Interview with the authors: High indirect fitness benefits for helpers across the nesting cycle in the tropical paper wasp Polistes canadensis

Understanding how complex social systems evolve is a long-standing and challenging goal of evolutionary biology. Many animal taxa are eusocial, where individuals forego reproduction in order to help raise young produced by another member their group. This behavior only makes sense if there are potential benefits – either direct or indirect – to the helpers. The genus of paper wasps Polistes is commonly used as a model system for studying sociality, however most of the research on this group has been conducted in temperate ecosystems, whereas relatively little is known about their behavior in tropical ecosystems where the genus likely originated. Does our understanding of how direct and indirect benefits contribute to the evolution of complex social systems depend on the environment where we conduct the research? Below, we go behind the scenes with Emily Bell and Robin Southon on their latest paper in Molecular Ecology to find out.

Link to the study: https://onlinelibrary.wiley.com/doi/10.1111/mec.15137

Polistes canadensis nest. Photo by RJ Southon.

What led to your interest in this topic / what was the motivation for this study? 
Wasps provide a fascinating insight into the evolution of sociality; among them you can find various levels of social complexity ranging from solitary to highly eusocial. Many studies have focused on the emerging model organism Polistes dominula. An interesting aspect of this wasp’s behaviour is that female ‘foundresses’ often form new nests in the spring with unrelated individuals – they help even though reproductive skew is monopolised by a single female. However, this is unlikely to be true in tropical species. Tropical Polistes do not experience an overwinter break in the colony cycle, and highly related foundresses may form new nests together directly from their previous natal nest. We wanted to highlight that although Polistes species share many traits, such as high reproductive skew, fitness payoffs for nonreproductive foundresses in temperate and tropical species are unlikely to be equivalent. This is an important consideration in making conclusions about the evolution of group living within the genus – Robin Southon

https://onlinelibrary.wiley.com/doi/10.1111/mec.15137

What difficulties did you run into along the way? 
With most field work it comes down to logistical issues. Along the Panama Canal are many ‘wasp havens’ – abandoned buildings containing a high density of nests. Which is great, unless you happen to live on the other side of the canal to your field site. There is one bridge that crosses the canal near our site, which is only accessible when boats are not passing through the nearby lock. This takes many hours for giant container ships! We’d spot ships approaching from afar and try to beat them to the lock in time to cross. Although if we missed our chance, at least all our datasheets were processed and backed up in the downtime – Robin Southon

Beating a container ship to the lock. Photo by EF Bell.

What is the biggest or most surprising finding from this study? 
In large established nests a reproductive monopoly is held by one queen. Our SNP and microsatellite pedigree analyses of post-emergence nests revealed in nearly all nests brood belonged to a single queen. Control over reproduction in Polistes is maintained primarily through physical aggression, and in the case of small temperate nests there is relatively few nestmates to dominate. However, nests of the tropical Polistes canadensis can reach 200+ in membership. It seems unlikely the queen could physically aggress that many wasps in a network! It would be interesting to see what other mechanisms may be at play, such as vibrational or odour signalling – Robin Southon

Moving forward, what are the next steps for this research?
We have established that there are differences in founding group structure between Polistes from different environments, but it’s rather simplistic to look at such differences as tropical vs temperate. There will be exceptions in both environments, for example montane ecosystems. The next step should look at what exactly is it about these environments that influence founding behaviour. Studying wasps in subtropical climates is likely to be key, especially in looking at species that have distributions which transition through tropical, subtropical, and temperate regions – Robin Southon

What would your message be for students about to start their first research projects in this topic?
Do your background research before you start a new project – literature reviews are an invaluable learning experience. They act as a chance for you to explore your new field and come up with lots of ideas for where you may want to take your project over your studies. Wasps are an amazing organism to work with, fascinating life history and captivating to watch in the field. I would wholehearted recommend taking a chance to observe your species in their natural habitat, if that is possible. It will enable you to learn what techniques might work for your project and novel ways that you could implement techniques in the field – Emily Bell

