ABSTRACT1. Effective conservation measures for endangered species require basic knowledge of habitat use and critical environmental variables influencing the species' occurrence. However, setting priorities may be difficult when multiple endangered species inhabit the same area and differ in habitat use.2. This study characterized the physical and chemical environment at 30 sites along a 2.5 km stretch of stream associated with the Baños del Azufre hydrogen sulphide spring complex, home to the only population of the widemouth gambusia (Gambusia eurystoma) in existence. Also present in the spring is Poecilia sulphuraria, a narrowly endemic and endangered species known from only a few populations. This study provides the most detailed report to date for physical and chemical drivers of species density in this small, extreme environment.3. Gambusia eurystoma were generally rare throughout the stream, and stream flow and substrate size were the best predictors of G. eurystoma densities. Conversely, P. sulphuraria were considerably more abundant, but no physical or chemical variables predicted their density among sites. Size distributions of adult P. sulphuraria were significantly influenced by stream flow and water chemistry, indicating a cost to living in greater proximity to toxic springs with high hydrogen sulphide concentrations.4. Overall, the results provide the first extensive report of environmental variation and factors associated with G. eurystoma densities, and indicate that conservation measures prioritizing environmental conditions for G. eurystoma will also benefit P. sulphuraria. Fish in sulphide springs provide prime examples of narrowly endemic species in desperate need of conservation, and their habitats face conservation challenges similar to those found in other springs with sympatric, endemic species in the world's arid regions.

22Extreme environments test the limits of life. Still, some organisms thrive in harsh conditions, 23 begging the question whether the repeated colonization of extreme environments is facilitated by 24 predictable and repeatable evolutionary innovations. We identified the mechanistic basis underlying 25 convergent evolution of tolerance to hydrogen sulfide (H2S)-a potent toxicant that impairs 26 mitochondrial function-across evolutionarily independent lineages of a fish (Poecilia mexicana, 27 Poeciliidae) from H2S-rich freshwater springs. We found that mitochondrial function is maintained 28 in the presence of H2S in sulfide spring P. mexicana, but not ancestral lineages in adjacent nonsulfidic 29 habitats, due to convergent adaptations in both the primary toxicity target and a major detoxification 30 enzyme. Additionally, we show that H2S tolerance in 10 independent lineages of sulfide spring fishes 31 across multiple genera of Poeciliidae is mediated by convergent modification and expression changes 32 of genes associated with H2S toxicity and detoxification. Our results demonstrate that the repeated 33 modification of highly conserved physiological pathways associated with essential mitochondrial 34 processes enabled the colonization of novel environments. 35 36 3 Stephen J. Gould was a fierce proponent of the importance of contingency in evolution, famously 37 quipping that replaying the "tape of life" would lead to different outcomes every time (1). 38Mitochondrial genomes were historically thought to be a prime example of such contingency 39 evolution, because alternative genetic variants were assumed to be selectively neutral (2). This 40 paradigm has been shifting, with mounting evidence that mitochondria-and genes encoded in the 41 mitochondrial genome-play an important role in adaptation, especially in the context of 42 physiochemical stress (3). However, it often remains unclear how genetic variation in mitochondrial 43 genomes and nuclear genes that contribute to mitochondrial function translates to variation in 44 physiological and organismal function. Furthermore, it is not known whether exposure to similar 45 selective regimes may cause convergent modifications of mitochondrial genomes and emergent 46 biochemical and physiological functions in evolutionarily independent lineages. Extreme 47 environments that represent novel ecological niches are natural experiments to address questions 48 about mechanisms underlying mitochondrial adaptations and illuminate the predictability of adaptive 49 evolution of mitochondria. Among the most extreme freshwater ecosystems are springs with high 50 levels of hydrogen sulfide (H2S), a potent respiratory toxicant lethal to metazoans due to its 51 inhibition of mitochondrial ATP production (4). Multiple lineages of livebearing fishes (Poeciliidae) 52 have colonized H2S-rich springs throughout the Americas and independently evolved tolerance to 53 sustained H2S concentrations orders of magnitudes higher than those encountered by ancestral 54 lineages in nonsulfid...

