Genetic and phenotypic diversification in a widespread fish, the Sailfin Molly (Poecilia latipinna).

Widespread species often experience significant environmental clines over the area they naturally occupy. We investigated a widespread livebearing fish, the Sailfin molly (Poecilia latipinna) combining genetic, life-history, and environmental data, asking how structured populations are. Sailfin mollies can be found in coastal freshwater and brackish habitats from roughly Tampico, Veracruz in Mexico to Wilmington, North Carolina, in the USA. In addition, they are found inland on the Florida peninsula. Using microsatellite DNA, we genotyped 168 individuals from 18 populations covering most of the natural range of the Sailfin molly. We further determined standard life-history parameters for both males and females for these populations. Finally, we measured biotic and abiotic parameters in the field. We found six distinct genetic clusters based on microsatellite data, with very strong indication of isolation by distance. However, we also found significant numbers of migrants between adjacent populations. Despite genetic structuring we did not find evidence of cryptic speciation. The genetic clusters and the migration patterns do not match paleodrainages. Life histories vary between populations but not in a way that is easy to interpret. We suggest a role of humans in migration in the sailfin molly, for example in the form of a ship channel that connects southern Texas with Louisiana which might be a conduit for fish migration.

Nascent transcription reveals regulatory changes in extremophile fishes inhabiting hydrogen sulfide-rich environments.

Regulating transcription allows organisms to respond to their environment, both within a single generation (plasticity) and across generations (adaptation). We examined transcriptional differences in gill tissues of fishes in the Poecilia mexicana species complex (family Poeciliidae), which have colonized toxic springs rich in hydrogen sulfide (H2S) in southern Mexico. There are gene expression differences between sulfidic and non-sulfidic populations, yet regulatory mechanisms mediating this gene expression variation remain poorly studied. We combined capped-small RNA sequencing (csRNA-seq), which captures actively transcribed (i.e. nascent) transcripts, and messenger RNA sequencing (mRNA-seq) to examine how variation in transcription, enhancer activity, and associated transcription factor binding sites may facilitate adaptation to extreme environments. csRNA-seq revealed thousands of differentially initiated transcripts between sulfidic and non-sulfidic populations, many of which are involved in H2S detoxification and response. Analyses of transcription factor binding sites in promoter and putative enhancer csRNA-seq peaks identified a suite of transcription factors likely involved in regulating H2S-specific shifts in gene expression, including several key transcription factors known to respond to hypoxia. Our findings uncover a complex interplay of regulatory processes that reflect the divergence of extremophile populations of P. mexicana from their non-sulfidic ancestors and suggest shared responses among evolutionarily independent lineages.

Sub-acute exposure of male guppies (Poecilia reticulata) to environmentally relevant concentrations of PFOA and GenX induces significant changes in the testis transcriptome and reproductive traits.

Poly- and perfluoroalkyl substances (PFAS) are frequently detected in the environment and are linked to adverse reproductive health outcomes in humans. Although legacy PFAS have been phased out due to their toxicity, alternative PFAS are increasingly used despite the fact that information on their toxic effects on reproductive traits is particularly scarce. Here, we exposed male guppies (Poecilia reticulata) for a short period (21 days) to an environmentally realistic concentration (1 ppb) of PFOA, a legacy PFAS, and its replacement compound, GenX, to assess their impact on reproductive traits and gene expression. Exposure to PFAS did not impair survival but instead caused sublethal effects. Overall, PFAS exposure caused changes in male sexual behaviour and had detrimental effects on sperm motility. Sublethal variations were also seen at the transcriptional level, with the modulation of genes involved in immune regulation, spermatogenesis, and oxidative stress. We also observed bioaccumulation of PFAS, which was higher for PFOA than for GenX. Our results offer a comprehensive comparison of these two PFAS and shed light on the toxicity of a newly emerging alternative to legacy PFAS. It is therefore evident that even at low concentrations and with short exposure, PFAS can have subtle yet significant effects on behaviour, fertility, and immunity. These findings underscore the potential ramifications of pollution under natural conditions and their impact on fish populations.

Evolutionary Rate Shifts in Coding and Regulatory Regions Underpin Repeated Adaptation to Sulfidic Streams in Poeciliid Fishes.

