Lenin Arias-Rodriguez

EVOLUTION IN EXTREME ENVIRONMENTS: REPLICATED PHENOTYPIC DIFFERENTIATION IN LIVEBEARING FISH INHABITING SULFIDIC SPRINGS

We investigated replicated ecological speciation in the livebearing fish Poecilia mexicana and P. sulphuraria (Poeciliidae), which inhabit freshwater habitats and have also colonized multiple sulfidic springs in southern Mexico. These springs exhibit extreme hypoxia and high concentrations of hydrogen sulfide, which is lethal to most metazoans. We used phylogenetic analyses to test whether springs were independently colonized, performed phenotypic assessments of body and gill morphology variation to identify convergent patterns of trait differentiation, and conducted an eco‐toxicological experiment to detect differences in sulfide tolerances among ecotypes. Our results indicate that sulfidic springs were colonized by three different lineages, two within P. mexicana and one representing P. sulphuraria. Colonization occurred earlier in P. sulphuraria, whereas invasion of sulfidic springs in P. mexicana was more recent, such that each population is more closely related to neighboring populations from adjacent nonsulfidic habitats. Sulfide spring fish also show divergence from nonsulfidic phenotypes and a phenotypic convergence toward larger heads, larger gills, and increased tolerance to H2S. Together with previous studies that indicated significant reproductive isolation between fish from sulfidic and nonsulfidic habitats, this study provides evidence for repeated ecological speciation in the independent sulfide spring populations of P. mexicana and P. sulphuraria.

Genetic differentiation and selection against migrants in evolutionarily replicated extreme environments.

We investigated mechanisms of reproductive isolation in livebearing fishes (genus Poecilia) inhabiting sulfidic and nonsulfidic habitats in three replicate river drainages. Although sulfide spring fish convergently evolved divergent phenotypes, it was unclear if mechanisms of reproductive isolation also evolved convergently. Using microsatellites, we found strongly reduced gene flow between adjacent populations from different habitat types, suggesting that local adaptation to sulfidic habitats repeatedly caused the emergence of reproductive isolation. Reciprocal translocation experiments indicate strong selection against immigrants into sulfidic waters, but also variation among drainages in the strength of selection against immigrants into nonsulfidic waters. Mate choice experiments revealed the evolution of assortative mating preferences in females from nonsulfidic but not from sulfidic habitats. The inferred strength of sexual selection against immigrants (RIs) was negatively correlated with the strength of natural selection (RIm), a pattern that could be attributed to reinforcement, whereby natural selection strengthens behavioral isolation due to reduced hybrid fitness. Overall, reproductive isolation and genetic differentiation appear to be replicated and direct consequences of local adaptation to sulfide spring environments, but the relative contributions of different mechanisms of reproductive isolation vary across these evolutionarily independent replicates, highlighting both convergent and nonconvergent evolutionary trajectories of populations in each drainage.

Effects of male sexual harassment on female time budgets, feeding behavior, and metabolic rates in a tropical livebearing fish (Poecilia mexicana)

Sexual harassment by males has the potential to affect almost any aspect of female behavior and life history. Using Atlantic mollies (Poecilia mexicana) as a model—a species in which males do not court but almost constantly try to forcefully mate with females—we asked whether and how male harassment influences (a) females’ time budgets and (b) feeding rates (e.g., through frequent flight from male approaches), and (c) whether metabolic rates are increased as a response to stress. Field observations in a natural P. mexicana population revealed that males (average feeding rate 15%) spent far less time feeding than females (60%), and clearly traded off frequent pursuit of females with foraging. Most importantly, females’ feeding times were dramatically reduced when being pursued by a harassing male. Also in standardized lab experiments, females spent significantly less time feeding when accompanied by a male as compared to being in the presence of another female. This effect was also observed when partner fish (male or female) were presented only visually, but could not interact physically with the focal female. It seems, therefore, that females increase vigilance when a harassing male is around, which keeps them from feeding even before males actually approach them. Based on the latter result, we asked whether a stress-induced increase in metabolic rates would be discernible. We measured oxygen consumption and gill ventilation frequencies (opercular rates) of females in different social contexts (alone, with another female, or a male). The predicted, strong body mass dependency of both physiological parameters was uncovered, but no evidence for an effect of social context was detected. We argue that male harassment represents such a constant (but non-lethal) stressor for poeciliid females that their metabolic stress responses have adapted to this through habituation.

Unique evolutionary trajectories in repeated adaptation to hydrogen sulphide-toxic habitats of a neotropical fish (Poecilia mexicana).

