Interview with the authors: Genomic signatures of sympatric speciation with historical and contemporary gene flow in a tropical anthozoan (Hexacorallia: Actiniaria)

Though increasing numbers of empirical studies suggest that sympatric speciation may be more common than previously thought, it is difficult to quantify the prevalence of sympatric speciation, since many different processes may lead to co-distributed sister species pairs. This difficulty is particularly pronounced in marine systems where there are relatively few barriers to dispersal. A recent paper by Benjamin Titus, Paul Blischak, and Marymegan Daly provides one of the first model-based investigations of sympatric speciation in a reef system. Titus and colleagues find support for cryptic diversity in the corkscrew anemone (Bartholomea annulata), and the two lineages that they recover co-occur. Model-based analyses support isolation with migration or secondary contact, suggesting that sympatric speciation may have occurred between these lineages. Finally, Titus and colleagues identify six loci that are putatively under divergent selection between these two lineages. Below, we go behind the scenes with lead author Benjamin Titus. Read the full article here.

Photo credit: Benjamin Titus.

What led to your interest in this topic / what was the motivation for this study? 
The motivation for this study evolved quite a bit from when I initially started the project. Initially, this work was part of a broader comparative phylogeographic study. However, like many poorly studied marine inverts, the anemone turned out to be a cryptic species complex that was fully co-distributed throughout its range. Since we found no obvious ecological differences between the cryptic taxa, the project shifted focus towards testing competing biogeographic diversification scenarios. Marine systems are highly dynamic, and species that diversify in allopatry can readily become co-distributed following secondary contact. Ultimately, we wanted to use model selection analyses to make objective inferences regarding the likelihood that this species diversified sympatrically versus allopatrically followed by secondary contact.

What difficulties did you run into along the way? 
Tropical anthozoans (e.g. corals, sea anemones, zoanthids, corallimorpharians) generally harbor endosymbiotic dinoflagellates, which allow these animals to thrive in the nutrient-poor waters of the tropics. Unfortunately, there is no avoiding them in field-collected samples, and the resulting DNA extractions harbor an unknown mix of anthozoan and dinoflagellate DNA. When I started this work no universal population-level markers existed for the Class Anthozoa, so we used a reduced representation sequencing approach. Thus, our resulting RADseq dataset is, presumably, an unknown mix of target and dinoflagellate DNA. Ultimately, we were really lucky there was a full genome from a closely related species that we could map our reads to so we could be confident that we were only left with anthozoan sequences.  

What is the biggest or most surprising finding from this study? 
I think there are a couple of important takeaways. The first is that coral reefs harbor an immense amount of biodiversity on a small fraction of seafloor, and in a setting with few hard barriers to dispersal. Sympatric speciation should be a major evolutionary process on coral reefs, but it’s rarely tested for explicitly. Given that different evolutionary processes can lead to similar biogeographic outcomes, our study is a rare empirical example demonstrating the importance of sympatric speciation on reefs.
The second is that this is the first range-wide phylogeographic study for a tropical sea anemone species, and our finding that Bartholomea annulata is a species complex underscores just how underdescribed sea anemone diversity likely is.

Moving forward, what are the next steps for this research? 
Our sampling here was necessarily coarse in order to cover the entire range of this species complex in the Tropical Western Atlantic. Fine scale sampling and sequencing would be nice to try and pin down any ecological differences between these cryptic taxa that may exist. Broadly, the field of marine phylogeography needs more evolutionary studies that incorporate demographic modeling into their analyses so we can better understand the relative contributions of allopatric and sympatric speciation on coral reefs.

What would your message be for students about to start their first research projects in this topic? 
Some of the most widely recognized species are actually cryptic species complexes. If you work on a poorly studied group and want to conduct population-level research, make sure you take the time to confirm you are only dealing with a single species. This is true for any group, but is especially true for marine invertebrates.

What have you learned about science over the course of this project? 
Staying on the poorly studied taxa theme, if you work on one, there’s an immense amount of basic systematic research that needs to be done. This project came out of my dissertation research, which I developed on what I thought were common and widely recognized species. A lot of my work turned into disentangling the systematics of cryptic species complexes. This is time consuming, but important so that downstream studies are framed in the proper taxonomic context.

Describe the significance of this research for the general scientific community in one sentence.
Sympatric speciation is an important, but difficult to demonstrate, evolutionary process in the marine environment.