Adaptation to extreme environments often involves the evolution of dramatic physiological changes. To better understand how organisms evolve these complex phenotypic changes, the repeatability and predictability of evolution, and possible constraints on adapting to an extreme environment, it is important to understand how adaptive variation has evolved. Poeciliid fishes represent a particularly fruitful study system for investigations of adaptation to extreme environments due to their repeated colonization of toxic hydrogen sulfide-rich springs across multiple species within the clade. Previous investigations have highlighted changes in the physiology and gene expression in specific species that are thought to facilitate adaptation to hydrogen sulfide-rich springs. However, the presence of adaptive nucleotide variation in coding and regulatory regions and the degree to which convergent evolution has shaped the genomic regions underpinning sulfide tolerance across taxa are unknown. By sampling across seven independent lineages in which nonsulfidic lineages have colonized and adapted to sulfide springs, we reveal signatures of shared evolutionary rate shifts across the genome. We found evidence of genes, promoters, and putative enhancer regions associated with both increased and decreased convergent evolutionary rate shifts in hydrogen sulfide-adapted lineages. Our analysis highlights convergent evolutionary rate shifts in sulfidic lineages associated with the modulation of endogenous hydrogen sulfide production and hydrogen sulfide detoxification. We also found that regions with shifted evolutionary rates in sulfide spring fishes more often exhibited convergent shifts in either the coding region or the regulatory sequence of a given gene, rather than both.

Evolutionary divergence of plasticity in brain morphology between ecologically divergent habitats of Trinidadian guppies.

Phenotypic plasticity is critical for organismal performance and can evolve in response to natural selection. Brain morphology is often developmentally plastic, affecting animal performance in a variety of contexts. However, the degree to which the plasticity of brain morphology evolves has rarely been explored. Here, we use Trinidadian guppies (Poecilia reticulata), which are known for their repeated adaptation to high-predation (HP) and low-predation (LP) environments, to examine the evolution and plasticity of brain morphology. We exposed second-generation offspring of individuals from HP and LP sites to 2 different treatments: predation cues and conspecific social environment. Results show that LP guppies had greater plasticity in brain morphology compared to their ancestral HP population, suggesting that plasticity can evolve in response to environmentally divergent habitats. We also show sexual dimorphism in the plasticity of brain morphology, highlighting the importance of considering sex-specific variation in adaptive diversification. Overall, these results may suggest the evolution of brain morphology plasticity as an important mechanism that allows for ecological diversification and adaptation to divergent habitats.

Aesthetic preference for artificially selected color variant affects mate choice copying behavior in female Poecilia latipinna.

Three experiments were conducted examining whether an artificially selected "gold" color variant in female "models" affects mate choice copying behavior in sailfin mollies (Poecilia latipinna). Experiment I consisted of a pair of female preference assays, first assessing preference for male body size, followed by a mate choice copying assay that paired a model female with the smaller, non-preferred male from the initial preference test. Female subjects were divided into three groups that used either a wildtype female model, an artificially selected "gold" variant (cultivated within the aquarium fish trade) model, or control wherein no model was presented. Results showed females consistently copied the model's choice, switching preferences from the larger to smaller male when paired with a model regardless of color. In the second experiment wildtype females were presented with a pair of size-matched dummy males both of which paired with model females (one gold and the other wild type). Subjects consistently preferred the male previously paired with the gold- over the male with the wildtype-model, suggesting pre-existing sensory/perceptual biases may have affected their mate choice copying behavior. Previous studies have offered evidence for the spread of novel traits in males via sensory exploitation. However, these results indicate such biases may influence courtship behavior in circumstances where the novel trait is expressed in females as well. For the third experiment, wildtype females were presented with a choice between gold vs wildtype dummy males, the results of which revealing significant preferences for gold. In a follow-up assay pairing a wild type model with the non-preferred wildtype male, females maintained their preference for gold males despite the conflicting social driver of mate choice copying. These data offer evidence for the existence of a perceptual/cognitive bias in the context of mate choice copying, favoring the gold phenotype and/or novelty in general.

Identification of Philaster apodigitiformis in aquaculture and functional characterization of its ß-PKA gene and expression analysis of infected Poecilia reticulata.

Cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) is a distinctive member of the serine­threonine protein AGC kinase family and an effective kinase for cAMP signal transduction. In recent years, scuticociliate has caused a lot of losses in domestic fishery farming, therefore, we have carried out morphological and molecular biological studies. In this study, diseased guppies (Poecilia reticulata) were collected from an ornamental fish market, and scuticociliate Philaster apodigitiformis Miao et al., 2009 was isolated. In our prior transcriptome sequencing research, we discovered significant expression of the ß-PKA gene in P. apodigitiformis during its infection process, leading us to speculate its involvement in pathogenesis. A complete sequence of the ß-PKA gene was cloned, and quantified by quantitative reverse transcription-polymerase chain reaction to analyse or to evaluate the functional characteristics of the ß-PKA gene. Morphological identification and phylogenetic analysis based on small subunit rRNA sequence, infection experiments and haematoxylin­eosin staining method were also carried out, in order to study the pathological characteristics and infection mechanism of scuticociliate. The present results showed that: (1) our results revealed that ß-PKA is a crucial gene involved in P. apodigitiformis infection in guppies, and the findings provide valuable insights for future studies on scuticociliatosis; (2) we characterized a complete gene, ß-PKA, that is generally expressed in parasitic organisms during infection stage and (3) the present study indicates that PKA plays a critical role in scuticociliate when infection occurs by controlling essential steps such as cell growth, development and regulating the activity of the sensory body structures and the irritability system.

Trophic niche drives the evolution of craniofacial shape in Trinidadian guppies.

Diverse clades of fishes adapted to feeding on the benthos repeatedly converge on steep craniofacial profiles and shorter, wider heads. But in an incipient radiation, to what extent is this morphological evolution measurable and can we distinguish the relative genetic vs. plastic effects? We use the Trinidadian guppy (Poecilia reticulata) to test the repeatability of adaptation and the alignment of genetic and environmental effects shaping poecilid craniofacial morphology. We compare wild-caught and common garden lab-reared fish to quantify the genetic and plastic components of craniofacial morphology across 4 populations from 2 river drainage systems (n = 56 total). We first use micro-computed tomography to capture 3D morphology, then place both landmarks and semilandmarks to perform size-corrected 3D morphometrics and quantify shape space. We find a measurable, significant, and repeatable divergence in craniofacial shape between high-predation invertivore and low-predation detritivore populations. As predicted from previous examples of piscine adaptive trophic divergence, we find increases in head slope and craniofacial compression among the benthic detritivore foragers. Furthermore, the effects of environmental plasticity among benthic detritivores produce exaggerated craniofacial morphological change along a parallel axis to genetic morphological adaptation from invertivore ancestors. Overall, many of the major patterns of benthic-limnetic craniofacial evolution appear convergent among disparate groups of teleost fishes.

Identification of Candidate Genes for Red-Eyed (Albinism) Domestic Guppies Using Genomic and Transcriptomic Analyses.

Guppies are small tropical fish with brightly colored bodies and variable tail shapes. There are two phenotypes of domestic guppy eye color: red and black. The wild type is black-eyed. The main object of this study was to identify candidate genes for the red-eyed phenotype in domestic guppies. We hope to provide molecular genetic information for the development of new domestic guppy strains. Additionally, the results also contribute to basic research concerning guppies. In this study, 121 domestic guppies were used for genomic analysis (GWAS), and 44 genes were identified. Furthermore, 21 domestic guppies were used for transcriptomic analysis, and 874 differentially expressed genes (DEGs) were identified, including 357 upregulated and 517 downregulated genes. Through GO and KEGG enrichment, we identified some important terms or pathways mainly related to melanin biosynthesis and ion transport. qRT-PCR was also performed to verify the differential expression levels of four important candidate genes (TYR, OCA2, SLC45A2, and SLC24A5) between red-eyed and black-eyed guppies. Based on the results of genomic and transcriptomic analyses, we propose that OCA2 is the most important candidate gene for the red-eyed phenotype in guppies.

Identification of a Tetrahymena species infecting guppies, pathology, and expression of beta-tubulin during infection.

