Understanding the molecular basis of adaptation to freshwater environments by prawns in the genus Macrobrachium

Abstract

Understanding the processes that drive adaptive change in response to environmental variation and their consequences for speciation have long been key questions in evolutionary biology. Following origins in seawater, a number of animal groups invaded and colonized freshwater successfully over various evolutionary timeframes. Crustaceans represent a group of relatively recent colonizers of freshwater that now show extensive diversity with representative taxa found in virtually all aquatic environments. Macrobrachium (Family: Palaemonidae) are one of the most speciose and diversified of all crustacean lineages. Taxa in the genus Macrobrachium occupy a wide range of aquatic habitats, possess relatively large body size, and many are highly abundant. Macrobrachium species, as relatively recent freshwater colonizers, therefore provide excellent models for deciphering mechanisms that have facilitated freshwater adaptation. Modern genomic technologies now allow identification of genomic regions influencing adaptation and adaptive diversification (or speciation) at a finer scale. The current study employed a comparative genomics approach to investigate the molecular basis of freshwater adaptation in this decapod crustacean group. In the first study, a transcriptomic scan was performed to identify potential candidate genes involved in freshwater adaptation using an obligate freshwater species, M. koombooloomba as a model. M. koombooloomba was used essentially as the „control‟ because this species completes its entire life in freshwater; thus, all of the important genes affecting freshwater adaptation should be highly expressed in this species. We identified 43 candidate genes (based on BLAST matching with other species) that are likely to be important genes for adapting to a freshwater lifestyle in this species. Identified genes fell under seven broad biological categories including: osmoregulation, cell volume regulation, hemolymph regulation, water channel regulation, osmotic stress response, egg size control and control of larval developmental stage number. We used this gene list as the foundation for future studies. In the second study, we performed a comparative transcriptomics analysis of three Macrobrachium species (M. australiense, M. novaehollandiae and M. tolmerum) representing a range of salinity tolerances at various stages of the life cycle. The three species were maintained under two experimental salinity levels (0‰ and 15‰) over a period of six weeks. The study identified 59 candidate genes (all 43 identified in study 1) including 16 novel „lineage specific orphan transcripts/genes‟. A number of candidate genes (associated with osmoregulation, osmotic stress response, cell volume regulation, water channel and hemolymph regulation) showed different expression patterns between experimental salinities, while expression of others (associated with egg size control and larval development number) remained stable between salinities. Novel genes/transcripts also showed salinity induced differential gene expression patterns. Neutrality tests on all 59 genes revealed that differentially expressed genes showed signatures of purifying selection, but other genes (those that were not differentially expressed) showed patterns consistent with strong positive selection. A few genes (osmotic stress response, cell volume and hemolymph regulatory) showed both differential expression patterns and signatures of positive selection, depending on whether the comparison was between species with similar or dissimilar life history traits. Sequences were highly conserved across species for genes that were differentially expressed between salinities. Results suggest that both plasticity of gene expression and sequence divergence in coding regions (functional mutations), act in a co-ordinated way to promote adaptation. We argue that changes to gene expression pattern play a vital role in the initial adaptive response, while efficient adaptation via mutation/s act over prolonged evolutionary time. In the third study, we conducted a physiological genomic study of the same three species used in the previous study to investigate how regulation of gene expression and body fluid (hemolymph) change with salinity level over time. Individuals from each of the three species were maintained at three experimental salinity levels (0‰, 6‰ and 12‰) for 28 days after an initial acclimation phase to a common condition (6‰) for 14 days. In total, 12 genes were investigated in this study that are involved with different biological functions (based on study 2). For the majority of genes studied (10 out of 12), expression patterns were found to be significantly different among salinity treatments. Differentially expressed genes followed a common pattern; an initial rise in expression level up to 48 hours, followed by a fall in expression up to 96 hours after which expression levels stabilized until the end of the experimental period. Changes to hemolymph osmolality showed a similar pattern to gene expression, with significant differences in hemolymph osmolality evident among salinity treatments. Results demonstrate that salinity level has a strong influence on both hemolymph osmolality and gene expression pattern in the target Macrobrachium species. We conclude that rapid changes to physiological and genomic responses likely shape initial adaptive response to variable environmental salinities. In the final experiment, we employed a comparative genomics analysis using genotyping-by-sequencing (GBS) that screened sequences in 34 Macrobrachium species representing all life history character types from different continents (i.e., replicates of independent freshwater invasions). The study identified 5,018 single nucleotide polymorphisms (SNPs) from ≈310,000 aligned nucleotides in each species. Blasting of genotypes against both the Daphnia genome and Macrobrachium transcriptomes (sourced from studies 1 and 2) showed 65% sequence matching. Blast results revealed that the matched SNPs were located in 176 discrete genes. These genes are involved in an array of diversified functional roles including osmoregulation, hemolymph regulation, cell volume regulation, water channel regulation, egg size control, larval development pattern, energy budget, metabolism, and immune response. This suggests that many interacting genes and/or genomic regions are involved with adaptation to different environmental conditions. SNPs and aligned sequences were used to construct a maximum likelihood phylogenetic tree in addition to a „neutral‟ tree for the same 34 species using the mitochondrial (mtDNA) 16S gene. Topologies of the two trees were substantially different only at the within major clade level (distinct clade for each continent) and support an earlier hypothesis of multiple independent invasions in freshwater environments by ancestral Macrobrachium species. In the GBS tree, all continental freshwater species formed monophyletic groups indicating independent invasions of freshwater, following which Macrobrachium taxa underwent adaptive genomic divergence with respect to the environments they colonized. The GBS tree strongly supported the hypothesis that freshwater adaptation across the Macrobrachium genus likely involved convergent evolution of the same set of traits; so that all global freshwater Macrobrachium species evolved similar suites of phenotypic traits due to common selection pressures associated with a freshwater lifestyle. Overall, this study provides a comprehensive data set for resolving the genomic basis of freshwater adaptation by Palaemonid prawns in the genus Macrobrachium. We infer that response from the genome (rearrangement of the whole genome) is required for successful adaptation to a novel environment with major changes in phenotypic traits (morphology, physiology and overall organismal biology.