What have you learned about science over the course of this project? 
Molecular work is never straight forwards, it takes time (lots of time) to perfect methodologies – problem solving is a key skill that will always help with this. Sharing troubleshooting ideas with your lab group and other experts in departments is a brilliant way to learn new ways to analyse data. Once you have got a technique that works it really is such a satisfying experience and so exciting to be able to explore your species in a totally new way
– Emily Bell

Describe the significance of this research for the general scientific community in one sentence.
Environment influences the membership composition of new groups, resulting in varied group genetic structure and fitness payoffs for nonreproductive helpers in Polistes paper wasps – Robin Southon

Citation
Robin J. Southon, Emily F. Bell, Peter Graystock, Christopher D. R. Wyatt, Andrew N. Radford, & Seirian Sumner (2019). High indirect fitness benefits for helpers across the nesting cycle in the tropical paper wasp Polistes canadensis. Molecular Ecology, 28(13), 3271-3284. https://onlinelibrary.wiley.com/doi/10.1111/mec.15137

Interview with the authors: Phylogeography of a cryptic speciation continuum in Eurasian spadefoot toads

Understanding how species form, and the factors that contribute to reproductive isolation has been a long-standing goal of evolutionary biology. Cryptic radiations can provide insight into these questions. Dufresnes and colleagues investigate these questions in a cryptic radiation of Eurasian spadefoot toads (Genus Pelobates). They find a correlation between the amount of time spent in allopatry and the level of reproductive isolation between lineages experiencing secondary contact. Get a behind-the-scenes look at the research below with first author Christophe Dufresnes.

Aquatic portrait of the Common Spadefoot Pelobates fuscus. This species belongs to a cryptic speciation continuum new to science, deciphered through a fine-scaled genomic phylogeography. Credit: Edvárd MIZSEI.

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

This study was part of my post-doc efforts to compare geographic and genomic patterns of introgression across hybrid zones from several amphibian radiations, in order to understand the pace and the genetic mechanisms of allopatric speciation. For Pelobates, we originally intended to focus only on the P. fuscus/vespertinus hybrid zone in Ukraine/Russia, but inadvertently discovered that our outgroup taxon (the traditionally-recognized P. cf. syriacus) consisted of a cryptic diversification involving several phylogeographic transitions. Instead of one contact, it turned out we could study as many as six within a single radiation. We thus seized that rare opportunity to understand the relationship between genetic divergence and reproductive isolation under natural settings, which had only been attempted in a handful of systems so far.

What difficulties did you run into along the way? 

Not much actually, except perhaps time constrains and taxonomic issues. Because we were willing to describe the new Pelobates species/subspecies discovered and use the appropriate names in our Mol. Ecol. article for clarity, we had to synchronize the peer-reviewing and publication of an accompanying paper (ZooKeys 859: 131–158). This was successful thanks to the support of the two journals and both papers were released just a few hours apart. The scientific stages per se went remarkably smoothly. Colleagues from many countries were enthusiastic to send us samples, the RAD-seq wet lab and bioinformatics performed admirably, and data analysis was straightforward. At the end, it took only about a year and a half from project initiation to publication.

What is the biggest or most surprising finding from this study? 

We were astonished by the strong divergence (>5My) between the Asian and European populations of P. cf. syriacus, and their lack of interbreeding despite parapatric (and perhaps even sympatric?) distributions near the Bosphorus. While they clearly represent two different species (coined P. balcanicus and P. syriacus), no phenotypic differences have been reported (despite several morphometric surveys), so this was not suspected. But more globally, our big finding is the very neat link between genetic divergence and admixture across phylogeographic transitions. It was beyond expectations since hybridization at contact zones often depends on local factors (dispersal constraints, etc.), which blurs the link. Such a clear relationship supports the Darwinian view of a gradual and dynamic speciation continuum, and extends it to cryptic radiations of eco-morphologically similar species.

Moving forward, what are the next steps for this research? 

