Thermal plasticity of the kelp Laminaria digitata: latitudinal and transgenerational effects

In temperate and polar rocky coastal ecosystems, kelps form the base of complexly structured and highly diverse species associations. In recent years, kelp forests worldwide have faced extinction at their equatorward limits and models have predicted a poleward shift of kelp ecosystems during climate change. To gain an understanding of local thermal adaptation and response plasticity in a forest-forming kelp species, we assessed populations of Laminaria digitata along its entire European distribution range from Brittany to Spitsbergen for their capacity to withstand high temperature stress, and connected physiological results to population genetics. Although the overall heat response was similar across the distribution range, we identified subtle adaptions reflecting the long-term local temperature history in one northern and one southern population. Additionally, we investigated the potential role of transgenerational plasticity (TGP, i.e. effects of parental environment on offspring traits) as a means of fast response to a warming environment between haploid gametophyte parents and diploid sporophyte offspring of L. digitata. Our results show TGP for the first time in a kelp, in that not the 15°C, but the 5°C gametophyte parent treatment enhanced juvenile sporophyte offspring growth. In this talk, I will conceptualize the thermal response of L. digitata and discuss our results within the framework of global warming

Cold thermal priming of Laminaria digitata (Laminariales, Phaeophyceae) gametophytes enhances gametogenesis and thermal performance of sporophytes

Thermal characteristics of kelp species have been studied in many ways, but potentially persistent effects of temperature across generations are yet poorly understood. In this context, the effect of thermal priming on fertility and growth of the N-Atlantic kelp species Laminaria digitata was investigated within and across life cycle generations in a two-step common garden experiment. Using vegetative clonal gametophytes from cold (5°C) and warm (15°C) pre-experimental cultivation (3 years), we first quantified gametogenesis and recruitment over two weeks at a common temperature of 10°C. Then, recruited sporophytes were transferred to a temperature gradient spanning the tolerance range of the species from 0°C to 20°C. We hypothesized that a warm gametophyte preexperimental cultivation promotes performance of sporophytes at warm temperatures and vice versa. Interestingly, gametogenesis speed and sporophyte recruitment were higher in gametophytes following cold compared to warm pre-experimental cultivation, which indicates carry-over effects of temperature within the gametophyte generation. Compared to warm pre-experimental cultivation of gametophytes, a cold preexperimental cultivation enhanced growth of juvenile Laminaria digitata sporophytes by more than 69% at the extreme low and high temperatures of 0 and 20°C. This is the first evidence for a cross-generational effect between gametophyte parents and offspring sporophytes. As cold gametophyte cultivation increased the trait performance of gametogenesis, recruitment and thermal tolerance of juvenile sporophytes, priming of early life cycle stages may be used to increase resilience and productivity of kelps in marine forest restoration efforts and kelp mariculture

Transgenerational temperature effects in the kelp Laminaria digitata

Transgenerational effects (effects of parental environment on offspring traits) have recently gained attention as a means of fast response to changing environmental conditions, e.g. under climate change. In temperate and polar rocky coastal ecosystems, kelps form the base of complexly structured and highly diverse species associations. This study investigates the potential for temperature-mediated transgenerational plasticity along the development from haploid parents (gametophytes) to their juvenile diploid offspring (sporophytes) in a geographically isolated population of the brown alga Laminaria digitata from the island of Helgoland (North Sea). We sampled spores from wild donor sporophytes, which we raised at 5 and 15 °C during gametogenesis, and reared the resulting young sporophytes for three months in a full-factorial split design while keeping genetic lineages separate. A concluding 12-day experiment on growth, biochemistry (carbon, mannitol content) and photosynthetic characteristics (maximum quantum yield Fv/Fm, maximum electron transport rate rETRmax) of five genetic lineages allowed for the separation of late temperature (12 day) responses, within-generation plasticity (early temperature) and transgenerational plasticity (gametogenesis temperature) in response to 5 and 15 °C. We observed significant two- and three-way interactions between gametogenesis temperature and early and late sporophyte temperatures for all parameters. While interactive effects between early and late experimental temperatures probably represent acclimation processes, interactions involving gametogenesis temperature indicate transgenerational effects. The direction of these effects differed between parameters. A main effect is that only with a history of 5 °C as gametogenesis and early temperature, sporophytes are growing faster at 5 than 15 °C over 12 days late temperature. According to our results, the temperature experienced by parents during gametogenesis influences temperature reaction norms in three- to four-month-old Laminaria digitata sporophytes. This, to our knowledge, is the first evidence for transgenerational plasticity in kelps