Tetrahymenosis is caused by the ciliated protozoan Tetrahymena and is responsible for serious economic losses to the aquaculture industry worldwide. However, information regarding the molecular mechanism leading to tetrahymenosis is limited. In previous transcriptome sequencing work, it was found that one of the two ß-tubulin genes in T. pyriformis was significantly expressed in infected fish, we speculated that ß-tubulin is involved in T. pyriformis infecting fish. Herein, the potential biological function of the ß-tubulin gene in Tetrahymena species when establishing infection in guppies was investigated by cloning the full-length cDNA of this T. pyriformis ß-tubulin (BTU1) gene. The full-length cDNA of T. pyriformis BTU1 gene was 1873 bp, and the ORF occupied 1134 bp, whereas 5' UTR 434 bp, and 3' UTR 305 bp whose poly (A) tail contained 12 bases. The predicted protein encoded by T. pyriformis BTU1 gene had a calculated molecular weight of 42.26 kDa and pI of 4.48. Moreover, secondary structure analysis and tertiary structure prediction of BTU1 protein were also conducted. In addition, morphology, infraciliature, phylogeny, and histopathology of T. pyriformis isolated from guppies from a fish market in Harbin were also investigated. Furthermore, qRT-PCR analysis and experimental infection assays indicated that the expression of BTU1 gene resulted in efficient cell proliferation during infection. Collectively, our data revealed that BTU1 is a key gene involved in T. pyriformis infection in guppies, and the findings discussed herein provide valuable insights for future studies on tetrahymenosis.

Has recombination changed during the recent evolution of the guppy Y chromosome?

Genome sequencing and genetic mapping of molecular markers have demonstrated nearly complete Y-linkage across much of the guppy (Poecilia reticulata) XY chromosome pair. Predominant Y-linkage of factors controlling visible male-specific coloration traits also suggested that these polymorphisms are sexually antagonistic (SA). However, occasional exchanges with the X are detected, and recombination patterns also appear to differ between natural guppy populations, suggesting ongoing evolution of recombination suppression under selection created by partially sex-linked SA polymorphisms. We used molecular markers to directly estimate genetic maps in sires from 4 guppy populations. The maps are very similar, suggesting that their crossover patterns have not recently changed. Our maps are consistent with population genomic results showing that variants within the terminal 5 Mb of the 26.5 Mb sex chromosome, chromosome 12, are most clearly associated with the maleness factor, albeit incompletely. We also confirmed occasional crossovers proximal to the male-determining region, defining a second, rarely recombining, pseudo-autosomal region, PAR2. This fish species may therefore have no completely male-specific region (MSY) more extensive than the male-determining factor. The positions of the few crossover events suggest a location for the male-determining factor within a physically small repetitive region. A sex-reversed XX male had few crossovers in PAR2, suggesting that this region's low crossover rate depends on the phenotypic, not the genetic, sex. Thus, rare individuals whose phenotypic and genetic sexes differ, and/or occasional PAR2 crossovers in males can explain the failure to detect fully Y-linked variants.

Disentangling historical relationships within Poeciliidae (Teleostei: Cyprinodontiformes) using ultraconserved elements.

Poeciliids (Cyprinodontiformes: Poeciliidae), commonly known as livebearers, are popular fishes in the aquarium trade (e.g., guppies, mollies, swordtails) that are widely distributed in the Americas, with 274 valid species in 27 genera. This group has undergone various taxonomic changes recently, spurred by investigations using traditional genetic markers. Here we used over 1,000 ultraconserved loci to infer the relationships within Poeciliidae in the first attempt at understanding their diversification based on genome-scale data. We explore gene tree discordance and investigate potential incongruence between concatenation and coalescent inference methods. Our aim is to examine the influence of incomplete lineage sorting and reticulate evolution on the poeciliids' evolutionary history and how these factors contribute to the observed gene tree discordace. Our concatenated and coalescent phylogenomic inferences recovered four major clades within Poeciliidae. Most supra-generic level relationships we inferred were congruent with previous molecular studies, but we found some disagreements; the Middle American taxa Phallichthys and Poecilia (Mollienesia) were recovered as non-monophyletic, and unlike other recent molecular studies, we recovered Brachyrhaphis as monophyletic. Our study is the first to provide signatures of reticulate evolution in Poeciliidae at the family level; however, continued finer-scale investigations are needed to understand the complex evolutionary history of the family along with a much-needed taxonomic re-evaluation.

