Field application of de novo transcriptomic analysis to evaluate the effects of sublethal freshwater salinization on Gasterosteus aculeatus in urban streams.

Freshwater salinization poses global challenges for aquatic organisms inhabiting urban streams, impacting their physiology and ecology. However, current salinization research predominantly focuses on mortality endpoints in limited model species, overlooking the sublethal effects on a broader spectrum of organisms and the exploration of adaptive mechanisms and pathways under natural field conditions. To address these gaps, we conducted high-throughput sequencing transcriptomic analysis on the gill tissue of the euryhaline fish Gasterosteus aculeatus, investigating its molecular response to salinity stress in the highly urbanized river Boye, Germany. We found that in stream sections with sublethal concentrations of chloride costly osmoregulatory systems were activated, evidenced by the differential expression of genes related to osmoregulation. Our enrichment analysis revealed differentially expressed genes (DEGs) related to transmembrane transport and regulation of transport and other osmoregulation pathways, which aligns with the crucial role of these pathways in maintaining biological homeostasis. Notably, we identified candidate genes involved in increased osmoregulatory activity under salinity stress, including those responsible for moving ions across membranes: ion channels, ion pumps, and ion transporters. Particularly, genes from the solute carrier family SLC, aquaporin AQP1, chloride channel CLC7, ATP-binding cassette transporter ABCE1, and ATPases member ATAD2 exhibited prominent differential expression. These findings provide insights into the potential molecular mechanisms underlying the adaptive response of euryhaline fish to salinity stress and have implications for their conservation and management in the face of freshwater salinization.

The Asymmetric Response Concept explains ecological consequences of multiple stressor exposure and release.

Our capacity to predict trajectories of ecosystem degradation and recovery is limited, especially when impairments are caused by multiple stressors. Recovery may be fast or slow and either complete or partial, sometimes result in novel ecosystem states or even fail completely. Here, we introduce the Asymmetric Response Concept (ARC) that provides a basis for exploring and predicting the pace and magnitude of ecological responses to, and release from, multiple stressors. The ARC holds that three key mechanisms govern population, community and ecosystem trajectories. Stress tolerance is the main mechanism determining responses to increasing stressor intensity, whereas dispersal and biotic interactions predominantly govern responses to the release from stressors. The shifting importance of these mechanisms creates asymmetries between the ecological trajectories that follow increasing and decreasing stressor intensities. This recognition helps to understand multiple stressor impacts and to predict which measures will restore communities that are resistant to restoration.

Linking local movement and molecular analysis to explore philopatry and population connectivity of the southern stingray Hypanus americanus.

Limited data pertaining to life history and population connectivity of the data-deficient southern stingray (Hypanus americanus) are available. To determine potential vulnerabilities of their populations, this study aimed to analyse their movement patterns and genetic variability. A population of southern stingrays encompassing nine sites around Cape Eleuthera, the Bahamas, has been monitored using mark-recapture, spanning a 2.5 year period. Out of 200 individual stingrays, more than a third were encountered again. The home range of the females appears to be restricted, which supports the notion of high site residency. As resident populations of stingrays could suffer from a lack of population connectivity and be predestined for genetic isolation and local extirpation, this study further investigated the genetic connectivity of four sample sites in the central and western Bahamas. A haplotype analysis from the mitochondrial D-loop region showed that no distinct population structure strictly correlated with the sample site. These findings were complemented by five microsatellite loci that revealed high degrees in genotypic variability and little population differentiation. The results suggest gene flow mediated by both males and females.

Red Queen revisited: Immune gene diversity and parasite load in the asexual Poecilia formosa versus its sexual host species P. mexicana.

In accordance with the Red Queen hypothesis, the lower genotypic diversity in clonally reproducing species should make them easier targets for pathogen infection, especially when closely related sexually reproducing species occur in close proximity. We analyzed two populations of clonal P. formosa and their sexual parental species P. mexicana by correlating individual parasite infection with overall and immune genotype. Our study revealed lower levels of overall genotypic diversity and marginally fewer MHC class I alleles in P. formosa individuals compared to sexually reproducing P. mexicana. Parasite load, however, differed only between field sites but not between species. We hypothesize that this might be due to slightly higher genotypic diversity in P. formosa at the innate immune system (toll like receptor 8) which is likely due to the species' hybrid origin. In consequence, it appears that clonal individuals do not necessarily suffer a disadvantage compared to sexual individuals when fighting parasite infection.

