Repeated Divergence in Opsin Gene Expression Mirrors Photic Habitat Changes in Rapidly Evolving Crater Lake Cichlid Fishes.

AbstractSelection pressures differ along environmental gradients, and traits tightly linked to fitness (e.g., the visual system) are expected to track such variation. Along gradients, adaptation to local conditions might be due to heritable and nonheritable environmentally induced variation. Disentangling these sources of phenotypic variation requires studying closely related populations in nature and in the laboratory. The Nicaraguan lakes represent an environmental gradient in photic conditions from clear crater lakes to very turbid great lakes. From two old, turbid great lakes, Midas cichlid fish (Amphilophus cf. citrinellus) independently colonized seven isolated crater lakes of varying light conditions, resulting in a small adaptive radiation. We estimated variation in visual sensitivities along this photic gradient by measuring cone opsin gene expression among lake populations. Visual sensitivities observed in all seven derived crater lake populations shifted predictably in direction and magnitude, repeatedly mirroring changes in photic conditions. Comparing wild-caught and laboratory-reared fish revealed that 48% of this phenotypic variation is genetically determined and evolved rapidly. Decreasing intrapopulation variation as environments become spectrally narrower suggests that different selective landscapes operate along the gradient. We conclude that the power to predict phenotypic evolution along gradients depends on both the magnitude of environmental change and the selective landscape shape.

Gut Microbiota Uniqueness Is Associated with Lake Size, a Proxy for Diet Diversity, in Stickleback Fish.

AbstractOrganismal divergence can be driven by differential resource use and adaptation to different trophic niches. Variation in diet is a major factor shaping the gut microbiota, which is crucial for many aspects of their hosts' biology. However, it remains largely unknown how host diet diversity affects the gut microbiota, and it could be hypothesized that trophic niche width is positively associated with gut microbiota diversity. To test this idea, we sequenced the 16S ribosomal RNA gene from intestinal tissue of 14 threespine stickleback populations from lakes of varying size on Vancouver Island, Canada, that have been shown to differ in trophic niche width. Using lake size as a proxy for trophic ecology, we found evidence for higher gut microbiota uniqueness among individuals from populations with broader trophic niches. While these results suggest that diet diversity might promote gut microbiota diversity, additional work investigating diet and gut microbiota variation of the same host organisms will be necessary. Yet our results motivate the question of how host population diversity (e.g., ecological, morphological, genetic) might interact with the gut microbiota during the adaptation to ecological niches.

The effects of host ecology and phylogeny on gut microbiota (non)parallelism across birds and mammals.

IMPORTANCE: What are the roles of determinism and contingency in evolution? The paleontologist and evolutionary biologist Stephen J. Gould raised this question in his famous thought experiment of "replaying life's tape." Settings where independent lineages have repeatedly adapted to similar ecological niches (i.e., parallel evolution) are well suited to address this question. Here, we quantified whether repeated ecological shifts across 53 mammalian and 50 avian host species are associated with parallel gut microbiota changes. Our results indicate that parallel shifts in host diet are associated with greater gut microbiota parallelism (i.e., more deterministic). While further research will be necessary to obtain a comprehensive picture of the circumstances under which deterministic gut microbiota changes might be expected, our study can be instrumental in motivating the use of more quantitative methods in microbiota research. This, in turn, can help us better understand microbiota dynamics during adaptive evolution of their hosts.

Assessing the validity of fecal sampling for characterizing variation in threespine stickleback's gut microbiota.

The gut microbiota is crucial for many aspects of their hosts' biology, and it has been characterized for many species across the animal kingdom. Yet, we still don't have a good understanding of whether non-lethal sampling can accurately capture the diversity of gut-associated bacterial communities, as estimated from lethal sampling of intestinal tissue. We further lack knowledge on whether non-lethal sampling methods are suitable for detecting gut microbiota shifts associated with changes in environmental factors (e.g., diet). We addressed these questions in threespine stickleback fish, a model system for evolutionary ecology, by comparing bacterial communities from intestinal tissue and feces. Despite some differences in community composition between the two sample types and considerable temporal variation among fecal samples, bacterial communities appear to largely overlap. Further, we detected consistent and significant changes of fecal bacterial communities associated with an experimental diet manipulation. This suggests that fecal sampling can represent an adequate non-lethal method to characterize the gut microbiota of threespine stickleback, but additional studies will be necessary before drawing general conclusions regarding the validity of fecal sampling for gut microbiota studies. To this end, we give recommendations to improve the characterization of the gut microbiota via fecal sampling. Fecal sampling allows studying temporal gut microbiota shifts associated with environmental change at the individual level, which increases opportunities for future experimental gut microbiota research.