Describe the significance of this research for your scientific community in one sentence.
Explicit tests of competing diversification scenarios are important to disentangle different evolutionary processes that can lead to similar biogeographic outcomes on coral reefs

Interview with the authors: Latitudinal divergence in a wide-spread amphibian: contrasting patterns of neutral and adaptive genomic variation

It is difficult to parse the effects of demography and historic processes and the effects of selection, particularly in species that are widespread over heterogeneous environments. In this paper, Patrik Rödin‐Mörch and colleagues use reduced-representation genomic data to investigate the demographic and selective forces driving patterns of genetic diversity in the moor frog. They find evidence of two refugial linages with support for gene flow between lineages, and they find striking differences between neutral and putatively adaptive markers. Read the full article here, and see below for an interview with the authors.

What led to your interest in this topic / what was the motivation for this study? 
We are generally interested in how amphibian populations diverge along environmental gradients, in particular relating to latitude. We have previously focused on adaptive divergence in phenotypic traits relating to growth and development in this system. Amphibians occurring at higher latitudes are very constrained by seasonality and differences in thermal regimes as well as other aspects of the environment, and this should result in strong selection to cope with these constraints. In northern Europe, populations also have a history of glacially mediated range expansions and we are very interested in how this influences divergence along the gradient. Amphibians are very good organisms to study local adaptation as a number of species have quite wide distributions where they occur in different habitat types and thermal regimes with large differences in season length. We wanted to build on previous research by taking a more genome-wide approach that would enable us to detect signatures of divergent selection, explore the distribution of genetic variation along the gradient and model the post-glacial demographic history of the populations.

What difficulties did you run into along the way?
Applying a custom ddRAD library prep protocol on R.arvalis for the first time was a bit challenging in the beginning, as the protocol was put together in another lab for another organism. Because of the large genome of this species, it was challenging settling on which restriction enzyme combination to use and how many fragments that would result in, as we wanted to multiplex ~150 individuals and had limited funds for sequencing. We also wanted to sample populations over the contact zone to get a more comprehensive look at what’s going on there, but finding populations in between the two edge regions of the contact zone was ultimately unsuccessful.

What is the biggest or most surprising finding from this study? 
The findings that intrigued us the most was the contrasting way neutral and putatively adaptive  genetic variation is distributed along the gradient. Particularly so over the post-glacial contact zone, both in terms of nucleotide diversity and based on hybrid index estimation. We were also very pleased that we obtained good support for a model that describes what we initially thought was the correct post-glacial demographic scenario, involving two lineages diverging before the last glacial maximum. After divergence they colonized Scandinavia from two different directions, with gene flow occurring over a contact zone that we could place further south than previously proposed.

Moving forward, what are the next steps for this research? 
In order to continue this work, the next step will be to replicate the latitudinal gradient on the eastern side of the Baltic sea, as well as obtaining samples across the contact zone. The plan is also to move away from ddRAD seq to RNA-seq, and eventually whole genome sequencing. We are currently planning to look at how gene expression as well as SNP variation differs with latitude and combine that information with common garden experiments on larval life-history variation. Ultimately we want to understand the genetic basis of local adaptation based on larval life-history variation and how the demographic effects of post-glacial range expansion has influenced that.

What would your message be for students about to start their first research projects in this topic? 
Make sure you know the literature. Many previous studies have investigated adaptive divergence along various environmental gradients for a number of species, including amphibians in different settings. Also, be prepared to conduct extensive field work, common garden experiments, lab work and bioinformatics, and make sure you have collaborators that can help you out.

What have you learned about science over the course of this project? 
That things usually never work out like you first planned, and sometimes you need to adjust your conceptual and methodological approach as you go along. Another important lesson is the value of collaboration and relying on other people’s expertise and skills.

Describe the significance of this research for the general scientific community in one sentence.
Amphibian populations extending their distribution range northwards after the last ice age have adapted to the environmental constraints experienced at higher latitudes and this has influenced the distribution of genetic variation along the gradient.

Describe the significance of this research for your scientific community in one sentence.
We find neutral and putatively adaptive gene flow over a post-glacial contact zone within a single species and together with strong environmental constraints and historical range dynamics this has shaped patterns of contrasting genetic variation and adaptive divergence along the gradient.

Full article:

Rödin‐Mörch P, Luquet E, Meyer‐Lucht Y, Richter‐Boix A, Höglund J, Laurila A. Latitudinal divergence in a widespread amphibian: Contrasting patterns of neutral and adaptive genomic variation. Mol Ecol. 2019;28:2996–3011. https://doi.org/10.1111/mec.15132

Interview with the author: Sociality, hyenas and DNA methylation

Adding of methyl groups to a DNA molecule or methylation has the interesting ability to alter the activity of a DNA segment without changing the sequence.  In this behind the scenes look, Zachary Laubach and colleagues test if this valuable biomarker is impacted by differences in hyena social status or other ecological factors early in life. What’s particularly impressive is that they garnered insights into methylation from a wild population. They find some surprising results, such as that high ranking mums can confer higher levels of methylation to their cubs that disappears when they get older. Why? Find out below and read the full article here.

