Does the loss of diadromy imply the loss of salinity tolerance? A gene expression study with replicate nondiadromous populations of Galaxias maculatus.

The recurrent colonization of freshwater habitats and subsequent loss of diadromy is a major ecological transition that has been reported in many ancestrally diadromous fishes. Such residency is often accompanied by a loss of tolerance to seawater. The amphidromous Galaxias maculatus has repeatedly colonized freshwater streams with evidence that freshwater-resident populations exhibit stark differences in their tolerance to higher salinities. Here, we used transcriptomics to gain insight into the mechanisms contributing to reduced tolerance to higher salinities in freshwater resident populations. We conducted an acute salinity challenge (0 ppt to 23-25 ppt) and measured osmoregulatory ability (muscle water content) over 48 h in three populations: diadromous, saltwater intolerant resident (Toltén), and saltwater tolerant resident (Valdivia). RNA sequencing of the gills identified genes that were differentially expressed in association with the salinity change and associated with the loss of saltwater tolerance in the Toltén population. Key genes associated with saltwater acclimation were characterized in diadromous G. maculatus individuals, some of which were also expressed in the saltwater tolerant resident population (Valdivia). We found that some of these "saltwater acclimation" genes, including the cystic fibrosis transmembrane conductance regulator gene (CFTR), were not significantly upregulated in the saltwater intolerant resident population (Toltén), suggesting a potential mechanism for the loss of tolerance to higher salinities. As the suite of differentially expressed genes in the diadromous-resident comparison differed between freshwater populations, we hypothesize that diadromy loss results in unique evolutionary trajectories due to drift, so the loss of diadromy does not necessarily lead to a loss in upper salinity tolerance.

La colonización recurrente de hábitats de agua dulce y la subsecuente pérdida de diadromía es una transición ecológica importante que ha sido reportada en varias especies de peces con ancestros diádromos. Esta residencia está acompañada frecuentemente por la pérdida de tolerancia a ambientes de agua salada. Galaxias maculatus, especie anfídroma, ha colonizado ríos repetidamente y existe evidencia que las poblaciones residentes presentan diferencias respecto a la tolerancia al agua salada. En este estudio, usamos transcriptómica para dilucidar los mecanismos que contribuyen a la reducida tolerancia a altas salinidades en las poblaciones residentes de agua dulce. Realizamos un desafío agudo de salinidad (0 ppt a 23-2 ppt) y medimos la habilidad osmoreguladora (contenido de agua en músculo) por 48 horas en individuos de tres poblaciones: una diádroma, una intolerante a agua salada (Toltén) y una tolerante a agua salada (Valdivia). Con el secuenciamiento de ARN de las branquias identificamos los genes expresados diferencialmente al cambio de salinidad y cuales están asociados a la pérdida de tolerancia a agua salada en la población de Toltén. Genes claves asociados a la aclimatación al agua salada fueron caracterizados en individuos diádromos, algunos de ellos también se expresaron en la población residente tolerante al agua salada (Valdivia). Sin embargo, algunos genes involucrados en la aclimatación al agua salada, incluyendo el gen regulador de la conductancia transmembrana de la fibrosis quística (CFTR), no se diferenciaron significativamente en la población residente intolerante al agua salada (Toltén), sugiriendo un mecanismo potencial de la pérdida de tolerancia a ambientes con salinidad elevada. Como el conjunto de genes expresados difiere entre las dos poblaciones residentes al compararse con la población diádroma, hipotetizamos que la pérdida de diadromía resulta en trayectorias evolutivas únicas debido a deriva génica, por lo que la pérdida de la diadromía no necesariamente conlleva a la pérdida de la tolerancia a aguas saladas.

Alternative splicing: An overlooked mechanism contributing to local adaptation?

Identifying the molecular mechanisms contributing to phenotypic variation in natural populations is a major goal of molecular ecology. However, the multiple regulatory steps between genotype and phenotype mean that many potential mechanisms can lead to trait divergence. To date, the role of transcriptional regulation in local adaptation has received much focus, as we can readily measure mRNA quantity and have a reasonable grasp of how variation in the expression of many protein-coding genes can influence phenotype. Thus, studying the evolution of protein-coding gene mRNA abundance in candidate tissues has led to successes in detecting the molecular mechanisms underlying local adaptation (reviewed by Hill et al., 2021). However, the contribution of differential splicing of precursor mRNA (pre-mRNA) to adaptive differentiation, as well as the loci controlling this variation, remains largely unexplored in wild populations. In their "From the Cover'" article in this issue of Molecular Ecology, Jacobs and Elmer (2021) reanalyse muscle RNA sequencing (RNA-seq) data to quantify the relative contributions of variation in mRNA quantity (differentially expressed "DE" genes) and splice variant identity (differentially spliced "DS" genes) to parallel divergence of wild "benthic" and "pelagic" ecotypes of a salmonid fish, the Arctic charr (Salvelinus alpinus). They found little overlap in the identity and biological functions of DE and DS genes, suggesting that these two regulatory mechanisms act on different cellular traits to complementarily alter organismal phenotype. Furthermore, many DE and DS genes could be mapped to cis-acting QTL, arguing that some of this regulatory divergence is genetically based. DE and DS genes were also more likely to be "hub genes" than their nondivergent counterparts, hinting that this regulatory variation may have a variety of phenotypic effects. The comparison of three independently evolved pairs of benthic and pelagic charr uncovered greater than expected parallelism in both expression and splicing between ecotypes across different lakes, supporting a role for these molecular phenotypes in adaptive divergence. Overall, the findings of Jacobs and Elmer (2021) highlight the importance of alternative splicing as a potential mechanism underlying local adaptation and provide a framework for others hoping to make the most of their RNA-seq data.