Gut microbiota parallelism and divergence associated with colonisation of novel habitats.

An organism's gut microbiota can change in response to novel environmental conditions, in particular when colonisation of new habitats is accompanied by shifts in the host species' ecology. Here, we investigated the gut microbiota of three lizard species (A. inornata, H. maculata and S. cowlesi) from their ancestral-like habitat in the Chihuahuan desert and two colonised habitats with contrasting geological and ecological compositions: the White Sands and Carrizozo lava flow. The host species and the lizards' environment both shape gut microbiota composition, but host effects were overall stronger. Further, we found evidence that colonisation of the same environment by independent host species led to parallel changes of the gut microbiota, whereas the colonisation of two distinct environments by the same host species led to gut microbiota divergence. Some of the gut microbiota changes that accompanied the colonisation of the White Sands were associated with shifts in diet (based on diet information from previous studies), which is congruent with the general observation that trophic ecology has a strong effect on gut microbiota composition. Our study provides insights into how shifts in host ecology accompanying colonisation of novel environments can affect gut microbiota composition and diversity.

Host biology, ecology and the environment influence microbial biomass and diversity in 101 marine fish species.

Fish are the most diverse and widely distributed vertebrates, yet little is known about the microbial ecology of fishes nor the biological and environmental factors that influence fish microbiota. To identify factors that explain microbial diversity patterns in a geographical subset of marine fish, we analyzed the microbiota (gill tissue, skin mucus, midgut digesta and hindgut digesta) from 101 species of Southern California marine fishes, spanning 22 orders, 55 families and 83 genera, representing ~25% of local marine fish diversity. We compare alpha, beta and gamma diversity while establishing a method to estimate microbial biomass associated with these host surfaces. We show that body site is the strongest driver of microbial diversity while microbial biomass and diversity is lowest in the gill of larger, pelagic fishes. Patterns of phylosymbiosis are observed across the gill, skin and hindgut. In a quantitative synthesis of vertebrate hindguts (569 species), we also show that mammals have the highest gamma diversity when controlling for host species number while fishes have the highest percent of unique microbial taxa. The composite dataset will be useful to vertebrate microbiota researchers and fish biologists interested in microbial ecology, with applications in aquaculture and fisheries management.

Thyroid hormone tinkering elicits integrated phenotypic changes potentially explaining rapid adaptation of color vision in cichlid fish.

Vision is critical for most vertebrates, including fish. One challenge that aquatic habitats pose is the high variability in spectral properties depending on depth and the inherent optical properties of the water. By altering opsin gene expression and chromophore usage, cichlid fish modulate visual sensitivities to maximize sensory input from the available light in their respective habitat. Thyroid hormone (TH) has been proposed to play a role in governing adaptive diversification in visual sensitivity in Nicaraguan Midas cichlids, which evolved in less than 4000 generations. As suggested by indirect measurements of TH levels (i.e., expression of deiodinases), populations adapted to short wavelength light in clear lakes have lower TH levels than ones inhabiting turbid lakes enriched in long-wavelength light. We experimentally manipulated TH levels by exposing 2-week-old Midas cichlids to exogenous TH or a TH inhibitor and measured opsin gene expression and chromophore usage (via cyp27c1 expression). Although exogenous TH induces long-wavelength sensitivity by changing opsin gene expression and chromophore usage in a concerted manner, TH-inhibited fish exhibit a visual phenotype with sensitivities shifted to shorter wavelengths. Tinkering with TH levels in eyes results in concerted phenotypic changes that can provide a rapid mechanism of adaptation to novel light environments.

An intronic transposon insertion associates with a trans-species color polymorphism in Midas cichlid fishes.