Photo credit: Zach Laubach

What led to your interest in this topic / what was the motivation for this study? 

Across a broad taxonomic spectrum, social experiences, particularly those early in life, seem to have a profound impact on organisms’ development. The idea that during sensitive periods of development, social experiences and early life environment can have lasting impacts on the later life phenotype and health is known as the Developmental Origins of Health and Disease (DOHaD) hypothesis, and was formalized in the 1980s by epidemiologists, namely David Barker and his research on cardiovascular disease. Among social mammals, including humans and non-human primates, an individual’s social rank affects their behavior, physiology, and related health outcomes. For example, in humans, low socioeconomic status is widely recognized as a risk factor for cardiovascular complications and other chronic diseases. In non-human primates, low social rank is risk factor for elevated chronic stress and immune dysregulation. So, although we observe that social status affects biology, we still know little about how this all works. To better understand a potential mechanism for how early life environment affects biology, we investigated possible early environmental determinants of a molecular biomarker (DNA methylation) over the course of development in a population of wild spotted hyenas. Similar to many primates, hyenas live in groups organized by a social dominance hierarchy, and whether or not a hyena is born high or low ranking has lifelong consequences.

What difficulties did you run into along the way? 

In this study, we focused on measuring DNA methylation, which is generally of interest to researchers because it is responsive to environmental stimuli and associated with gene expression. Still, while spotted hyenas present a unique opportunity to investigate how various social experiences and ecological factors early in life are associated with biological characteristics later in life, there were no previous studies (at least of which we were aware) that measured DNA methylation in this species. In other words, this was not like working with a well characterized molecular biology model organism, like fruit flies or lab rats. In fact, when we were conducting our lab work there was no publicly available draft hyena genome. In our attempt to assess a potentially informative biomarker in hyenas, we measured multiple types of DNA methylation with varying degrees of success. Finally, the hyenas we study live freely in a large reserve in Kenya, so much of our data were observational and collected under a variety of field conditions making collection of samples non-trivial.

Photo credit: Zach Laubach

What is the biggest or most surprising innovation highlighted in this study? 

This work represents one of a handful of studies conducted in a wild population that measures DNA methylation to better understand how early life environment may influence organisms’ biology over the course of development. Taking advantage of our approximately 30 years’ worth of continuously collected data on individually recognizable hyenas from the Masai Mara Hyena Project, we not only amassed a particularly large sample size for a long-lived, wild mammal, but we were also able to compare patterns of DNA methylation at various stages of development with respect to multiple early life environmental factors. We found that being born to a higher-ranking mom corresponded with greater global DNA methylation in young but not older hyenas. One interpretation of this result is that high ranking moms confer some advantage to their cubs early in life, but that the effect of maternal rank per se is not evident in global DNA methylation of subadult or adult hyenas. We also found some associations between global DNA methylation and litter size, human disturbance, and prey availability in the year a hyena was born, and these associations were strongest in the youngest age group of hyenas.

Moving forward, what are the next steps in this area of research?

In our next steps we are working to understand whether specific types of early life social environments, like maternal care and how well socially connected an animal is within its group, correspond with variation in DNA methylation and adult stress. We are also utilizing more advanced techniques for measuring DNA methylation, so that we might home in on functional pathways that are involved in the development of an adverse stress phenotype. As part of our broader research agenda looking at general biological principles related to DOHaD hypothesis, we have also teamed up with epidemiologists to ask how social status in humans affects biology. In fact, we have recently published another a paper looking at the associations between maternal socioeconomic status and patterns of DNA methylation over the course of development in children who are part of the Project Viva pre-birth cohort study (check out the paper here).

Photo credit: Zach Laubach

What would your message be for students about to start developing or using novel techniques in Molecular Ecology?

This project was part of my PhD work, and from this experience I have learned just how fast molecular biology advances as a field. Given that this technology is constantly changing, it is critical to find mentors and collaborators with up-to-date expertise who are willing to support you. I was fortunate to work in a cutting-edge molecular laboratory, and to receive training from internationally recognized experts in Dr. Dana Dolinoy’s lab who specialize in studying DNA methylation. Additionally, in studies like these that involve large observational data sets and that aim to understand biological mechanisms, the value of clearly defined study questions, hypotheses and a complimentary analytical strategy cannot be understated. In my opinion, novel technology will not substitute for a thoughtful and well-planned analysis.

