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The catalytic α-subunits of both the Na + ,K + -ATPase and the gastric H + ,K + -ATPase possess lysine-rich N-termini which project into the cytoplasm. Due to conflicting experimental results, it is currently unclear whether the N-termini play a role in ion pump function or regulation, and, if they do, by what mechanism. Comparison of the lysine frequencies of the N-termini of both proteins with those of all of their extramembrane domains showed that the N-terminal lysine frequencies are far higher than one would expect simply from exposure to the aqueous solvent. The lysine frequency was found to vary significantly between different vertebrate classes, but this is due predominantly to a change in N-terminal length. As evidenced by a comparison between fish and mammals, an evolutionary trend towards an increase of the length of the N-terminus of the H + ,K + -ATPase on going from an ancestral fish to mammals could be identified. This evolutionary trend supports the hypothesis that the N-terminus is important in ion pump function or regulation. In placental mammals, one of the lysines is replaced by serine (Ser-27), which is a target for protein kinase C. In most other animal species, a lysine occupies this position and hence no protein kinase C target is present. Interaction with protein kinase C is thus not the primary role of the lysine-rich N-terminus. The disordered structure of the N-terminus may, via increased flexibility, facilitate interaction with another binding partner, e.g. the surrounding membrane, or help to stabilise particular enzyme conformations via the increased entropy it produces. Graphical Abstract

There is wide interest in understanding how genetic diversity is generated and maintained in parthenogenetic lineages, as it will help clarify the debate of the evolution and maintenance of sexual reproduction. There are three mechanisms that can be responsible for the generation of genetic diversity of parthenogenetic lineages: contagious parthenogenesis, repeated hybridization and microorganism infections (e.g. Wolbachia). Brine shrimps of the genus Artemia (Crustacea, Branchiopoda, Anostraca) are a good model system to investigate evolutionary transitions between reproductive systems as they include sexual species and lineages of obligate parthenogenetic populations of different ploidy level, which often co-occur. Diploid parthenogenetic lineages produce occasional fully functional rare males, interspecific hybridization is known to occur, but the mechanisms of origin of asexual lineages are not completely understood. Here we sequenced and analysed fragments of one mitochondrial and two nuclear genes from an extensive set of populations of diploid parthenogenetic Artemia and sexual species from Central and East Asia to investigate the evolutionary origin of diploid parthenogenetic Artemia, and geographic origin of the parental taxa. Our results indicate that there are at least two, possibly three independent and recent maternal origins of parthenogenetic lineages, related to A. urmiana and Artemia sp. from Kazakhstan, but that the nuclear genes are very closely related in all the sexual species and parthenogegetic lineages except for A. sinica, who presumable took no part on the origin of diploid parthenogenetic strains. Our data cannot rule out either hybridization between any of the very closely related Asiatic sexual species or rare events of contagious parthenogenesis via rare males as the contributing mechanisms to the generation of genetic diversity in diploid parthenogenetic Artemia lineages.

Sodium pumps (EC 3.6.3.9, Na + -ATPase), which mediate excretion of Na + from the cell, play a crucial role in Na + homeostasis in eukaryotic cells. The objective of this study is to understand the Na + efflux system in a marine red alga. We identified a novel sodium pump gene, PyKPA2 , from the marine red alga Porphyra yezoensis . The amino acid sequence of PyKPA2 shares 65 % identity with PyKPA1, a previously identified P. yezoensis sodium pump. Similar to PyKPA1, PyKPA2 contains conserved sequences for functions such as phosphorylation, ATP binding, and cation binding. Phylogenetic analysis revealed that the two genes cluster with sodium pumps from algae. Reverse-transcription polymerase chain reaction (RT-PCR) analysis showed that PyKPA1 is expressed preferentially in sporophytes, whereas PyKPA2 is expressed specifically in gametophytes. RT-PCR and quantitative real-time PCR analysis revealed that PyKPA1 and PyKPA2 transcripts were upregulated and downregulated, respectively, in gametophytes during exposure to alkali stress. In addition, transcription of both genes in gametophytes was also induced by cold stress. These results suggest that PyKPA1 and PyKPA2 play an important role in alkali and cold stress tolerance.