We have several major follow-ups ongoing. Our Pelobates speciation genomic framework is now being implemented into multi-system comparative analyses aiming to understand how the genetic architecture of reproductive isolation evolves as speciation progresses, by re-analyzing transitions at the locus scale. In parallel, we are characterizing the homomorphic sex chromosomes of Pelobates to gauge their importance in hybrid incompatibilities. Following this research, our co-author Ilias Strachinis has now just started a PhD on Pelobates from the Balkan Peninsula to study the diversity and distribution of the new lineages unraveled. It should provide significant insights for their biogeography and conservation, notably of the mysterious Peloponnese endemic P. balcanicus chloeae.

What would your message be for students about to start their first research projects in this topic? 

Even in supposedly well-studied biogeographic regions like the Western Palearctic, do not take the established phylogenies, species delimitations and taxonomies for granted! Significant diversifications may have been overlooked, especially since previous work relied mostly on mitochondrial and poorly-informative nuclear markers, which can be deceptive to disentangle among closely-related lineages. Nowadays, RAD-seq provides affordable, powerful and straightforward resources to address many questions with a combination of population genetic and phylogenetic analyses, so it appears a tool of choice to study the phylogeography of speciation in many taxonomic groups.

What have you learned about science over the course of this project? 

That unexpected results are worth exploiting and may lead to fascinating scientific discoveries. An unthinkable amount of biodiversity still lies unnoticed right under our noses. Moreover, our study was only possible (especially in such an efficient timeframe) thanks to a great collective effort bringing together renowned teams of herpetological researchers. From my personal perspective, this human aspect emphasizes how science is best appreciated collaboratively rather than through competitive emulation, and I look forward to reiterate the experience.

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

In cryptic diversifications, whether the continuous nature of speciation leads to discrete, reproductively-isolated entities is mostly dependent on the time they spent in allopatry.

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

Our study provides empirical evidence within a single radiation that speciation is a dynamic and reversible process where phylogeographic lineages can merge together upon secondary contact, unless a threshold of evolutionary divergence is reached (>3My in amphibians), in which case they can quickly build up reproductive isolation and become incipient species.

Read the full article here: Dufresnes C, Strachinis I, Suriadna N, et al. Phylogeography of a cryptic speciation continuum in Eurasian spadefoot toads (Pelobates). Mol Ecol. 2019;28:3257–3270. https://doi.org/10.1111/mec.15133

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 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

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.

Interview with the author: A hierarchical Bayesian Beta regression approach to study the effects of geographical genetic structure and spatial autocorrelation on species distribution range shifts

Forecasting the effects of global climate change on species distributions is a major challenge with direct relevance for conservation, management, and policy. Incorporating genetic information into these models may help improve these predictions by accounting for within-species variation in demographic history and adaptation to local environments. In a recent issue of Molecular Ecology Resources, Dr. Joaquín Martínez-Minaya and colleagues present a new approach to forecasting species distributions accounting for within-species genetic variation and spatial autocorrelation using the model species Arabidopsis thaliana. Here, co-author Dr. F. Xavier Picó gives us a behind-the-scenes look at the study.

Link to study: https://onlinelibrary.wiley.com/doi/10.1111/1755-0998.13024

Arabidopsis thaliana in the wild. Photo provided by Xavier Picó

What led to your interest in this topic / what was the motivation for this study? 
Climate change models ought to incorporate more realistic species’ attributes to better understand their response in predicted and probably inevitable warming scenarios for the near future. In other words, we need to model the demographic, ecological, genetic and evolutionary processes that account for changes in distribution range mediated by warming. However, these data are hard to obtain at large spatial scales even for a single species, which seriously limits our understanding of the impact of global climate change on biodiversity. Our motivation was to develop a model to overcome such limitations using long-term existing data for an annual plant that, interestingly, may be the result of the suite of processes mentioned above, such as genetic structure and spatial autocorrelation of data.

What difficulties did you run into along the way? 
This work is the result of a collaborative effort among scientists from different disciplines, including genetics, ecology and mathematics, that in some cases worked together for the very first time. We had to find the way to speak the same language to achieve a common objective from planning to execution. In a way, we taught each other as learned from each other throughout the development of this work. Although this can be regarded as a difficulty, it was also an extremely rewarding process to see how we all pulled off the project.

