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:

Method summary: Mapping genetic patterns across landscapes with PHYLIN

            The spatial representation of species’ data is needed in most areas of biodiversity related research. In fact, mapping the species’ continuum to guide the prioritization of areas for conservation was the main driver for PHYLIN development, but the possible application is far more vast.

            Spatial representation of distances between georeferenced samples is challenging. The PHYLIN input are distance matrices and a table of samples classified in groups (lineages, for instance) with locations. PHYLIN relates a matrix measuring a particular distance between samples (for example, a genetic distance) with a matrix representing spatial distance between the same samples. PHYLIN then applies a kriging interpolation: models the relation by means of a variogram and uses that information as weights to interpolate to other locations a probability of belonging to each of the groups

Different applications of PHYLIN with randomly generated data. a) using a simple euclidean distance with 3 dimensions is possible to interpolate over 3d environments; b) using a layer of climate as resistance to movement it is possible to analyse the impact of climate change on connectivity; c) using a Jaccard distance matrix instead of genetic distance to map the contact zone between two species (click on the image for source code).

The latest version of PHYLIN adds the possibility of using multiple spatial distance metrics, opening an exciting avenue with different applications. In our recent paper in Molecular Ecology Resources, we showed how different mechanisms of genetic isolation can be represented in space by PHYLIN. The application of the method is not limited to that and we show here three other possible applications: using 3 dimensional distance (similarly to an ocean environment), climate change connectivity and species distributions/contact zone.

Pedro Tarroso, Guillermo Velo-Antón and Silvia Carvalho  

See the full paper here: https://onlinelibrary.wiley.com/doi/abs/10.1111/1755-0998.13010

A step by step tutorial can be found here: https://cran.r-project.org/web/packages/phylin/vignettes/phylin_tutorial.pdf

Using transcriptomics to investigate the Circadian clock

Circadian clocks provide a mechanism that allows organisms to anticipate environmental rhythms, like light-dark cycles. Nematostella vectensis, an estuarine sea anemone, has a surprising degree of overlap in genomic complexity with vertebrates, including circadian clock genes. These genes are predicted to serve a similar role in driving circadian patterns in sea anemones, but we have not worked out the exact mechanism they use.

Photo courtesy of Whitney Leach

In this study, we utilize next-generation sequencing to investigate the time-course transcriptional profiles of animals over 3 days, to dissociate true circadian gene expression vs. photo-responsiveness, by exposing animals to regular light-dark cycles for one month, then abruptly removing the light cue. Hypothesized ‘clock’ genes were rhythmic in the presence of light-dark cycles; however, several of these genes lost their characteristic oscillation after 1 or 2 days in the dark, suggesting lack of endogenous circadian regulation. One would expect a truly circadian gene to continue to cycle in the absence of light, however our results indicate either: 1) the hypothesized ‘clock’ genes simply respond directly to light cues, which implies they are not circadian, or 2) a circadian regulator resides in specific cell types, and the expression signal is too dampened when measuring in the whole animal.

Whitney Leach, Doctoral Candidate, The Reitzel Laboratory, University of North Carolina at Charlotte

Read the full article here: https://onlinelibrary.wiley.com/doi/abs/10.1111/mec.15163

Finding the diamonds in the rough: using genomics and climate data to re-explore crop collections

Global crop collections carry a wealth of native genes and alleles of immense potential value for farmers and consumers. Equally, within their DNA lies variation of negative value. The challenge is finding the diamonds in the rough – this is such a difficult task that the vast majority of collections remains underutilized and under-explored. As genotyping methods have evolved to generate larger densities of data for lower costs, comprehensive genotypic fingerprinting of collections is now within reach.

Photo courtesy of CIMMYT’s Flickr account

Phenotypic data (field, greenhouse and chemical analysis data), the stalwart of plant breeding, and counterpart data used to determine the value of genes for breeding is now more expensive and complex to obtain than genotypic data. In our study, we used climate data from the sites of origin of the maize collections studied – a cheap proxy for phenotypic data related to constraint such as acid soils and high temperatures. Applying innovative analyses to fingerprinting and climatic data we identified genes, genomic regions and maize of potential value for breeding. This approach highlights an opportunity to use genomics and climate data to re-explore crop collections, excluding large numbers of irrelevant materials and identifying the potential gems that will contribute to feeding and nourishing future generations.       

Sarah Hearne and HuiHui Li (International Maize and Wheat Improvement Center)

Read the full article here: https://onlinelibrary.wiley.com/doi/full/10.1111/mec.15169

Interview with the author: Detecting pathogens in koalas – dogs versus qPCR

Detecting pathogens in wild populations can be an enormous challenge. This is particularly the case for one of Australia’s most iconic but threatened animals – the koala. Koala populations face numerous threats, but one threat that has been difficult to quantify is infections from Chlamydia bacteria that can cause significant mortality in this species. Romane Cristescu (a postdoc at the University of the Sunshine Coast) gives us a behind the scenes look at the innovative paper she published recently with colleagues in Molecular Ecology Resources.

Find the full paper here: https://onlinelibrary.wiley.com/doi/full/10.1111/1755-0998.12999

Lead author Dr Romane Cristescu routinely works with detection dogs for ecological surveys, so naturally when the molecular tools proved imperfect, the team thought of training a disease detection dog (credits Marie Colibri)

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

Koalas are a very iconic species – in fact they are one of the most recognised animals on Earth. Yet they are also threatened, with most of their populations now in decline. They face many threats – some are easy to see and study, such as habitat loss, but some are harder. This is the case of the disease Chlamydia. To study the impact of Chlamydia on koalas, in the wild, at a broader landscape level, has been difficult. This is because koalas are hard to find (they are both cryptic and often occur at low density), and expensive to catch and sample. This led our team’s interest in Chlamydia detection from non-invasive samples: koala scats (droppings).