Background: Flatfish metamorphosis is a hormone regulated post-embryonic developmental event that transforms a symmetric larva into an asymmetric juvenile. In altricial-gastric teleost fish, differentiation of the stomach takes place after the onset of first feeding, and during metamorphosis dramatic molecular and morphological modifications of the gastrointestinal (GI-) tract occur. Here we present the functional ontogeny of the developing GI-tract from an integrative perspective in the pleuronectiforme Atlantic halibut, and test the hypothesis that the multiple functions of the teleost stomach develop synchronously during metamorphosis. Results: Onset of gastric function was determined with several approaches (anatomical, biochemical, molecular and in vivo observations). In vivo pH analysis in the GI-tract lumen combined with quantitative PCR (qPCR) of α and β subunits of the gastric proton pump (H+/K+-ATPase) and pepsinogen A2 indicated that gastric proteolytic capacity is established during the climax of metamorphosis. Transcript abundance of ghrelin, a putative orexigenic signalling molecule produced in the developing stomach, correlated (p < 0.05) with the emergence of gastric proteolytic activity, suggesting that the stomach’s role in appetite regulation occurs simultaneously with the establishment of proteolytic function. A 3D models series of the GI-tract development indicated a functional pyloric sphincter prior to first feeding. Observations of fed larvae in vivo confirmed that stomach reservoir function was established before metamorphosis, and was thus independent of this event. Mechanical breakdown of food and transportation of chyme through the GI-tract was observed in vivo and resulted from phasic and propagating contractions established well before metamorphosis. The number of contractions in the midgut decreased at metamorphic climax synchronously with establishment of the stomach’s proteolytic capacity and its increased peristaltic activity. Putative osmoregulatory competence of the GI-tract, inferred by abundance of Na+/K +-ATPase α transcripts, was already established at the onset of exogenous feeding and was unmodified by metamorphosis. Conclusions: The functional specialization of the GI-tract was not exclusive to metamorphosis, and its osmoregulatory capacity and reservoir function were established before first feeding. Nonetheless, acid production and the proteolytic capacity of the stomach coincided with metamorphic climax, and also marked the onset of the stomach’s involvement in appetite regulation via ghrelin.

Here we show a complete list of the P-type ATPase genes in Caenorhabditis elegans and Drosophila melanogaster. A detailed comparison of the deduced amino-acid sequences in combination with phylogenetic and chromosomal analyses has revealed the following: (1) The diversity of this gene family has been achieved by two major evolutionary steps; the establishment of the major P-type ATPase subgroups with distinct substrate (ion) specificities in a common ancestor of vertebrate and invertebrate, followed by the evolution of multiple isoforms occurring independently in vertebrate and invertebrate phyla. (2) Pairs of genes that have intimate phylogenetic relationship are frequently found in proximity on the same chromosome. (3) Some of the Na,K- and H,K-ATPase isoforms in D. melanogaster and C. elegans lack motifs shown to be important for alpha/beta-subunit assembly, suggesting that such alpha- and beta-subunits might exist by themselves (lonely subunits). The mutation rates for these subunits are much faster than those for the subunits with recognizable assembly domains. (4) The lonely alpha-subunits also lack the major site for ouabain binding that apparently arose before the separation of vertebrates and invertebrates and thus well before the separation of vertebrate Na,K-ATPases and H,K-ATPases. These findings support the idea that a relaxation of functional constraints would increase the rate of evolution and provide clues for identifying the origins of inhibitor sensitivity, subunit assembly, and separation of Na,K- and H,K-ATPases.

The repeated evolutionary specialization of distantly related insects to cardenolide-containing host plants provides a stunning example of parallel adaptation. Hundreds of herbivorous insect species have independently evolved insensitivity to cardenolides, which are potent inhibitors of the alpha-subunit of Na + ,K + -ATPase (ATPα). Previous studies investigating ATPα-mediated cardenolide insensitivity in five insect orders have revealed remarkably high levels of parallelism in the evolution of this trait, including the frequent occurrence of parallel amino acid substitutions at two sites and recurrent episodes of duplication followed by neo-functionalization. Here we add data for a sixth insect order, Orthoptera, which includes an ancient group of highly aposematic cardenolide-sequestering grasshoppers in the family Pyrgomorphidae. We find that Orthopterans exhibit largely predictable patterns of evolution of insensitivity established by sampling other insect orders. Taken together the data lend further support to the proposal that negative pleiotropic constraints are a key determinant in the evolution of cardenolide insensitivity in insects. Furthermore, analysis of our expanded taxonomic survey implicates positive selection acting on site 111 of cardenolide-sequestering species with a single-copy of ATPα, and sites 115, 118 and 122 in lineages with neo-functionalized duplicate copies, all of which are sites of frequent parallel amino acid substitution. This article is part of the theme issue ‘Convergent evolution in the genomics era: new insights and directions’.

