Hybridization has been recognized to play important roles in evolution, however studies of the genetic consequence are still lagging behind in vertebrates due to the lack of appropriate experimental systems. Fish of the genus Xiphophorus are proposed to have evolved with multiple ancient and ongoing hybridization events. They have served as an informative research model in evolutionary biology and in biomedical research on human disease for more than a century. Here, we provide the complete genomic resource including annotations for all described 26 Xiphophorus species and three undescribed taxa and resolve all uncertain phylogenetic relationships. We investigate the molecular evolution of genes related to cancers such as melanoma and for the genetic control of puberty timing, focusing on genes that are predicted to be involved in pre-and postzygotic isolation and thus affect hybridization. We discovered dramatic size-variation of some gene families. These persisted despite reticulate evolution, rapid speciation and short divergence time. Finally, we clarify the hybridization history in the entire genus settling disputed hybridization history of two Southern swordtails. Our comparative genomic analyses revealed hybridization ancestries that are manifested in the mosaic fused genomes and show that hybridization often preceded speciation.

Freshwater ecosystems are under threat from rising pharmaceutical pollution. While such pollutants are known to elicit biological effects on organisms, we have limited knowledge on how these effects might cascade through food-webs, disrupt ecological processes, and shape freshwater communities. In this study, we used a mesocosm experiment to explore how the community impacts of a top-order predator, the eastern mosquitofish (Gambusia holbrooki), are mediated by exposure to environmentally relevant low (measured concentration: ∼10 ng/L) and high concentrations (∼110 ng/L) of the pervasive pharmaceutical pollutant fluoxetine. We found no evidence that exposure to fluoxetine altered the consumptive effects of mosquitofish on zooplankton. However, once mosquitofish were removed from the mesocosms, zooplankton abundance recovered to a greater extent in control mesocosms compared to both low and high fluoxetine-exposed mesocosms. By the end of the experiment, this resulted in fundamental differences in community structure between the control and fluoxetine-treated mesocosms. Specifically, the control mesocosms were characterized by higher zooplankton abundances and lower algal biomass, whereas mesocosms exposed to either low or high concentrations of fluoxetine had lower zooplankton abundances and higher algal biomass. Our results suggest that fluoxetine, even at very low concentrations, can alter aquatic communities and hinder their recovery from disturbances.

We explored the relationship between gestational states, fecundity, and steroid hormone levels in three species of live-bearing fish with different maternal provisioning strategies. We studied two lecithotrophic species, Gambusia affinis and Xiphophorus couchianus, where embryos feed exclusively on yolk stored in the eggs, and one matrotrophic species, Heterandria formosa, which actively transfers nutrients to embryos through a follicular placenta. We measured water-borne cortisol, estradiol, and progesterone along with brood size (fecundity) and gestational stage(s). We examined the physiological costs of both maternal provisioning modes. Matrotrophy likely imposes energetic demands due to active nutrient transfer, while lecithotrophy may incur costs from carrying many large embryos. We hypothesized that fecundity, gestational stage, and hormones would covary differently in lecithotrophic vs. matrotrophic species. We found no relationships between hormones and fecundity or gestational stage in any species. However, in H. formosa, we found a positive relationship between estradiol levels and female mass, and a negative relationship between progesterone levels and female mass indicating a change in the circulating levels of both hormones as females grow. We observed differences in average hormone levels among species: the matrotrophic species had higher progesterone and lower estradiol compared to lecithotrophic species. Higher estradiol in lecithotrophic species may relate to egg yolk formation, while placental structures could play a role in progesterone production in matrotrophic species. Elevated cortisol in H. formosa suggests either higher energetic costs or a preparative role for reproduction. Our findings highlight progesterone\'s importance in maintaining gestation in matrotrophic species, like other placental species.