What have you learned about methods and resources development over the course of this project? 

Working in a novel system, like investigating DNA methylation in wild spotted hyenas, presents challenges and limitations that are unique from those encountered in laboratory settings and when working with model organisms. However, there are deep insights and rich perspective to be gained at the three-way interface between molecular biology, behavioral ecology and evolutionary biology from study populations with intact life histories and that are subject to natural selection. I have also learned that long-term field studies with uninterrupted data collection, like the Masai Mara Hyena Project, provide an invaluable resource and an unmatched opportunity to combine molecular techniques with vast collections of behavioral, demographic and ecological data. In addition, while long-term field studies represent a substantial investment of time and resources, they also present a chance for comparative research that can help elucidate basic biological principals that span taxa, like the DOHaD hypothesis. As such, I believe I have been fortunate to work with Dr. Kay Holekamp’s hyenas and that these types of long-term field studies are an asset to be prioritized and preserved.

Describe the significance of this research for the general scientific community in one sentence.

Social and ecological factors experienced early in life can correspond to changes in molecular biomarkers, like DNA methylation, that are detected over the course of development, and that may affect patterns of gene expression.

Photo credit: Zach Laubach

Describe the significance of this research for your scientific community in one sentence.

Findings from this research suggests that maternal rank, anthropogenic disturbance, and prey availability around the time of birth are associated with later life global DNA methylation in spotted hyenas, particularly in cubs.

Citation: Laubach, ZM, Faulk, CD, Dolinoy, DC, et al. Early life social and ecological determinants of global DNA methylation in wild spotted hyenas. Mol Ecol. 2019; 28: 3799– 3812. https://doi.org/10.1111/mec.15174

Interview with the authors: Evidence for rapid evolution in a grassland biodiversity experiment

Our ability to detect rapid evolution at the level of the genome has improved dramatically over the past decades, driven in part by advances in sequencing technology. It is now possible to detect small genetic differences in a population in just a few generations. This ability has stimulated many questions surrounding the causes and processes of rapid evolution. For example, how is the evolutionary trajectory of a species affected by the diversity of the surrounding community? In their recent Molecular Ecology paper, Dr. Sofia J. van Moorsel and colleagues quantify genetic and epigenetic differences across a set of plant species in the long-term Jena Experiment in Germany, which aims to test the effects of biodiversity on ecosystem functioning. Read below for a behind-the-scenes look at their study.

Link to the study: https://onlinelibrary.wiley.com/doi/full/10.1111/mec.15191

The Jena Experiment in Jena, Germany. This is where the plants had been growing for a decade in their respective communities. The mixtures are not only more productive, but also more photogenic.

What led to your interest in this topic / what was the motivation for this study? 
Previously we had found that offspring of plants from the same species that had been growing either in monoculture or mixture for an extended period of time showed clear phenotypic differences in common environments. We thought that selection in response to community diversity was driving these observations. If selection was occurring, we would find genetic differences between individuals of the same species either from a monoculture or mixture background. However, at the time it was suggested that potentially epigenetics were the source of the observed effects (Tilman & Snell-Rodd 2014). Considering the current interest in the potential role of epigenetics in ecology we wanted to state an example by analyzing our monoculture and mixture phenotypes with a new combined method.

Measuring the traits of the plants in our glasshouse experiment. These are the plants we took the samples from for subsequent sequencing.

What difficulties did you run into along the way? 
In terms of the lab work and bioinformatic analysis, the method we used was still very new, so we needed to update and improve it along the way. Also, we focused perhaps too much on the hypothesis that the observed evolutionary differentiations could have “simply reflected epigenetic effects”. However, when we found clear genetic effects, we realized that this makes it more difficult to detect independent epigenetic effects, in particular because we could not analyze whole epi-/genomes. Further this research was a collaboration between two labs from two different countries. Consequently, we had to organized exchange visits to do lab work and discuss results. Lastly, the publication process is always accompanied with frustrations and hurdles, but thanks to fantastic teamwork and a healthy dose of perseverance we made it!

What is the biggest or most surprising finding from this study? 
The most surprising finding was that for four out of five perennial (!) plant species selected in monoculture vs. mixture were genetically distinct already after 10 years (with at least two experimentally ensured reproductive cycles). We showed that rapid evolution can happen in plant communities after only a small number of generations. Previously it was thought that evolution happening at ecological time scales was either largely limited to organisms with very short generation times (i.e., microbial species) or in macro-organisms like plants limited to non-genetic effects. Even though some of us were critical about the role of epigenetics to start with, most of us were still intrigued that genetic divergence was so clear and that it could explain almost all epigenetic variation.

