FIRST RECORD OF BATRACHOCHYTRIUM DENDROBATIDIS IN THE NORTHERN NETHERLANDS.

Batrachochytrium dendrobatidis (Bd) infects amphibians and has been linked to the decline of hundreds of anuran amphibians all over the world. In the province of Groningen in the Netherlands, this fungal pathogen was not detected before this study. To determine whether Groningen was Bd-free, we surveyed 12 locations in this province in 2020 and 2021. Samples were then used to quantify the presence of Bd with a qPCR assay. In total, 2 out of 110 (∼0.02%) collected in 2020 and 11 out of 249 samples collected in 2021 tested positive for Bd. Infected amphibians were found in 4 out of the 12 sites, and the prevalence of Bd was estimated at 4% for both years combined. Our study provides the first record of Bd in Groningen, and we hypothesize that Bd is present throughout the Netherlands in regions currently considered "Bd-free." Furthermore, we warn scientists and policymakers to be apprehensive when calling a site free from Bd when sampling is limited or not recent.

Developmental and environmental plasticity in opsin gene expression in Lake Victoria cichlid fish.

In many organisms, sensory abilities develop and evolve according to the changing demands of navigating, foraging, and communication across different environments and life stages. Teleost fish inhabit heterogeneous light environments and exhibit a large diversity in visual system properties among species. Cichlids are a classic example of this diversity; visual system variation is generated by different tuning mechanisms that involve both genetic factors and phenotypic plasticity. Here, we document the developmental progression of visual pigment gene expression in Lake Victoria cichlids and test if these patterns are influenced by variation in light conditions. We reared two sister species of Pundamilia to adulthood in two distinct visual conditions that resemble the light environments that they naturally inhabit in Lake Victoria. We also included interspecific first-generation hybrids. We focused on the four opsins that are expressed in Pundamilia adults (using real-time quantitative polymerase chain reaction (RT-qPCR)) (SWS2B, SWS2A, RH2A, and LWS) at 17 time points. We find that opsin expression profiles progress from shorter-wavelength sensitive opsins to longer-wavelength sensitive opsins with increasing age, in both species and their hybrids. The developmental trajectories of opsin expression also responded plastically to the visual conditions. Developmental and environmental plasticity in opsin expression may provide an important stepping stone in the evolution of cichlid visual system diversity.

Selection on Visual Opsin Genes in Diurnal Neotropical Frogs and Loss of the SWS2 Opsin in Poison Frogs.

Amphibians are ideal for studying visual system evolution because their biphasic (aquatic and terrestrial) life history and ecological diversity expose them to a broad range of visual conditions. Here, we evaluate signatures of selection on visual opsin genes across Neotropical anurans and focus on three diurnal clades that are well-known for the concurrence of conspicuous colors and chemical defense (i.e., aposematism): poison frogs (Dendrobatidae), Harlequin toads (Bufonidae: Atelopus), and pumpkin toadlets (Brachycephalidae: Brachycephalus). We found evidence of positive selection on 44 amino acid sites in LWS, SWS1, SWS2, and RH1 opsin genes, of which one in LWS and two in RH1 have been previously identified as spectral tuning sites in other vertebrates. Given that anurans have mostly nocturnal habits, the patterns of selection revealed new sites that might be important in spectral tuning for frogs, potentially for adaptation to diurnal habits and for color-based intraspecific communication. Furthermore, we provide evidence that SWS2, normally expressed in rod cells in frogs and some salamanders, has likely been lost in the ancestor of Dendrobatidae, suggesting that under low-light levels, dendrobatids have inferior wavelength discrimination compared to other frogs. This loss might follow the origin of diurnal activity in dendrobatids and could have implications for their behavior. Our analyses show that assessments of opsin diversification in across taxa could expand our understanding of the role of sensory system evolution in ecological adaptation.

Variation in parasite infection between replicates of speciation in Lake Victoria cichlid fish.