Polymorphisms have fascinated biologists for a long time, but their genetic underpinnings often remain elusive. Here, we aim to uncover the genetic basis of the gold/dark polymorphism that is eponymous of Midas cichlid fish (Amphilophus spp.) adaptive radiations in Nicaraguan crater lakes. While most Midas cichlids are of the melanic "dark morph", about 10% of individuals lose their melanic pigmentation during their ontogeny and transition into a conspicuous "gold morph". Using a new haplotype-resolved long-read assembly we discover an 8.2 kb, transposon-derived inverted repeat in an intron of an undescribed gene, which we term goldentouch in reference to the Greek myth of King Midas. The gene goldentouch is differentially expressed between morphs, presumably due to structural implications of inverted repeats in both DNA and/or RNA (cruciform and hairpin formation). The near-perfect association of the insertion with the phenotype across independent populations suggests that it likely underlies this trans-specific, stable polymorphism.

Quantifying (non)parallelism of gut microbial community change using multivariate vector analysis.

Parallel evolution of phenotypic traits is regarded as strong evidence for natural selection and has been studied extensively in a variety of taxa. However, we have limited knowledge of whether parallel evolution of host organisms is accompanied by parallel changes of their associated microbial communities (i.e., microbiotas), which are crucial for their hosts' ecology and evolution. Determining the extent of microbiota parallelism in nature can improve our ability to identify the factors that are associated with (putatively adaptive) shifts in microbial communities. While it has been emphasized that (non)parallel evolution is better considered as a quantitative continuum rather than a binary phenomenon, quantitative approaches have rarely been used to study microbiota parallelism. We advocate using multivariate vector analysis (i.e., phenotypic change vector analysis) to quantify direction and magnitude of microbiota changes and discuss the applicability of this approach for studying parallelism, and we compiled an R package for multivariate vector analysis of microbial communities ('multivarvector'). We exemplify its use by reanalyzing gut microbiota data from multiple fish species that exhibit parallel shifts in trophic ecology. We found that multivariate vector analysis results were largely consistent with other statistical methods, parallelism estimates were not affected by the taxonomic level at which the microbiota is studied, and parallelism might be stronger for gut microbiota function compared to taxonomic composition. This approach provides an analytical framework for quantitative comparisons across host lineages, thereby providing the potential to advance our capacity to predict microbiota changes. Hence, we emphasize that the development and application of quantitative measures, such as multivariate vector analysis, should be further explored in microbiota research in order to better understand the role of microbiota dynamics during their hosts' adaptive evolution, particularly in settings of parallel evolution.

Diversity in visual sensitivity across Neotropical cichlid fishes via differential expression and intraretinal variation of opsin genes.

The visual system of vertebrates has greatly contributed to our understanding of how different molecular mechanisms shape adaptive phenotypic diversity. Extensive work on African cichlid fishes has shown how variation in opsin gene expression mediates diversification as well as convergent evolution in colour vision. This trait has received less attention in Neotropical cichlids, the sister lineage to African cichlids, but the work done so far led to the conclusion that colour vision is much less variable in Neotropical species. However, as only few taxa have been investigated and as recent work found contradicting patterns, the diversity in meotropical cichlids might be greatly underestimated. Here, we survey patterns of opsin gene expression in 35 representative species of Neotropical cichlids, revealing much more variation than previously known. This diversity can be attributed to two main mechanisms: (i) differential expression of the blue-sensitive sws2a, the green-sensitive rh2a, and the red-sensitive lws opsin genes, and (ii) simultaneous expression of up to five opsin genes, instead of only three as commonly found, in a striking dorsoventral pattern across the retina. This intraretinal variation in opsin genes expression results in steep gradients in visual sensitivity that may represent a convergent adaptation to clear waters with broad light environments. These results highlight the role and flexibility of gene expression in generating adaptive phenotypic diversification.

The genomic signature of ecological divergence along the benthic-limnetic axis in allopatric and sympatric threespine stickleback.

The repeated occurrence of similar phenotypes in independent lineages (i.e., parallel evolution) in response to similar ecological conditions can provide compelling insights into the process of adaptive evolution. An intriguing question is to what extent repeated phenotypic changes are underlain by repeated changes at the genomic level and whether patterns of genomic divergence differ with the geographic context in which populations evolve. Here, we combined genomic, morphological and ecological data sets to investigate the genomic signatures of divergence across populations of threespine stickleback (Gasterosteus aculeatus) that adapted to contrasting ecological niches (benthic or limnetic) in either sympatry or allopatry. We found that genome-wide differentiation (FST ) was an order of magnitude higher and substantially more repeatable for sympatric benthic and limnetic specialists compared to allopatric populations with similar levels of ecological divergence. We identified genomic regions consistently differentiated between sympatric ecotypes that were also differentiated between or associated with benthic vs. limnetic niche in allopatric populations. These candidate regions were enriched on three chromosomes known to be involved in the benthic-limnetic divergence of threespine stickleback. Some candidate regions overlapped with QTL for body shape and trophic traits such as gill raker number, traits that strongly differ between benthic and limnetic ecotypes. In summary, our study shows that magnitude and repeatability of genomic signatures of ecological divergence in threespine stickleback highly depend on the geographic context. The identified candidate regions provide starting points to identify functionally important genes for the adaptation to benthic and limnetic niches.