Measuring traits, harvesting the biomass and taking samples was a big team effort. Which also made it more fun.

Moving forward, what are the next steps for this research?
Reduced-representation sequencing will never be able to exclude with certainty that epigenetic effects are entirely due to genetic differences at a place in the genome far away and thus possibly not sequenced. Ideally, we could do whole-genome bisulfite sequencing to get more to the bottom of all of this. We only sequenced about 2% of the genome, so potentially we have overlooked some important genes affecting DNA methylation. One next step would be selection experiments with clonal replicates of our perennial plants. However, this would also set epigenetic variation to zero and selection would have to use variation arising by new epigenetic mutations, whereas it may be more conceivable that epigenetic differentiation results from “sorting out” standing epigenetic variation.

What would your message be for students about to start their first research projects in this topic?
First of all: forge collaborations. This paper would not have been possible, if we had not met at a conference. If you hear a talk of somebody at a conference or at your department, even if you do not see an immediate potential for collaboration, approach the speaker and tell them about your research. They are likely equally interested in your things as you are in theirs. Further, following the more unconventional research avenue pays off, even when it sometimes might take a little longer getting a paper accepted for publication. Specific to our topic, we would definitely recommend adding an evolutionary twist to classic plant community ecology, it’s an emerging field and it’s always exciting to be among the first researchers to enter a new topic.

Measuring traits in the glasshouse with help of the amazing Enrica De Luca and Nadia Castro.

What have you learned about science over the course of this project? 
Interdisciplinarity, even the small one between ecologists, molecular biologists and bioinformaticians is challenging but highly rewarding. Clearly, hot topics, such as epigenetics in ecology, are not free from differences in beliefs. Here we were juggling many different perspectives both among co-authors and among reviewers. It forced us to find a balance, which is also testimony for the importance of a broad-scale review process (five reviewers and a very engaged associate editor).

Describe the significance of this research for the general scientific community in one sentence.
Rapid genetic but not epigenetic adaptation among plant species in mixtures means that we cannot predict community functioning by studying species in isolation and that we should conserve and restore entire communities and not individual species.

Citation
van Moorsel SJ, Schmid MW, Wagemaker CA, van Gurp T, Schmid B, Vergeer P. (2019). Evidence for rapid evolution in a grassland biodiversity experiment. Molecular Ecology, 28(17), 4097-4117. https://onlinelibrary.wiley.com/doi/full/10.1111/mec.15191

Interview with the author: Understanding reef bacterial responses to climate change

Climate change is causing dramatic changes to coral reefs and the eukaryotic life they provide habitat for, but what about the bacterial communities? In this interview with the author, Susana Carvalho from the Red Sea Research Center gives us a behind the scenes take on the paper she and colleagues published using autonomous reef monitoring structures (ARMS) to uncover insights into how bacterial communities respond to environmental stress.

See her bio here and the paper here.

An ARMS unit in action.  Photo credits:  Jessica Bouwmeester

What led to your interest in this topic / what was the motivation for this study? 

Coral reefs face unprecedented decline due to local pressures and climate change, making assessment of species diversity and responses to environmental change a priority. Despite the critical roles of bacteria in reef functioning, their communities remain largely overlooked, partially due to the lack of standardized tools and protocols. Fostered by the recently developed Autonomous Reef Monitoring Structures (ARMS), the team – already studying eukaryotes associated with ARMS – decided to expand the research to bacterial communities. This step was further motivated based on the knowledge previously gathered on the eukaryotic reef benthic communities as well as the fact that the Red Sea can be seen as a natural laboratory for ‘Future Oceans’ due to clear environmental gradients in sea surface temperature and salinity. 

What difficulties did you run into along the way? 

The biggest difficulties for this project were logistical and also the fact that it was an exploratory study. Firstly, the reefs which were studied were spread across 2000km of the Saudi Arabian coastline. This posed problems not only in getting permissions from a number of different agencies to visit the reefs but also in organising the logistics so equipment and people could undertake the sampling safely over such a wide spatial extent and across numerous years. Secondly, the nature of the experiment was exploratory, as very little knowledge is known about reefs in the Red Sea. While this makes the experiment vital to further knowledge it also complicates the ability to understand the trends observed as no prior knowledge is available.

What is the biggest or most surprising innovation highlighted in this study? 