Urban stream syndrome alters stream habitat complexity. We define habitat complexity as the degree of variation in physical habitat structure, with increasing variation equating to higher complexity. Habitat complexity affects species composition and shapes animal ecology, physiology, behaviour and cognition. We used a delayed detour test to measure whether cognitive processes (motor self-regulation) and behaviour (risk-taking) of female Western mosquitofish, Gambusia affinis, varied with habitat structural complexity (low, moderate and high) that was quantified visually for nine populations. We predicted that motor self-regulation and risk-taking behaviour would increase with increasing habitat complexity, yet we found support for the opposite. Lower complexity habitats offer less refuge potentially leading to higher predation pressure and selecting for greater risk-taking by fish with higher motor self-regulation. Our findings provide insight into how habitat complexity can shape cognitive processes and behaviour and offers a broader understanding of why some species may tolerate conditions of urbanized environments.

We considered the relationship between the benefits and costs of territorial defense in a wild population of the Leon Springs pupfish, Cyprinodon bovinus. We defined benefit as the number of eggs deposited on an artificial substratum placed within the defender\'s territory. Costs included two defensive behaviors. First, males frequently \"patrolled\" their territories, swimming back-and-forth across their area. Second, males chased intruding Pecos gambusia (Gambusia nobilis) as well as small male and female conspecific C. bovinus from their territories. Both of these species prey on the territorial defenders\' eggs; additionally, small male C. bovinus will attempt to \"steal\" spawns from the territorial defender by spawning with females in the territory. Our analyses revealed that only patrol frequency was related to the reproductive benefit of the territory. Neither chases against gambusia nor conspecifics were predicted by egg numbers on the breeding substrata. We speculate that the frequency of patrolling is an indicator of territorial value and note the qualitative differences in chasing behavior against the different species of intruder.

Natural temperature variation in many marine ecosystems is stochastic and unpredictable, and climate change models indicate that this thermal irregularity is likely to increase. Temperature acclimation may be more challenging when conditions are highly variable and stochastic, and there is a need for empirical physiological data in these thermal environments. Using the hermaphroditic, amphibious mangrove rivulus (Kryptolebias marmoratus), we hypothesized that compared with regular, warming diel thermal fluctuations, stochastic warm fluctuations would negatively affect physiological performance. To test this, we acclimated fish to: (1) non-stochastic and (2) stochastic thermal fluctuations with a similar thermal load (27-35°C), and (3) a stable/consistent control temperature at the low end of the cycle (27°C). We determined that fecundity was reduced in both cycles, with reproduction ceasing in stochastic thermal environments. Fish acclimated to non-stochastic thermal cycles had growth rates lower than those of control fish. Exposure to warm, fluctuating cycles did not affect emersion temperature, and only regular diel cycles modestly increased critical thermal tolerance. We predicted that warm diel cycling temperatures would increase gill surface area. Notably, fish acclimated to either thermal cycle had a reduced gill surface area and increased intralamellar cell mass when compared with control fish. This decreased gill surface area with warming contrasts with what is observed for exclusively aquatic fish and suggests a preparatory gill response for emersion in these amphibious fish. Collectively, our data reveal the importance of considering stochastic thermal variability when studying the effects of temperature on fishes.

BACKGROUND: To unravel the evolutionary history of a complex group, a comprehensive reconstruction of its phylogenetic relationships is crucial. This requires meticulous taxon sampling and careful consideration of multiple characters to ensure a complete and accurate reconstruction. The phylogenetic position of the Orestias genus has been estimated partly on unavailable or incomplete information. As a consequence, it was assigned to the family Cyprindontidae, relating this Andean fish to other geographically distant genera distributed in the Mediterranean, Middle East and North and Central America. In this study, using complete genome sequencing, we aim to clarify the phylogenetic position of Orestias within the Cyprinodontiformes order.
RESULTS: We sequenced the genome of three Orestias species from the Andean Altiplano. Our analysis revealed that the small genome size in this genus (~ 0.7 Gb) was caused by a contraction in transposable element (TE) content, particularly in DNA elements and short interspersed nuclear elements (SINEs). Using predicted gene sequences, we generated a phylogenetic tree of Cyprinodontiformes using 902 orthologs extracted from all 32 available genomes as well as three outgroup species. We complemented this analysis with a phylogenetic reconstruction and time calibration considering 12 molecular markers (eight nuclear and four mitochondrial genes) and a stratified taxon sampling to consider 198 species of nearly all families and genera of this order. Overall, our results show that phylogenetic closeness is directly related to geographical distance. Importantly, we found that Orestias is not part of the Cyprinodontidae family, and that it is more closely related to the South American fish fauna, being the Fluviphylacidae the closest sister group.
CONCLUSIONS: The evolutionary history of the Orestias genus is linked to the South American ichthyofauna and it should no longer be considered a member of the Cyprinodontidae family. Instead, we submit that Orestias belongs to the Orestiidae family, as suggested by Freyhof et al. (2017), and that it is the sister group of the Fluviphylacidae family, distributed in the Amazonian and Orinoco basins. These two groups likely diverged during the Late Eocene concomitant with hydrogeological changes in the South American landscape.

