance with functional predictions: freshwater ion-uptake genes (most notably, the 'freshwater paralog' of Na+ /K+ -ATPase α-subunit) were more highly expressed in landlocked forms, whereas genes that regulate saltwater ion secretion (e.g. the 'saltwater paralog' of NKAα) exhibited a blunted response to saltwater. Parallel divergence of ion transport gene expression is associated with shifts in salinity tolerance limits among landlocked forms, suggesting that changes to the gill's transcriptional response to salinity facilitate freshwater adaptation.

Relaxed selection causes microevolution of seawater osmoregulation and gene expression in landlocked Alewives.

Ecological transitions from marine to freshwater environments have been important in the creation of diversity among fishes. Evolutionary changes associated with these transitions likely involve modifications of osmoregulatory function. In particular, relaxed selection on hypo-osmoregulation should strongly affect animals that transition into novel freshwater environments. We used populations of the Alewife (Alosa pseudoharengus) to study evolutionary shifts in hypo-osmoregulatory capacity and ion regulation associated with freshwater transitions. Alewives are ancestrally anadromous, but multiple populations in Connecticut have been independently restricted to freshwater lakes; these landlocked populations complete their entire life cycle in freshwater. Juvenile landlocked and anadromous Alewives were exposed to three salinities (1, 20 and 30 ppt) in small enclosures within the lake. We detected strong differentiation between life history forms: landlocked Alewives exhibited reduced seawater tolerance and hypo-osmoregulatory performance compared to anadromous Alewives. Furthermore, gill Na(+)/K(+)-ATPase activity and transcription of genes for seawater osmoregulation (NKCC-Na(+)/K(+)/2Cl(-) cotransporter and CFTR-cystic fibrosis transmembrane conductance regulator) exhibited reduced responsiveness to seawater challenge. Our study demonstrates that adaptations of marine-derived species to completely freshwater life cycles involve partial loss of seawater osmoregulatory performance mediated through changes to ion regulation in the gill.

Introduction to "HaloDaSH: The Deep and Shallow History of Aquatic Life's Passages Between Marine and Freshwater Habitats".

This series of papers highlights research into how biological exchanges between salty and freshwater habitats have transformed the biosphere. Life in the ocean and in freshwaters have long been intertwined; multiple major branches of the tree of life originated in the oceans and then adapted to and diversified in freshwaters. Similar exchanges continue to this day, including some species that continually migrate between marine and fresh waters. The series addresses key themes of transitions, transformations, and current threats with a series of questions: When did major colonizations of fresh waters happen? What physiographic changes facilitated transitions? What organismal characteristics facilitate colonization? Once a lineage has colonized freshwater, how frequently is there a return to the sea? Have transitions impelled diversification? How do organisms adapt physiologically to changes in halohabitat, and are such adaptive changes predictable? How do marine and freshwater taxa differ in morphology? How are present-day global changes in the environment influencing halohabitat and how are organisms contending with them? The purpose of the symposium and the papers in this volume is to integrate findings at multiple levels of biological organization and from disparate fields, across biological and geoscience disciplines.

Identification of supraoptimal temperatures in juvenile blueback herring (Alosa aestivalis) using survival, growth rate and scaled energy reserves.

For young fishes, growth of somatic tissues and energy reserves are critical steps for survival and progressing to subsequent life stages. When thermal regimes become supraoptimal, routine metabolic rates increase and leave less energy for young fish to maintain fitness-based activities and, in the case of anadromous fishes, less energy to prepare for emigration to coastal habitats. Thus, understanding how energy allocation strategies are affected by thermal regimes in young anadromous fish will help to inform climate-ready management of vulnerable species and their habitat. Blueback herring (Alosa aestivalis) are an anadromous fish species that remain at historically low population levels and are undergoing southern edge-range contraction, possibly due to climate change. We examined the effects of temperature (21°C, 24°C, 27°C, 30°C, 33°C) on survival, growth rate and energy reserves of juveniles collected from the mid-geographic range of the species. We identified a strong negative relationship between temperature and growth rate, resulting in smaller juveniles at high temperatures. We observed reduced survival at both 21°C and 33°C, increased fat and lean mass-at-length at high temperatures, but no difference in energy density. Juveniles were both smaller and contained greater scaled energy reserves at higher temperatures, indicating growth in length is more sensitive to temperature than growth of energy reserves. Currently, mid-geographic range juvenile blueback herring populations may be well suited for local thermal regimes, but continued warming could decrease survival and growth rates. Blueback herring populations may benefit from mitigation actions that maximize juvenile energy resources by increasing the availability of cold refugia and food-rich habitats, as well as reducing other stressors such as hypoxic zones.