Many distantly related insect species are specialized feeders of cardenolide-containing host plants such as milkweed (Asclepias spp.). Previous studies have revealed frequent, parallel substitution of a functionally important amino acid substitution (N122H) in the alpha subunit of Na⁺,K⁺-ATPase in a number of these species. This substitution facilitates the ability of these insects to feed on their toxic hosts and sequester cardenolides for their own use in defense. Among milkweed butterflies of the genus Danaus, the previously established phylogeny for this group suggests that N122H arose independently and fixed in two distinct lineages. We reevaluate this conclusion by examining Danaus phylogenetic relationships using >400 orthologous gene sequences assembled from transcriptome data. Our results indicate that the three Danaus species known to harbor the N122H substitution are more closely related than previously thought, consistent with a single, common origin for N122H. However, we also find evidence of both incomplete lineage sorting and post-speciation genetic exchange among these butterfly species, raising the possibility of collateral evolution of cardenolide-insensitivity in this species group.

Felix Beuschlein, Martin Reincke and colleagues identify recurrent somatic mutations in ATP1A1 and ATP2B3 in aldosterone-producing adenomas with wild-type KCNJ5 . The ATP1A1 and ATP2B3 mutations alter conserved residues and lead to impaired sodium, potassium and calcium ion homeostasis. Primary aldosteronism is the most prevalent form of secondary hypertension. To explore molecular mechanisms of autonomous aldosterone secretion, we performed exome sequencing of aldosterone-producing adenomas (APAs). We identified somatic hotspot mutations in the ATP1A1 (encoding an Na + /K + ATPase α subunit) and ATP2B3 (encoding a Ca 2+ ATPase) genes in three and two of the nine APAs, respectively. These ATPases are expressed in adrenal cells and control sodium, potassium and calcium ion homeostasis. Functional in vitro studies of ATP1A1 mutants showed loss of pump activity and strongly reduced affinity for potassium. Electrophysiological ex vivo studies on primary adrenal adenoma cells provided further evidence for inappropriate depolarization of cells with ATPase alterations. In a collection of 308 APAs, we found 16 (5.2%) somatic mutations in ATP1A1 and 5 (1.6%) in ATP2B3 . Mutation-positive cases showed male dominance, increased plasma aldosterone concentrations and lower potassium concentrations compared with mutation-negative cases. In summary, dominant somatic alterations in two members of the ATPase gene family result in autonomous aldosterone secretion.

Among the few crustacean taxa that managed to inhabit terrestrial environments, Oniscidea includes the most successful colonizers in terms of species richness and abundance. However, neither morphological traits nor molecular markers have definitively resolved phylogenetic relationships among major Oniscidea clades or established the monophyly of the taxon. Herein, we employed the highly conserved, nuclear protein-coding genes Sodium-Potassium Pump (NAK) and Phosphoenolpyruvate Carboxykinase (PEPCK), along with the traditionally used 18 s and 28 s ribosomal RNA genes, in an attempt to clarify these questions. Our dataset included sequences representing all major Oniscidea clades and closely related aquatic taxa, as suggested by previous studies. We applied Bayesian Inference and Maximum Likelihood methods and produced a robust and fully resolved phylogenetic tree that offers strong evidence against the monophyly of Oniscidea. The amphibious genus Ligia appears to be more closely related to representatives of marine suborders, while the phylogenetic pattern of the remaining Oniscidea implies a complex history of the transition from the marine environment to land. With the exception of the basal clade, all other established major clades have been recovered as monophyletic, even though relationships within these clades call for a revised interpretation of morphological characters used in terrestrial isopod taxonomy.

Light-activated, ion-pumping rhodopsins are broadly distributed among many different bacteria and archaea inhabiting the photic zone of aquatic environments. Bacterial proton- or sodium-translocating rhodopsins can convert light energy into a chemiosmotic force that can be converted into cellular biochemical energy, and thus represent a widespread alternative form of photoheterotrophy. Here we report that the genome of the marine flavobacterium Nonlabens marinus S1-08 ᵀ encodes three different types of rhodopsins: Nonlabens marinus rhodopsin 1 (NM-R1), Nonlabens marinus rhodopsin 2 (NM-R2), and Nonlabens marinus rhodopsin 3 (NM-R3). Our functional analysis demonstrated that NM-R1 and NM-R2 are light-driven outward-translocating H ⁺ and Na ⁺ pumps, respectively. Functional analyses further revealed that the light-activated NM-R3 rhodopsin pumps Cl ⁻ ions into the cell, representing the first chloride-pumping rhodopsin uncovered in a marine bacterium. Phylogenetic analysis revealed that NM-R3 belongs to a distinct phylogenetic lineage quite distant from archaeal inward Cl ⁻-pumping rhodopsins like halorhodopsin, suggesting that different types of chloride-pumping rhodopsins have evolved independently within marine bacterial lineages. Taken together, our data suggest that similar to haloarchaea, a considerable variety of rhodopsin types with different ion specificities have evolved in marine bacteria, with individual marine strains containing as many as three functionally different rhodopsins.