DOI: 10.5061/dryad.pg4f4qrzv
title: Data from: Integrative analyses of convergent adaptation in sympatric extremophile fishes

description: 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.
description: <strong>Study sites and sample
 collection</strong> 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. <strong>Water
 chemistry</strong> 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.
 <strong>Analysis of body shape</strong>
 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) <strong>Tolerance to acute H2S
 exposure</strong> 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.
 <strong>Reciprocal translocation
 experiments</strong> 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
 (&lt;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 (&lt;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.
 <strong><em> </em></strong><strong>References</strong> 1.     Tobler, M., Palacios, M., Chapman, L.J., Mitrofanov, I., Bierbach, D., Plath, M., Arias-Rodriguez, L., de León, F.J.G., and Mateos, M. (2011). Evolution in extreme environments: replicated phenotypic differentiation in livebearing fish inhabiting sulfidic springs. Evolution <em>65</em>, 2213–2228. 2.     Greenway, R., Arias-Rodriguez, L., Diaz, P., and Tobler, M. (2014). Patterns of macroinvertebrate and fish diversity in freshwater sulphide springs. Diversity <em>6</em>, 597–632. 3.     Palacios, M., Arias-Rodriguez, L., Plath, M., Eifert, C., Lerp, H., Lamboj, A., Voelker, G., and Tobler, M. (2013). The rediscovery of a long described species reveals additional complexity in speciation patterns of poeciliid fishes in sulfide springs. PLoS One <em>8</em>, e71069. 4.     Álvarez del Villar, J. (1948). Descripción de una nueva especie de <em>Mollienisia</em> capturada en Baños del Azufre, Tabasco (Pisces, Poeciliidae). Anales de la Escuela Nacional de Ciencias Biológicas <em>5</em>, 275–281. 5.     Rohlf, F.J. (2004). tpsDig. Available from http://life.bio.sunysb.edu/morph/. 6.     Butler, I.B., Schoonen, M.A.A., and Rickard, D.T. (1994). Removal of dissolved oxygen from water: a comparison of four common techniques. Talanta <em>41</em>, 211–215. 7.     Kawecki, T.J., and Ebert, D. (2004). Conceptual issues in local adaptation. Ecol. Lett. <em>7</em>, 1225–1241. 8.     Nosil, P., Vines, T.H., and Funk, D.J. (2005). Perspective: Reproductive isolation caused by natural selection against immigrants from divergent habitats. Evolution <em>59</em>, 705–719. 9.     Tobler, M., Riesch, R., Tobler, C.M., Schulz-Mirbach, T., and Plath, M. (2009). Natural and sexual selection against immigrants maintains differentiation among micro-allopatric populations. J. Evol. Biol. <em>22</em>, 2298–2304. 10.   Plath, M., Pfenninger, M., Lerp, H., Riesch, R., Eschenbrenner, C., Slattery, P.A., Bierbach, D., Herrmann, N., Schulte, M., Arias-Rodriguez, L., et al. (2013). Genetic differentiation and selection against migrants in evolutionarily replicated extreme environments. Evolution <em>67</em>, 2647–2661.
description: # Data from: Integrative analyses of convergent adaptation in sympatric
 extremophile fishes
 [https://doi.org/10.5061/dryad.pg4f4qrzv](https://doi.org/10.5061/dryad.pg4f4qrzv) ## Description of the data and file structure Methods for all data sets are described in the methods section of the repository. **waterchemistry.csv** Environmental parameters measured in the adjacent sulfidic and non-sulfidic habitats. **gloriatoerance.csv** Results of the H2S tolerance trials.  **Translocation_sulf2.csv** Results of the reciprocal translocation experiment. **lagloria_geomorph.csv** Results of the geometric morphometric analysis. ### Files and variables #### File: waterchemistry.csv **Description:** Environmental parameters measured in the adjacent sulfidic and non-sulfidic habitats. ##### Variables * Habitat: NS (nonsulfidic) vs. S (sulfidic) * Width: maximum stream width (in meters) * Depth: maximum stream depth (in meters) * Flow: maximum water flow (in meters per second) * DO: dissolved oxygen concentration (in milligrams per liter) * Temperature: water temperature (in degrees Celsius) * pH: pH (unitless) * SpCond: specific conductivity (in microsiemens per centimeter) * H2Smol: H2S concentration (in millimol) #### File: Translocation\_sulf2.csv **Description:** Results of the reciprocal translocation experiment. ##### Variables * Species: bimac (P. bimaculatus), hellerii (X. hellerii), or mexicana (P. mexicana) * Origin: habitat type of origin, NS (nonsulfidic) vs. S (sulfidic) * Location: habitat type of testing, NS (nonsulfidic) vs. S (sulfidic) * Replicate: mesocosm id * Sex: M (male) vs. F (female) * SL: standard length (in millimeter) * Survival: 0 (died) vs. 1 (lived) #### File: gloriatolerance.csv **Description:** Results of the H2S tolerance trials. ##### Variables * ID: individual identifier * Group: combination of species and habitat type (see below) * Species: Pbim (Pseudoxiphophorus bimaculatus), Pmex (Poecilia mexicana), or Xhell (Xiphophorus hellerii) * Type: habitat type of origin, NS (nonsulfidic) vs. S (sulfidic) * Sex: M (male) vs. F (female) * Mass: body wet mass (in grams) * LoE: time of loss of equilibrium (in seconds) * H2S1: estimated H2S concentration at LoE (replicate 1) * H2S2: estimated H2S concentration at LoE (replicate 1) * H2Smean: average of H2S1 and H2S2 * Censoring: wether an individual reached LoE; 0 (reached LoE) vs. 1 (did not reach LoE) #### File: lagloria\_geomorph.csv **Description:** Results of the geometric morphometric analysis ##### Variables * ID: individual identifier * Species: PB  (P. bimaculatus), XH (X. hellerii), or PM (P. mexicana) * Site: Fieldsite ID * Habitat: habitat type, NS (nonsulfidic) vs. S (sulfidic) * Sex: M (male) vs. F (female) * CS: centroid size (in pixels) * X1/Y1 to X14/Y14: raw coordinates of landmark points * X15/Y15 and X16/Y16: raw coordinates on size standard * CF: correction factor for size correction * Xsc1/Ysc1 to Xsc14/Ysc14: size-corrected coordinates of landmark points ## Code/software csv (comma-separate value) files do not require any proprietary software to view and can be opened in any text editor, spreadsheet editor, or statistical software.