While ARMS have previously been used to study the small cryptic eukaryotes (small-sized organisms that are hidden in the reef structure), they had not been utilised to study bacterial communities. For the first time, we employed ARMS to investigate structure and composition of bacterial reef communities across pronounced environmental gradients spanning 16 degrees of latitude. Using this standardized framework, we found that bacterial community structure and diversity aligned with environmental differences. We also found that the decrease in taxonomic diversity was not mirrored by a decrease in functional diversity, suggesting that resilience is not a direct function of taxonomic biodiversity. Importantly, the structure of ARMS devices feature crevices and light-/dark-exposed surfaces as well as exterior and interiors surfaces; in a word it displays many different microhabitats perfectly designed to sample a large diversity of bacteria. This is not possible using other sampling protocols (e.g. water sampling or sediment sampling). The current approach can be expanded to other regions allowing global questions such as the effect of climate change in coral reefs to be addressed and to build a standardized comparable ARMS-based database that allows for meta-analyses beyond the insight from single studies. 

Moving forward, what are the next steps in this area of research?

To fully understand how coral reef microbial communities are being affected by local and global stressors and how the functioning of the reef changes, long term time series are required. Thus, repeated sampling over a number of years is critical. Currently, we have 19 reefs along the Red Sea where ARMS are deployed and retrieved every two-years along with traditional reef surveys (photo-transects for benthos and fish visual censuses). To properly understand the function within the reef we also aim to undertake a metatranscriptomic analysis of the sessile community to see which genes are active. This would ideally be expanded to include manipulative experiments to understand how specific stressors affect not only the community composition but also the functional activity of the biological communities.

What would your message be for students about to start developing or using novel techniques in Molecular Ecology? 

First of all, we would recommend to consider fully the question/issue that they would like to address with these novel techniques. It should be taken in consideration whether the use of molecular tools will benefit the answering of the desired question in comparison with more traditional methods. While novel techniques can bring substantial improvements in understanding questions, such as the current study, sometimes improvements are just marginal, or can just be in cost and speed.

What have you learned about methods and resources development over the course of this project? 

The aim of this project was to characterize the taxonomic and functional diversity of coral reef-associated bacteria using ARMS. Whilst the team had previous experience working with bacterial taxonomic characterization, a ‘deep dive’ had to be undertaken to perform the functional analysis. These areas are rapidly developing with a wealth of new algorithms appearing in the literature. While there is no perfect approach, during the course of this project we learnt that one of the most crucial aspects when developing a new method is to understand both the benefits and the limitations of it. Only by gaining this understanding can you confidentially present your results and highlight the areas in which these techniques can be applied.

Describe the significance of this research for your scientific community in one sentence.

ARMS provide a standardized platform to investigate the response of coral reef-associated bacteria to environmental change, with current results suggesting that this research should be conducted from taxonomic and functional perspectives.

Describe the significance of this research for the general scientific community in one sentence.

This study lays the foundation for a holistic understanding of how reef communities respond to environmental changes and proposes a framework that, if applied worldwide, can be vital in providing answers to global questions such as the impacts of climate change on ecosystem diversity and functioning.

Citation: Pearman, JK, Aylagas, E, Voolstra, CR, Anlauf, H, Villalobos, R, Carvalho, S. Disentangling the complex microbial community of coral reefs using standardized Autonomous Reef Monitoring Structures (ARMS). Mol Ecol. 2019; 28: 3496– 3507. https://doi.org/10.1111/mec.15167

The story behind the article: Climate change impacts on below-ground communities

Free‐air carbon dioxide enrichment (FACE) experiments have dramatically increased our understanding of how plants may respond to future climate change scenarios. These experiments also provide unique opportunities to better predict how below-ground symbiont communities, crucial to plant health, may respond to climate change. Dr Irena Maček and Prof. Alex Dumbrell give us their behind the scenes insights into their paper that combines Illumina HiSeq sequencing and one of the longest running FACE experiment to find novel insights into mycorrhizal fungi communities and climate change.

Check out the full study here.

The Giessen FACE experimental setup. Photo courtesy of Irena Macek

What led to your interest in this topic / what was the motivation for this study?

Arbuscular mycorrhizal (AM) fungi form mutualistic associations with over two‐thirds of plant species, providing numerous benefits in exchange for carbon. Importantly, different AM fungi provide different plant species with different resources. Thus changes to AM fungal communities can alter plant competition and above-ground productivity. This functional differentiation has motivated both of us to explore the ecological mechanisms regulating AM fungal communities, and it was apparent there was a lack of knowledge on how AM fungal communities respond to elevated atmospheric CO2. This is a major research gap, as AM fungi are entirely dependent on their hosts for carbon and changes in photosynthesis in a high CO2 world may influence this. Thus the chance to sample plant roots from one of the longest running free‐air carbon dioxide enrichment (FACE) experiment in the northern hemisphere in Giessen (Gi-FACE) was an excellent opportunity to address this gap in global climate-change research.

What difficulties did you run into along the way? 