Because of potentially strong eco-evolutionary interactions with their hosts, parasites may initiate or enhance host diversification. The adaptive radiation of cichlid fish in Lake Victoria provides a good system to study the role of parasites at different stages of host speciation. We analyzed the macroparasite infection of four replicates of sympatric blue and red Pundamilia species pairs that vary in their age and extent of differentiation. Sympatric host species differed in parasite community composition and in the infection levels of some of these parasite taxa. Most infection differences were consistent between sampling years, indicating temporal consistency in parasite-mediated divergent selection between species. Infection differentiation increased linearly with genetic differentiation. However, significant infection differences between sympatric species were only found in the oldest, most strongly differentiated Pundamilia species pair. This is inconsistent with parasite-driven speciation. Next, we identified five distinct species of Cichlidogyrus, a genus of highly specific gill parasites that has radiated elsewhere in Africa. Infection profiles of species of Cichlidogyrus differed between sympatric cichlid species only in the oldest and most differentiated pair, again inconsistent with parasite-mediated speciation. To conclude, parasites may contribute to host differentiation after speciation, but do not initiate host speciation.

Lack of alignment across yeast-dependent life-history traits may limit Drosophila melanogaster dietary specialization.

Heterogeneity in food resources is a major driver of local adaptation and speciation. Dietary specialization typically involves multiple life-history traits and may thus be limited by the extent to which these traits adapt in concert. Here, we use Drosophila melanogaster, representing an intermediate state in the generalist-specialist continuum, to explore the scope for dietary specialization. D. melanogaster has a close association with yeast, an essential but heterogeneous food resource. We quantify how different D. melanogaster strains from around the globe respond to different yeast species, across multiple yeast-dependent life-history traits including feeding, mating, egg-laying, egg development and survival. We find that D. melanogaster strains respond to different yeast species in different ways, indicating distinct fly strain-yeast interactions. However, we detect no evidence for trade-offs: fly performance tends to be positively rather than negatively correlated across yeast species. We also find that the responses to different yeast species are not aligned across traits: different life-history traits are maximized on different yeast species. Finally, we confirm that D. melanogaster is a resource generalist: it can grow, reproduce and survive on all the yeast species we tested. Together, these findings provide a possible explanation for the limited extent of dietary specialization in D. melanogaster.

Contribution of opsins and chromophores to cone pigment variation across populations of Lake Victoria cichlids.

Adaptation to heterogeneous sensory environments has been implicated as a key parameter in speciation. Cichlid fish are a textbook example of divergent visual adaptation, mediated by variation in the sequences and expression levels of cone opsin genes (encoding the protein component of visual pigments). In some vertebrates including fish, visual sensitivity is also tuned by the ratio of vitamin A1 /A2 -derived chromophores (i.e., the light-sensitive component of the visual pigment bound to the opsin protein), where higher proportions of A2 cause a more red-shifted wavelength absorbance. This study explores the variation in chromophore ratios across multiple cichlid populations in Lake Victoria, using as a proxy the expression of the gene Cyp27c1, which has been shown to regulate the conversion of vitamin A1 into vitamin A2 in several vertebrates. This study focuses on sympatric Pundamilia cichlids, where species with blue or red male coloration co-occur at multiple islands but occupy different depths and consequently different visual habitats. In the red species, we found higher cyp27c1 expression in populations from turbid waters than from clear waters, but there was no such pattern in the blue species. Across populations, differences between the sympatric species in cyp27c1 expression had a consistent relationship with species differences in opsin expression patterns, but the red/blue identity reversed between clear and turbid waters. To assess the contribution of heritable vs. environmental causes of variation, we tested whether light manipulations induce a change in cyp27c1 expression in the laboratory. We found that cyp27c1 expression was not influenced by experimental light conditions, suggesting that the observed variation in the wild is due to genetic differences. Nonetheless, compared to other cichlid species, cyp27c1 is expressed at very low levels in Pundamilia, suggesting that it may not be relevant for visual adaptation in this species. Conclusively, establishing the biological importance of this variation requires testing of actual A1 /A2 ratios in the eye, as well as its consequences for visual performance.

Patterns of ectoparasite infection in wild-caught and laboratory-bred cichlid fish, and their hybrids, implicate extrinsic rather than intrinsic causes of species differences in infection.

