ing to the production of reactive oxygen species (ROS) (Bal et al. 2021). Taurine acts as a potent antioxidant that protects fish from this form of stress (Zeng et al. 2009). The levels of antioxidant enzymes, such as catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GR), and glutathione peroxidase (GPx) are good measures of salinity-induced oxidative stress. Certain types of aquaporin (AQP) also play a role in regulating the response to water salinity in fish (Hirose et al. 2003). As water salinity increases, the number of AQP channels decreases to mitigate the amount of water lost due to hyperosmotic conditions. In general, salinity tends to have an osmoprotective role against heavy metal ions, such as nickel, cadmium, zinc, and mercury (Gioda et al. 2007; Saglam et al. 2013; Blewett et al. 2016), which are mainly found inland, thus representing a challenge to fish during the landlocking process. Increased salinity induces AQP channels that facilitate salt ion transport, as well as the NKA ion channel to produce energy via ATP. Differences in salinity may be associated with a massive number of genes to be differentially expressed, numbering in the thousands. Gibbons et al. (2017) found that 2,515 genes were differentially expressed in threespine stickleback due to changes in salinity. These genes are involved in the migration of epithelial cells during gill remodeling, transmembrane ion transport, epithelial Ca2+ channels (ECaCs), the NKA and NHE3 channels. Claudins and occludins, genes that code for proteins that form tight junctions and reduce ion permeability also change their gene expression levels in freshwater conditions. AQP3 is also differentially regulated in varying salinity conditions, with its expression level changing up to thirteen-fold in freshwater conditions (Whitehead et al. 2011). Several genes that are differentially regulated between freshwater and saltwater conditions include the NF-κB family of transcription factors that respond to infection, stress, and injury (Xiao and Ghosh 2005). V-type H+ ATPase was also found to play an important role in osmoregulation in freshwater fish species, such as sticklebacks, but also in non-fish species, such as copepods (Lee et al. 2011; Kozak et al. 2014), by facilitating the uptake of sodium into the cell (Katoh et al. 2003). Slowing down the progression of the cell cycle is a way in which fish can repair DNA damage before cell duplication, as has been observed in fish kidney cells (Kammerer and Kültz 2009; Dowd et al. 2010). Several genes involved in energy production are also differentially expressed, such as the subunits of the NADH dehydrogenase, ATP synthase, and cytochrome B and C (Newmeyer and Ferguson-Miller 2003). Water salinity is such an important factor that it also affects other adaptational factors as well, such as food abundance, immunity, and exposure to parasites and predators (Saboret and Ingram 2019; Blasco-Costa et al. 2013), and can even lead to reproductive differences (Kozak et al. 2014). For example, Blanar et al. (2011) found that salinity played a role in the structure and composition of pathogen species infecting mummichog (Fundulus heteroclitus) in two polluted estuaries in New Brunswick, Canada. B. Mitochondrial DNA analysis of different fish groups In the following, the mtDNA of the nine fish groups around the level of the order listed in Table 1 will be analyzed to discover what kinds of putative baraminic relationships exist within them. 1. Acipenseriformes Acipenseriformes is an order of ray-finned fishes including sturgeons and paddlefishes. The mtDNA of 29 species was examined from this group according to the Materials and Methods section. The results can be seen in Figure 3, and are also available in Supplementary File 1. According to the heatmap (Figure 3A), there appear to be two groups within Acipenseriformes, four species from the genus Scaphirhynchus and the remaining 24 species from various genera, Acipenser, Huso, Polydon, Psephurus, and Pseudoscaphyrhynchus. The three Lampetra species formed a distinct outgroup compared to the two other clusters. The Hopkins clustering statistic was very good at 0.878. The Silhouette plot in Figure 3B shows a maximum silhouette value at two clusters, but there may be distortion in the data. All three groups had a statistically significant p-value. The smaller group (Scaphirhynchus) had a lower normalized entropy value (0.512), with information from FishBase for four out of five species, and among these three from freshwater. In contrast, the larger group of 24 species had a normalized entropy value of 0.999, amongst which an almost even number of species inhabit freshwater, brackish water, and saltwater. These results clearly show that several species from this group can inhabit habitats of varying salinities, and that transitioning between habitats is not difficult. Indeed, 15 of the 22 species which had migratory annotation in FishBase were anadromous, with the remaining seven being potamodromous (completing their entire life cycle in freshwater). 2. Anguilliformes Anguilliformes (eels) are long-bodied fish that use peristaltic movement to swim, undulatory waves that are propagated posteriorly through the animal’s body. These characteristics make these animals a distinct apobaramin compared to all other fish groups. A total of 61 species of Anguilliformes were studied. For the heatmap, the ‘average’ clustering method was used. Besides the three outlier lamprey species, there are two larger groups, with 19 and 41 species, respectively, as can be seen in Figure 4A. The Silhouette plot in Figure 4B shows a maximum silhouette value at three clusters. The Hopkins clustering statistic is 0.804, indicating good clustering. However, the larger group of 41 species is not statistically significant, with a p-value of 0.201. Furthermore, the species Neocyema erythrosoma does not fit in with either cluster. N. erythrosoma is also a deep-sea eel, found in depths between 6,600 and 7,200 ft. As such, it inhabits a different habitat than most eels. This is more evidence that this species belongs to its own holobaramin (Wise 1992). Poulsen et al. (2018) place members of the genus Neocyema into their own family, called Neocyematidae, based on divergent mtDNA sequences and mitochondrial gene order. In contrast with members of the family Cyematidae, the mitochondrial gene order of N. erythrosoma resembles that of Eurypharynx and Sacchopharynx but is significantly shorter in length (17,765 bp as opposed to 18,978 bp of Eurypharynx pelecanoides). When the gene order for the mtDNA of the four putative Anguilliformes groups was analyzed, it was found that some species in group #3 had a slightly different gene order than the rest of the species. Thus, group 3 was reassigned to groups 3 and 4, and group 4 was CSERHATI Molecular baraminology of fish 2023 ICC 186
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