Oxygen concentration drives local adaptation in the greenlip abalone (Haliotis laevigata).

Understanding adaptation has become one of the major biological questions especially in the light of rapid environmental changes induced by climate change. Ocean temperatures are rising which triggers massive changes in water chemistry and thereby alters the living environment of all marine organisms. Studying adaptation, however, can be tricky because spatial genetic patterns might also occur due to random effects, for example, genetic drift. Genetic drift is reduced in very large and well-connected populations, such as in broadcast marine spawning organisms. Here, spatial genetic divergence is likely to be produced by selection. In this issue of Molecular Ecology, Sandoval-Castillo et al. (2018) investigated patterns of spatial genetic divergence and their association with environmental factors in the greenlip abalone (Haliotis laevigata). This commercially important species of mollusc is a broadcast spawner with large population sizes, rendering genetic drift an unlikely factor in the genetic divergence of wild populations. Sandoval-Castillo et al. (2018) used a ddRAD genomic approach to test for genetic divergence between sampled populations while also measuring different environmental factors, for example, water temperature and oxygen content. The majority of identified SNPs was putatively neutral and showed only low levels of genetic divergence between field sites. However, 323 candidate adaptive markers were identified that clearly separated the individuals into five different clusters. These genetic clusters correlated with environmental clusters mainly determined by water temperature and (correlated) oxygen concentration. Gene annotation of the candidate SNPs revealed a large proportion of loci being involved in biological processes influenced by oxygen availability. The study by Sandoval-Castillo et al. (2018) in this issue of Molecular Ecology exemplifies the benefits of combining genomic studies with ecological data. It is a great starting point for more detailed (gene function, physiology) as well as broader (biodiversity) investigations that might help us to better understand adaptation and predict ecosystems' resilience and resistance to environmental disturbances. In addition, this information can be applied to implement optimal conservation regime policies and sustainable harvesting strategies, hopefully protecting biodiversity as well as commercial interests in marine life.

Clonal polymorphism and high heterozygosity in the celibate genome of the Amazon molly.

The extreme rarity of asexual vertebrates in nature is generally explained by genomic decay due to absence of meiotic recombination, thus leading to extinction of such lineages. We explore features of a vertebrate asexual genome, the Amazon molly, Poecilia formosa, and find few signs of genetic degeneration but unique genetic variability and ongoing evolution. We uncovered a substantial clonal polymorphism and, as a conserved feature from its interspecific hybrid origin, a 10-fold higher heterozygosity than in the sexual parental species. These characteristics seem to be a principal reason for the unpredicted fitness of this asexual vertebrate. Our data suggest that asexual vertebrate lineages are scarce not because they are at a disadvantage, but because the genomic combinations required to bypass meiosis and to make up a functioning hybrid genome are rarely met in nature.

More than one genotype: how common is intracolonial genetic variability in scleractinian corals?

In recent years, a few colonial marine invertebrates have shown intracolonial genetic variability, a previously unreported phenomenon. Intracolonial genetic variability describes the occurrence of more than a single genotype within an individual colony. This variability can be traced back to two underlying processes: chimerism and mosaicism. Chimerism is the fusion of two or more individuals, whereas mosaicism mostly derives from somatic cell mutations. Until now, it remained unclear to what degree the ecologically important group of hermatypic (reef building) corals might be affected. We investigate the occurrence of intracolonial genetic variability in five scleractinian corals: Acropora florida, Acropora hyacinthus, Acropora sarmentosa, Pocillopora species complex and Porites australiensis. The main focus was to test different genera for the phenomenon via microsatellite markers and to distinguish which underlying process caused the genetic heterogeneity. Our results show that intracolonial genetic variability was common (between 46.6% for A. sarmentosa and 23.8% for P. species complex) in all tested corals. The main process was mosaicism (69 cases of 222 tested colonies), but at least one chimera existed in every species. This suggests that intracolonial genetic variability is widespread in scleractinian corals and could challenge the view of a coral colony as an individual and therefore a unit of selection. However, it might also hold potential for colony survival under rapidly changing environmental conditions.

Single-male paternity in coelacanths.