Parasite-mediated selection may initiate or enhance differentiation between host populations that are exposed to different parasite infections. Variation in infection among populations may result from differences in host ecology (thereby exposure to certain parasites) and/or intrinsic immunological traits. Species of cichlid fish, even when recently diverged, often differ in parasite infection, but the contributions of intrinsic and extrinsic causes are unknown. Here, we compare infection patterns between two closely related host species from Lake Victoria (genus Pundamilia), using wild-caught and first-generation laboratory-reared fish, as well as laboratory-reared hybrids. Three of the commonest ectoparasite species observed in the wild were also present in the laboratory populations. However, the infection differences between the host species as observed in the wild were not maintained in laboratory conditions. In addition, hybrids did not differ in infection from either parental species. These findings suggest that the observed species differences in infection in the wild might be mainly driven by ecology-related effects (i.e. differential exposure), rather than by intrinsic species differences in immunological traits. Thus, while there is scope for parasite-mediated selection in Pundamilia in the wild, it has apparently not yet generated divergent evolutionary responses and may not enhance assortative mating among closely related species.

Predator biomass and vegetation influence the coastal distribution of threespine stickleback morphotypes.

Intraspecific niche differentiation can contribute to population persistence in changing environments. Following declines in large predatory fish, eutrophication, and climate change, there has been a major increase in the abundance of threespine stickleback (Gasterosteus aculeatus) in the Baltic Sea. Two morphotype groups with different levels of body armor-completely plated and incompletely plated-are common in coastal Baltic Sea habitats. The morphotypes are similar in shape, size, and other morphological characteristics and live as one apparently intermixed population. Variation in resource use between the groups could indicate a degree of niche segregation that could aid population persistence in the face of further environmental change. To assess whether morphotypes exhibit niche segregation associated with resource and/or habitat exploitation and predator avoidance, we conducted a field survey of stickleback morphotypes, and biotic and abiotic ecosystem structure, in two habitat types within shallow coastal bays in the Baltic Sea: deeper central waters and shallow near-shore waters. In the deeper waters, the proportion of completely plated stickleback was greater in habitats with greater biomass of two piscivorous fish: perch (Perca fluviatilis) and pike (Esox lucius). In the shallow waters, the proportion of completely plated stickleback was greater in habitats with greater coverage of habitat-forming vegetation. Our results suggest niche segregation between morphotypes, which may contribute to the continued success of stickleback in coastal Baltic Sea habitats.

Homage to Felsenstein 1981, or why are there so few/many species?

If there are no constraints on the process of speciation, then the number of species might be expected to match the number of available niches and this number might be indefinitely large. One possible constraint is the opportunity for allopatric divergence. In 1981, Felsenstein used a simple and elegant model to ask if there might also be genetic constraints. He showed that progress towards speciation could be described by the build-up of linkage disequilibrium among divergently selected loci and between these loci and those contributing to other forms of reproductive isolation. Therefore, speciation is opposed by recombination, because it tends to break down linkage disequilibria. Felsenstein then introduced a crucial distinction between "two-allele" models, which are subject to this effect, and "one-allele" models, which are free from the recombination constraint. These fundamentally important insights have been the foundation for both empirical and theoretical studies of speciation ever since.

Microhabitat distributions and species interactions of ectoparasites on the gills of cichlid fish in Lake Victoria, Tanzania.

Heterogeneous exposure to parasites may contribute to host species differentiation. Hosts often harbour multiple parasite species which may interact and thus modify each other's effects on host fitness. Antagonistic or synergistic interactions between parasites may be detectable as niche segregation within hosts. Consequently, the within-host distribution of different parasite taxa may constitute an important axis of infection variation among host populations and species. We investigated the microhabitat distributions and species interactions of gill parasites (four genera) infecting 14 sympatric cichlid species in Lake Victoria, Tanzania. We found that the two most abundant ectoparasite genera (the monogenean Cichlidogyrus spp. and the copepod Lamproglena monodi) were non-randomly distributed across the host gills and their spatial distribution differed between host species. This may indicate microhabitat selection by the parasites and cryptic differences in the host-parasite interaction among host species. Relationships among ectoparasite genera were synergistic: the abundances of Cichlidogyrus spp. and the copepods L. monodi and Ergasilus lamellifer tended to be positively correlated. In contrast, relationships among morphospecies of Cichlidogyrus were antagonistic: the abundances of morphospecies were negatively correlated. Together with niche overlap, this suggests competition among morphospecies of Cichlidogyrus. We also assessed the reproductive activity of the copepod species (the proportion of individuals carrying egg clutches), as it may be affected by the presence of other parasites and provide another indicator of the species specificity of the host-parasite relationship. Copepod reproductive activity did not differ between host species and was not associated with the presence or abundance of other parasites, suggesting that these are generalist parasites, thriving in all cichlid species examined from Lake Victoria.

