Genome scans reveal signals of selection associated with pollution in fish populations of Basilichthys microlepidotus, an endemic species of Chile.

The Maipo River catchment is one of Chile's most polluted basins. In recent decades, discharges of untreated sewage and organic matter have caused eutrophication and water quality degradation. We employed the indigenous silverfish species Basilichthys microlepidotus as a model organism to investigate the process of adaptation and selection on genes influenced by pollution. Using variation at single nucleotide polymorphisms (SNPs), we determined the temporal stability of the population structure patterns previously identified in this species by varying SNPs. We also examined local adaptation to pollution-selected genes. Using the genotypes of 7684 loci in 180 individuals, we identified 429 and 700 loci that may be undergoing selection. We detected these loci using the FSTHET and ARLEQUIN outlier detection software, respectively. Both software packages simultaneously identified a total of 250 loci. B. microlepidotus' population structure did not change over time at contaminated or unpolluted sites. In addition, our analysis found: (i) selection of genes associated with pollution, consistent with observations in other organisms; (ii) identification of candidate genes that are functionally linked to the same biological processes, molecular functions and/or cellular components that previously showed differential expression in the same populations; and (iii) a candidate gene with differential expression and a non-synonymous substitution.

Biomarker selection depends on gene function and organ: the case of the cytochrome P450 family genes in freshwater fish exposed to chronic pollution.

Pollution and its effects have been of major concern in recent decades. Many strategies and markers have been developed to assess their effects on biota. Cytochrome P450 (CYP) genes have received significant attention in this context because of their relationship with detoxification and activation of exogenous compounds. While their expression has been identified as a pollution exposure biomarker, in most cases, it has been tested only after acute exposures and for CYP genes associated with exogenous compounds. To elucidate CYP gene expression patterns under chronic pollution exposure, we have used the silverside Basilichthys microlepidotus as a model, which inhabits the Maipo River Basin, a freshwater system with different pollution levels. We performed next-generation RNA sequencing of liver and gill tissues from polluted and non-polluted populations. We found most CYP genes were not dysregulated by pollution, and the seven genes that were present and differentially expressed in liver and gill were mainly downregulated. Three CYP genes associated with exogenous compounds showed differential expression in the gill, while four CYP genes associated with endogenous compounds showed differential expression in the liver. The findings presented here highlight the importance of CYP genes, his family, tissues and his interaction in the context of pollution biomarkers use.

Spatial and temporal stability in the genetic structure of a marine crab despite a biogeographic break.

Elucidating the processes responsible for maintaining the population connectivity of marine benthic species mediated by larval dispersal remains a fundamental question in marine ecology and fishery management. Understanding these processes becomes particularly important in areas with a biogeographic break and unidirectional water movement along the sides of the break. Based on variability at 4209 single-nucleotide polymorphisms in 234 individuals, we determine the genetic structure, temporal genetic stability, and gene flow among populations of the commercially important mola rock crab Metacarcinus edwardsii in a system in southern Chile with a biogeographic break at latitude 42°S. Specimens were collected at eight sites within its geographic distribution, with collection at four of these sites was performed twice. Using population genetic approaches, we found no evidence of geographic or temporal population differentiation. Similarly, we found no evidence of an effect on gene flow of the biogeographic break caused by the the West Wind Drift Current. Moreover, migration analyses supported gene flow among all sites but at different rates for different pairs of sites. Overall, our findings indicate that M. edwardsii comprises a single large population with high levels of gene flow among sites separated by over 1700 km and demonstrate temporal stability in its genetic structure.

Geographical context outweighs habitat disturbance effects in explaining mistletoe population genetic differentiation at a regional scale.