Sperm specificity and potential paternal effects in gynogenesis in the Amazon Molly (Poecilia formosa).

The Amazon Molly (Poecilia formosa) reproduces by gynogenesis, a relatively rare form of asexual reproduction where sperm is required to trigger embryogenesis, but male genes are not incorporated into the genome of the embryo. Studying gynogenesis could isolate paternal non-genetic effects on reproduction. This study explored which of eleven related species can produce sperm to trigger gynogenesis through natural mating in P. formosa, and whether sympatry affects reproductive success in P. formosa. Reproductive outcomes measured were relative reproductive output (number of offspring in the first brood divided by female standard length), relative embryo output (number of embryos in the first brood divided by female standard length) and combined relative reproductive output (sum of relative reproductive output and relative embryo output). For large (>4 cm) P. formosa, combined relative reproductive output was higher with sympatric Atlantic Molly (Poecilia mexicana) males than with allopatric P. mexicana males. P. formosa produced live offspring or late-stage embryos with all species tested in the genera Poecilia and Limia but did not produce offspring or embryos with males from the genera Gambusia, Girardinus, Heterandria, Poeciliopsis, or Xiphophorus. This information, as well as the limitations characterized in this study, will set a foundation for use of P. formosa as a model for paternal effects and the species specificity of sperm on fertilization, embryogenesis, and reproductive success.

Whole Genome Assembly and Annotation of Blackstripe Livebearer Poeciliopsis prolifica.

The blackstripe livebearer Poeciliopsis prolifica is a live-bearing fish belonging to the family Poeciliidae with high level of postfertilization maternal investment (matrotrophy). This viviparous matrotrophic species has evolved a structure similarly to the mammalian placenta. Placentas have independently evolved multiple times in Poeciliidae from nonplacental ancestors, which provide an opportunity to study the placental evolution. However, there is a lack of high-quality reference genomes for the placental species in Poeciliidae. In this study, we present a 674 Mb assembly of P. prolifica in 504 contigs with excellent continuity (contig N50 7.7 Mb) and completeness (97.2% Benchmarking Universal Single-Copy Orthologs [BUSCO] completeness score, including 92.6% single-copy and 4.6% duplicated BUSCO score). A total of 27,227 protein-coding genes were annotated from the merged datasets based on bioinformatic prediction, RNA sequencing and homology evidence. Phylogenomic analyses revealed that P. prolifica diverged from the guppy (Poecilia reticulata) ∼19 Ma. Our research provides the necessary resources and the genomic toolkit for investigating the genetic underpinning of placentation.

Genetic and context-specific effects on individual inhibitory control performance in the guppy (Poecilia reticulata).

Among-individual variation in cognitive traits, widely assumed to have evolved under adaptive processes, is increasingly being demonstrated across animal taxa. As variation among individuals is required for natural selection, characterizing individual differences and their heritability is important to understand how cognitive traits evolve. Here, we use a quantitative genetic study of wild-type guppies repeatedly exposed to a 'detour task' to test for genetic variance in the cognitive trait of inhibitory control. We also test for genotype-by-environment interactions (GxE) by testing related fish under alternative experimental treatments (transparent vs. semi-transparent barrier in the detour-task). We find among-individual variation in detour task performance, consistent with differences in inhibitory control. However, analysis of GxE reveals that heritable factors only contribute to performance variation in one treatment. This suggests that the adaptive evolutionary potential of inhibitory control (and/or other latent variables contributing to task performance) may be highly sensitive to environmental conditions. The presence of GxE also implies that the plastic response of detour task performance to treatment environment is genetically variable. Our results are consistent with a scenario where variation in individual inhibitory control stems from complex interactions between heritable and plastic components.

Sperm production is negatively associated with muscle and sperm telomere length in a species subjected to strong sperm competition.

