Seagrass genomes reveal ancient polyploidy and adaptations to the marine environment.

We present chromosome-level genome assemblies from representative species of three independently evolved seagrass lineages: Posidonia oceanica, Cymodocea nodosa, Thalassia testudinum and Zostera marina. We also include a draft genome of Potamogeton acutifolius, belonging to a freshwater sister lineage to Zosteraceae. All seagrass species share an ancient whole-genome triplication, while additional whole-genome duplications were uncovered for C. nodosa, Z. marina and P. acutifolius. Comparative analysis of selected gene families suggests that the transition from submerged-freshwater to submerged-marine environments mainly involved fine-tuning of multiple processes (such as osmoregulation, salinity, light capture, carbon acquisition and temperature) that all had to happen in parallel, probably explaining why adaptation to a marine lifestyle has been exceedingly rare. Major gene losses related to stomata, volatiles, defence and lignification are probably a consequence of the return to the sea rather than the cause of it. These new genomes will accelerate functional studies and solutions, as continuing losses of the 'savannahs of the sea' are of major concern in times of climate change and loss of biodiversity.

Experimental mining plumes and ocean warming trigger stress in a deep pelagic jellyfish.

The deep pelagic ocean is increasingly subjected to human-induced environmental change. While pelagic animals provide important ecosystem functions including climate regulation, species-specific responses to stressors remain poorly documented. Here, we investigate the effects of simulated ocean warming and sediment plumes on the cosmopolitan deep-sea jellyfish Periphylla periphylla, combining insights gained from physiology, gene expression and changes in associated microbiota. Metabolic demand was elevated following a 4 °C rise in temperature, promoting genes related to innate immunity but suppressing aerobic respiration. Suspended sediment plumes provoked the most acute and energetically costly response through the production of excess mucus (at ≥17 mg L-1), while inducing genes related to aerobic respiration and wound repair (at ≥167 mg L-1). Microbial symbionts appeared to be unaffected by both stressors, with mucus production maintaining microbial community composition. If these responses are representative for other gelatinous fauna, an abundant component of pelagic ecosystems, the effects of planned exploitation of seafloor resources may impair deep pelagic biodiversity and ecosystem functioning.

Non-native hosts of an invasive seaweed holobiont have more stable microbial communities compared to native hosts in response to thermal stress.

Seaweeds are colonized by a microbial community, which can be directly linked to their performance. This community is shaped by an interplay of stochastic and deterministic processes, including mechanisms which the holobiont host deploys to manipulate its associated microbiota. The Anna Karenina principle predicts that when a holobiont is exposed to suboptimal or stressful conditions, these host mechanisms may be compromised. This leads to a relative increase of stochastic processes that may potentially result in the succession of a microbial community harmful to the host. Based on this principle, we used the variability in microbial communities (i.e., beta diversity) as a proxy for stability within the invasive holobiont Gracilaria vermiculophylla during a simulated invasion in a common garden experiment. Independent of host range, host performance declined at elevated temperature (22°C) and disease incidence and beta diversity increased. Under thermally stressful conditions, beta diversity increased more in epibiota from native populations, suggesting that epibiota from non-native holobionts are thermally more stable. This pattern reflects an increase in deterministic processes acting on epibiota associated with non-native hosts, which in the setting of a common garden can be assumed to originate from the host itself. Therefore, these experimental data suggest that the invasion process may have selected for hosts better able to maintain stable microbiota during stress. Future studies are needed to identify the underlying host mechanisms.

Immunological tolerance in the evolution of male pregnancy.