Contrasting signatures of genomic divergence during sympatric speciation.

The transition from 'well-marked varieties' of a single species into 'well-defined species'-especially in the absence of geographic barriers to gene flow (sympatric speciation)-has puzzled evolutionary biologists ever since Darwin1,2. Gene flow counteracts the buildup of genome-wide differentiation, which is a hallmark of speciation and increases the likelihood of the evolution of irreversible reproductive barriers (incompatibilities) that complete the speciation process3. Theory predicts that the genetic architecture of divergently selected traits can influence whether sympatric speciation occurs4, but empirical tests of this theory are scant because comprehensive data are difficult to collect and synthesize across species, owing to their unique biologies and evolutionary histories5. Here, within a young species complex of neotropical cichlid fishes (Amphilophus spp.), we analysed genomic divergence among populations and species. By generating a new genome assembly and re-sequencing 453 genomes, we uncovered the genetic architecture of traits that have been suggested to be important for divergence. Species that differ in monogenic or oligogenic traits that affect ecological performance and/or mate choice show remarkably localized genomic differentiation. By contrast, differentiation among species that have diverged in polygenic traits is genomically widespread and much higher overall, consistent with the evolution of effective and stable genome-wide barriers to gene flow. Thus, we conclude that simple trait architectures are not always as conducive to speciation with gene flow as previously suggested, whereas polygenic architectures can promote rapid and stable speciation in sympatry.

Parallel and non-parallel changes of the gut microbiota during trophic diversification in repeated young adaptive radiations of sympatric cichlid fish.

BACKGROUND: Recent increases in understanding the ecological and evolutionary roles of microbial communities have underscored the importance of their hosts' biology. Yet, little is known about gut microbiota dynamics during the early stages of ecological diversification and speciation. We sequenced the V4 region of the 16s rRNA gene to study the gut microbiota of Nicaraguan Midas cichlid fish (Amphilophus cf. citrinellus). Specifically, we tested the hypothesis that parallel divergence in trophic ecology in extremely young adaptive radiations from two crater lakes is associated with parallel changes of their gut microbiota. RESULTS: Bacterial communities of fish guts and lake water were highly distinct, indicating that the gut microbiota is shaped by host-specific factors. Among individuals of the same crater lake, differentiation in trophic ecology was weakly associated with gut microbiota differentiation, suggesting that diet, to some extent, affects the gut microbiota. However, differences in trophic ecology were much more pronounced across than within species whereas similar patterns were not observed for taxonomic and functional differences of the gut microbiota. Across the two crater lakes, we could not detect conclusive evidence for parallel changes of the gut microbiota associated with trophic ecology. CONCLUSIONS: A lack of clearly differentiated niches during the early stages of ecological diversification might result in non-parallel changes of gut microbial communities, as observed in our study system as well as in other recently diverged fish species. Video Abstract.

Reverting ontogeny: rapid phenotypic plasticity of colour vision in cichlid fish.

Phenotypic plasticity, particularly during development, allows organisms to rapidly adjust to different environmental conditions. Yet, it is often unclear whether the extent and direction of plastic changes are restricted by an individual's ontogeny. Many species of cichlid fishes go through ontogenetic changes in visual sensitivity, from short to long wavelengths, by switching expression of cone opsin genes crucial for colour vision. During this progression, individuals often exhibit phenotypic plasticity to the ambient light conditions. However, it is commonly assumed that once an adult visual phenotype is reached, reverting to an earlier ontogenetic state with higher sensitivity at shorter wavelengths is not common. In this study, we experimentally demonstrate that four-month-old Midas cichlid fish (Amphilophus astorquii) show plasticity in single cone opsin expression after experiencing drastic changes in light conditions. Resulting shifts of visual sensitivity occurred presumably in an adaptive direction-towards shorter or longer wavelengths when exposed to short- or long-wavelength light, respectively. Single cone opsin expression changed within only a few days and went through a transitional phase of co-expression. When the environment was experimentally enriched in long-wavelength light, the corresponding change occurred gradually along a dorsoventral gradient within the retina. This plasticity allowed individuals to revert earlier ontogenetic changes and return to a more juvenile visual phenotype demonstrating previously unrecognized insights into temporal and spatial dynamics of phenotypic plasticity of the visual system in response to ambient light.