Replicated ecological gradients are prime systems to study processes of molecular evolution underlying ecological divergence. Here, we investigated the repeated adaptation of the neotropical fish Poecilia mexicana to habitats containing toxic hydrogen sulphide (H2S) and compared two population pairs of sulphide‐adapted and ancestral fish by sequencing population pools of >200 individuals (Pool‐Seq). We inferred the evolutionary processes shaping divergence and tested the hypothesis of increase of parallelism from SNPs to molecular pathways. Coalescence analyses showed that the divergence occurred in the face of substantial bidirectional gene flow. Population divergence involved many short, widely dispersed regions across the genome. Analyses of allele frequency spectra suggest that differentiation at most loci was driven by divergent selection, followed by a selection‐mediated reduction of gene flow. Reconstructing allelic state changes suggested that selection acted mainly upon de novo mutations in the sulphide‐adapted populations. Using a corrected Jaccard index to quantify parallel evolution, we found a negligible proportion of statistically significant parallel evolution of Jcorr = 0.0032 at the level of SNPs, divergent genome regions (Jcorr = 0.0061) and genes therein (Jcorr = 0.0091). At the level of metabolic pathways, the overlap was Jcorr = 0.2545, indicating increasing parallelism with increasing level of biological integration. The majority of pathways contained positively selected genes in both sulphide populations. Hence, adaptation to sulphidic habitats necessitated adjustments throughout the genome. The largely unique evolutionary trajectories may be explained by a high proportion of de novo mutations driving the divergence. Our findings favour Goulds view that evolution is often the unrepeatable result of stochastic events with highly contingent effects.

A sperm cryopreservation protocol for the loach Misgurnus anguillicaudatus and its applicability for other related species

The aim of the present study was to establish a protocol of sperm cryopreservation in Misgurnus anguillicaudatus and verify the applicability of the obtained protocol in other loach species. We evaluated the following parameters: inseminating dose, thawing temperatures (20, 25 and 30 degrees C for 10s), extenders (loach or cyprinid extenders), internal cryoprotectants (dimethyl sulfoxide (DMSO), dimethylacetamide (DMA), glycerol (Gly), ethylene glycol (EG), and methanol (MeOH) at 0, 5, 10 and 15%), external cryoprotectants (bovine serum albumin 1 and 2%; sucrose 0.5 and 1%; glucose 0.5 and 1%; glycine 0.5 and 1%), activating solutions (distilled water, dechlorinated tap water, 25mM NaCl and 50mM NaCl), and hatchability of the eggs when fertilized with fresh or cryopreserved sperm. After the evaluation of these parameters, we optimized the cryopreservation using the following procedure: thawing temperature at 25 degrees C for 10s; loach or cyprinid extenders; methanol at 10 or 15% as internal cryoprotectants; glycine 0.5% or bovine serum albumin 1% as external cryoprotectants and 50mM NaCl for sperm activation. Using this procedure, the fertilizability of the post-thawed sperm was 47% in comparison to the fresh sperm, at the minimum inseminating dose (687.65 spermatozoa egg(-1)mL(-1)). Based on this protocol, sperm from other loach species Lefua nikkonis, Misgurnus mizolepis and Barbatula toni were cryopreserved successfully.

Mechanisms Underlying Adaptation to Life in Hydrogen Sulfide–Rich Environments

Hydrogen sulfide (H2S) is a potent toxicant interfering with oxidative phosphorylation in mitochondria and creating extreme environmental conditions in aquatic ecosystems. The mechanistic basis of adaptation to perpetual exposure to H2S remains poorly understood. We investigated evolutionarily independent lineages of livebearing fishes that have colonized and adapted to springs rich in H2S and compared their genome-wide gene expression patterns with closely related lineages from adjacent, nonsulfidic streams. Significant differences in gene expression were uncovered between all sulfidic and nonsulfidic population pairs. Variation in the number of differentially expressed genes among population pairs corresponded to differences in divergence times and rates of gene flow, which is consistent with neutral drift driving a substantial portion of gene expression variation among populations. Accordingly, there was little evidence for convergent evolution shaping large-scale gene expression patterns among independent sulfide spring populations. Nonetheless, we identified a small number of genes that was consistently differentially expressed in the same direction in all sulfidic and nonsulfidic population pairs. Functional annotation of shared differentially expressed genes indicated upregulation of genes associated with enzymatic H2S detoxification and transport of oxidized sulfur species, oxidative phosphorylation, energy metabolism, and pathways involved in responses to oxidative stress. Overall, our results suggest that modification of processes associated with H2S detoxification and toxicity likely complement each other to mediate elevated H2S tolerance in sulfide spring fishes. Our analyses allow for the development of novel hypotheses about biochemical and physiological mechanisms of adaptation to extreme environments.