Temporally consistent species differences in parasite infection but no evidence for rapid parasite-mediated speciation in Lake Victoria cichlid fish.

Parasites may have strong eco-evolutionary interactions with their hosts. Consequently, they may contribute to host diversification. The radiation of cichlid fish in Lake Victoria provides a good model to study the role of parasites in the early stages of speciation. We investigated patterns of macroparasite infection in a community of 17 sympatric cichlids from a recent radiation and 2 older species from 2 nonradiating lineages, to explore the opportunity for parasite-mediated speciation. Host species had different parasite infection profiles, which were only partially explained by ecological factors (diet, water depth). This may indicate that differences in infection are not simply the result of differences in exposure, but that hosts evolved species-specific resistance, consistent with parasite-mediated divergent selection. Infection was similar between sampling years, indicating that the direction of parasite-mediated selection is stable through time. We morphologically identified 6 Cichlidogyrus species, a gill parasite that is considered a good candidate for driving parasite-mediated speciation, because it is host species-specific and has radiated elsewhere in Africa. Species composition of Cichlidogyrus infection was similar among the most closely related host species (members of the Lake Victoria radiation), but two more distantly related species (belonging to nonradiating sister lineages) showed distinct infection profiles. This is inconsistent with a role for Cichlidogyrus in the early stages of divergence. To conclude, we find significant interspecific variation in parasite infection profiles, which is temporally consistent. We found no evidence that Cichlidogyrus-mediated selection contributes to the early stages of speciation. Instead, our findings indicate that species differences in infection accumulate after speciation.

Testing sensory drive speciation in cichlid fish: Linking light conditions to opsin expression, opsin genotype and female mate preference.

Ecological speciation is facilitated when divergent adaptation has direct effects on selective mating. Divergent sensory adaptation could generate such direct effects, by mediating both ecological performance and mate selection. In aquatic environments, light attenuation creates distinct photic environments, generating divergent selection on visual systems. Consequently, divergent sensory drive has been implicated in the diversification of several fish species. Here, we experimentally test whether divergent visual adaptation explains the divergence of mate preferences in Haplochromine cichlids. Blue and red Pundamilia co-occur across south-eastern Lake Victoria. They inhabit different photic conditions and have distinct visual system properties. Previously, we documented that rearing fish under different light conditions influences female preference for blue versus red males. Here, we examine to what extent variation in female mate preference can be explained by variation in visual system properties, testing the causal link between visual perception and preference. We find that our experimental light manipulations influence opsin expression, suggesting a potential role for phenotypic plasticity in optimizing visual performance. However, variation in opsin expression does not explain species differences in female preference. Instead, female preference covaries with allelic variation in the long-wavelength-sensitive opsin gene (LWS), when assessed under broad-spectrum light. Taken together, our study presents evidence for environmental plasticity in opsin expression and confirms the important role of colour perception in shaping female mate preferences in Pundamilia. However, it does not constitute unequivocal evidence for the direct effects of visual adaptation on assortative mating.

Geographic variation in opsin expression does not align with opsin genotype in Lake Victoria cichlid populations.

Sensory adaptation to the local environment can contribute to speciation. Aquatic environments are well suited for studying this process: The natural attenuation of light through water results in heterogeneous light environments, to which vision-dependent species must adapt for communication and survival. Here, we study visual adaptation in sympatric Pundamilia cichlids from southeastern Lake Victoria. Species with blue or red male nuptial coloration co-occur at many rocky islands but tend to be depth-differentiated, entailing different visual habitats, more strongly at some islands than others. Divergent visual adaptation to these environments has been implicated as a major factor in the divergence of P. pundamilia and P. nyererei, as they show consistent differentiation in the long-wavelength-sensitive visual pigment gene sequence (LWS opsin). In addition to sequence variation, variation in the opsin gene expression levels may contribute to visual adaptation. We characterized opsin gene expression and LWS genotype across Pundamilia populations inhabiting turbid and clear waters, to examine how different mechanisms of visual tuning contribute to visual adaptation. As predicted, the short-wavelength-sensitive opsin (SWS2b) was expressed exclusively in a population from clear water. Contrary to prediction however, expression levels of the other opsins were species- and island-dependent and did not align with species differences in LWS genotype. Specifically, in two locations with turbid water, the shallow-water dwelling blue species expressed more LWS and less RH2A than the deeper-dwelling red species, while the opposite pattern occurred in the two locations with clear water. Visual modeling suggests that the observed distribution of opsin expression profiles and LWS genotypes does not maximize visual performance, implying the involvement of additional visual tuning mechanisms and/or incomplete adaptation. OPEN RESEARCH BADGE: This article has earned an Open Data Badge for making publicly available the digitally-shareable data necessary to reproduce the reported results. The data is available at https://hdl.handle.net/10411/I1IUUQ.