Convergent phenotypic evolution of the visual system via different molecular routes: How Neotropical cichlid fishes adapt to novel light environments.

How predictable is evolution? This remains a fundamental but contested issue in evolutionary biology. When independent lineages colonize the same environment, we are presented with a natural experiment that allows us to ask if genetic and ecological differences promote species-specific evolutionary outcomes or whether species phenotypically evolve in a convergent manner in response to shared selection pressures. If so, are the molecular mechanisms underlying phenotypic convergence the same? In Nicaragua, seven species of cichlid fishes concurrently colonized two novel photic environments. Hence, their visual system represents a compelling model to address these questions, particularly since the adaptive value of phenotypic changes is well-understood. By analyzing retinal transcriptomes, we found that differential expression of genes responsible for color vision (cone opsins and cyp27c1) produced rapid and mostly convergent changes of predicted visual sensitivities. Notably, these changes occurred in the same direction in all species although there were differences in underlying gene expression patterns illustrating nonconvergence at the molecular level. Adaptive phenotypes evolved deterministically, even when species differ substantially in ecology and genetic variation. This provides strong evidence that phenotypic evolution of the visual system occurred in response to similar selective forces of the photic environment.

Heterochronic opsin expression due to early light deprivation results in drastically shifted visual sensitivity in a cichlid fish: Possible role of thyroid hormone signaling.

During early ontogeny, visual opsin gene expression in cichlids is influenced by prevailing light regimen. Red light, for example, leads to an early switch from the expression of short-wavelength sensitive to long-wavelength sensitive opsins. Here, we address the influence of light deprivation on opsin expression. Individuals reared in constant darkness during the first 14 days post-hatching (dph) showed a general developmental delay compared with fish reared under a 12:12 hr light-dark cycle (control group). Several characters including pigmentation patterns and eye development, appeared later in dark-reared individuals. Quantitative real-time PCR and fluorescent in situ hybridization at six time points during the 14 days period revealed that fish from the control group expressed opsin genes from 5 dph on and maintained a short-wavelength sensitive phenotype (sws1, rh2b, and rh2a). Onset of opsin expression in dark-reared Midas cichlids was delayed by 4 days and visual sensitivity rapidly progressed toward a long-wavelength sensitive phenotype (sws2b, rh2a, and lws). Shifts in visual sensitivities toward longer wavelengths are mediated by thyroid hormone (TH) in many vertebrates. Compared to control fish, dark-reared individuals showed elevated dio3 expression levels - a validated proxy for TH concentration - suggesting higher circulating TH levels. Despite decelerated overall development, ontogeny of opsin gene expression was accelerated, resulting in retinae with long-wavelength shifted predicted sensitivities compared to light-reared individuals. Indirect evidence suggests that this was due to altered TH metabolism.

Rapid adaptation to a novel light environment: The importance of ontogeny and phenotypic plasticity in shaping the visual system of Nicaraguan Midas cichlid fish (Amphilophus citrinellus spp.).

Colonization of novel habitats is typically challenging to organisms. In the initial stage after colonization, approximation to fitness optima in the new environment can occur by selection acting on standing genetic variation, modification of developmental patterns or phenotypic plasticity. Midas cichlids have recently colonized crater Lake Apoyo from great Lake Nicaragua. The photic environment of crater Lake Apoyo is shifted towards shorter wavelengths compared to great Lake Nicaragua and Midas cichlids from both lakes differ in visual sensitivity. We investigated the contribution of ontogeny and phenotypic plasticity in shaping the visual system of Midas cichlids after colonizing this novel photic environment. To this end, we measured cone opsin expression both during development and after experimental exposure to different light treatments. Midas cichlids from both lakes undergo ontogenetic changes in cone opsin expression, but visual sensitivity is consistently shifted towards shorter wavelengths in crater lake fish, which leads to a paedomorphic retention of their visual phenotype. This shift might be mediated by lower levels of thyroid hormone in crater lake Midas cichlids (measured indirectly as dio2 and dio3 gene expression). Exposing fish to different light treatments revealed that cone opsin expression is phenotypically plastic in both species during early development, with short and long wavelength light slowing or accelerating ontogenetic changes, respectively. Notably, this plastic response was maintained into adulthood only in the derived crater lake Midas cichlids. We conclude that the rapid evolution of Midas cichlids' visual system after colonizing crater Lake Apoyo was mediated by a shift in visual sensitivity during ontogeny and was further aided by phenotypic plasticity during development.

