Dr. Carol Eunmi LEE
430 Lincoln Drive, Birge Hall
Office: (608) 262-2675
Genetic and Physiological Targets of Selection During Habitat Transitions
As most animal phyla have evolved in the sea, fresh water imposes profound physiological challenges for most taxa (Hutchinson 1957; Lee and Bell 1999). In general, transitions away from the sea have led to the evolution of increased physiological regulation - in other words, the evolution of increased homeostasis (physiological constancy). For example, colonizations from marine to freshwater habitats, and from aquatic habitats onto land, have given rise to the evolution of increased ionic regulation in order to maintain an "internal ocean" in the face of environmental change.
Despite the challenges imposed by these invasions, species crossing such boundaries are overrepresented among successful contemporary invaders. In recent years, many species have invaded fresh water from saline habitats as a result of human activity (Lee and Bell 1999). For example, the vast majority of recent invaders into the Great Lakes, including zebra and quagga mussels, have originated from brackish waters of the Black and Caspian Seas (Lee and Bell 1999; Gelembiuk et al. 2006; May et al. 2006).
Such invasions are remarkable, as saline and freshwater invertebrates are typically separated by a biogeographic boundary (of ~5 PSU, ~150 mOsm/kg), across which most species are physiologically unable to penetrate (Khlebovich and Abramova 2000). How are these invaders able to survive these radical habitat shifts? Might common mechanisms govern the ability to invade fresh water from more saline sources?
My laboratory has been discovering striking evolutionary shifts in physiological traits across independent invasions by the copepod Eurytemora affinis species complex. For instance, our functional assays revealed physiological evolution following saline to freshwater invasions, including shifts in salinity tolerance (Lee et al. 2003; Lee et al. 2007), hemolymph osmolality (Lee et al. 2012), and ion transport activity and expression (Lee et al. 2011; Posavi et al. 2020). The evolutionary shifts were repeatable, with parallel evolutionary shifts across independent invasions in the wild, as well as in selection experiments in the laboratory. This parallelism suggests that labile evolutionary mechanisms underlie these rapid evolutionary events.
Most notably, across our multiple studies we found that ion transporter paralogs dominate as the category of genes under natural selection during salinity transitions. In particular, we found evolutionary shifts in activity and expression of key ion transporters (Lee et al. 2011; Posavi et al. 2020). Surprisingly, while salinity response was moderately polygenic, we found a striking pattern of parallel selection across replicate salinity transitions in wild populations and in laboratory selection lines (Stern & Lee 2020). Our simulations suggest that this striking parallelism is driven in part by selection from standing genetic variation and also synergistic epistasis acting among key ion transporter paralogs (Stern et al. In Revision).
The particular ion transporter paralogs under selection during salinity change suggest mechanisms of ion uptake from freshwater habitats (Fig. 1). While ion transport is a ubiquitous function performed by all cells across all domains of life, mechanisms of ion uptake from low salinities are still not well-characterized for any organism (Charmantier et al. 2009; McNamara and Faria 2012). And, still no consensus exists on the identities of the fundamental ion transporters involved in ion uptake, even for human kidneys (Chambrey et al. 2013).
Figure 1. Hypothetical models of ion uptake from fresh water by ionocytes in E. affinis complex populations. Shown are primary transporters for energizing ion transport (VHA, NKA) and hypothetical secondary transporters for sodium uptake (NHA, Na+ channel [Nach], or NHE). A: Model 1 (Wieczorek’s Model): VHA (blue) pumps H+ out of the cell and creates a proton gradient, through which Na+ is transported into the cell through an electrogenic NHA or Na+ channel. The stoichiometry of ion transport for NHA is not fully known, even for Drosophila. B: Model 2: Ammonia is transported out of the cell by an ammonia transporter (Rh), which then drives electroneutral NHE to export H+, and consequently import Na+. In both models, Na+ is transported to the hemolymph via NKA. Carbonic anhydrase (CA) supplies protons to VHA or NHE and HCO3- to anion exchanger (AE). The Na+/K+/2Cl- cotransporter (NKCC) might also play a role in ion uptake, but the localization of NKCC paralogs is unknown for E. affinis complex. In response to salinity change, certain paralogs of these ion transporters show evolutionary changes in gene expression and signatures of natural selection. Alternative models have also been proposed and not all potentially relevant ion transporters are shown. Adapted from Lee et al. (2011), Stern & Lee (2020), and Posavi et al. (2020).
Our results tend to give stronger support for Model 1 as the mechanism for ion uptake in freshwater, given that ion transporter genes involved in this model show the most prominent signals of evolutionary change during salinity decline. In freshwater populations, we found evolutionary increases in activity and expression of the proton pump V-type H+ ATPase (VHA) (Lee et al. 2011), which could energize ion uptake by creating a proton gradient across the cell membrane (Fig. 1A). However, the ion transporter that utilizes this proton gradient to transport cations into the cell had been unknown, and had been dubbed the mystery “Wieczorek exchanger” (Wieczorek et al. 1991; Beyenbach and Wieczorek 2006).