What is the biggest or most surprising innovation highlighted in this study? 
From a conceptual viewpoint, the heterogeneity that any species has – due to historical, demographic, ecological and genetic factors – can no longer be overlooked whatever the research question and goal. Molecular markers allow us to tackle such inherent heterogeneity beyond our full comprehension of the underlying forces accounting for it. From a technical viewpoint, spatial Bayesian models take spatial autocorrelation of data into account. Ignoring spatial autocorrelation is a huge problem when it comes to the correct interpretation of spatial models. Handling these two key elements at once represents the most remarkable innovation of this study.

Moving forward, what are the next steps for this research?
With no doubt, the most important next step is to include demographic and evolutionary processes explicitly into global climate change models. We need to expand our current Bayesian framework to include dispersal, establishment of new populations, and local adaptation in a context of rapid environmental and land-use changes fuelled by global climate change. These fundamental processes, which can be modelled and/or parameterized with empirical data, will confer realism and power to model predictions. Only realistic models will generate an array of likely global climate change scenarios upon which we will be able to pose working hypotheses and appropriate actions to mitigate the impacts of global climate change on biodiversity.

Photo provided by Xavier Picó

What would your message be for students about to start their first research projects in this topic? 
Learning in science is a tough process, but extremely rewarding. Try to learn from the best to acquire solid foundations. Nevertheless, do not forget that your particular view on a given problem may open up new paths to keep making progress on the discipline. Recall what all renowned artists normally do: they first copy the old masters to learn the techniques to end up innovating and developing their own artistic style. Innovation can only be done when one understands the potential and limitations that any technique has.

What have you learned about methods and resources development over the course of this project? 
Complex problems require imaginative solutions that, for common people, can only be addressed by gathering together professionals from different disciplines sharing similar interests. In addition, it is important to bear in mind that the biological knowledge of the study species is of paramount importance to assess the value and impact of new methodologies. Inevitably, we believe quite often that any new method developed by us might become a panacea to solve multiple problems. Although this enthusiasm is necessary to move forward, it is very important to clearly detect the caveats and limitations of the methods and resources developed. In our particular case, the knowledge of the study species across the region allowed us to identify when the model outcomes were interpretable and when they could be biased.

Describe the significance of this research for the general scientific community in one sentence.
Promising progress is being made to improve models that will allow us to figure out realistically the future of biodiversity in a context of warming, which seems to be inevitable.

Describe the significance of this research for your scientific community in one sentence.
Genetic heterogeneity and spatial autocorrelation, the result of multiple forces acting in concert, can be handled and interpreted to better understand the response of any species to global climate change.

Citation
Joaquín Martínez‐Minaya, David Conesa, Marie‐Josée Fortin, Carlos Alonso‐Blanco, F. Xavier Picó, & Arnald Marcer. (2019). A hierarchical Bayesian Beta regression approach to study the effects of geographical genetic structure and spatial autocorrelation on species distribution range shifts. Molecular Ecology Resources, 19(4), 929-943. https://onlinelibrary.wiley.com/doi/10.1111/1755-0998.13024

Interview with the author: Seed and pollen dispersal distances in two African legume timber trees and their reproductive potential under selective logging

Seed and pollen dispersal, which are critical processes that influence the regeneration of trees, are likely affected by human activities like logging. However, determining how far pollen and seed move is notoriously difficult in the field. Thankfully, modern genetic techniques have provided us new approaches to estimating pollen and seed dispersal, which alleviates some of this issue. Using genetic markers, Dr. Olivier Hardy and colleagues assessed how the movement of pollen and seed in two African timber species is affected by selective logging, where one or two trees per hectare is removed on a 25-30 year cycle. Read below for a behind the scenes interview with Olivier.

