S = − Σ pi · log2pi i=F,B,S where F stands for freshwater, B for brackish water, and S for saltwater. The variable pi stands for the proportion of fish that fall into each of the three categories. In order to get a normalized entropy value, the entropy was divided by the maximum entropy value for three categories, which is approximately 1.585, where pi = 1/3. Supplementary files containing the results of the analysis of the nine fish groups are available on Zenodo at https://zenodo.org/ record/7227028#.Y1C9zEzMLrc. RESULTS AND DISCUSSION A. Literature review Adaptation to differing salinities is a process that entails large-scale genetic and morphological changes. One example is landlocking whereby diadromous fish lose their capability of migrating back to the ocean and permanently end up in a lacustrine environment. This may be accompanied by the loss of genes that allow the fish to adapt to marine environments. During this process, the differential expression of a large repertoire of genes may take place. Due to the changes in selective pressures and possible ensuing differential genetic regulation and gene loss, the newly landlocked species are rendered less capable of adapting to future changes in its new environment (Hunt et al. 2011). The landlocking process is similar to how eyeless fish lose genetic information associated with sight in caves. In the dark recesses of aquatic caves, fish lose the need for organs involving sight, and thereby lose the genes that are necessary for eyes. Evolutionists assert that landlocking is evidence of natural selection producing new species, but this claim is rather problematic. The mere fact that the number of freshwater fish species is the same as marine species, despite the greater volume of inhabitable marine water implies a much faster diversification rate in freshwater fishes (Rabosky 2020). This also implies selection factors that lead to the diversification of fish species during their adaptation to lacustrine environments. The landlocking process happens at a much faster rate than expected according to the evolutionary timescale. Palkovacs et al. (2008) found, based on mtDNA and microsatellite analysis, that the alewife (Alosa pseudoharengus) adapted to saltwater multiple times within as little as 300, but up to 5,000 years (both within the biblical timescale). Hendry et al. (2000) found, based on microsatellite data and phenotypic variation, that two populations of sockeye salmon (Oncorhynchus nerka) became reproductively isolated from one another and inhabited a lacustrine and a river environment after only 13 generations. Besides fish, Lee and Bell (1999) list 18 marine species that have adapted to freshwater environments over only the past 200 years. This leaves little time and chance for many new genes to arise during adaptation to lacustrine environments, as per the evolutionary models. This is all the more significant, since the ion composition of saltwater is largely in the range of that of body fluids, and maintaining this ion composition in freshwater has high energy costs (Lee and Bell 1999). Adapting to freshwater demands quite a bit of adaptation on the part of fish, since the cell would have to expel superfluous water entering it. It would make more sense if the expression of a wide range of already existing genes is either differentially expressed or these genes undergo differential epigenetic regulation. These processes have been found to occur fairly rapidly. The discovery of such differentially expressed gene (DEG) repertoires is greatly facilitated by RNA-seq technology, whereby dozens, or even hundreds of genes can be identified that take part in the transition between saltwater and freshwater. Several factors can play a role during landlocking, and the adaptation to different water salinities in general. These include salt concentration, temperature, dissolved oxygen levels, new pathogens, parasites and predators, lighting, sex-associated differences, anthropogenic factors, geographical location, food sources, and also epigenetic factors. The most important factor that plays a role in the landlocking process is water salinity. As anadromous fish species invade lacustrine environments, their bodies must get accustomed to hypo-osmolarity, whereas catadromous species must contend with hyper-osmolarity. The most important cellular components that regulate osmotic relationships within the body are ion channels, and transmembrane transport systems. Ions that are the focus of establishing osmotic relationships within the cell are K+, Na+, and Cl-, and to a lesser extent Ca2+ and NH 4 +. Teleost fish adjust to varying salinity levels by either secreting or absorbing ions via special mitochondrion-rich cells called ‘ionocytes’ that line the gill epithelium and the intestines, the main sites of water uptake (Velselvi et al. 2021). Since the gills also play a major role in fish immunity, parasites are often found in this area of the fish’s body. These ionocytes contain several ion-transport proteins, including sodium-potassium ATPase (NKA), Na+/K+/2Cl- cotransporter 1 (NKCC1), cystic fibrosis transmembrane conductance regulator (CFTR), apical Na+/H+ exchanger 3 (NHE3), and Na+/Cl- cotransporter (NCC) (Hiroi and McCormick 2012), the first three being the most important in the regulation of osmolarity in fish. The function of NKA is to maintain a Na+ gradient across the membrane by exuding Na+ ions to facilitate ATP production. NKCC1 admits Cl- ions into the cell, whereas the CFTR channel allows Cl- out of the cell in saltwater conditions (Evans 2008). Hypotonic conditions inhibit Cl- secretion by NKCC1. The efflux of Cl- from the cell in turn enables the secretion of Na+ from the blood vessels towards the external seawater (Marshall 2010). The regulation and the coordination of these ion channels all work together to maintain cell volume, which is critical when transitioning between freshwater and saltwater (Whitehead et al. 2012). Saltwater fish can also adapt to freshwater if calcium is present in significant amounts since intracellular Ca2+ is important for activating these osmolyte channels (Whitehead et al. 2011). Euryhaline fish are capable of differentially regulating the expression of these three ion channels when adjusting between freshwater and saltwater environments. Euryhalinity may have been the ancestral state, allowing fish to adapt to various salt concentrations, whereas stenohalinity is more derived due to the loss of ion channel regulation. This was found to be the case in killifish (Fundulus), which adapted to a freshwater environment from a saltwater environment CSERHATI Molecular baraminology of fish 2023 ICC 184
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