Mitochondria have been known to be involved in speciation through the generation of Dobzhansky–Muller incompatibilities, where functionally neutral co-evolution between mitochondrial and nuclear genomes can cause dysfunction when alleles are recombined in hybrids. We propose that adaptive mitochondrial divergence between populations can not only produce intrinsic (Dobzhansky–Muller) incompatibilities, but could also contribute to reproductive isolation through natural and sexual selection against migrants, post-mating prezygotic isolation, as well as by causing extrinsic reductions in hybrid fitness. We describe how these reproductive isolating barriers can potentially arise through adaptive divergence of mitochondrial function in the absence of mito-nuclear coevolution, a departure from more established views. While a role for mitochondria in the speciation process appears promising, we also highlight critical gaps of knowledge: (1) many systems with a potential for mitochondrially-mediated reproductive isolation lack crucial evidence directly linking reproductive isolation and mitochondrial function; (2) it often remains to be seen if mitochondrial barriers are a driver or a consequence of reproductive isolation; (3) the presence of substantial gene flow in the presence of mito-nuclear incompatibilities raises questions whether such incompatibilities are strong enough to drive speciation to completion; and (4) it remains to be tested how mitochondrial effects on reproductive isolation compare when multiple mechanisms of reproductive isolation coincide. We hope this perspective and the proposed research plans help to inform future studies of mitochondrial adaptation in a manner that links genotypic changes to phenotypic adaptations, fitness, and reproductive isolation in natural systems, helping to clarify the importance of mitochondria in the formation and maintenance of biological diversity.

Hydrogen sulfide (HS) is a natural toxicant in some aquatic environments that has diverse molecular targets. It binds to oxygen transport proteins, rendering them non-functional by reducing oxygen-binding affinity. Hence, organisms permanently inhabiting HS-rich environments are predicted to exhibit adaptive modifications to compensate for the reduced capacity to transport oxygen. We investigated 10 lineages of fish of the family Poeciliidae that have colonized freshwater springs rich in HS-along with related lineages from non-sulfidic environments-to test hypotheses about the expression and evolution of oxygen transport genes in a phylogenetic context. We predicted shifts in the expression of and signatures of positive selection on oxygen transport genes upon colonization of HS-rich habitats. Our analyses indicated significant shifts in gene expression for multiple hemoglobin genes in lineages that have colonized HS-rich environments, and three hemoglobin genes exhibited relaxed selection in sulfidic compared to non-sulfidic lineages. However, neither changes in gene expression nor signatures of selection were consistent among all lineages in HS-rich environments. Oxygen transport genes may consequently be predictable targets of selection during adaptation to sulfidic environments, but changes in gene expression and molecular evolution of oxygen transport genes in HS-rich environments are not necessarily repeatable across replicated lineages.

In this paper, we outline the use of a mitochondria-targeted ratiometric mass spectrometry probe, MitoA, to detect in vivo changes in mitochondrial hydrogen sulfide (H 2 S) in Poecilia mexicana (family Poeciliidae). MitoA is introduced via intraperitoneal injection into the animal and is taken up by mitochondria, where it reacts with H 2 S to form the product MitoN. The MitoN/MitoA ratio can be used to assess relative changes in the amounts of mitochondrial H 2 S produced over time. We describe the use of MitoA in the fish species P. mexicana to illustrate the steps for adopting the use of MitoA in a new organism, including extraction and purification of MitoA and MitoN from tissues followed by tandem mass spectrometry. In this proof-of-concept study we exposed H 2 S tolerant P. mexicana to 59 µM free H 2 S for 5 h, which resulted in increased MitoN/MitoA in brain and gills, but not in liver or muscle, demonstrating increased mitochondrial H 2 S levels in select tissues following whole-animal H 2 S exposure. This is the first time that accumulation of H 2 S has been observed in vivo during whole-animal exposure to free H 2 S using MitoA.