The evolution of independent lineages along replicated environmental transitions frequently results in convergent adaptation, yet the degree to which convergence is present across multiple levels of biological organization is often unclear. Additionally, inherent biases associated with shared ancestry and variation in selective regimes across geographic replicates often pose challenges for confidently identifying patterns of convergence. We investigated a system in which three species of poeciliid fishes sympatrically occur in a toxic spring rich in hydrogen sulfide (H₂S) and an adjacent nonsulfidic stream to examine patterns of adaptive evolution across levels of biological organization. We found convergence in morphological and physiological traits and genome-wide patterns of gene expression among all three species. In addition, there were shared signatures of selection on genes encoding H₂S toxicity targets in the mitochondrial genomes of each species. However, analyses of nuclear genomes revealed neither evidence for substantial genomic islands of divergence around genes involved in H₂S toxicity and detoxification nor substantial congruence of strongly differentiated regions across population pairs. These non-convergent, heterogenous patterns of genomic divergence may indicate that sulfide tolerance is highly polygenic, with shared allele frequency shifts present at many loci with small effects along the genome. Alternatively, H₂S tolerance may involve substantial genetic redundancy, with non-convergent lineage-specific variation at multiple loci along the genome underpinning similar changes in phenotypes and gene expression. Overall, we demonstrate variability in the extent of convergence across organizational levels and highlight the challenges of linking patterns of convergence across scales.

Study sites and sample collection

In the foothills of the Sierra Madre de Chiapas, several freshwater springs rich in naturally occurring H2S form the La Gloria spring complex.1,2 The spring complex is located near the city of Teapa, Mexico, and is part of the Río Pichucalco drainage. Habitats with high H2S concentrations are spatially restricted (~200 m in length), and the spring run flows directly into a nearby nonsulfidic stream (Arroyo Caracol). There are no physical barriers that prevent fish movement between the sulfidic spring run and the nonsulfidic stream, but there are stark differences in water chemistry. Populations of the poeciliid fishes Poecilia mexicana, Pseudoxiphophorus bimaculatus, and Xiphophorus hellerii have colonized H2S-rich habitats at La Gloria, and ancestral populations also occur in adjacent nonsulfidic habitats.3 Note that the population of P. mexicana at La Gloria likely belongs to an endemic species described from sulfide springs, P. sulphuraria.3,4 All samples used in this study were collected by seine from the La Gloria sulfide spring complex and nonsulfidic habitats in the adjacent Arroyo Caracol. The sole exception was Pseudoxiphophorus gill tissues for the nonsulfidic population, which were collected from another nearby stream.

Water chemistry

Physical and chemical water parameters were analyzed for several sites within the La Gloria spring complex and Arroyo Caracol. Temperature, specific conductivity, pH, and oxygen content were measured using a Hydrolab Multisonde 4A (Hach Environmental). Environmental H2S concentrations were measured with a methylene blue assay using a Hach DR1900 Portable Spectrophotometer (Hach Company, Loveland, CO, USA). Measurements and calibration of probes were conducted according to the manufacturer’s recommendations. At least three Hydrolab readings and two H2S samples were taken at each site.

Analysis of body shape

To test for divergence in body shape among populations in sulfidic and nonsulfidic habitats, adult Poecilia, Pseudoxiphophorus, and Xiphophorus were collected from the two sites using seines. Lateral photographs were taken for all specimens using a Canon EOS 400D digital camera (Canon USA Inc., Lake Success, NY, USA) mounted on a copy stand. We digitized 14 morphological landmarks on each photograph using the software program tpsDig.(5)

Tolerance to acute H2S exposure

To test for differences in H2S tolerance between individuals collected from the sulfidic and nonsulfidic populations, we conducted acute H2S exposure trials, subjecting wild-caught fish to logarithmically increasing concentrations of H2S.(1) We collected adult Poecilia, Pseudoxiphophorus, and Xiphophorus from the two sites using seines, transferred them to insulated coolers filled with water from the collection site, and transported them to a nearby field station. Fish were kept in population and species-specific holding tanks with aeration for at least 24 hours prior to testing. To standardize experimental conditions, water from the collection sites was slowly replaced with H2S-free well water over the first 8 hours in the holding tanks. Water was continuously aerated and filtered during this time, and the fish received no food.

For the experiment, we prepared stock solutions of 10 mM aqueous H2S solution by dissolving 2.4 g sodium sulfide hydrate (Na2S·6H2O) into 1 L of well water deoxygenated through purging with nitrogen gas.6 Individual fish were placed into clear plastic containers with 150 mL water from the holding tanks and allowed to acclimate for 5 minutes. Following acclimation, 10 mL of H2S solution were added to the experimental container at 2-minute intervals using a syringe placed under the water surface. Each fish was observed as H2S concentration increased in the experimental container. We measured the time until the fish lost equilibrium, at which point the fish was removed from the container, sexed, weighed, and placed into a heavily aerated recovery tank. Experiments were ended after 32 minutes (15 sulfide additions) if a fish did not lose equilibrium.

Reciprocal translocation experiments

To experimentally validate local adaptation7 and test for natural selection against migrants between sulfidic and nonsulfidic populations,(8) we conducted reciprocal translocation experiments using previously established approaches. (9,10) Large (20-liter) plastic buckets were placed into the two habitat types as experimental mesocosms. Two holes (18 × 32 cm) were cut into opposite sides of each bucket and sealed with 1.5 mm plastic mesh to allow the free exchange of water with the environment. Approximately 50 small holes (<1 mm) were drilled into the bucket lids to facilitate air exchange. Mesocosms were placed into shallow areas of the sulfidic and nonsulfidic streams, seeded with a 3–4 cm layer of natural substrate, and allowed to settle overnight before the start of the experiment. We established ten mesocosms in each habitat. Water conditions in these mesocosms have been shown to closely match conditions in the surrounding stream. (9)

We collected adult Poecilia, Pseudoxiphophorus, and Xiphophorus by seine and placed them in insulated coolers with aerated water for transport to the mesocosm locations. Five haphazardly chosen individuals of the same species and habitat type were introduced into a mesocosm. Half of the mesocosms in each habitat were used as controls to test the survival of resident fish, and the other half to test survival of fish from the opposite habitat type. Transportation and handling times were minimal (<1 hour) and were balanced for resident and translocated individuals. Fish were measured for standard length prior to introduction. Experiments ran for ~20 hours before termination. Following the experimental period, we quantified survival and returned surviving individuals to their original collection site.

 References

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