Microscopic life stages of North Atlantic Laminaria digitata (Phaeophyceae) exhibit trait-depedent thermal adaptation along latitudes

Kelp forests in the North Atlantic are at risk of decline at their warm temperature distribution margins due to anthropogenic temperature rise and more frequent marine heat waves. To investigate the thermal adaptation of the cold-temperate kelp Laminaria digitata, we sampled six populations, from the Arctic to Brittany (Spitsbergen, Tromsø, Bodø [all Norway], Helgoland [Germany], Roscoff and Quiberon [both France]), across the species’ entire distribution range, spanning 31.5° latitude and 12-13°C difference in mean summer sea surface temperature. We used pooled vegetative gametophytes derived from several sporophytes to approximate the genetic diversity of each location. Gametophytes were exposed to (sub-) lethal high (20-25°C) and (sub-) optimal low (0-15°C) temperature gradients in two full-factorial, common-garden experiments, subjecting subsets of populations from different origins to the same conditions. We assessed survival of gametophytes, their ability to develop microscopic sporophytes, and subsequent growth. We hypothesized that the thermal performance of gametophytes and microscopic sporophytes corresponds to their local long-term thermal history. Integrated gametophyte survival revealed a uniform upper survival temperature (UST) of 24°C among five tested populations (Tromsø to Quiberon). In contrast, following two weeks of thermal priming of gametophytes at 20-22°C, sporophyte formation at 15°C was significantly higher in southern populations (Quiberon and Roscoff) compared to the high-latitude population of Tromsø. Between 0-15°C, survival of the Arctic population (Spitsbergen) was negatively correlated with increasing temperatures, while the southernmost population (Quiberon) showed the opposite. Thus, responses of survival at low, and sporophyte formation at high temperatures, support the concept of local adaption. On the other hand, sporophyte formation between 0-15°C peaked at 6-9°C in the Quiberon and at 9-12°C in the Spitsbergen population. Sporophyte growth rates (GR) both in length and width were similar for Spitsbergen, Tromsø and Quiberon; all had maximum GRs at 12-15°C and low GRs at 0-6°C. Therefore, responses of sporophyte formation and growth at low temperatures do not reflect ecotypic adaptation. We conclude that L. digitata populations display trait-dependent adaptation, partly corresponding to their local temperature histories and partly manifesting uniform or unpredictable responses. This suggests differential selection pressures on the ontogenetic development of kelps such as L. digitata

Thermal Plasticity of the Kelp Laminaria digitata (Phaeophyceae) Across Life Cycle Stages Reveals the Importance of Cold Seasons for Marine Forests

Phenotypic plasticity (genotype × environment interaction) is an especially important means for sessile organisms to cope with environmental variation. While kelps, the globally most productive group of seaweeds, generally possess a wide thermal performance range, kelp populations at their warm distribution limits are threatened by ocean warming. Here, we investigated effects of temperature during ontogeny of the kelp Laminaria digitata across haploid gametophyte and diploid sporophyte life cycle stages in five distinct genetic lines. We hypothesized that thermal plasticity increases trait performance of juvenile sporophytes in experimental temperatures that match the temperature experienced during gametogenesis and recruitment, and that plasticity differs among genetic lines (genetic variation for plasticity). We applied a full-factorial experimental design to generate different temperature histories by applying 5 and 15°C during meiospore germination, gametogenesis of parental gametophytes and recruitment of offspring sporophytes (19–26 days), and juvenile sporophyte rearing (91–122 days). We then tested for thermal plasticity among temperature history treatments at 5 and 15°C in a final 12-day experiment assessing growth, the storage compound mannitol, carbon and nitrogen contents, and fluorometric responses in 3–4 month old sporophytes for five genetic lines. Our study provides evidence for the importance of cold temperatures at early development on later sporophyte performance of L. digitata. Gametogenesis and recruitment at 5°C promoted higher growth of offspring sporophytes across experimental temperatures. While photosynthetic capacity was higher at 15°C, carbon and nitrogen storage were higher at 5°C, both showing fast acclimation responses. We identified an important role of genetic variation for plasticity in shaping L. digitata’s thermal plasticity. Trait performance at 5 or 15°C (reaction norm slopes) differed among genetic lines, even showing opposite response patterns. Interestingly, genetic variation for plasticity was only significant when sporophytes were reared at 5°C. Thus, we provide evidence that the cold-temperate to Arctic kelp species, L. digitata, which possesses a wide temperature tolerance between 0 and 23°C, is impaired by warm temperature during gametogenesis and recruitment, reducing growth of juvenile sporophytes and expression of variable thermal plasticity in the wild