The unique male pregnancy in pipefishes and seahorses ranges from basic attachment (pouch-less species: Nerophinae) of maternal eggs to specialized internal gestation in pouched species (e.g. Syngnathus and Hippocampus) with many transitions in between. Due to this diversity, male pregnancy offers a unique platform for assessing physiological and molecular adaptations in pregnancy evolution. These insights will contribute to answering long-standing questions of why and how pregnancy evolved convergently in so many vertebrate systems. To understand the molecular congruencies and disparities in male pregnancy evolution, we compared transcriptome-wide differentially expressed genes in four syngnathid species, at four pregnancy stages (nonpregnant, early, late and parturition). Across all species and pregnancy forms, metabolic processes and immune dynamics defined pregnancy stages, especially pouched species shared expression features akin to female pregnancy. The observed downregulation of adaptive immune genes in early-stage pregnancy and its reversed upregulation during late/parturition in pouched species, most notably in Hippocampus, combined with directionless expression in the pouch-less species, suggests immune modulation to be restricted to pouched species that evolved placenta-like systems. We propose that increased foeto-paternal intimacy in pouched syngnathids commands immune suppression processes in early gestation, and that the elevated immune response during parturition coincides with pouch opening and reduced progeny reliance. Immune response regulation in pouched species supports the recently described functional MHC II pathway loss as critical in male pregnancy evolution. The independent co-option of similar genes and pathways both in male and female pregnancy highlights immune modulation as crucial for the evolutionary establishment of pregnancy.

Invasion genomics uncover contrasting scenarios of genetic diversity in a widespread marine invader.

Invasion rates have increased in the past 100 y irrespective of international conventions. What characterizes a successful invasion event? And how does genetic diversity translate into invasion success? Employing a whole-genome perspective using one of the most successful marine invasive species world-wide as a model, we resolve temporal invasion dynamics during independent invasion events in Eurasia. We reveal complex regionally independent invasion histories including cases of recurrent translocations, time-limited translocations, and stepping-stone range expansions with severe bottlenecks within the same species. Irrespective of these different invasion dynamics, which lead to contrasting patterns of genetic diversity, all nonindigenous populations are similarly successful. This illustrates that genetic diversity, per se, is not necessarily the driving force behind invasion success. Other factors such as propagule pressure and repeated introductions are an important contribution to facilitate successful invasions. This calls into question the dominant paradigm of the genetic paradox of invasions, i.e., the successful establishment of nonindigenous populations with low levels of genetic diversity.

Draft genome and description of Waterburya agarophytonicola gen. nov. sp. nov. (Pleurocapsales, Cyanobacteria): a seaweed symbiont.

This work introduces Waterburya agarophytonicola Bonthond and Shalygin gen. nov., sp. nov, a baeocyte producing cyanobacterium that was isolated from the rhodophyte Agarophyton vermiculophyllum (Ohmi) Gurgel et al., an invasive seaweed that has spread across the northern hemisphere. The new species genome reveals a diverse repertoire of chemotaxis and adhesion related genes, including genes coding for type IV pili assembly proteins and a high number of genes coding for filamentous hemagglutinin family (FHA) proteins. Among a genetic basis for the synthesis of siderophores, carotenoids and numerous vitamins, W. agarophytonicola is potentially capable of producing cobalamin (vitamin B12), for which A. vermiculophyllum is an auxotroph. With a taxonomic description of the genus and species and a draft genome, this study provides as a basis for future research, to uncover the nature of this geographically independent association between seaweed and cyanobiont.

A novel metabarcoding primer pair for environmental DNA analysis of Cephalopoda (Mollusca) targeting the nuclear 18S rRNA region.

Cephalopods are pivotal components of marine food webs, but biodiversity studies are hampered by challenges to sample these agile marine molluscs. Metabarcoding of environmental DNA (eDNA) is a potentially powerful technique to study oceanic cephalopod biodiversity and distribution but has not been applied thus far. We present a novel universal primer pair for metabarcoding cephalopods from eDNA, Ceph18S (Forward: 5'-CGC GGC GCT ACA TAT TAG AC-3', Reverse: 5'-GCA CTT AAC CGA CCG TCG AC-3'). The primer pair targets the hypervariable region V2 of the nuclear 18S rRNA gene and amplifies a relatively short target sequence of approximately 200 bp in order to allow the amplification of degraded DNA. In silico tests on a reference database and empirical tests on DNA extracts from cephalopod tissue estimate that 44-66% of cephalopod species, corresponding to about 310-460 species, can be amplified and identified with this primer pair. A multi-marker approach with the novel Ceph18S and two previously published cephalopod mitochondrial 16S rRNA primer sets targeting the same region (Jarman et al. 2006 Mol. Ecol. Notes. 6, 268-271; Peters et al. 2015 Mar. Ecol. 36, 1428-1439) is estimated to amplify and identify 89% of all cephalopod species, of which an estimated 19% can only be identified by Ceph18S. All sequences obtained with Ceph18S were submitted to GenBank, resulting in new 18S rRNA sequences for 13 cephalopod taxa.