Understanding the phylogeographic history of a group and identifying the factors contributing to speciation is an important challenge in evolutionary biology. The Goodeinae are a group of live-bearing fishes endemic to Mexico. Here, we develop genomic resources for species within the Goodeinae and use phylogenomic approaches to characterise their evolutionary history. We sequenced, assembled and annotated the genomes of four Goodeinae species, including Ataeniobius toweri, the only matrotrophic live-bearing fish without a trophotaenia in the group. We estimated timings of species divergence and examined the extent and timing of introgression between the species to assess if this may have occurred during an early radiation, or in more recent episodes of secondary contact. We used branch-site models to detect genome-wide positive selection across Goodeinae, and we specifically asked whether this differs in A. toweri, where loss of placental viviparity has recently occurred. We found evidence of gene flow between geographically isolated species, suggesting vicariant speciation was supplemented by limited post-speciation gene flow, and gene flow may explain previous uncertainties about Goodeid phylogeny. Genes under positive selection in the group are likely to be associated with the switch to live-bearing. Overall, our studies suggest that both volcanism-driven vicariance and changes in reproductive mode influenced radiation in the Goodeinae.

Both sexually selected traits and mate preferences for these traits can be context dependent, yet how variation in preferred traits could select for context dependent preferences has rarely been examined. The signal reliability hypothesis predicts that mate preferences vary across contexts (e.g., environments) in relation to the reliability of the information preferred traits provide in those contexts. Extensive variation in copy number of mc4r B alleles on the Y-chromosome that associates with male size in Xiphophorus multilineatus allowed us to use a split-sibling design to determine if male size is more likely to provide information about male genotype (i.e., dam) when males were reared in a warm as compared to a cold environment. We then examined strength of preference for male size by females reared in the same two environments. We found that males were larger in the cold environment, but male size was more variable across dams in the warm environment, and therefore male size would be a more reliable indicator of dam (i.e., genetics) in the warm environment. Females reared in the warm environment had stronger mate preferences based on male size than cold reared females, with a significant influence of dam on strength of preference. Therefore, strength of female preference for male size was influenced by the temperature in which they were reared, with the direction of the difference across treatments supporting the signal reliability hypothesis. Understanding how the reliability of male traits can select for contextual variation in the strength of the female mate preferences will further our discovery of adaptive mate preferences. For example, a relationship between the strength of a female\'s mate preference and their growth rates was detected in the context where females had a preference based on male size, supporting a hypothesis from previous work with this species of disassortative mating in relation to growth rates to mitigate a documented growth-mortality tradeoff.

Non-genetic sources of phenotypic variation, such as the epigenome and the microbiome, could be important contributors to adaptive variation for species with low genetic diversity. However, little is known about the complex interaction between these factors and the genetic diversity of the host, particularly in wild populations. Here, we examine the skin microbiome composition of two closely-related mangrove killifish species with different mating systems (self-fertilising and outcrossing) under sympatric and allopatric conditions. This allows us to partition the influence of the genotype and the environment on their microbiome and (previously described) epigenetic profiles. We find the diversity and community composition of the skin microbiome are strongly shaped by the environment and, to a lesser extent, by species-specific influences. Heterozygosity and microbiome alpha diversity, but not epigenetic variation, are associated with the fluctuating asymmetry of traits related to performance (vision) and behaviour (aggression). Our study identifies that a proportion of the epigenetic diversity and microbiome differentiation is unrelated to genetic variation, and we find evidence for an associative relationship between microbiome and epigenetic diversity in these wild populations. This suggests that both mechanisms could potentially contribute to variation in species with low genetic diversity.