Link to the study: https://onlinelibrary.wiley.com/doi/10.1111/mec.15138

Inflorescence of Distemonanthus benthamianus. Photograph by Xander van der Burgt

What led to your interest in this topic / what was the motivation for this study? 
Tropical forests are fascinating but threatened ecosystems. Logging is usually perceived as one of their major threat by the public but selective logging might also be a solution when adequate management plans allows the regeneration of logged tree species, while the revenue generated prevents land conversion. However, forest regeneration is complex because many biotic and abiotic factors can be involved in the process. The extent of seed and pollen dispersal is one of the factors for which few data exist, and it can vary among species or locations. Fundamental research has developed methodologies based on genetic markers to quantify seed and pollen dispersal, so it is a great satisfaction to apply these methods for generating data that should help improving the management of tropical forests.

What difficulties did you run into along the way? 
The study required sampling exhaustively all adult trees of our focal species over several square kilometres in very remote sites of Central African forests, where visibility can be limited to a few tens of meters. This is the kind of field work that would have been very difficult to achieve for a small team of researchers. The collaboration with logging companies, FSC-certified, was a great advantage because we could rely on their field technicians who are trained to conduct similar inventories for planning logging activities.

What is the biggest or most surprising finding from this study? 
First that pollen dispersal distances were so different between two insect-pollinated tree species from the same family but bearing different flower types. It suggests that different flower types could attract different guilds of pollinators with contrasted flight abilities, but for now we ignore who are the pollinators. Second that about 25% of the seeds of the wind-dispersed species were transported over >500 m while their pods do not seem to be efficient gliders and, indeed, the majority falls within <100 m. It suggests that storm winds could play a crucial role in the dissemination of wind-dispersed canopy trees.

Moving forward, what are the next steps for this research?
First, we need to identify the pollinators and seed dispersers. This is challenging, in particular for pollinators because flowers open in the canopy, 20 to 40 meters above the ground and we must invent devices to observe or capture pollinators. Sporty, adventurous and creative PhD or MSc students are welcome! Second, we need to replicate these studies on numerous tree species with different contrasted reproductive characters to be able to derive some generalizations about seed and pollen dispersal according to species traits and environmental conditions. Third, we need to translate these results into recommendations for the sustainable management of tropical forests in collaboration with the forestry sector, and disseminate the messages to policy makers. Fourth, beyond seed and pollen dispersal, many other key processes affect the regeneration cycle and must be considered as well.

What would your message be for students about to start their first research projects in this topic? 
Tropical biology is fascinating for those attracted by fundamental research on biotic interactions, and it is full of opportunities for those inclined towards applied research of societal importance. Some students might be afraid by the remote and little equipped field conditions, and this is obviously constraining. But most students who discovered tropical Africa for the first time returned enthusiastic by their experiences, and some decided to pursue scientific research there.

Describe the significance of this research for the general scientific community in one sentence.
Seed and pollen dispersal investigations are relevant to assess the sustainability of selective logging in tropical forests.

Describe the significance of this research for your scientific community in one sentence.
Seed and pollen dispersal appears to be non-limiting for the natural regeneration of two African tree species under selective logging, while the minimal cutting diameter should be defined by accounting of the relationship between reproductive success and tree size

Citation
Olivier J. Hardy, Boris Delaide, Hélène Hainaut, Jean‐François Gillet, Pauline Gillet, Esra Kaymak, Nina Vankerckhove, Jérôme Duminil, & Jean‐Louis Doucet (2019). Seed and pollen dispersal distances in two African legume timber trees and their reproductive potential under selective logging. Molecular Ecology, 28(12), 3119-3134. https://onlinelibrary.wiley.com/doi/10.1111/mec.15138

Interview with the author: Landscape genomic signatures indicate reduced gene flow and forest‐associated adaptive divergence in an endangered neotropical turtle

The Dahl’s Toad-headed turtle (Mesoclemmys dahli) is one of the 25 most endangered turtle species on Earth and one of the unlucky few that is constantly being compared to a toad. A previous study has shown that extensive habitat conversion and fragmentation in its native range in Colombia has led to genetic differentiation among populations. However, critical questions remain for the conservation and management of this species: how does habitat type and quality in this rapidly changing region influence gene flow? How can genomic data inform the management of this endangered species?