Latimeria chalumnae, a 'living fossil,' is of great scientific interest, as it is closely related to the aquatic ancestors of land-living tetrapods. Latimeria show internal fertilization and bear live young, but their reproductive behaviour is poorly known. Here we present for the first time a paternity analysis of the only available material from gravid females and their offspring. We genotype two L. chalumnae females and their unborn brood for 14 microsatellite loci. We find that the embryos are closely related to each other and never show more than three different alleles per locus, providing evidence for a single father siring all of the offspring. We reconstruct the father's genotype but cannot identify it in the population. These data suggest that coelacanths have a monogamous mating system and that individual relatedness is not important for mate choice.

Exploring Pandora's box: potential and pitfalls of low coverage genome surveys for evolutionary biology.

High throughput sequencing technologies are revolutionizing genetic research. With this "rise of the machines", genomic sequences can be obtained even for unknown genomes within a short time and for reasonable costs. This has enabled evolutionary biologists studying genetically unexplored species to identify molecular markers or genomic regions of interest (e.g. micro- and minisatellites, mitochondrial and nuclear genes) by sequencing only a fraction of the genome. However, when using such datasets from non-model species, it is possible that DNA from non-target contaminant species such as bacteria, viruses, fungi, or other eukaryotic organisms may complicate the interpretation of the results. In this study we analysed 14 genomic pyrosequencing libraries of aquatic non-model taxa from four major evolutionary lineages. We quantified the amount of suitable micro- and minisatellites, mitochondrial genomes, known nuclear genes and transposable elements and searched for contamination from various sources using bioinformatic approaches. Our results show that in all sequence libraries with estimated coverage of about 0.02-25%, many appropriate micro- and minisatellites, mitochondrial gene sequences and nuclear genes from different KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways could be identified and characterized. These can serve as markers for phylogenetic and population genetic analyses. A central finding of our study is that several genomic libraries suffered from different biases owing to non-target DNA or mobile elements. In particular, viruses, bacteria or eukaryote endosymbionts contributed significantly (up to 10%) to some of the libraries analysed. If not identified as such, genetic markers developed from high-throughput sequencing data for non-model organisms may bias evolutionary studies or fail completely in experimental tests. In conclusion, our study demonstrates the enormous potential of low-coverage genome survey sequences and suggests bioinformatic analysis workflows. The results also advise a more sophisticated filtering for problematic sequences and non-target genome sequences prior to developing markers.

Population divergence in East African coelacanths.

The coelacanth, Latimeria chalumnae, occurs at the Eastern coast of Africa from South Africa up to Kenya. It is often referred to as a living fossil mainly because of its nearly unchanged morphology since the Middle Devonian. As it is a close relative to the last common ancestor of fish and tetrapods, molecular studies mostly focussed on their phylogenetic relationships. We now present a population genetic study based on 71 adults from the whole known range of the species. Despite an overall low genetic diversity, there is evidence for divergence of local populations. We assume that originally the coelacanths at the East African Coast derived from the Comoros population, but have since then diversified into additional independent populations: one in South Africa and another in Tanzania. Unexpectedly, we find a split of the Comoran coelacanths into two sympatric subpopulations. Despite its undeniably slow evolutionary rate, the coelacanth still diversifies and is therefore able to adapt to new environmental conditions.

Induction of male production in clones of Daphnia pulex by the juvenoid hormone methyl farnesoate under short photoperiod.

The juvenile hormone, methyl farnesoate (MF), and its analog insecticides have been used successfully to induce the production of males in cladocerans under long-day conditions in the laboratory. However, without hormone addition Daphnia do not usually produce male offspring under long photoperiods, while short photoperiods are a stimulus for the induction of males. We used 21 clones of Daphnia pulex differing in their propensity to produce males under short-day conditions to test whether the treatment with MF would result in an additive effect of shifting the sex ratio towards males. Contrary to our expectations, clones with a high tendency of male production showed a reduced sex ratio in response to MF treatment under short-day conditions, but clones that produced normally few males or did not produce males were stimulated by 700 nM MF to produce up to 40% males. We suggest that the endocrine disruptive effect of MF or juvenile hormone analogs in the field may depend on the clonal composition of the cladoceran population and on the natural photoperiod. This may affect the seasonal occurrence of sexual reproduction and eventually cause a mismatch between the presence of males and ephippial females.

Monophyletic origin of multiple clonal lineages in an asexual fish (Poecilia formosa).