The role of host promiscuity in the invasion process of a seaweed holobiont.

Invasive species are co-introduced with microbiota from their native range and also interact with microbiota found in the novel environment to which they are introduced. Host flexibility toward microbiota, or host promiscuity, is an important trait underlying terrestrial plant invasions. To test whether host promiscuity may be important in macroalgal invasions, we experimentally simulated an invasion in a common garden setting, using the widespread invasive macroalga Agarophyton vermiculophyllum as a model invasive seaweed holobiont. After disturbing the microbiota of individuals from native and non-native populations with antibiotics, we monitored the microbial succession trajectories in the presence of a new source of microbes. Microbial communities were strongly impacted by the treatment and changed compositionally and in terms of diversity but recovered functionally by the end of the experiment in most respects. Beta-diversity in disturbed holobionts strongly decreased, indicating that different populations configure more similar -or more common- microbial communities when exposed to the same conditions. This decline in beta-diversity occurred not only more rapidly, but was also more pronounced in non-native populations, while individuals from native populations retained communities more similar to those observed in the field. This study demonstrates that microbial communities of non-native A. vermiculophyllum are more flexibly adjusted to the environment and suggests that an intraspecific increase in host promiscuity has promoted the invasion process of A. vermiculophyllum. This phenomenon may be important among invasive macroalgal holobionts in general.

How do microbiota associated with an invasive seaweed vary across scales?

Communities are shaped by scale dependent processes. To study the diversity and variation of microbial communities across scales, the invasive and widespread seaweed Agarophyton vermiculophyllum presents a unique opportunity. We characterized pro- and eukaryotic communities associated with this holobiont across its known distribution range, which stretches over the northern hemisphere. Our data reveal that community composition and diversity in the holobiont vary at local but also larger geographic scales. While processes acting at the local scale (i.e., within population) are the main structuring drivers of associated microbial communities, changes in community composition also depend on processes acting at larger geographic scales. Interestingly, the largest analysed scale (i.e., native and non-native ranges) explained variation in the prevalence of predicted functional groups, which could suggest a functional shift in microbiota occurred over the course of the invasion process. While high variability in microbiota at the local scale supports A. vermiculophyllum to be a generalist host, we also identified a number of core taxa. These geographically independent holobiont members imply that cointroduction of specific microbiota may have additionally promoted the invasion process.

Somatic genetic drift and multilevel selection in a clonal seagrass.

All multicellular organisms are genetic mosaics owing to somatic mutations. The accumulation of somatic genetic variation in clonal species undergoing asexual (or clonal) reproduction may lead to phenotypic heterogeneity among autonomous modules (termed ramets). However, the abundance and dynamics of somatic genetic variation under clonal reproduction remain poorly understood. Here we show that branching events in a seagrass (Zostera marina) clone or genet lead to population bottlenecks of tissue that result in the evolution of genetically differentiated ramets in a process of somatic genetic drift. By studying inter-ramet somatic genetic variation, we uncovered thousands of single nucleotide polymorphisms that segregated among ramets. Ultra-deep resequencing of single ramets revealed that the strength of purifying selection on mosaic genetic variation was greater within than among ramets. Our study provides evidence for multiple levels of selection during the evolution of seagrass genets. Somatic genetic drift during clonal propagation leads to the emergence of genetically unique modules that constitute an elementary level of selection and individuality in long-lived clonal species.

Evolution of male pregnancy associated with remodeling of canonical vertebrate immunity in seahorses and pipefishes.