Dr. Natalia Gallego García (@NataGalle) and colleagues set out to answer these questions. Read below for an interview with Natalia about the challenges of working with such a rare species and how this study provides the foundation for a genetic rescue program for the Dahl’s Toad-headed turtle.

The critically endangered Dahl’s Toad‐headed turtle (Mesoclemmys dahli). Photo by N. Gallego García

What led to your interest in this topic / what was the motivation for this study? 
We conducted a previous population genetics study using microsatellite loci on this highly endangered species, in which we found significant genetic evidence of population fragmentation. In this follow-up study, we wanted to know how the current landscape, now composed of open grasslands for cattle instead of tropical dry forest, might be restricting gene flow and thus causing the observed fragmentation. We also wanted to know how this new anthropogenic environment was potentially driving local adaptation.

What difficulties did you run into along the way? 
The main difficulty was finding this rare species across its range to get the samples. Another difficulty was standardizing the RADseq protocol, as our research was the first genomic-level study on any side-necked (pleurodiran) turtle. Also, there is no reference genome for this species or for any closely related one, making the analysis of our data difficult. We had to assembly a de novo genome, which prevented us from running other analyses that could have allowed us to learn more about adaptive mechanisms to new environments.

What is the biggest or most surprising finding from this study? 
First of all, we were able to show that population fragmentation was related to habitat loss. However, we were expecting movement through grasslands to be costly, given that this is a forest species, but we found that from a scale of 1 (easy) to 1000 (hard) the cost of traversing grassland was only 13. We believe that this low cost is associated with the presence of water ponds built in the pastures for the cattle to drink, which are increasingly being used by this species. These ponds might be serving as a sort of stepping stone array of lower quality but still usable aquatic habitat, enabling movement over an otherwise hostile matrix. Our second surprising finding was observing possible adaptive divergence between populations occupying areas with more forest than populations in areas with almost no forest. This result suggests that the populations might be adapting to this new transformed environment. However, adaptation alone is not rescuing this species from the negative effects of fragmentation, and currently the species is facing a high risk of extinction.

Moving forward, what are the next steps for this research?
The next steps can be divided in terms of management and research. In terms of management, we are currently designing a genetic rescue program to reduce inbreeding and increase population genetic diversity, without disrupting the potential ongoing adaptation that we observed. In terms of research, we are currently assembling the genome of a closely related species, which will allow us to map the putatively adaptive loci found, and better understand how this species is adapting to its new transformed environment. This will also allow us to design a field and/or laboratory experiment to further explore the possibility of adaptation to altered, and degraded habitat.

What would your message be for students about to start their first research projects in this topic? 
Working with non-model, rare, and threatened organisms, although challenging, can lead to valuable information that is vital in their conservation. So, accept the challenge and stand up for those forgotten species. Any new information on a data deficient species will increase its chance of survival, which in itself already makes the research worthwhile.

What have you learned about science over the course of this project? 
Science always comes with exciting surprises that do not always comply with our expectations, and it usually leaves more questions than answers. But it is gratifying to contribute, even in a small way, to the understanding of complex processes that can eventually be applied to solve difficult problems, such as the conservation of an endangered species.

Describe the significance of this research for the general scientific community in one sentence.
Adaptation to habitat change can happen, but perhaps not quickly or completely enough to overcome the negative effects of population reduction and fragmentation.

Describe the significance of this research for your scientific community in one sentence.
Landscape genomics analyses provide evidence of reduced gene flow in a fragmented habitat, leading to harmful effects on a critically endangered neotropical turtle, despite its possible adaptation to the new anthropogenically created environment.