Repeated Genetic Targets of Natural Selection Underlying Adaptation of Fishes to Changing Salinity.

Ecological transitions across salinity boundaries have led to some of the most important diversification events in the animal kingdom, especially among fishes. Adaptations accompanying such transitions include changes in morphology, diet, whole-organism performance, and osmoregulatory function, which may be particularly prominent since divergent salinity regimes make opposing demands on systems that maintain ion and water balance. Research in the last decade has focused on the genetic targets underlying such adaptations, most notably by comparing populations of species that are distributed across salinity boundaries. Here, we synthesize research on the targets of natural selection using whole-genome approaches, with a particular emphasis on the osmoregulatory system. Given the complex, integrated and polygenic nature of this system, we expected that signatures of natural selection would span numerous genes across functional levels of osmoregulation, especially salinity sensing, hormonal control, and cellular ion exchange mechanisms. We find support for this prediction: genes coding for V-type, Ca2+, and Na+/K+-ATPases, which are key cellular ion exchange enzymes, are especially common targets of selection in species from six orders of fishes. This indicates that while polygenic selection contributes to adaptation across salinity boundaries, changes in ATPase enzymes may be of particular importance in supporting such transitions.

Reduced Swimming Performance Repeatedly Evolves on Loss of Migration in Landlocked Populations of Alewife.

Whole-organism performance tasks are accomplished by the integration of morphological traits and physiological functions. Understanding how evolutionary change in morphology and physiology influences whole-organism performance will yield insight into the factors that shape its own evolution. We demonstrate that nonmigratory populations of alewife (Alosa pseudoharengus) have evolved reduced swimming performance in parallel, compared with their migratory ancestor. In contrast to theoretically and empirically based predictions, poor swimming among nonmigratory populations is unrelated to the evolution of osmoregulation and occurs despite the fact that nonmigratory alewives have a more fusiform (torpedo-like) body shape than their ancestor. Our results suggest that elimination of long-distance migration from the life cycle has shaped performance more than changes in body shape and physiological regulatory capacity.

Trade-offs in osmoregulation and parallel shifts in molecular function follow ecological transitions to freshwater in the Alewife.

Adaptation to freshwater may be expected to reduce performance in seawater because these environments represent opposing selective regimes. We tested for such a trade-off in populations of the Alewife (Alosa pseudoharengus). Alewives are ancestrally anadromous, and multiple populations have been independently restricted to freshwater (landlocked). We conducted salinity challenge experiments, whereby juvenile Alewives from one anadromous and multiple landlocked populations were exposed to freshwater and seawater on acute and acclimation timescales. In response to acute salinity challenge trials, independently derived landlocked populations varied in the degree to which seawater tolerance has been lost. In laboratory-acclimation experiments, landlocked Alewives exhibited improved freshwater tolerance, which was correlated with reductions in seawater tolerance and hypo-osmotic balance, suggesting that trade-offs in osmoregulation may be associated with local adaptation to freshwater. We detected differentiation between life-history forms in the expression of an ion-uptake gene (NHE3), and in gill Na(+) /K(+) -ATPase activity. Trade-offs in osmoregulation, therefore, may be mediated by differentiation in ion-uptake and salt-secreting pathways.

The allometry of energy reserve depletion: test of a mechanism for size-dependent winter mortality.

We experimentally tested the hypothesis that energy reserve depletion varies inversely with size in the fish Menidia menidia, an estuarine fish known to exhibit size-dependent winter mortality. Individuals in two size groups were starved at two winter temperatures (4°and 8°C) and sacrificed at a range of time intervals (up to 127 days). Lipid levels and lean tissue were analyzed to estimate somatic energy storage. As predicted, energy depletion was greater at high temperatures, and proportionally greater in small than in large fish. After 60 days of starvation at 4°C, small fish retained an average of 67% of their original energy reserves (vs 53% at 8°C), while large fish retained an average of 80% (vs 66% at 8°C). At 4°C, fish that were fed depleted their energy reserves as rapidly as unfed fish, but at 8°C, fish that were fed maintained reserves at higher levels than unfed fish. A high proportion of unfed fish (56% at 4°C, 27% at 8°C) died before they were to be sacrificed. Survival probability did not vary with size, nor was it influenced by the amount of energy reserves. The rate of energy depletion (equivalent to routine metabolic rate) decreased gradually over time, particularly in small fish. Routine metabolism did not conform to a single scaling relationship. Within each temperature-size group, the routine rate declined more rapidly than metabolically active mass (lean mass). At 8°C, the difference between size groups in energy depletion rate conformed closely to the expected allometry exponent of 0.8. In contrast, at 4°C, the estimated allometry exponent increased over the experiment (-0.19 to 2.5). We conclude that strategies to minimize energy loss may often modify bioenergetic scaling relationships.