A fundamental problem for the evolution of pregnancy, the most specialized form of parental investment among vertebrates, is the rejection of the nonself-embryo. Mammals achieve immunological tolerance by down-regulating both major histocompatibility complex pathways (MHC I and II). Although pregnancy has evolved multiple times independently among vertebrates, knowledge of associated immune system adjustments is restricted to mammals. All of them (except monotremata) display full internal pregnancy, making evolutionary reconstructions within the class mammalia meaningless. Here, we study the seahorse and pipefish family (syngnathids) that have evolved male pregnancy across a gradient from external oviparity to internal gestation. We assess how immunological tolerance is achieved by reconstruction of the immune gene repertoire in a comprehensive sample of 12 seahorse and pipefish genomes along the "male pregnancy" gradient together with expression patterns of key immune and pregnancy genes in reproductive tissues. We found that the evolution of pregnancy coincided with a modification of the adaptive immune system. Divergent genomic rearrangements of the MHC II pathway among fully pregnant species were identified in both genera of the syngnathids: The pipefishes (Syngnathus) displayed loss of several genes of the MHC II pathway while seahorses (Hippocampus) featured a highly divergent invariant chain (CD74). Our findings suggest that a trade-off between immunological tolerance and embryo rejection accompanied the evolution of unique male pregnancy. That pipefishes survive in an ocean of microbes without one arm of the adaptive immune defense suggests a high degree of immunological flexibility among vertebrates, which may advance our understanding of immune-deficiency diseases.

Microbial embryonal colonization during pipefish male pregnancy.

While originally acquired from the environment, a fraction of the microbiota is transferred from parents to offspring. The immune system shapes the microbial colonization, while commensal microbes may boost host immune defences. Parental transfer of microbes in viviparous animals remains ambiguous, as the two transfer routes (transovarial vs. pregnancy) are intermingled within the maternal body. Pipefishes and seahorses (syngnathids) are ideally suited to disentangle transovarial microbial transfer from a contribution during pregnancy due to their maternal egg production and their unique male pregnancy. We assessed the persistency and the changes in the microbial communities of the maternal and paternal reproductive tracts over proceeding male pregnancy by sequencing microbial 16S rRNA genes of swabs from maternal gonads and brood pouches of non-pregnant and pregnant fathers. Applying parental immunological activation with heat-killed bacteria, we evaluated the impact of parental immunological status on microbial development. Our data indicate that maternal gonads and paternal brood pouches harbor distinct microbial communities, which could affect embryonal development in a sex-specific manner. Upon activation of the immune system, a shift of the microbial community was observed. The activation of the immune system induced the expansion of microbiota richness during late pregnancy, which corresponds to the time point of larval mouth opening, when initial microbial colonization must take place.

Effects of parental care on resource allocation into immune defense and buccal microbiota in mouthbrooding cichlid fishes.

Sexual dimorphism is founded upon a resource allocation trade-off between investments in reproduction versus other life-history traits including the immune system. In species with conventional parental care roles, theory predicts that males maximize their lifetime reproductive success by allocating resources toward sexual selection, while females achieve this through prolonging their lifespan. Here, we examine the interrelation between sexual dimorphism and parental care strategies in closely related maternal and biparental mouthbrooding cichlid fishes from East African Lake Tanganyika. We measured cellular immune parameters, examined the relative expression of 28 immune system and life history-related candidate genes and analyzed the microbiota composition in the buccal cavity. According to our predictions, maternal mouthbrooders are more sexually dimorphic in immune parameters than biparental mouthbrooders, which has possibly arisen through a differential resource allocation into parental care versus secondary sexual traits. Biparental mouthbrooders, on the other hand, which share the costs of parental care, feature an upregulated adaptive immune response and stronger antiviral properties, while their inflammation response is reduced. Overall, our results suggest a differential resource allocation trade-off between the two modes of parental investment.

Poly-ß-hydroxybutyrate administration during early life: effects on performance, immunity and microbial community of European sea bass yolk-sac larvae.

The reliable production of marine fish larvae is one of the major bottlenecks in aquaculture due to high mortalities mainly caused by infectious diseases. To evaluate if the compound poly-ß-hydroxybutyrate (PHB) might be a suitable immunoprophylactic measure in fish larviculture, its capacity to improve immunity and performance in European sea bass (Dicentrarchus labrax) yolk-sac larvae was explored. PHB was applied from mouth opening onwards to stimulate the developing larval immune system at the earliest possible point in time. Larval survival, growth, microbiota composition, gene expression profiles and disease resistance were assessed. PHB administration improved larval survival and, furthermore, altered the larva-associated microbiota composition. The bacterial challenge test using pathogenic Vibrio anguillarum revealed that the larval disease resistance was not influenced by PHB. The expression profiles of 26 genes involved e.g. in the immune response showed that PHB affected the expression of the antimicrobial peptides ferritin (fer) and dicentracin (dic), however, the response to PHB was inconsistent and weaker than previously demonstrated for sea bass post-larvae. Hence, the present study highlights the need for more research focusing on the immunostimulation of different early developmental stages for gaining a more comprehensive picture and advancing a sustainable production of high quality fry.