Investigating Diadromy in Fishes and Its Loss in an -Omics Era.

Diadromy, the predictable movements of individuals between marine and freshwater environments, is biogeographically and phylogenetically widespread across fishes. Thus, despite the high energetic and potential fitness costs involved in moving between distinct environments, diadromy appears to be an effective life history strategy. Yet, the origin and molecular mechanisms that underpin this migratory behavior are not fully understood. In this review, we aim first to summarize what is known about diadromy in fishes; this includes the phylogenetic relationship among diadromous species, a description of the main hypotheses regarding its origin, and a discussion of the presence of non-migratory populations within diadromous species. Second, we discuss how recent research based on -omics approaches (chiefly genomics, transcriptomics, and epigenomics) is beginning to provide answers to questions on the genetic bases and origin(s) of diadromy. Finally, we suggest future directions for -omics research that can help tackle questions on the evolution of diadromy.

Genomic basis of the loss of diadromy in Galaxias maculatus: Insights from reciprocal transplant experiments.

Diadromy is known for having major effects on the distribution and richness of aquatic species, and so does its loss. The loss of diadromy has led to the diversification of many species, yet research focusing on understanding its molecular basis and consequences are limited. This is particularly true for amphidromous species despite being the most abundant group of diadromous species. Galaxias maculatus, an amphidromous species and one of the most widely distributed fishes in the Southern Hemisphere, exhibits many instances of nonmigratory or resident populations. The existence of naturally replicated resident populations in Patagonia can serve as an ideal system for the study of the mechanisms that lead to the loss of the diadromy and its ecological and evolutionary consequences. Here, we studied two adjacent river systems in which resident populations are genetically differentiated yet derived from the same diadromous population. By combining a reciprocal transplant experiment with genomic data, we showed that the two resident populations followed different evolutionary pathways by exhibiting a differential response in their capacity to survive in salt water. While one resident population was able to survive salt water, the other was not. Genomic analyses provided insights into the genes that distinguished (a) migratory from nonmigratory populations; (b) populations that can vs those that cannot survive a saltwater environment; and (c) between these resident populations. This study demonstrates that the loss of diadromy can be achieved by different pathways and that environmental (selection) and random (genetic drift) forces shape this dynamic evolutionary process.

The effects of diadromy and its loss on genomic divergence: The case of amphidromous Galaxias maculatus populations.

Understanding the evolutionary mechanisms that affect the genetic divergence between diadromous and resident populations across heterogeneous environments is a challenging task. While diadromy may promote gene flow leading to a lack of genetic differentiation among populations, resident populations tend to be affected by local adaptation and/or plasticity. Studies on these effects on genomic divergence in nonmodel amphidromous species are scarce. Galaxias maculatus, one of the most widespread fish species in the Southern Hemisphere, exhibits two life histories, an ancestral diadromous, specifically, amphidromous form, and a derived freshwater resident form. We examined the genetic diversity and divergence among 20 estuarine and resident populations across the Chilean distribution of G. maculatus and assessed the extent to which selection is involved in the differentiation among resident populations. We obtained nearly 4,400 SNP markers using a RADcap approach for 224 individuals. As expected, collections from estuarine locations typically consist of diadromous individuals. Diadromous populations are highly differentiated from their resident counterparts by both neutral and putative adaptive markers. While diadromous populations exhibit high gene flow and lack site fidelity, resident populations appear to be the product of different colonization events with relatively low genetic diversity and varying levels of gene flow. In particular, the northernmost resident populations were clearly genetically distinct and reproductively isolated from each other suggesting local adaptation. Our study provides insights into the role of life history differences in the maintenance of genetic diversity and the importance of genetic divergence in species evolution.

Intestinal Microbial Community Dynamics of White-Tailed Deer (Odocoileus virginianus) in an Agroecosystem.

The intestinal microbiota has important functions that contribute to host health. The compositional dynamics of microbial communities are affected by many factors, including diet and presence of pathogens. In contrast to humans and domestic mammals, the composition and seasonal dynamics of intestinal microbiota of wildlife species remain comparatively understudied. White-tailed deer (Odocoileus virginianus) is an ecologically and economically important wildlife species that inhabits agricultural ecosystems and is known to be a reservoir of enteric pathogens. Nevertheless, there is a lack of knowledge of white-tailed deer intestinal microbiota diversity and taxonomic composition. This study's first objective was to characterize and compare the intestinal microbiota of 66 fecal samples from white-tailed deer collected during two sampling periods (March and June) using 16S rDNA pyrosequencing. Associations between community diversity and composition and factors including season, sex, host genetic relatedness, and spatial location were quantified. Results revealed that white-tailed deer intestinal microbiota was predominantly comprised of phyla Firmicutes and Proteobacteria, whose relative frequencies varied significantly between sampling periods. The second objective was to examine the associations between the presence of Escherichia coli and Salmonella, and microbiota composition and diversity. Results indicated that relative abundance of some microbial taxa varied when a pathogen was present. This study provides insights into microbial compositional dynamics of a wildlife species inhabiting coupled natural and agricultural landscapes. Data focus attention on the high prevalence of Proteobacteria particularly during the summer and highlight the need for future research regarding the role of white-tailed deer as a natural pathogen reservoir in agroecosystems.