Despite the advantage of avoiding the costs of sexual reproduction, asexual vertebrates are very rare and often considered evolutionarily disadvantaged when compared to sexual species. Asexual species, however, may have advantages when colonizing (new) habitats or competing with sexual counterparts. They are also evolutionary older than expected, leaving the question whether asexual vertebrates are not only rare because of their 'inferior' mode of reproduction but also because of other reasons. A paradigmatic model system is the unisexual Amazon molly, Poecilia formosa, that arose by hybridization of the Atlantic molly, Poecilia mexicana, as the maternal ancestor, and the sailfin molly, Poecilia latipinna, as the paternal ancestor. Our extensive crossing experiments failed to resynthesize asexually reproducing (gynogenetic) hybrids confirming results of previous studies. However, by producing diploid eggs, female F(1) -hybrids showed apparent preadaptation to gynogenesis. In a range-wide analysis of mitochondrial sequences, we examined the origin of P. formosa. Our analyses point to very few or even a single origin(s) of its lineage, which is estimated to be approximately 120,000 years old. A monophyletic origin was supported from nuclear microsatellite data. Furthermore, a considerable degree of genetic variation, apparent by high levels of clonal microsatellite diversity, was found. Our molecular phylogenetic evidence and the failure to resynthesize the gynogenetic P. formosa together with the old age of the species indicate that some unisexual vertebrates might be rare not because they suffer the long-term consequences of clonal reproduction but because they are only very rarely formed as a result of complex genetic preconditions necessary to produce viable and fertile clonal genomes and phenotypes ('rare formation hypothesis').

Determination of onset of sexual maturation and mating behavior by melanocortin receptor 4 polymorphisms.

Polymorphisms in reproductive strategies are among the most extreme and complex in nature. A prominent example is male body size and the correlated reproductive strategies in some species of platyfish and swordtails of the genus Xiphophorus. This polymorphism is controlled by a single Mendelian locus (P) that determines the onset of sexual maturity of males. Because males cease growth after reaching puberty, this results in a marked size polymorphism. The different male size classes show pronounced behavioral differences (e.g., courtship versus sneak mating), and females prefer large over small males. We show that sequence polymorphisms of the melanocortin receptor 4 gene (mc4r) comprise both functional and non-signal-transducing versions and that variation in copy number of mc4r genes on the Y chromosome underlies the P locus polymorphism. Nonfunctional Y-linked mc4r copies in larger males act as dominant-negative mutations and delay the onset of puberty. Copy number variation, as a regulating mechanism, endows this system with extreme genetic flexibility that generates extreme variation in phenotype. Because Mc4r is critically involved in regulation of body weight and appetite, a novel link between the physiological system controlling energy balance and the regulation of reproduction becomes apparent.

A little bit is better than nothing: the incomplete parthenogenesis of salamanders, frogs and fish.

A re-examination of the mitochondrial genomes of unisexual salamander lineages, published in BMC Evolutionary Biology, shows them to be the oldest unisexual vertebrates known, having been around for 5 million years. This presents a challenge to the prediction that lack of genetic recombination is a fast track to extinction. See research article http://www.biomedcentral.com/1471-2148/10/238.

A tetraploid Amazon molly, Poecilia formosa.

Polyploidization is thought to be an important driving force in evolution as it increases the genetic material on which mutation and selection can act. In the Amazon molly, Poecilia formosa, triploid genotypes can be found in the field and frequently arise from diploid breeding stocks, a tetraploid individual, however, was so far never documented. Here, we report the first tetraploid Amazon molly. Flow cytometry clearly showed the tetraploid DNA content, whereas microsatellite analysis not only confirmed the tetraploidy but also pointed to allotetraploidy. Most likely the fourth genome was received through paternal leakage, namely, by fertilization of a triploid egg with a haploid sperm. The existence of tetraploid individuals offers new explanations for the enormous clonal diversity observed in wild populations of P. formosa.

Automictic reproduction in interspecific hybrids of poeciliid fish.