Local differentiation in heat response of Laminaria digitata at the range edges

In recent years, kelp populations worldwide have faced decline and extirpation at their equatorward limits, while models predict a poleward shift of kelp ecosystems during climate change. To gain an understanding of local thermal adaptation and response plasticity in a forest-forming kelp species, we assessed populations of Laminaria digitata along its entire European distribution range for their capacity to withstand high temperature stress, and analysed population structure and diversity with microsatellite markers (n=12). We sampled wild meristematic L. digitata material (n=30) at six locations ranging from Kongsfjorden, Spitsbergen, to the southernmost distribution limit in Quiberon, France. In a heatwave experiment, we subjected samples from all locations to the same, sublethal temperature treatments (15–23°C for eight days including acclimation) and assessed growth, storage compounds, photosynthetic efficiency and pigment contents as response traits. Recovery was assessed following seven days at 15°C. Microsatellite genotyping revealed all sampled populations to be genetically distinct entities, underlying strong regional structuring between southern and northern clades. Genetic diversity was highest at the southern distribution limit in Quiberon and lowest in the geographically isolated population on the island of Helgoland in the North Sea. The physiological response of L. digitata to temperature was similar over the entire distribution range and did not reflect the mean temperature gradient along the latitudinal gradient. However, material from Spitsbergen and Helgoland presented subtle differentiations in their temperature responses, which reflect long-term local temperature histories at these sites. Finally, a heatwave reaching 23°C for five days led to a cessation of growth, from which none of the sampled populations recovered. Our results suggest that the heat stress response of L. digitata is generally stable across its distribution range, despite strong genetic structuring of the populations. Slight local differentiation occurred in populations from the most distinct thermal environments, but 23°C posed a growth limit for all populations. This implies that local adaptation in trailing edge populations of L. digitata might not alleviate detrimental effects of global warming

Increased Heat Resilience of Intraspecific Outbred Compared to Inbred Lineages in the Kelp Laminaria digitata: Physiology and Transcriptomics

Marine forests and kelps as their foundation species are threatened by ocean warming especially at the warm distributional edges. Previously identified genetic divergence and ecotypic differentiation within kelp species may allow to produce more resilient lineages by intraspecific outbreeding among populations. In a mechanistic investigation of heat stress, heterosis (hybrid vigour), and underlying gene expression patterns, we assessed the thermal performance of inbred (selfings) and outbred (reciprocal crosses) sporophytes of the N-Atlantic kelp Laminaria digitata among clonal isolates from two divergent populations; one from the temperate North Sea (Helgoland) and one from the Arctic (Spitsbergen). First, we investigated the upper thermal tolerance of microscopic sporophytes in a 14-day experiment applying sublethal to lethal 20–23°C. The upper survival temperature of microscopic sporophytes was lower for the inbred Arctic selfing (21°C) than for the temperate selfing and the reciprocal crosses (22°C). Only in the temperate selfing, 4.5% of sporophytes survived 23°C. We then subjected 4–7 cm long sporophytes to a control temperature (10°C), moderate (19°C) and sublethal to lethal heat stress (20.5°C) for 18 days to assess gene expression in addition to physiological parameters. Growth and optimum quantum yield decreased similarly in the reciprocal crosses and the temperate selfing at 19 and 20.5°C, while inbred Arctic sporophytes died within seven days at both 19 and 20.5°C. In response to 20.5°C, 252 genes were constitutively regulated across all surviving lineages, which we use to describe metabolic regulation patterns in response to heat stress in kelp. At sublethal 20.5°C, ca. 150 genes were differentially expressed by either crossed lineage in comparison to the temperate selfing, indicating that they maintained a growth response similar to the temperate selfing with differential metabolic regulation during sublethal heat stress. Subtle differences in physiology and the differential expression of nine genes between the reciprocal crosses at 20.5°C indicate that female and male gametophytes may contribute differently to offspring traits. We consider potential inbreeding depression in the Spitsbergen selfing and quantify the better performance of both crosses using heterosis-related parameters. We discuss the potential and risks of outbreeding to produce more resilient crops for mariculture and marine forest restoration