What difficulties did you run into along the way?

In this research project, we were interested in ascertaining both the sensitivity (the ability to correctly identify individuals with a pathogen) and the specificity (the ability to correctly identify individuals without a pathogen) of Chlamydia detection from koala scats using a well-proven method: qPCR. We unexpectedly found that qPCR had quite low sensitivity in our study (58% for koalas with urogenital infection, 78% for koalas with urogenital disease). This led us to try another molecular method: next generation sequencing. Again, we were taken aback when sensitivity was low. We then had to think outside the box. We already work with detection dogs, in fact they are the method we use to collect the scats to start with. Logically (for us), we decided to train and test a Chlamydia detection dog.

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

When comparing two molecular (qPCR and next generation sequencing) methods and a detection dog to detect Chlamydia from koala scats, we did not expect that the dog would come out on top! The dog had both 100% sensitivity and specificity. This was a sobering outcome – no matter how fancy and high tech we have become in the laboratory, sometimes we are still no match to our old best friend – a dog. We were absolutely flabbergasted when we also found that the dog could detect ocular infection from the scats – we still do not know how this happens. Just that somehow the smell of the scat is affected by the eye infection. The volatile organic compounds (our likely suspects) involved are still unknown.

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

There are two areas our team is keen to move on to. First, next generation sequencing using a specific Chlamydia panel. We expect this will dramatically increase the ability of next generation sequencing to detect Chlamydia. Second, and because not everyone can have a detection dog, we’d be keen to team up (please contact us!) and investigate ‘artificial noses’ to detect the volatile compounds – as these might be more robust against environmental conditions than DNA (non-invasive samples, such as scats, can be degraded by the elements). But beyond the method used to detect the presence of a chlamydial infection, we are still at the infancy of understanding the link between infection and disease (i.e. clinical signs). This is the most important aspect requiring clarification before we can effectively study the impact of chlamydial disease at the landscape level and, in particular, under which conditions this disease can threaten a population’s survival.

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

Our strongest advice in molecular ecology is to understand, and test, your methodology, especially if it is novel, but also if you are using a well-proven method in a novel way. An essential step is to quantify a novel technique’s characteristics and limitations (in our example, sensitivity and specificity of chlamydial detection tests from non-invasive (scat) samples). A risk here is to be tempted to compare results that have different methods to draw general conclusions. If you compare the results of one population using a particular test to another study of a different population and test, you must remember to also compare both methods using samples with known outcomes: how consistent are the results of different tests compared to the reality? This is costly and time consuming, but nonetheless necessary before novel methods can be widely applied.

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

Resilience is key – the first method you try might not give you the answer you want, nor the second. You haven’t failed yet – you have found many ways that do not work. Keep on keeping on, think outside the box, collaborate with others outside your area of expertise, who will bring skills and ideas you would never think of: this is how scientific advances are made.

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

This research highlights that sometimes the best method can be unexpected – to do successful research takes both creativity and perseverance.

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

We need to continue developing non-invasive, affordable, accurate tools if we are to understand how hosts, pathogens and landscapes interact to create a perfect extinction vortex; for koalas and Chlamydia – we are not there yet.

Summary from the authors: Finding the multi-host origins of foot and mouth disease virus

Although foot-and-mouth disease virus (FMDV) is among the most economically important livestock diseases in the world, our understanding of its epidemiology in regions where it is endemic is poorly understood. FMDV serotypes A, O, SAT1 and SAT 2 are endemic in Africa with African buffalo known as carriers of SAT serotypes. Epidemiological and genetic evidence is fairly conclusive that buffalo populations are the source of the disease for cattle in the Southern Africa regions. Little is known about the situation in East Africa yet it has amongst the most complex FMDV situations in the world due to high viral diversity, unrestricted livestock movement, and presence of wildlife reservoirs. Although wildlife-livestock contact in East Africa is more frequent and intimate due to shared rangelands, the role of buffalo as an FMDV reservoir has not been resolved due to lack of information on the genetic diversity of FMDV circulating in buffalo.    

Photo credit: K. VanderWaal

We sequenced 80 buffalo-origin FMD viruses and examined the evolutionary epidemiology of currently circulating clades of SAT1 and SAT2 FMDV in East Africa.  Our analyses suggest that currently circulating SAT1 viruses in East Africa originated in Zimbabwe, whereas Kenya is the likely country of origin for contemporary SAT2 viruses.

We also show that cattle are the likely source of the SAT1 and SAT2 clades currently circulating in East Africa, though buffalo may still have an ancestral role deeper in the past. Our results suggest that the majority of SAT1 and SAT2 in cattle comes from  other livestock rather than buffalo, with limited evidence that buffalo serve as reservoirs for cattle. Insights from the present study highlight the role of transboundary spread, most likely through cattle movements and other anthropogenic activities, in shaping the evolutionary history of FMDV in East Africa.

Map illustrating chronological space-time genetic evolution and spread of SAT1 in Eastern and Southern Africa. The genetic tree below is color coded by the affected host species and shows the cattle origin of currently circulating clades of SAT1 FMDV.

Blog by Moh Alkhamis and Kim VanderWaal (Kuwait University & University of Minnesota)

For the full article, see:

Omondi, G, Alkhamis, MA, Obanda, V, et al. Phylogeographical and cross‐species transmission dynamics of SAT1 and SAT2 foot‐and‐mouth disease virus in Eastern Africa. Mol Ecol. 2019; 28: 2903– 2916. https://doi.org/10.1111/mec.15125