Tengchong County, located in Yunnan Province, Southwestern China, is known for its geothermal features. Tectonically, Tengchong is located at the collision boundary between the India and Eurasia plates. Subduction of oceanic crust leads to extensive volcanism. The hot springs of Tengchong are a result of such volcanism with more than 50 volcanoes and 140 geothermal areas throughout the county. In terms of the diversity and scale, the Tengchong springs are comparable to the geothermal systems in Yellowstone National Park (YNP).

The Rehai (“Hot Sea”) geothermal field in Tengchong harbors intense hydrothermal activity with numerous springs and pools. Physicochemical conditions span a wide range of temperature (58 to ∼97°C) and pH (<1.8 to ≥9.3) (Hou, Wang et al. 2013, He, Wang et al. 2021) and these conditions provide numerous niches to support phylogenetically and functionally specialized microbial communities. Inhabiting these hot springs are microorganisms that have evolved exceptional adaptations to cope with the harsh and fluctuating conditions. As relatively isolated ecosystems, terrestrial hot springs present a unique opportunity for microbial diversification and specialization. Therefore, the Tengchong geothermal fields likely represent a biodiversity hotspot for thermophiles and provide an opportunity to study the ecological and evolutionary control of thermophiles. That is why the mechanisms of their diversity formation are of great interest to researchers.

 The entrance of the Tengchong Rehai geothermal park (left) and people joining in the field work (right). Image credit: Dr. Shang Wang.

 The field work was carried out in 2018. Dr. Weiguo Hou and Dr. Shang Wang (in the middle of the photo above) started to do research in Reihai since 2011 and they came to Tengchong once or twice every year. The left of us were the first time to do field work and we were so excited and full of energy to take different tasks during the whole field sampling, even though we have to run up and down following the mountain trail (elevation difference about 100 m) several times by carrying dry ice box, Hach in-situ measurement toolbox, liquid nitrogen tank and other materials for collecting and storage of samples. Once the sampling begins, we do the field measurements first and then start the water and sediment collection.

Dr. Shang Wang is measuring the in-situ water temperature and pH (left) and The students are mixing and putting the sediments in the brown serum bottles (right). Image credit: Dr. Weiguo Hou and Dr. Shang Wang.

Of all the environmental factors, temperature-driven microbial diversity has attracted more attention (Inskeep, Rusch et al. 2010, Shock, Holland et al. 2010, Hou, Wang et al. 2013). Similarly, temperature is one of the most important niche axis in shaping microbial community structure, carbon fixation rate and community assembly in Tengchong hot springs (Hou, Wang et al. 2013, Zhang, Qi et al. 2020, He, Wang et al. 2021). This is mainly because the geothermal springs are an analog to the ancient early earth. As we know, earth experienced a process of cooling down and environmental temperature has been the most prominent factor driving the thermophile’s diversity expansion (Boussau, Blanquart et al. 2008, Groussin and Gouy 2011). Therefore, temperature was of particular interest in its effect on microbial diversity and efforts to understand the mechanisms by which temperature regulates diversity have yielded two perspectives. Firstly, high temperatures could act as a selective force, leading to environmental filtering, wherein species not adapted to the extreme conditions are eliminated, thus reducing microbial diversity (Guo, Wang et al. 2020, He, Wang et al. 2021). This ecological process is driven by the differential thermal tolerance of species, where only those capable of surviving and thriving in the high-temperature environment persist. Secondly, high temperatures could also facilitate the speciation and diversification of new species through evolutionary processes (Bromham and Cardillo 2003, Allen, Gillooly et al. 2006). As organisms are exposed to challenging thermal conditions, genetic mutations and adaptations may occur, giving rise to novel traits and specialized thermal adaptations, which ultimately contribute to increased microbial diversity. Despite advances in understanding these individual processes, the integration of ecological and evolutionary mechanisms regulating microbial diversity in high-temperature environments remains a complex and understudied area. Further research is essential to elucidate how these processes interact and jointly shape the microbial communities in response to temperature.

Conceptual diagram of high-temperature driven speciation and environmental filtering, leading to contrasting trends of microbial diversity. This figure is drawn by Qing He.

In order to address the research questions at hand, we conducted a comprehensive investigation into the ecological and evolutionary characteristics of microbiota in hot springs with temperatures ranging from 54.8 to 80°C. Distinguishing the phylogenetic evolution of microbial taxa in their natural habitat presents a significant challenge. We chose the full-length 16S rRNA genes by PacBio RSII sequencing to obtain finer and more accurate microbial phylogenetic classification (Callahan, Grinevich et al. 2021). Subsequently, we have selected what we believe to be the most appropriate analysis methods to address the quesitons. First, the mean-nearest-taxon-distance (MNTD) and nearest-taxon-index (NTI) irrelative to richness were calculated to describe phylogenetic distinctiveness of communities (Webb, Ackerly et al. 2002, Horner-Devine and Bohannan 2006). We found that the community phylogenetic relatedness became closer with temperature and higher temperature promoted stronger phylogenetic clustering at finer taxonomic level near the tips of the phylogenetic tree. Second, the binary-state speciation and extinction model (BiSSE), a phylogenetic tree-based model was used to reveal the evolutionary features of microbial taxa (Maddison, Midford et al. 2007). We found high speciation for niche-specialists and high extinction for niche-generalists. At last, Metabolic theory of ecology (MTE) (Allen, Brown et al. 2002, Brown, Gillooly et al. 2004) offers a framework to link temperature and speciation and microbial diversity, so we used the MTE framework to quantify the influence of temperature on diversification potential (DP), environmental-filtering potential (EP) and their relative strength (RS_{DP vs EP}). The results showed that EP is dominant over DP across temperatures, partially explaining the decreased trend of microbial diversity with temperature.

 By integrating these results, we have gained a comprehensive understanding of the intricate interplay between ecological and evolutionary dynamics governing niche specialists and niche generalists in microbial communities. On the thermal tolerance niche axis, thermal (T) sensitive (at a specific temperature) versus T-resistant (at least in five temperatures) species were characterized by different niche breadth, community abundance and dispersal potential, consequently differing in potential evolutionary trajectory. T-sensitive species, being specialists, exhibited strong temperature barriers, resulting in complete shifts in species composition and the formation of low-abundance but high-fitness communities in their respective temperatures ("home niche”). This trade-off was shown to reinforce peak performance through high speciation rates across temperatures and an increasing potential for diversification with rising temperatures. On the other hand, T-resistant species, being generalists, possessed the advantage of niche expansion but suffered from poor local performance, as indicated by their wide niche breadth with high extinction rates. This suggested that these niche generalists were akin to "jack-of-all-trades, master-of-none" microorganisms. Despite their differences, we observed evolutionary interactions between T-sensitive and T-resistant species. The continuous transition from T-sensitive to T-resistant species ensured a relatively constant richness of T-resistant species across temperatures. This co-evolution and co-adaptation between the two types of species aligned with the principles of the red queen theory. In conclusion, our findings revealed that the high speciation rates of niche specialists played a crucial role in alleviating the negative impact of environmental filtering on diversity. The interplay between niche specialists and niche generalists sheds light on the intricate mechanisms governing microbial community dynamics and their adaptive responses to changing environmental conditions.

Conceptual diagram of the dynamic balance between microbial speciation and environmental filtering in a stressful environment such as high temperature. This is the Figure 6 from the published paper.

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