Biophysical and Population Genetic Models Predict the Presence of "Phantom" Stepping Stones Connecting Mid-Atlantic Ridge Vent Ecosystems.

Deep-sea hydrothermal vents are patchily distributed ecosystems inhabited by specialized animal populations that are textbook meta-populations. Many vent-associated species have free-swimming, dispersive larvae that can establish connections between remote populations. However, connectivity patterns among hydrothermal vents are still poorly understood because the deep sea is undersampled, the molecular tools used to date are of limited resolution, and larval dispersal is difficult to measure directly. A better knowledge of connectivity is urgently needed to develop sound environmental management plans for deep-sea mining. Here, we investigated larval dispersal and contemporary connectivity of ecologically important vent mussels (Bathymodiolus spp.) from the Mid-Atlantic Ridge by using high-resolution ocean modeling and population genetic methods. Even when assuming a long pelagic larval duration, our physical model of larval drift suggested that arrival at localities more than 150 km from the source site is unlikely and that dispersal between populations requires intermediate habitats ("phantom" stepping stones). Dispersal patterns showed strong spatiotemporal variability, making predictions of population connectivity challenging. The assumption that mussel populations are only connected via additional stepping stones was supported by contemporary migration rates based on neutral genetic markers. Analyses of population structure confirmed the presence of two southern and two hybridizing northern mussel lineages that exhibited a substantial, though incomplete, genetic differentiation. Our study provides insights into how vent animals can disperse between widely separated vent habitats and shows that recolonization of perturbed vent sites will be subject to chance events, unless connectivity is explicitly considered in the selection of conservation areas.

Evaluating the adaptive potential of the European eel: is the immunogenetic status recovering?

The recent increased integration of evolutionary theory into conservation programs has greatly improved our ability to protect endangered species. A common application of such theory links population dynamics and indices of genetic diversity, usually estimated from neutrally evolving markers. However, some studies have suggested that highly polymorphic adaptive genes, such as the immune genes of the Major Histocompatibility Complex (MHC), might be more sensitive to fluctuations in population dynamics. As such, the combination of neutrally- and adaptively-evolving genes may be informative in populations where reductions in abundance have been documented. The European eel (Anguilla anguilla) underwent a drastic and well-reported decline in abundance in the late 20th century and still displays low recruitment. Here we compared genetic diversity indices estimated from neutral (mitochondrial DNA and microsatellites) and adaptive markers (MHC) between two distinct generations of European eels. Our results revealed a clear discrepancy between signatures obtained for each class of markers. Although mtDNA and microsatellites showed no changes in diversity between the older and the younger generations, MHC diversity revealed a contemporary drop followed by a recent increase. Our results suggest ongoing gain of MHC genetic diversity resulting from the interplay between drift and selection and ultimately increasing the adaptive potential of the species.

The genome of the seagrass Zostera marina reveals angiosperm adaptation to the sea.

Seagrasses colonized the sea on at least three independent occasions to form the basis of one of the most productive and widespread coastal ecosystems on the planet. Here we report the genome of Zostera marina (L.), the first, to our knowledge, marine angiosperm to be fully sequenced. This reveals unique insights into the genomic losses and gains involved in achieving the structural and physiological adaptations required for its marine lifestyle, arguably the most severe habitat shift ever accomplished by flowering plants. Key angiosperm innovations that were lost include the entire repertoire of stomatal genes, genes involved in the synthesis of terpenoids and ethylene signalling, and genes for ultraviolet protection and phytochromes for far-red sensing. Seagrasses have also regained functions enabling them to adjust to full salinity. Their cell walls contain all of the polysaccharides typical of land plants, but also contain polyanionic, low-methylated pectins and sulfated galactans, a feature shared with the cell walls of all macroalgae and that is important for ion homoeostasis, nutrient uptake and O2/CO2 exchange through leaf epidermal cells. The Z. marina genome resource will markedly advance a wide range of functional ecological studies from adaptation of marine ecosystems under climate warming, to unravelling the mechanisms of osmoregulation under high salinities that may further inform our understanding of the evolution of salt tolerance in crop plants.