Our results suggest that the Na+,H+-antiporter (NHA) serves as the mystery "Wieczorek" ion transporter that cooperates with VHA to uptake Na+ in low salinity conditions (Fig. 1A) (Posavi et al. 2020; Lee & Stern 2020; Popp et al. In Prep.). Based on our results, NHA is likely a major player that facilitates adaptation to freshwater habitats. For instance, freshwater populations of the E. affinis complex show increases in expression of certain NHA paralogs, relative to their saline ancestors (Posavi et al. 2020). Interestingly, the highest density of SNPs under selection occur in the genomic region containing seven tandem NHA paralogs (Fig. 2) (Stern & Lee 2020). The high numbers of NHA paralogs in the genome of this copepod is unusual, with a greater number than any other metazoan, based on a survey of over 40 genomes (Mathers et al. In Prep.).
Figure 2. The Na+/H+ Antiporter (NHA) gene family in the E. affinis complex (Stern & Lee 2020). A: Tandem paralogs of the NHA gene family on Scaffold 68 in the E. affinis complex genome. B: Support for directional selection between saline and freshwater populations estimated with BayeScan 3 by comparing SNP frequencies. The horizontal dotted line marks the significance threshold for directional selection. Blue dots correspond to SNPs with highest support for parallel directional selection (using BayeScan 3). Black dots above the dotted line correspond to SNPs with support for directional selection in one clade alone. Size of the dots corresponds to the level of support for association with salinity (Bayes Factor). C: Signatures of balancing selection based on an excess of SNPs at similar frequencies (Beta(2) scores) in four native ange saline populations from the Atlantic (red) and Gulf (green) clades. Colored data points represent SNPs in the top 1% of ?(2) scores in each population. D: Drawing of the copepod E. affinis complex, showing five pairs of swimming legs (L1-L5). E: Immunolocalization of NHA-7 (bright green) in the swimming legs of an individual E. affinis complex copepod from a freshwater population. Photograph by Catherine Lorin-Nebel.
Additionally, using in situ immunohistochemistry, we have localized the expression of three critical ion transporters (VHA, NHA, and NKA) in the swimming legs of the copepod E. affinis, at novel osmoregulatory organs that we named the “Crusalis organs” (Johnson et al. 2014; Gerber et al. 2016). Different paralogs of NHA are localized in different tissues, suggesting differences in function among paralogs. We are currently examining evolutionary shifts in localization and expression of specific ion transporter paralogs during the shift from saline to freshwater populations (Popp et al. In Prep.). The in situ immunolocalization work is being performed in collaboration with our colleagues at the Université de Montpellier (e.g. Catherine Lorin-Nebel), funded by a grant from the French government awarded to Carol Lee ("Make Our Planet Great Again" award).
Stern, DB, CE Lee. 2020. Evolutionary origins of genomic adaptations in an invasive copepod. Nature Ecology & Evolution. 4:1084-1094. https://doi.org/10.1038/s41559-020-1201-y Supplementary Tables
Posavi M, D Gulisija, JC Silva, CE Lee. 2020. Rapid evolution of genome-wide expression and plasticity during saline to freshwater invasions by a copepod. Molecular Ecology. 29:4835-4856. DOI: 10.1111/mec.15681
Gerber L, CE Lee, E Grousset, E Blondeau-Bidet, NB Boucheker, C Lorin-Nebel, M Charmantier-Daures, G Charmantier. 2016. The Legs Have It: Localizing and quantifying expression of ion transporters V-Type H+ ATPase and Na+/K+-ATPase in the swimming legs of the freshwater invading copepod Eurytemora affinis. Physiological and Biochemical Zoology. 89:233-250.
Johnson KE, L Perreau, G Charmantier, M Charmantier-Daures, CE Lee. 2014. Without Gills: Exploring the localization of osmoregulatory function in the copepod Eurytemora affinis. Physiological and Biochemical Zoology. 87:310-324.
Lee CE, M Posavi, G Charmantier. 2012. Rapid evolution of body fluid regulation following independent invasions into freshwater habitats. Journal of Evolutionary Biology. 25:625-633.
Lee CE, M Kiergaard, BD Eads, GW Gelembiuk, M Posavi. 2011. Pumping ions: Rapid parallel evolution of ionic regulation following habitat invasions. Evolution. 65:2229-2244.
Lee, CE. 2017. Evolutionary mechanisms of habitat invasions, using the copepod Eurytemora affinis as a model system. Evolutionary Applications. 9:248-270.