Rapid and Parallel Adaptive Evolution of the Visual System of Neotropical Midas Cichlid Fishes.

Midas cichlid fish are a Central American species flock containing 13 described species that has been dated to only a few thousand years old, a historical timescale infrequently associated with speciation. Their radiation involved the colonization of several clear water crater lakes from two turbid great lakes. Therefore, Midas cichlids have been subjected to widely varying photic conditions during their radiation. Being a primary signal relay for information from the environment to the organism, the visual system is under continuing selective pressure and a prime organ system for accumulating adaptive changes during speciation, particularly in the case of dramatic shifts in photic conditions. Here, we characterize the full visual system of Midas cichlids at organismal and genetic levels, to determine what types of adaptive changes evolved within the short time span of their radiation. We show that Midas cichlids have a diverse visual system with unexpectedly high intra- and interspecific variation in color vision sensitivity and lens transmittance. Midas cichlid populations in the clear crater lakes have convergently evolved visual sensitivities shifted toward shorter wavelengths compared with the ancestral populations from the turbid great lakes. This divergence in sensitivity is driven by changes in chromophore usage, differential opsin expression, opsin coexpression, and to a lesser degree by opsin coding sequence variation. The visual system of Midas cichlids has the evolutionary capacity to rapidly integrate multiple adaptations to changing light environments. Our data may indicate that, in early stages of divergence, changes in opsin regulation could precede changes in opsin coding sequence evolution.

The imperiled fish fauna in the Nicaragua Canal zone.

Large-scale infrastructure projects commonly have large effects on the environment. The planned construction of the Nicaragua Canal will irreversibly alter the aquatic environment of Nicaragua in many ways. Two distinct drainage basins (San Juan and Punta Gorda) will be connected and numerous ecosystems will be altered. Considering the project's far-reaching environmental effects, too few studies on biodiversity have been performed to date. This limits provision of robust environmental impact assessments. We explored the geographic distribution of taxonomic and genetic diversity of freshwater fish species (Poecilia spp., Amatitlania siquia, Hypsophrys nematopus, Brycon guatemalensis, and Roeboides bouchellei) across the Nicaragua Canal zone. We collected population samples in affected areas (San Juan, Punta Gorda, and Escondido drainage basins), investigated species composition of 2 drainage basins and performed genetic analyses (genetic diversity, analysis of molecular variance) based on mitochondrial cytb. Freshwater fish faunas differed substantially between drainage basins (Jaccard similarity = 0.33). Most populations from distinct drainage basins were genetically differentiated. Removing the geographic barrier between these basins will promote biotic homogenization and the loss of unique genetic diversity. We found species in areas where they were not known to exist, including an undescribed, highly distinct clade of live bearing fish (Poecilia). Our results indicate that the Nicaragua Canal likely will have strong impacts on Nicaragua's freshwater biodiversity. However, knowledge about the extent of these impacts is lacking, which highlights the need for more thorough investigations before the environment is altered irreversibly.

Critical Uncertainties and Gaps in the Environmental- and Social-Impact Assessment of the Proposed Interoceanic Canal through Nicaragua.

The proposed interoceanic canal will connect the Caribbean Sea with the Pacific Ocean, traversing Lake Nicaragua, the major freshwater reservoir in Central America. If completed, the canal would be the largest infrastructure-related excavation project on Earth. In November 2015, the Nicaraguan government approved an environmental and social impact assessment (ESIA) for the canal. A group of international experts participated in a workshop organized by the Academy of Sciences of Nicaragua to review this ESIA. The group concluded that the ESIA does not meet international standards; essential information is lacking regarding the potential impacts on the lake, freshwater and marine environments, and biodiversity. The ESIA presents an inadequate assessment of natural hazards and socioeconomic disruptions. The panel recommends that work on the canal project be suspended until an appropriate ESIA is completed. The project should be resumed only if it is demonstrated to be economically feasible, environmentally acceptable, and socially beneficial.