Phenotypic Characterization of EIF2AK4 Mutation Carriers in a Large Cohort of Patients Diagnosed Clinically With Pulmonary Arterial Hypertension.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a rare disease with an emerging genetic basis. Heterozygous mutations in the gene encoding the bone morphogenetic protein receptor type 2 (BMPR2) are the commonest genetic cause of PAH, whereas biallelic mutations in the eukaryotic translation initiation factor 2 alpha kinase 4 gene (EIF2AK4) are described in pulmonary veno-occlusive disease/pulmonary capillary hemangiomatosis. Here, we determine the frequency of these mutations and define the genotype-phenotype characteristics in a large cohort of patients diagnosed clinically with PAH. METHODS: Whole-genome sequencing was performed on DNA from patients with idiopathic and heritable PAH and with pulmonary veno-occlusive disease/pulmonary capillary hemangiomatosis recruited to the National Institute of Health Research BioResource-Rare Diseases study. Heterozygous variants in BMPR2 and biallelic EIF2AK4 variants with a minor allele frequency of <1:10 000 in control data sets and predicted to be deleterious (by combined annotation-dependent depletion, PolyPhen-2, and sorting intolerant from tolerant predictions) were identified as potentially causal. Phenotype data from the time of diagnosis were also captured. RESULTS: Eight hundred sixty-four patients with idiopathic or heritable PAH and 16 with pulmonary veno-occlusive disease/pulmonary capillary hemangiomatosis were recruited. Mutations in BMPR2 were identified in 130 patients (14.8%). Biallelic mutations in EIF2AK4 were identified in 5 patients with a clinical diagnosis of pulmonary veno-occlusive disease/pulmonary capillary hemangiomatosis. Furthermore, 9 patients with a clinical diagnosis of PAH carried biallelic EIF2AK4 mutations. These patients had a reduced transfer coefficient for carbon monoxide (Kco; 33% [interquartile range, 30%-35%] predicted) and younger age at diagnosis (29 years; interquartile range, 23-38 years) and more interlobular septal thickening and mediastinal lymphadenopathy on computed tomography of the chest compared with patients with PAH without EIF2AK4 mutations. However, radiological assessment alone could not accurately identify biallelic EIF2AK4 mutation carriers. Patients with PAH with biallelic EIF2AK4 mutations had a shorter survival. CONCLUSIONS: Biallelic EIF2AK4 mutations are found in patients classified clinically as having idiopathic and heritable PAH. These patients cannot be identified reliably by computed tomography, but a low Kco and a young age at diagnosis suggests the underlying molecular diagnosis. Genetic testing can identify these misclassified patients, allowing appropriate management and early referral for lung transplantation

Comprehensive Rare Variant Analysis via Whole-Genome Sequencing to Determine the Molecular Pathology of Inherited Retinal Disease

Inherited retinal disease is a common cause of visual impairment and represents a highly heterogeneous group of conditions. Here, we present findings from a cohort of 722 individuals with inherited retinal disease, who have had whole-genome sequencing (n = 605), whole-exome sequencing (n = 72), or both (n = 45) performed, as part of the NIHR-BioResource Rare Diseases research study. We identified pathogenic variants (single-nucleotide variants, indels, or structural variants) for 404/722 (56%) individuals. Whole-genome sequencing gives unprecedented power to detect three categories of pathogenic variants in particular: structural variants, variants in GC-rich regions, which have significantly improved coverage compared to whole-exome sequencing, and variants in non-coding regulatory regions. In addition to previously reported pathogenic regulatory variants, we have identified a previously unreported pathogenic intronic variant in $\textit{CHM}$ in two males with choroideremia. We have also identified 19 genes not previously known to be associated with inherited retinal disease, which harbor biallelic predicted protein-truncating variants in unsolved cases. Whole-genome sequencing is an increasingly important comprehensive method with which to investigate the genetic causes of inherited retinal disease.This work was supported by The National Institute for Health Research England (NIHR) for the NIHR BioResource – Rare Diseases project (grant number RG65966). The Moorfields Eye Hospital cohort of patients and clinical and imaging data were ascertained and collected with the support of grants from the National Institute for Health Research Biomedical Research Centre at Moorfields Eye Hospital, National Health Service Foundation Trust, and UCL Institute of Ophthalmology, Moorfields Eye Hospital Special Trustees, Moorfields Eye Charity, the Foundation Fighting Blindness (USA), and Retinitis Pigmentosa Fighting Blindness. M.M. is a recipient of an FFB Career Development Award. E.M. is supported by UCLH/UCL NIHR Biomedical Research Centre. F.L.R. and D.G. are supported by Cambridge NIHR Biomedical Research Centre