The covariance of routine and compensatory juvenile growth rates over a seasonality gradient in a coastal fish.

Compensatory growth is widespread in juvenile animals; it refers to the ability to grow at faster-than-routine rates, following intervals of slow growth due to reduced food supply or temperature. Whether the ability to grow rapidly under routine conditions is associated with enhanced or reduced compensatory growth performance is unknown. We examine the among-population covariance in routine and compensatory growth ability in juvenile Atlantic silverside, Menidia menidia. Routine growth rate in this species positively varies with latitude, indicating selection for more rapid increase in size in highly seasonal environments. High-latitude fish may also show strong compensatory ability, considering the selective value of rapid recovery from winter for growth and breeding in the spring. Alternatively, low-latitude fish may be more readily able to compensate because they have more energetic scope for growth. To test these alternatives, laboratory-reared juveniles from three populations [Nova Scotia (NS), New York (NY), and South Carolina (SC)] were maintained at temperatures promoting maximum growth. In each population, control fish were furnished ad libitum rations, while treatment fish were subject to 5 or 10 days of maintenance ration before returning to unlimited ration. Treatment fish grew slightly in length, but not mass, over the period of limited ration. We observed compensatory growth in all populations. NS fish recovering from 10 days of limited ration grew 12% faster in length and 46% faster in dry mass than NS control fish, over 15 days of unrestricted growth. In contrast, recovering SC fish grew 1.4% faster in length and 22% faster in dry mass than SC control fish. A period of starvation (mass loss) is not a required condition for a compensatory response, as has been assumed in theoretical work. The positive covariance between routine and compensatory growth performance indicates that the growth rate strategy of high latitude fish includes rapid recovery from periods of depletion, adding to the suite of characteristics that have differentiated along an environmental gradient in seasonality.

Beta-thymosin gene polymorphism associated with freshwater invasiveness of alewife (Alosa pseudoharengus).

Predicting the success of a species' colonization into a novel environment is routinely considered to be predicated on niche-space similarity and vacancy, as well as propagule pressure. The role genomic variation plays in colonization success (and the interaction with environment) may be suggested, but has not rigorously been documented. To test an hypothesis that previously observed ecotype-specific polymorphisms between anadromous and landlocked alewife (Alosa pseudoharengus) populations are an adaptive response to osmoregulatory challenges rather than a result of allele sampling at founding, we examined multiple anadromous and landlocked (colonized) populations for their allelic profiles at a conserved region (3'-UTR end) of a ß-thymosin gene whose protein product plays a central role in the organization of cytoskeleton. The putatively ancestral ß-thymosin allele was prevalent in anadromous populations, whereas a newly derived allele was overrepresented in landlocked populations; a third allele was exclusive to the anadromous populations. We also conducted a complementary set of salinity exposure experiments to test osmoregulatory performance of the alewife ecotypes in contrasting saline environments. The pattern of variation and results from these challenges indicate a strong association of ß-thymosin with colonization success and a transition from species with an anadromous life history to one with only a freshwater component.

DISTINGUISHING MULTIPLE LINEAGES OF PEDIASTRUM DUPLEX WITH MORPHOMETRICS AND A PROPOSAL FOR LACUNASTRUM GEN. NOV(1).

Accurately defining species boundaries in the green algae (Chlorophyta) is integral for studies of biodiversity and conservation, water-quality assessments, and the use of particular species as paleoindicators. Recent molecular phylogenetic and SEM analyses of the family Hydrodictyaceae (Chlorophyta) resolved three phylogenetic lineages of isolates with the Pediastrum duplex Meyen 1829 phenotype. The present study employed analyses of cell shape and cell wall ultrastructure to determine if the three lineages possessing the P. duplex morphotype were distinguishable. Only one of the groups, containing isolates with the P. duplex var. gracillimum West et G. S. West phenotype, was shown to be morphologically distinct from the other two P. duplex groups. The erection of a new genus, Lacunastrum, is proposed to recognize this group as a separate taxon.

THE EFFECT OF BIRTH DATE ON FITNESS OF FEMALE DWARF PERCH, MICROMETRUS MINIMUS (PERCIFORMES: EMBIOTOCIDAE).