Citation
Natalia Gallego‐García, Germán Forero‐Medina, Mario Vargas‐Ramírez, Susana Caballero, & Howard Bradley Shaffer. (2019). Landscape genomic signatures indicate reduced gene flow and forest‐associated adaptive divergence in an endangered neotropical turtle. Molecular Ecology, 28(11), 2757-2771. https://onlinelibrary.wiley.com/doi/10.1111/mec.15112

Interview with the author: A guide to the application of Hill numbers to DNA based diversity analyses

image
Diversity assessment procedures in traditional and DNA sequencing‐based approaches. Recorded entities need to be classified into types, before each type is weighed according to its relative abundance and the order of diversity (q). Note the example refers to an abundance‐based, rather than incidence‐based, approach

What are Hill Numbers? What do they have to do with estimating biodiversity? How can you use them as a Molecular Ecologist? Read the recent review in Molecular Ecology Resources by Antton Alberti and Thomas Gilbert on this topic, and read the interview with Antton below to learn how they think about Hill numbers and their applications to metabarcoding. Also, check hilldiv, “an R package to assist analysis of diversity for diet reconstruction, microbial community profiling or more general ecosystem characterisation analyses based on Hill numbers, using OTU tables and associated phylogenetic trees as inputs. The package includes functions for (phylo)diversity measurement, (phylo)diversity profile plotting, (phylo)diversity comparison between samples and groups, (phylo)diversity partitioning and (dis)similarity measurement. All of these grounded in abundance-based and incidence-based Hill numbers.”

What led to your interest in this topic / what was the motivation for this study? 
Measuring, estimating and contrasting biological diversity are central operations in most ecological studies. In the last decades, dozens of diversity indices and metrics have been proposed, each with their individual strengths and weaknesses, and specific mathematical assumptions. The measures that many of them yield are difficult to interpret, because the values might refer to abstract units, which lack an straightforward interpretation for non-specialists. We believe that the statistical framework developed around the Hill numbers overcomes many of these problems, and provides a statistical toolset that is extremely useful for ecologists. Besides, Hill numbers enable incorporating complementary information, such as phylogenetic dissimilarities across organisms, which are really handy for molecular ecologists who can easily build phylogenetic trees from metabarcoding data.

What difficulties did you run into along the way? 
We are a molecular ecologist and an evolutionary biologist that use many different mathematical tools, but are not expert mathematicians. Hence, of the main challenges was to make sure that all the statements and mathematical interpretations were correct!

What is the biggest or most surprising innovation highlighted in this study?
The aim of our review was to demonstrate to ecologists, who like us might have a limited mathematical background, that implementing the framework developed around the Hill numbers is not difficult, and has big potential gains. In our review we gathered information and tools generated by others, mainly Lou Jost, Anne Chao and Chun-Huo Chiu, and displayed them in a comprehensive way for molecular ecologists. We have tried to explain complex mathematical formulations in layman terms, exactly as we would like others to explain us other contents we are not familiar with. We have provided examples and pieces of code, that we hope will encourage other researches to use these tools.

Moving forward, what are the next steps in this area of research?
Our article mainly focuses on diversity measurement from data generated using DNA metabarcoding. While bioinformatic methods to generate metabarcoding data have received much attention in the last decade, the impact of the statistical approaches used to analyse diversity has been less studied. Assessing their impact and providing guidelines for selecting the tool best suited to address specific questions with specific types of data, will be an important next step in the area of metabarcoding-based diversity analyses.

What would your message be for students about to start developing or using novel techniques in Molecular Ecology? 
Despite the fact that they might at first seem complex and abstract, bioinformatic and statistical tools are necessary to address ecological questions. Hence, we would encourage students to try to understand the basic bioinformatic and statistical procedures, so as to be able to select the best tools to address their research questions.

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Differences between abundance‐based and incidence‐based Hill numbers. The Hill numbers yielded for the entire system are different depending on the approach employed. In abundance‐based approaches, the DNA sequence is the unit that the diversity is computed on, while in incidence‐based approaches, it is the sample the unit upon which the diversity is measured. (*) The asterisk indicates that the equations are undefined for q = 1, thus in practice either the 1D formula shown in Table 1 or a limit of the unity must be used, for example, q = 0.9999. However, q = 1 is used for the sake of simplicity

What have you learned about methods and resources development over the course of this project?
That its not the most broadly-employed tools that are always the best way to address scientific questions!

Describe the significance of this research for your scientific community in one sentence.
Hill numbers provide powerful, solid and versatile tools with which to carry out most of the analyses that are needed to assess biological diversity within a common statistical framework.