Automixis, the process whereby the fusion of meiotic products restores the diploid state of the egg, is a common mode of reproduction in plants but has also been described in invertebrate animals. In vertebrates, however, automixis has so far only been discussed as one of several explanations for isolated cases of facultative parthenogenesis. Analyzing oocyte formation in F1 hybrids derived from Poecilia mexicana limantouri and P. latipinna crosses (the cross that led to the formation of the gynogenetic Poecilia formosa), we found molecular evidence for automictic oocyte production. The mechanism involves the random fusion of meiotic products after the second meiotic division. The fertilization of diploid oocytes gives rise to fully viable triploid offspring. Although the automictic production of diploid oocytes as seen in these F1 hybrids clearly represents a preadaptation to parthenogenetic reproduction, it is also a powerful intrinsic postzygotic isolation mechanism because the resulting next generation triploids were always sterile. The mechanism described here can explain facultative parthenogenesis, as well as varying ploidy levels reported in different animal groups. Most importantly, at least some of the reported cases of triploidy in humans can now be traced back to automixis.

Stable inheritance of host species-derived microchromosomes in the gynogenetic fish Poecilia formosa.

B chromosomes are additional, usually unstable constituents of the genome of many organisms. Their origin, however, is often unclear and their evolutionary relevance is not well understood. They may range from being deleterious to neutral or even beneficial. We have followed the genetic fate of B chromosomes in the asexual, all-female fish Poecilia formosa over eight generations. In this species, B chromosomes come in the form of one to three tiny microchromosomes derived from males of the host species that serve as sperm donors for this gynogenetic species. All microchromosomes have centromeric heterochromatin but usually only one has a telomere. Such microchromosomes are stably inherited, while the telomereless are prone to be lost in both the soma and germline. In some cases the stable microchromosome carries a functional gene lending support to the hypothesis that the B chromosomes in P. formosa could increase the genetic diversity of the clonal lineage in this ameiotic organism and to some degree counteract the genomic decay that is supposed to be connected with the lack of recombination.

Analysis of a possible independent origin of triploid P. formosa outside of the Río Purificación river system.

BACKGROUND: Unisexuality, or all female reproduction, is rare among vertebrates. Studying these exceptional organisms may give useful information with respect to the evolution and maintenance of sexual reproduction. Poecilia formosa was the first unisexual vertebrate species to be detected and since then has served as a paradigmatic organism for unisexuality and studies on the evolution of sex. It reproduces through gynogenesis, using sperm of males from related species to trigger parthenogenetic development of the unreduced diploid eggs. Like in other unisexual vertebrates, triploids occur in a certain range of P. formosa. It has been suggested that the addition of the host species derived third chromosome set is evolutionary important. Clonal organisms lack sufficient genotypic diversity for adaptive changes to variable environments. Also non-recombining genomes cannot purge deleterious mutations and therefore unisexual organisms should suffer from a genomic decay. Thus, polyploidization leading to triploidy should bring "fresh" genetic material into the asexual lineage. To evaluate the importance of triploidy for maintaining the asexual species, it is important to know whether such an introgression event happens at a reasonable frequency. RESULTS: In an earlier study it was found that all triploid P. formosa in the Rio Purificación river system are of monophyletic origin. Here we have analyzed fish from a different river system. Using microsatellite analysis we can show that the triploids from this new location are genetically divergent and most probably of an independent origin. CONCLUSION: Our data support the hypothesis that triploidy was not a single chance event in the evolutionary history of P. formosa and hence might be a relevant mechanism to increase genotypic divergence and at least partially counteract the genetic degeneration connected to asexuality. It is, however, much rarer than in other asexual vertebrates analyzed so far and thus probably only of moderate evolutionary importance for the maintenance of the asexual breeding complex.

Evidence for a monophyletic origin of triploid clones of the Amazon molly, Poecilia formosa.

Asexual reproduction in vertebrates is rare and generally considered an evolutionary dead end. Asexuality is often associated with polyploidy, and several hypotheses have been put forward to explain this relationship. So far, it remains unclear whether polyploidization in asexual organisms is a frequent or a rare event. Here we present a field study on the gynogenetic Amazon molly, Poecilia formosa. We used multilocus fingerprints and microsatellites to investigate the genetic diversity in 339 diploid and 55 triploid individuals and in 25 P. mexicana, its sexual host. Although multilocus DNA fingerprints found high clonal diversity in triploids, microsatellites revealed only two very similar clones in the triploids. Phylogenetic analysis of microsatellite data provided evidence for a monophyletic origin of the triploid clones of P. formosa. In addition, shared alleles within the triploid clones between the triploid and diploid genotypes and between asexual and sexual lineages indicate a recent origin of triploid clones in Poecilia formosa.