Lifetime reproductive success may vary considerably with birth date. I measured phenotypic selection on female birth date in a viviparous teleost fish (Embiotocidae: Micrometrus minimus) by sampling birth-date cohorts over time in Tomales Bay, California. Four episodes of selection were measured: survival from birth to first reproduction, reproductive success in the first breeding season, survival to second reproduction, and reproductive success in the second season. Birth date had a significant impact on fitness in the first two episodes. Early born females were more successful in their first breeding season than late born females (directional selection on birth date), but early born females were less likely to survive the period between birth and first reproduction, relative to females born in the middle of the season (stabilizing selection on birth date). The final two episodes of selection had no detectable effect on birth date. Because of the relationship between birth date and survival in the first year, overall selection on female birth date was stabilizing.

SEXUAL SELECTION AND MALE CHARACTERISTICS IN THE BLUEHEAD WRASSE, THALASSOMA BIFASCIATUM: MATING SITE ACQUISITION, MATING SITE DEFENSE, AND FEMALE CHOICE.

Through a series of replacement experiments with the bluehead wrasse, Thalassoma bifasciatum, we have identified male morphological characteristics that appear to be under phenotypic sexual selection. We were particularly interested in whether the various sources of sexual selection (male-male competition for unoccupied mating sites, defense of mating sites against small males, and female choice of males) were (1) independently associated with different phenotypic characteristics; (2) jointly affected the same characteristic in the same way; or (3) jointly affected the same characteristic in an antagonistic fashion. We replaced the resident large, brightly colored Terminal Phase (TP) males on a reef with the same number of TP males from other reefs. When transplanted, these males contest with each other to take over mating sites. The transplanted group of males were then scored for three components of fitness: (1) the quality of the site obtained through competition with other large males; (2) the male's ability to defend arriving females from small intruding males; and (3) changes in female visits to the site once the new male takes over. The first and second components are part of intrasexual selection; the third represents intersexual selection. We measured the opportunity for selection by partitioning variance in mating success, and measured the direct effects of sexual selection by estimating the covariance between morphology and fitness components. Opportunities for selection: Because females generally remain faithful to particular mating sites, most (54%) of the explainable variation in male mating success is due to the acquisition of a particular mating territory, which is the outcome of competition among TP males. There was less variation in mating success due to shifts in site use by females and defense of females against the intrusions of smaller males, but all components were significant. Effects of selection: Success in male-male competition among TP males, estimated by the quality of the territory acquired, was positively associated with body length and the relative length of the pectoral fin. Success in territorial defense against small males was primarily related to body length, with lesser contributions from body depth and the area of a white band on the flank. Contribution to fitness through female choice of males was positively associated with white band area. In the two instances where a character was associated with two fitness components, the direction of selection was the same. While body length was positively associated with winning intrasexual contests, it was not correlated to any behavioral measures of aggression. Similarly, the white band associated with attractiveness was not correlated with any aspect of courtship or aggression. Parasite load was uncorrelated with other morphological characters, and did not appear to affect any aspect of sexual selection. There was no evidence for stabilizing selection or significant additional contributions from second-order effects to the fitness surfaces. Fitness functions calculated using cubic splines were generally linear except for body length, which appeared sigmoid in its effect on site acquisition ability; this same feature tended to plateau in its effect on site defense. Analyses of the interactions of selection gradients with reef or experiment indicated that the effect of particular male characters on estimates of fitness was generally homogeneous in both time and space.

PHENOTYPIC PLASTICITY IN LIFE-HISTORY TRAITS OF FEMALE THALASSOMA BIFASCIATUM (PISCES: LABRIDAE). 1. MANIPULATIONS OF SOCIAL STRUCTURE IN TESTS FOR ADAPTIVE SHIFTS OF LIFE-HISTORY ALLOCATIONS.

Optimal-life-history theory is based on the relative benefit of immediate versus future reproduction. We apply this theory to the life-history tactics of female Thalassoma bifasciatum, a sex-changing coral-reef fish. Local social structure varies in this species and influences a female's chances of achieving high future reproductive success as a territorial terminal-phase male. We predicted that female life-history allocations would be flexible and responsive to variation in future reproductive prospects. We altered population size structure on four reefs, removing large fish and adding small fish. These alterations enhanced the residual reproductive value of the remaining larger female residents. The predicted response to the manipulation was a reduction in reproductive activity and an increase in growth. Our results do not support this hypothesis. Possible interpretations are that: 1) the theory, or our application of the theory, is flawed; 2) female Thalassoma are unresponsive to changes in future reproductive prospects; or 3) the design of our study was ineffectual at detecting a response. We report here an approach to the study of adaptive life-history strategies that deserves implementation in other systems.