Life-history theory suggests that ageing is one of the costs of reproduction. Accordingly, a higher reproductive allocation is expected to increase the deterioration of both the somatic and the germinal lines through enhanced telomere attrition. In most species, males' reproductive allocation mainly regards traits that increase mating and fertilization success, that is sexually selected traits. In this study, we tested the hypothesis that a higher investment in sexually selected traits is associated with a reduced relative telomere length (RTL) in the guppy (Poecilia reticulata), an ectotherm species characterized by strong pre- and postcopulatory sexual selection. We first measured telomere length in both the soma and the sperm over guppies' lifespan to see whether there was any variation in telomere length associated with age. Second, we investigated whether a greater investment in pre- and postcopulatory sexually selected traits is linked to shorter telomere length in both the somatic and the sperm germinal lines, and in young and old males. We found that telomeres lengthened with age in the somatic tissue, but there was no age-dependent variation in telomere length in the sperm cells. Telomere length in guppies was significantly and negatively correlated with sperm production in both tissues and life stages considered in this study. Our findings indicate that telomere length in male guppies is strongly associated with their reproductive investment (sperm production), suggesting that a trade-off between reproduction and maintenance is occurring at each stage of males' life in this species.

Selection on standing genetic variation mediates convergent evolution in extremophile fish.

Hydrogen sulfide is a toxic gas that disrupts numerous biological processes, including energy production in the mitochondria, yet fish in the Poecilia mexicana species complex have independently evolved sulfide tolerance several times. Despite clear evidence for convergence at the phenotypic level in these fishes, it is unclear if the repeated evolution of hydrogen sulfide tolerance is the result of similar genomic changes. To address this gap, we used a targeted capture approach to sequence genes associated with sulfide processes and toxicity from five sulfidic and five nonsulfidic populations in the species complex. By comparing sequence variation in candidate genes to a reference set, we identified similar population structure and differentiation, suggesting that patterns of variation in most genes associated with sulfide processes and toxicity are due to demographic history and not selection. But the presence of tree discordance for a subset of genes suggests that several loci are evolving divergently between ecotypes. We identified two differentiation outlier genes that are associated with sulfide detoxification in the mitochondria that have signatures of selection in all five sulfidic populations. Further investigation into these regions identified long, shared haplotypes among sulfidic populations. Together, these results reveal that selection on standing genetic variation in putatively adaptive genes may be driving phenotypic convergence in this species complex.

Extensive variation in germline de novo mutations in Poecilia reticulata.

The rate of germline mutation is fundamental to evolutionary processes, as it generates the variation upon which selection acts. The guppy, Poecilia reticulata, is a model of rapid adaptation, however the relative contribution of standing genetic variation versus de novo mutation (DNM) to evolution in this species remains unclear. Here, we use pedigree-based approaches to quantify and characterize germline DNMs in three large guppy families. Our results suggest germline mutation rate in the guppy varies substantially across individuals and families. Most DNMs are shared across multiple siblings, suggesting they arose during early embryonic development. DNMs are randomly distributed throughout the genome, and male-biased mutation rate is low, as would be expected from the short guppy generation time. Overall, our study shows remarkable variation in germline mutation rate and provides insights into rapid evolution of guppies.

The quest for good genes: Epigamic traits, fitness, MHC and multilocus heterozygosity in the guppy.

The 'good genes' hypothesis for the evolution of male secondary sexual traits poses that female preferences for such traits are driven by indirect genetic benefits. However, support for the hypothesis remains ambiguous, and, in particular, the genetic basis for the benefits has rarely been investigated. Here, we use seminatural populations of Trinidadian guppies to investigate whether sexually selected traits (orange, black and iridescent colouration, gonopodium length and body size) predict fitness measured as the number of grandoffspring, a metric that integrates across fitness components and sexes. Furthermore, we tested whether two potential sources of genetic benefits-major histocompatibility complex (MHC) genotypes and multilocus heterozygosity (MLH)-are significant predictors of fitness and of the size of sexually selected traits. We found a significant, nonlinear effect of the area of black pigmentation and male body size on the number of grandoffspring, suggesting stabilizing selection on black area, and nonlinear selection favouring small body size. MLH was heritable (h2 = 0.14) and significantly predicted the number of grandoffspring, indicating the potential for genetic benefits based on heterozygosity. We also found support for local heterozygosity effects, which may reflect a noneven distribution of genetic load across the genome. MHC genotype was not significantly associated with any tested fitness component, or with the load of Gyrodactylus parasites. Neither MHC nor MLH was significant predictor of sexually selected traits. Overall, our results highlight the role of heterozygosity in determining fitness, but do not provide support for male sexually selected traits being indicators of genetic quality.