Visual adaptation and microhabitat choice in Lake Victoria cichlid fish.

When different genotypes choose different habitats to better match their phenotypes, genetic differentiation within a population may be promoted. Mating within those habitats may subsequently contribute to reproductive isolation. In cichlid fish, visual adaptation to alternative visual environments is hypothesized to contribute to speciation. Here, we investigated whether variation in visual sensitivity causes different visual habitat preferences, using two closely related cichlid species that occur at different but overlapping water depths in Lake Victoria and that differ in visual perception (Pundamilia spp.). In addition to species differences, we explored potential effects of visual plasticity, by rearing fish in two different light conditions: broad-spectrum (mimicking shallow water) and red-shifted (mimicking deeper waters). Contrary to expectations, fish did not prefer the light environment that mimicked their typical natural habitat. Instead, we found an overall preference for the broad-spectrum environment. We also found a transient influence of the rearing condition, indicating that the assessment of microhabitat preference requires repeated testing to control for familiarity effects. Together, our results show that cichlid fish exert visual habitat preference but do not support straightforward visual habitat matching.

Developmental effects of environmental light on male nuptial coloration in Lake Victoria cichlid fish.

BACKGROUND: Efficient communication requires that signals are well transmitted and perceived in a given environment. Natural selection therefore drives the evolution of different signals in different environments. In addition, environmental heterogeneity at small spatial or temporal scales may favour phenotypic plasticity in signaling traits, as plasticity may allow rapid adjustment of signal expression to optimize transmission. In this study, we explore signal plasticity in the nuptial coloration of Lake Victoria cichlids, Pundamilia pundamilia and Pundamilia nyererei. These two species differ in male coloration, which mediates species-assortative mating. They occur in adjacent depth ranges with different light environments. Given the close proximity of their habitats, overlapping at some locations, plasticity in male coloration could contribute to male reproductive success but interfere with reproductive isolation. METHODS: We reared P. pundamilia, P. nyererei, and their hybrids under light conditions mimicking the two depth ranges in Lake Victoria. From photographs, we quantified the nuptial coloration of males, spanning the entire visible spectrum. In experiment 1, we examined developmental colour plasticity by comparing sibling males reared in each light condition. In experiment 2, we assessed colour plasticity in adulthood, by switching adult males between conditions and tracking coloration for 100 days. RESULTS: We found that nuptial colour in Pundamilia did respond plastically to our light manipulations, but only in a limited hue range. Fish that were reared in light conditions mimicking the deeper habitat were significantly greener than those in conditions mimicking shallow waters. The species-specific nuptial colours (blue and red) did not change. When moved to the opposing light condition as adults, males did not change colour. DISCUSSION: Our results show that species-specific nuptial colours, which are subject to strong divergent selection by female choice, are not plastic. We do find plasticity in green coloration, a response that may contribute to visual conspicuousness in darker, red-shifted light environments. These results suggest that light-environment-induced plasticity in male nuptial coloration in P. pundamilia and P. nyererei is limited and does not interfere with reproductive isolation.

Community assembly in Lake Tanganyika cichlid fish: quantifying the contributions of both niche-based and neutral processes.