Reduction of Energetic Demands through Modification of Body Size and Routine Metabolic Rates in Extremophile Fish

Variation in energy availability or maintenance costs in extreme environments can exert selection for efficient energy use, and reductions in organismal energy demand can be achieved in two ways: reducing body mass or metabolic suppression. Whether long-term exposure to extreme environmental conditions drives adaptive shifts in body mass or metabolic rates remains an open question. We studied body size variation and variation in routine metabolic rates in locally adapted populations of extremophile fish (Poecilia mexicana) living in toxic, hydrogen sulfide–rich springs and caves. We quantified size distributions and routine metabolic rates in wild-caught individuals from four habitat types. Compared with ancestral populations in nonsulfidic surface habitats, extremophile populations were characterized by significant reductions in body size. Despite elevated metabolic rates in cave fish, the body size reduction precipitated in significantly reduced energy demands in all extremophile populations. Laboratory experiments on common garden–raised fish indicated that elevated routine metabolic rates in cave fish likely have a genetic basis. The results of this study indicate that adaptation to extreme environments directly impacts energy metabolism, with fish living in cave and sulfide spring environments expending less energy overall during routine metabolism.

Improvement of gamete quality and its short-term storage: an approach for biotechnology in laboratory fish

In fish, in vitro fertilization is an important reproductive tool used as first step for application of others biotechniques as chromosome and embryo manipulation. In this study, we aimed to optimize gamete quality and their short-term storage from the yellowtail tetra Astyanax altiparanae, for future application in laboratory studies. Working with sperm, we evaluated the effects of spawning inducers (carp pituitary gland and Ovopel® [(D-Ala6, Pro9-NEt) - mGnRH+metoclopramide]) and the presence of female on sperm motility. Additionally, we developed new procedures for short-term storage of sperm and oocytes. Briefly, sperm motility was higher when male fish were treated with carp pituitary gland (73.1 ± 4.0%) or Ovopel® (79.5 ± 5.5%) when compared with the control group treated with 0.9% NaCl (55.6 ± 27.2%; P=0.1598). Maintenance of male fish with an ovulating female fish also improved sperm motility (74.4 ± 7.4%) when compared with untreated male fish (42.1 ± 26.1%; P=0.0018). Storage of sperm was optimized in modified Ringer solution, in which the sperm was kept motile for 18 days at 2.5°C. The addition of antibiotics or oxygen decreased sperm motility, but partial change of supernatant and the combination of those conditions improve storage ability of sperm. Fertilization ability of oocytes decreased significantly after storage for 30, 60 90 and 120 min at 5, 10, 15 and 20°C when compared with fresh oocytes (P=0.0471), but considering only the stored samples, the optimum temperature was 15°C. Those data describe new approaches to improve semen quality and gametes short-term storage in yellowtail tetra A. altiparanae and open new possibilities in vitro fertilization.

Females prefer males with superior fighting abilities but avoid sexually harassing winners when eavesdropping on male fights

Selection imposed by male competition (intrasexual selection) and female choice (intersexual selection) can be con- or discordant. Specifically, females may or may not prefer mating with dominant males, and direct costs of interacting with dominant (and possibly more harassing) males have been suggested to explain avoidance of dominant males. Here, we exemplify that inter- and intrasexual selection may normally act in the same direction, but can be temporarily conflicting when social information becomes available. Using video playback techniques, we presented females of the Mexican livebearing fish Poecilia mexicana with two size-matched males and established association preferences. Half of the females could then observe the same two males fight and establish dominance, while control females saw both males side by side, but physically separated, and female preferences were subsequently re-evaluated. Females in the control group showed a significant preference for future winners in the subsequent testing, confirming an innate or acquired preference for male traits that are indicative of physical superiority, even when body size as a choice criterion is excluded. When allowed to eavesdrop on male fights, however, females did not show a preference for observed winners and even decreased time spent with them relative to the control treatment in which no fight was shown. A subsequent experiment found contest winners to show elevated levels of sexual behavior, so we argue that the temporary offset of the intrinsic female preference for dominant males after having observed a fight is indeed driven by direct costs females expect from more harassing contest winners.

Shared and unique patterns of phenotypic diversification along a stream gradient in two congeneric species

Stream ecosystems show gradual variation of various selection factors, which can result in a zonation of species distributions and gradient evolution of morphological and life-history traits within species. Identifying the selective agents underlying such phenotypic evolution is challenging as different species could show shared and/or unique (species-specific) responses to components of the river gradient. We studied a stream gradient inhabited by two mosquitofishes (genus Gambusia) in the Río Grijalva basin in southern Mexico and found a patchy distribution pattern of both congeners along a stretch of 100 km, whereby one species was usually dominant at a given site. We uncovered both shared and unique patterns of diversification: some components of the stream gradient, including differences in piscine predation pressure, drove shared patterns of phenotypic divergence, especially in females. Other components of the gradient, particularly abiotic factors (max. annual temperature and temperature range) resulted in unique patterns of divergence, especially in males. Our study highlights the complexity of selective regimes in stream ecosystems. It exemplifies that even closely related, congeneric species can respond in unique ways to the same components of the river gradient and shows how both sexes can exhibit quite different patterns of divergence in multivariate phenotypic character suites.