Microbiome structure of the fungid coral Ctenactis echinata aligns with environmental differences.

The significance of bacteria for eukaryotic functioning is increasingly recognized. Coral reef ecosystems critically rely on the relationship between coral hosts and their intracellular photosynthetic dinoflagellates, but the role of the associated bacteria remains largely theoretical. Here, we set out to relate coral-associated bacterial communities of the fungid host species Ctenactis echinata to environmental settings (geographic location, substrate cover, summer/winter, nutrient and suspended matter concentrations) and coral host abundance. We show that bacterial diversity of C. echinata aligns with ecological differences between sites and that coral colonies sampled at the species' preferred habitats are primarily structured by one bacterial taxon (genus Endozoicomonas) representing more than 60% of all bacteria. In contrast, host microbiomes from lower populated coral habitats are less structured and more diverse. Our study demonstrates that the content and structure of the coral microbiome aligns with environmental differences and denotes habitat adequacy. Availability of a range of coral host habitats might be important for the conservation of distinct microbiome structures and diversity.

Assessing Symbiodinium diversity in scleractinian corals via next-generation sequencing-based genotyping of the ITS2 rDNA region.

The persistence of coral reef ecosystems relies on the symbiotic relationship between scleractinian corals and intracellular, photosynthetic dinoflagellates in the genus Symbiodinium. Genetic evidence indicates that these symbionts are biologically diverse and exhibit discrete patterns of environmental and host distribution. This makes the assessment of Symbiodinium diversity critical to understanding the symbiosis ecology of corals. Here, we applied pyrosequencing to the elucidation of Symbiodinium diversity via analysis of the internal transcribed spacer 2 (ITS2) region, a multicopy genetic marker commonly used to analyse Symbiodinium diversity. Replicated data generated from isoclonal Symbiodinium cultures showed that all genomes contained numerous, yet mostly rare, ITS2 sequence variants. Pyrosequencing data were consistent with more traditional denaturing gradient gel electrophoresis (DGGE) approaches to the screening of ITS2 PCR amplifications, where the most common sequences appeared as the most intense bands. Further, we developed an operational taxonomic unit (OTU)-based pipeline for Symbiodinium ITS2 diversity typing to provisionally resolve ecologically discrete entities from intragenomic variation. A genetic distance cut-off of 0.03 collapsed intragenomic ITS2 variants of isoclonal cultures into single OTUs. When applied to the analysis of field-collected coral samples, our analyses confirm that much of the commonly observed Symbiodinium ITS2 diversity can be attributed to intragenomic variation. We conclude that by analysing Symbiodinium populations in an OTU-based framework, we can improve objectivity, comparability and simplicity when assessing ITS2 diversity in field-based studies.

Bacterial profiling of White Plague Disease in a comparative coral species framework.

Coral reefs are threatened throughout the world. A major factor contributing to their decline is outbreaks and propagation of coral diseases. Due to the complexity of coral-associated microbe communities, little is understood in terms of disease agents, hosts and vectors. It is known that compromised health in corals is correlated with shifts in bacterial assemblages colonizing coral mucus and tissue. However, general disease patterns remain, to a large extent, ambiguous as comparative studies over species, regions, or diseases are scarce. Here, we compare bacterial assemblages of samples from healthy (HH) colonies and such displaying signs of White Plague Disease (WPD) of two different coral species (Pavona duerdeni and Porites lutea) from the same reef in Koh Tao, Thailand, using 16S rRNA gene microarrays. In line with other studies, we found an increase of bacterial diversity in diseased (DD) corals, and a higher abundance of taxa from the families that include known coral pathogens (Alteromonadaceae, Rhodobacteraceae, Vibrionaceae). In our comparative framework analysis, we found differences in microbial assemblages between coral species and coral health states. Notably, patterns of bacterial community structures from HH and DD corals were maintained over species boundaries. Moreover, microbes that differentiated the two coral species did not overlap with microbes that were indicative of HH and DD corals. This suggests that while corals harbor distinct species-specific microbial assemblages, disease-specific bacterial abundance patterns exist that are maintained over coral species boundaries.