Background

Red algae have thrived on Earth for more than 1.2 billion years, evolving into a unique lineage of photosynthetic eukaryotes that were the first to adapt to intertidal environments. They are characterized by their low evolutionary status, rich diversity, structurally simple yet diverse forms, strong resistance to stress, and economic value. Moreover, red algae play a dual role as both the first eukaryotic hosts involved in primary endosymbiosis and as the plastid donors involved in secondary endosymbiosis, thereby endowing them with a combination of ancestral land plant-like stress resistance mechanisms and their own distinct adaptations. Interestingly, numerous studies have highlighted the crucial role of horizontal gene transfer (HGT) in biological adaptive evolution. Here, we focus on Pyropia haitanensis, a representative intertidal red alga with economic value and a model for studying adaptation. We propose that within the intricate phycosphere community surrounding P. haitanensis, specific microbial taxa may play pivotal roles as potential donors or facilitators of HGT events. These HGT events drive adaptation, enhancing P. haitanensis's stress tolerance and allowing it to thrive in the intertidal zone's harsh conditions.

Approach

To validate our hypothesis, this study meticulously assembled a high-quality genome of P. haitanensis and delved into the identification of horizontal gene transfer (HGT) events and their donors. Furthermore, we employed a bulked segregant analysis (BSA) approach to pinpoint quantitative trait loci (QTLs) and candidate HGT genes that are intricately linked to the high-temperature tolerance of P. haitanensis. Additionally, we delved deeply into the intricacies of microbial community structural changes in heat-sensitive and heat-resistant strains following high temperature stress. Through the use of selective culture media, we were able to successfully isolate a beneficial bacterium, Saccharothrix sp., that notably enhances the high-temperature adaptability of P. haitanensis.

Results

Horizontal gene transfer contributed significantly to the evolution of symbiont species like algae. Across 10 species, we found 2,325 HGT genes, with 54 gene families linked to early HGTs (Fig. 1a). A total of 286 HGT genes were detected in P. haitanensis, accounting for approximately 2.25% of the entire genome. Notably, 251 of these HGT genes were associated with transposable element (TE) insertions nearby (Fig. 1b), suggesting TEs may facilitate HGT. Further, 97.9% of HGT genes originated from bacteria, mainly Pseudomonadota, Actinomycetota, Bacteroidota, and Planctomycetota (54.9%) (Fig. 1c).

Fig. 1. Comparative analysis of horizontal gene transfer (HGT). a) Phylogenetic tree presenting the relationships among ten algal species. b) Genomic collinearity demonstrated by the comparison of P. yezoensis, P. haitanensis, and P. umbilicalis. c) Sankey diagram illustrating the flow of HGT genes from donor species to TE classes in P. haitanensis.

To pinpoint HGT genes enabling P. haitanensis heat tolerance, we conducted BSA-seq on heat-resistant (HR-Pool) and heat-sensitive (HS-Pool) algae pools.  Based on 95% confidence interval of Δ(SNP-index), 2 genomic regions (1.46 Mb on chr2, 0.22 Mb on chr3) were identified with 468 candidate genes. Notably, among these candidate genes, ten HGT genes, such as sirohydrochlorin ferrochelatase (SIRB) and peptide-methionine (R)-S-oxide reductase (MSRB), were HGT genes related to the adaptation of P. haitanensis to heat stress.

To further examine how the symbiotic microbial community affected the tolerance of P. haitanensis to heat stress, we conducted a 16S rDNA sequencing analysis to determine the differences in the microbial communities of the heat-resistant (HR) strain and the heat-sensitive (HS) strain under normal (21 °C) and high-temperature (30 °C) conditions (Fig. 2a-b). Interestingly, we found that seven bacteria, including Saccharothrix sp., are unique to the surface of HR strains, with Saccharothrix sp. being the most abundant (Fig. 2c). The presence (experimental group) and absence (control group) of Saccharothrix sp. in the culture medium influenced the phenotype and cell morphology of the HS strain (Fig. 2d). To identify the genes responsive to high temperatures, we analyzed the association between the composition of algal symbiotic bacteria and gene transcription levels in the HR strain (HRH), HS strain (control group, HSH), and HS strain supplemented with Saccharothrix sp. (experimental group, PHSH). The experimental group showed a significant positive correlation with the HGT genes proC, ggt, HSP20, fabd, uvrD, AMY, and aceA (P < 0.05), while the control group exhibited the negative correlation with these genes (Fig. 2e). Among these genes, proC is a key gene that involved in the proline synthesis pathway. Surprisingly, treating the HS strain with proline significantly increased the relative growth rate under high-temperature conditions (Fig. 2f-i). Overall, our findings demonstrate that the introduction of Saccharothrix sp. isolated from the HR strain substantially increased the heat resistance of the HS strain.

Fig. 2. Screening, isolation, and regulation of key HGT donor bacteria in P. haitanensis and verification of their ability to enhance the thermal tolerance of thalli. a) Changes in the Chao1 and Shannon indices in water and thallus samples the from the HS strain and HR strain under high-temperature conditions. T and W represent thallus and water samples, respectively, whereas C and H represent normal- and high-temperature treatments, respectively. b) Relative abundance of the community composition (phylum) in the algal intermicrobial environment. c) Venn diagram and heatmap illustrating the differences in the number and abundance of shared and unique bacteria between the thallus samples of the HS strain and HR strain. d) Analysis of the composition of the macrogenomic community (phylum) and sample clusters. P represents the addition of Saccharothrix sp. e) Analysis of the correlation between different treatments of intermicrobial microorganisms and HGT homologous genes. 6. f–i) Effects of the addition of proline (Pro) and Saccharothrix sp. (PHS) on the physiological and biochemical indices of the HS strain (CK) thallus under high-temperature conditions. Measurements include (f) Relative growth rate, (g) maximum quantum yield (Fv/Fm), (h) superoxide dismutase (SOD) activity, and (i) free proline content.

In summary, this study reveals the critical role of HGT and microbial symbionts in promoting the high-temperature tolerance of P. haitanensis. It not only provides genetic resources for the cultivation of new heat-tolerant seaweed varieties, but also contributes to the understanding of how organisms evolve to adapt to extreme environments, thereby having broader implications for fields such as ecology, and evolutionary biology.