The cichlid family features some of the most spectacular examples of adaptive radiation. Evolutionary studies have highlighted the importance of both trophic adaptation and sexual selection in cichlid speciation. However, it is poorly understood what processes drive the composition and diversity of local cichlid species assemblages on relatively short, ecological timescales. Here, we investigate the relative importance of niche-based and neutral processes in determining the composition and diversity of cichlid communities inhabiting various environmental conditions in the littoral zone of Lake Tanganyika, Zambia. We collected data on cichlid abundance, morphometrics, and local environments. We analyzed relationships between mean trait values, community composition, and environmental variation, and used a recently developed modeling technique (STEPCAM) to estimate the contributions of niche-based and neutral processes to community assembly. Contrary to our expectations, our results show that stochastic processes, and not niche-based processes, were responsible for the majority of cichlid community assembly. We also found that the relative importance of niche-based and neutral processes was constant across environments. However, we found significant relationships between environmental variation, community trait means, and community composition. These relationships were caused by niche-based processes, as they disappeared in simulated, purely neutrally assembled communities. Importantly, these results can potentially reconcile seemingly contrasting findings in the literature about the importance of either niche-based or neutral-based processes in community assembly, as we show that significant trait relationships can already be found in nearly (but not completely) neutrally assembled communities; that is, even a small deviation from neutrality can have major effects on community patterns.

Differential Survival between Visual Environments Supports a Role of Divergent Sensory Drive in Cichlid Fish Speciation.

Identifying the selective forces that initiate ecological speciation is a major challenge in evolutionary biology. Sensory drive has been implicated in speciation in various taxa, largely based on phenotype-environment correlations and signatures of selection in sensory genes. Here, we present a reciprocal transplant experiment revealing species differences in performance in alternative visual environments, consistent with speciation by divergent sensory drive. The closely related cichlids Pundamilia pundamilia and Pundamilia nyererei inhabit different visual environments in Lake Victoria and show associated differences in visual system properties. Mimicking the two light environments in the laboratory, we find a substantial reduction in survival of both species when reared in the other species' visual environment. This implies that the observed differences in Pundamilia color vision are indeed adaptive and substantiates the implicit assumption in sensory drive speciation models that divergent environmental selection is strong enough to drive divergence in sensory properties.

Genomics and the origin of species.

Speciation is a fundamental evolutionary process, the knowledge of which is crucial for understanding the origins of biodiversity. Genomic approaches are an increasingly important aspect of this research field. We review current understanding of genome-wide effects of accumulating reproductive isolation and of genomic properties that influence the process of speciation. Building on this work, we identify emergent trends and gaps in our understanding, propose new approaches to more fully integrate genomics into speciation research, translate speciation theory into hypotheses that are testable using genomic tools and provide an integrative definition of the field of speciation genomics.

Colour variation in cichlid fish: developmental mechanisms, selective pressures and evolutionary consequences.

Cichlid fishes constitute one of the most species-rich families of vertebrates. In addition to complex social behaviour and morphological versatility, they are characterised by extensive diversity in colouration, both within and between species. Here, we review the cellular and molecular mechanisms underlying colour variation in this group and the selective pressures responsible for the observed variation. We specifically address the evidence for the hypothesis that divergence in colouration is associated with the evolution of reproductive isolation between lineages. While we conclude that cichlid colours are excellent models for understanding the role of animal communication in species divergence, we also identify taxonomic and methodological biases in the current research effort. We suggest that the integration of genomic approaches with ecological and behavioural studies, across the entire cichlid family and beyond it, will contribute to the utility of the cichlid model system for understanding the evolution of biological diversity.

Species-Specific Relationships between Water Transparency and Male Coloration within and between Two Closely Related Lake Victoria Cichlid Species.

Environmental variation in signalling conditions affects animal communication traits, with possible consequences for sexual selection and reproductive isolation. Using spectrophotometry, we studied how male coloration within and between populations of two closely related Lake Victoria cichlid species (Pundamilia pundamilia and P. nyererei) covaries with water transparency. Focusing on coloration patches implicated in sexual selection, we predicted that in clear waters, with broad-spectrum light, (1) colours should become more saturated and (2) shift in hue away from the dominant ambient wavelengths, compared to more turbid waters. We found support for these predictions for the red and yellow coloration of P. nyererei but not the blue coloration of P. pundamilia. This may be explained by the species difference in depth distribution, which generates a steeper gradient in visual conditions for P. nyererei compared to P. pundamilia. Alternatively, the importance of male coloration in intraspecific sexual selection may differ between the species. We also found that anal fin spots, that is, the orange spots on male haplochromine anal fins that presumably mimic eggs, covaried with water transparency in a similar way for both species. This is in contrast to the other body regions studied and suggests that, while indeed functioning as signals, these spots may not play a role in species differentiation.