Genetic differentiation depends on ecological and evolutionary processes that operate at different spatial and temporal scales. While the geographical context is likely to determine large-scale genetic variation patterns, habitat disturbance events will probably influence small-scale genetic diversity and gene flow patterns. Therefore, the genetic diversity patterns that we observe today result from the combination of both processes, but they are rarely assessed simultaneously. We determined the population structure and genetic diversity of a hemiparasitic mistletoe (Tristerix corymbosus) from the temperate rainforests of southern Chile to determine the effects of geographical context and habitat disturbance at a regional scale and if it is affected by the abundance and occurrence of its seed disperser mutualist (the arboreal marsupial Dromiciops gliroides). We genotyped 359 individuals from 12 populations using single nucleotide polymorphisms, across three different geographical contexts and four disturbance conditions. We also used camera traps to estimate the abundance and occurrence of the seed disperser. Our results suggest that genetic differences among populations are related more to geographical context than to habitat disturbance. However, as disturbance increased, D. gliroides abundance and occurrence decreased, and mistletoe inbreeding index (FIS ) increased. We also found highly uneven gene flow among study sites. Despite the high levels of disturbance that these temperate rainforests are facing, our results suggest that mistletoe genetic differentiation at a regional scale was more influenced by historical events. However, habitat disturbance can indirectly affect mistletoe population genetic differentiation via the seed dispersal process, which may increase levels of inbreeding.

Temporal changes in gene expression and genotype frequency of the ornithine decarboxylase gene in native silverside Basilichthys microlepidotus: Impact of wastewater reduction due to implementation of public policies.

Human activity has caused a deterioration in the health and population size of riverine species; thus, public policies have been implemented to mitigate the anthropogenic impacts of water use, watercourse transformation, and pollution. We studied the Maipo River Basin, one of the most polluted with untreated wastewater in Chile, for a period of 12 years (2007-2019). Since the implementation of new public policies, including the operation of a wastewater collector (2012), the Maipo River Basin is currently much less polluted by untreated water than before. To analyze the impact of wastewater reduction in this river basin, we studied the native silverside (Basilichthys microlepidotus), which inhabits both polluted and unpolluted areas of the river basin. Previous studies reported the overexpression of the ornithine decarboxylase (odc) gene, heterozygote deficit, and high frequency of a homozygote odc genotype in silverside populations that inhabit wastewater-polluted sites, suggesting a phenotypic change and genotypic selection in response to pollution. Here, a population affected and another population unaffected by wastewater were studied before and after implementing the wastewater collector. The physicochemical data of water samples, changes in odc expression and microsatellite variability, and odc genotype frequencies were analyzed. The results showed physicochemical changes in the affected site before and after the operation of the wastewater collector. The microsatellite loci showed no changes in either population. The odc expression in the affected site was higher before the operation of the wastewater collector. Significant changes in the genotype frequencies of the odc gene before and after the wastewater collector operation were detected only at the affected site, wherein the homozygous dominant genotype decreased from >59% to <25%. Our results suggest that public policies aimed at mitigating aquatic pollution can indirectly affect both gene expression and genotype frequencies of important functional genes.

Selection of suitable reference genes for gene expression analysis in gills and liver of fish under field pollution conditions.

To understand the role of gene expression in adaptive variation, it is necessary to examine expression variation in an ecological context. Quantitative real-time PCR (qPCR) is considered the most accurate and reliable technique to measure gene expression and to validate the data obtained by RNA-seq; however, accurate normalization is crucial. In Chile, the freshwater silverside fish Basilichthys microlepidotus inhabits both polluted and nonpolluted areas, showing differential gene expression related to pollution. In this study, we infer the stability of six potential reference genes (tubulin alpha, hypoxanthine-guanine phosphoribosyltransferase, glyceraldehyde-3-phosphate dehydrogenase, beta-actin, 60S ribosomal protein L13, and 60S ribosomal protein L8) in the gills and liver of silverside individuals inhabiting polluted and nonpolluted areas. To validate the reference genes selected, the most and least stable reference genes were used to normalize two target transcripts, one for each organ. The RefFinder tool was used to analyze and identify the most stably expressed genes. The 60S ribosomal protein L8 gene was ranked as the most stable gene for both organs. Our results show that reference gene selection influences the detection of differences in the expression levels of target genes in different organs and, also highlighting candidate reference genes that could be used in field studies.

The hidden costs of living in a transformed habitat: Ecological and evolutionary consequences in a tripartite mutualistic system with a keystone mistletoe.