Long-term FACE experiments in natural ecosystems are extremely rare, because such set-ups demand stable funding and the persistence of several generations of highly motivated researchers. This would have been a far greater current problem if Prof. Christoph Müller (Justus‐Liebig University Giessen) and his group had not continuously addressed these difficulties since the Gi-FACE experiment’s inception. Another difficulty that we are increasingly finding in metabarcoding research is how to present often extensive and complex data. Dr Dave Clark (University of Essex) was central in working on innovative ways to address this challenge and moving beyond coarse summaries of total community change.

What is the biggest or most surprising innovation highlighted in this study? 

For the first time, we have combined a long-term FACE experiment in a natural habitat with high-throughput molecular sequencing (Illumina HiSeq) and new ways of presenting community data. This has allowed us to see subtle population-level responses within broader community-level responses of AM fungi to elevated atmospheric CO2 across the course of a year.

Moving forward, what are the next steps in this area of research?

Understanding the impacts of global-climate change on terrestrial ecosystems requires an integrative approach that explores responses across all levels of biological organisation and spatiotemporal scales, both above- and below- ground. We still lack a comprehensive understanding about how interactions between the above- and below- ground components of biodiversity respond to both acute short-term (e.g. episodic heatwaves, drought etc.) and chronic longer-term climate changes (e.g. warming, elevated CO2). New experiments aimed at addressing these knowledge gaps with robust levels of replication and appropriate experimental durations for capturing longer or shorter-term responses are required, and these must allow for combined sampling of above- and below- ground biota.

Plant root with arbuscular mycorrhizal fungal structures (hyphae and arbuscules). Photo courtesy of Irena Macek

What would your message be for students about to start developing or using novel techniques in Molecular Ecology? 

The same as our message for all earlier career researchers – identify your research question, read around your research area, develop your hypotheses and plan an appropriate study to address them, and then choose the correct tools/techniques to conduct the research with. The novel techniques can have a lot of analytical power but can also produce a lot of erroneous data due to the rapid development, a lack of testing and a lack of experience. It is very important to initiate the study with clear questions resulting in hypotheses driven research. Do invest time in skills of data analyses and bioinformatics from the very beginning as there is never enough time to do that the later you are in a career, the more difficult it gets with many other obligations.

What have you learned about methods and resources development over the course of this project? 

The field of molecular ecology continuously develops at a rapid pace. It is crucial to have a good network of people with a multidisciplinary range of expertise to collaborate with and to capitalise on all these new and often varied developments. Essentially, good collaboration is crucial. It should also be fun, which is always a good recipe for it to be sustainable and develop into a long-term connection.

Describe the significance of this research for the general scientific community in one sentence.

Elevated levels of atmospheric CO2,reflective of those we will experience in the next ~100yrs, drive changes in symbiotic AM fungal populations with the potential to resonate throughout their associated plant communities, changing above-ground competition dynamics and ecosystem productivity in currently unpredictable ways.

Describe the significance of this research for your scientific community in one sentence.

Predictions regarding future terrestrial ecosystems must consider changes both above-ground and below-ground, but avoid relying on broad‐scale community‐level responses of soil microbes observed on single occasions.

Citation: Maček, I, Clark, DR, Šibanc, N, et al. Impacts of long‐term elevated atmospheric CO2 concentrations on communities of arbuscular mycorrhizal fungi. Mol Ecol. 2019; 28: 3445– 3458. https://doi.org/10.1111/mec.15160Citation:

Interview with the authors: Anthropization level of Lascaux Cave microbiome shown by regional‐scale comparisons of pristine and anthropized caves

Estimated to be around 17,000 years old, the Paleolithic paintings in the Lascaux cave of southwestern France give us a rare insight into the history and culture of communities that existed long before modern society. The conservation of caves such as Lascaux is a high priority for historians, scientists, and the general public. The anthropization, or human use, of caves may have dramatic effects on cave-dwelling macro- and micro-organisms, though few studies have been conducted on this topic. By comparing ‘pristine’ caves with anthropized caves frequently visited by humans, Dr. Lise Alonso and colleagues demonstrate that the anthropization of caves is associated with reduced microbial diversity for bacteria and archaea living on cave walls, though microeukaryotes and arthropods were not as strongly affected. In this post, we go behind-the-scenes with Dr. Yvan Moënne-Loccoz on their recent publication in Molecular Ecology and talk about the importance and challenges of working in cave ecosystems.