Land use change is one of the most important anthropogenic drivers of biodiversity loss. Nevertheless, the ecological and evolutionary consequences of habitat transformation remain less understood than those from habitat fragmentation. Transformed habitats are structurally simpler, altering species composition and their ecological interactions, potentially compromising gene flow and genetic diversity. We focused on a tripartite mutualistic system composed of a mistletoe (Tristerix corymbosus), its pollinator (Sephanoides sephaniodes) and its seed disperser (Dromiciops gliroides) to assess changes in their ecological and evolutionary dynamics as a result of habitat transformation. We used eight microsatellite markers to compare genetic diversity, relatedness and gene flow among five mistletoe groups inhabiting native and transformed habitats (abandoned Eucalyptus globulus plantations). We found that these groups were genetically structured, with greater allelic richness and genetic diversity in their native habitat. Also, we found higher relatedness among mistletoe individuals in transformed habitats, which varied as a function of the geographic distance among plants, probably as a result of larger resource availability, which influenced mutualist visitation rates. We did not find differences in the current migration patterns, which suggests that Tristerix corymbosus may be resilient to habitat transformation. Yet, its highly specialized interactions along with changes in its spatial configuration depict a more complex scenario, which probably impose a cost in terms of lower genetic diversity and increased relatedness that might compromise its long-term viability.

Development of ten microsatellite markers from the keystone mistletoe Tristerix corymbosus (Loranthaceae) using 454 next generation sequencing and their applicability to population genetic structure studies.

Tristerix corymbosus (Loranthaceae) is a keystone mistletoe from the South American temperate rainforests. As most mistletoes, T. corymbosus relies on biotic pollination and seed dispersal, which may cause population structure. For a better understanding of its ecology, we isolated and characterized ten polymorphic microsatellite loci for this species. We used 454 Next Generation Sequencing to build a microsatellite library from a high quality DNA sample. We tested 90 sequences from which we obtained ten polymorphic markers. In order to assess their variability, the novel markers were tested in 48 individuals from three locations of the Valdivian Coastal Reserve in Chile. We also estimated genetic differences between pairs of populations using the FST statistic. The mean number of alleles per locus in the 48 individuals studied was 7.1 (2-17 alleles per locus). The observed and expected heterozygosity ranged from 0.298 to 0.634 and from 0.310 to 0.881, respectively. There were genetic differences among the three populations assessed, according to the FST values (ranging from 0.048 to 0.100, all significant) and, the number of alleles per locus ranged from 3.9 to 5.1. These are the first microsatellite markers developed for T. corymbosus, and they arise as a powerful tool for studying population structure, genetic diversity and gene flow at the landscape scale, along its distribution.

Next generation sequencing yields the complete mitochondrial genome of the Endangered Chilean silverside Basilichthys microlepidotus (Jenyns, 1841) (Teleostei, Atherinopsidae), validated with RNA-seq.

The complete sequence of the mitochondrial genome for the Chilean silverside Basilichthys microlepidotus is reported for the first time. The entire mitochondrial genome was 16,544 bp in length (GenBank accession no. KM245937); gene composition and arrangement was conformed to that reported for most fishes and contained the typical structure of 2 rRNAs, 13 protein-coding genes, 22 tRNAs and a non-coding region. The assembled mitogenome was validated against sequences of COI and Control Region previously sequenced in our lab, functional genes from RNA-Seq data for the same species and the mitogenome of two other atherinopsid species available in Genbank.

Liver transcriptome characterization of the endangered freshwater silverside Basilichthys microlepidotus (Teleostei: Atherinopsidae) using next generation sequencing.

Twenty eight out of 44 Chilean freshwater fishes have been considered to fall within the vulnerable or threatened category. Thus, information about the biology, ecology and the effects of the anthropic activity on these species is fundamental to take appropriate conservation measures. In Chile the endemic silverside Basilichthys microlepidotus inhabits mainly rivers surrounded by cities; during the last 10years it was categorized as an endangered species, thus more basic information is needed in order to elaborate a plan of protection for this species. To this end, the transcriptome of B. microlepidotus was sequenced, assembled and characterized. A total of 7.8 million reads (1.05Gb) were obtained from the sequencing and 5.93 million reads (0.83Gb) were used for the de novo assembly, obtaining a total of 31,523 contigs. Of these, 13,724 contigs with expression in all the individuals used were retained for the functional annotation. 7938 sequences were successfully annotated; the biological processes class was the most highly represented, followed by molecular function and cellular component. These sequence data provide a useful new molecular resource for future studies on gene expression and the effects of the human activity on B. microlepidotus, which will facilitate obtaining more information about that, as well as the developing of appropriate conservation strategies for this species.