Link to the study: https://onlinelibrary.wiley.com/doi/10.1111/mec.15144

Great Hall of the Bulls in Lascaux Cave. The cables connect to monitoring probes. Source: DRAC Nouvelle Aquitaine

What led to your interest in this topic / what was the motivation for this study? 
Cave conservation is an important issue, especially when dealing with caves displaying Paleolithic artwork, as engravings and particularly paintings can be very fragile. There are many of these caves in Dordogne (South-West of France), some of them listed on the UNESCO World Heritage List (https://whc.unesco.org/en/list/85). The most famous Paleolithic cave in Dordogne is the Lascaux Cave, which was closed to the public in the 1960s for conservation reasons. To guide conservation efforts, it is important to understand the ecology and functioning of these caves, especially at the levels of microorganisms and arthropods, which form the main communities present. Against this background, the project was carried out to understand better the biotic communities residing in Lascaux Cave.

Entrance of Lascaux Cave. Source: DRAC Nouvelle Aquitaine

What difficulties did you run into along the way? 
When dealing with microorganisms and arthropods populating soils, sediments or water, in a majority of cases it is rather straightforward to collect samples and there is no restriction on sample size. In caves, taking samples from walls for microbial analyses, using a scalpel, may leave long-lasting marks. This is an issue in all caves, and particularly so in Paleolithic caves. In the Lascaux Cave, the sample list was prepared after discussions with the cave staff and approved by the cave conservator, and the samples were collected (away from ornate surfaces) by qualified restorers, under the guidance of microbial ecologists, so as to avoid any marks on the wall. It also means that only minute samples were available. Restrictions also apply for the type and location of arthropods traps, as sediments at the bottom of caves might contain historical artefacts.

Sampling of rock wall surface in a pristine cave, using a sterile scalpel. Source: B. Bigaï

What is the biggest or most surprising finding from this study? 
Caves are oligotrophic environments, so it is always a surprise to find diversified, rather large microbial communities on cave walls. In this study, the Lascaux Cave was compared with eight other caves from the same region, and these caves were quite different from one another in terms of size, architecture, distance from the soil surface, presence/absence of stream underground, human frequentation patterns, etc. Yet, there were clear distinctions in terms of microbial and arthropod communities when comparing anthropized caves versus non-anthropized (almost pristine) caves, which suggests that anthropization was more influential than these cave-specific features. Finally, we were rather surprised to find that prokaryotes (bacteria and archaea) were comparatively more impacted than eukaryotic residents (fungi, other micro-eukaryotes, arthropods) by cave anthropization.

Pristine cave used for sampling. Source: Y. Moënne-Loccoz

Moving forward, what are the next steps for this research?
This work was carried out with the Lascaux Cave and eight other caves from Dordogne, which corresponds to a relatively small area. There were at the most 35 km between two caves in this study. Therefore, it remains to be seen whether the results of the current investigation are also relevant elsewhere. At a larger geographic scale, several differences in cave properties can be expected, for instance in geological features (e.g. limestone type) and climatic conditions, which have the potential to influence cave biotic communities. In addition, we evidenced parallel variations in the diversity of microbial and arthropod communities, and it will be important to explore and understand better the ecological interactions between both types of cave inhabitants.

What would your message be for students about to start their first research projects in this topic?
First of all, the underground world and the interface between ecology and artwork conservation issues are fascinating, so welcome to the field! More importantly, each cave is different and represents a complex situation of its own, so one can be very busy focusing on a single cave only. This is reflected by the literature on cave microbial ecology, where often a single cave is considered at a time. However, we found that the comparison of different caves, following the path of various groups (e.g. Campbell et al. 2011 J Cave Karst Stud 73:75 ; Hathaway et al. 2014 Geomicrobiol J 31:205 ; De Mandal et al. 2017 BMC Microbiol 17:90 ; Pfendler et al. 2018 Sci Tot Environ 615:1207), brought very interesting insights, so comparative assessments are worth the effort.

What have you learned about science over the course of this project? 
The majority of participants to this project usually work on soil or aquatic ecosystems, and we found (once again) that concepts and methodology are applicable across different types of ecosystems. More specifically, we realized that underground systems represent interesting models to investigate ecological perturbations, because they are rather confined environments, where community fluctuations in response to mild environmental variations can be documented.

Describe the significance of this research for the general scientific community in one sentence.
This research shows that microbiome diversity can be used as a bioindicator of the level of cave anthropization.

Citation
Alonso L, Pommier T, Kaufmann B, Dubost A, Chapulliot D, Doré J, Douady CJ, Moënne‐Loccoz Y. Anthropization level of Lascaux Cave microbiome shown by regional‐scale comparisons of pristine and anthropized caves. Molecular Ecology, 28(14), 3383-3394. https://onlinelibrary.wiley.com/doi/10.1111/mec.15144