Summary from the authors: Detection of environmental and morphological adaptation despite high landscape genetic connectivity in a pest grasshopper

Male and female Phaulacridium vittatum. Photo credit: Sonu Yadav.

The Australian native grasshopper, Phaulacridium vittatum, known as the wingless grasshopper, is a common pest of pastures and crops in Australia, with outbreaks recorded every four or five years. With climate change and the expansion of agricultural land use, there is concern that grasshopper outbreaks could increase in frequency and severity. We used both neutral analysis of landscape genetic resistance combined with detection of selection using Environmental Association Analysis (EAA) to investigate common and disparate environmental drivers of  genetic dispersal and local adaptation in this grasshopper pest. With SNP data collected across a 900km gradient, we found that gene flow was best predicted by temperature, with only urban areas and water bodies limiting genetic dispersal. Although there was considerable admixture across the study area, local adaptation was evident and similarly driven by temperature, with additional evidence of morphological adaptation (body size and stripe polymorphism). Gene annotations revealed functions linked to UV shielding, and detoxification processes. Our study indicates that P. vittatum has high potential to adapt to heterogenous environments under high gene flow, and that temperature is the primary driver of both neutral and adaptive genetic structure. Thus, P. vittatum may become a more serious pest in the future as temperatures become warmer, and agricultural land use expands. 

Yadav S, Stow AJ, Dudaniec RY. Detection of environmental and morphological adaptation despite high landscape genetic connectivity in a pest grasshopper (Phaulacridium vittatum). Mol Ecol. 2019;28:3395–3412.

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:

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

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

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.

Summary from the authors: Dispersal limitations and historical factors determine the biogeography of specialized terrestrial protists

The diversity and geographical distribution of plants and animals are well documented and this information was essential to understand the factors that generate biodiversity, the most famous example being Darwin and Wallace’s theory of evolution. However, we know much less about microbial diversity and distribution, and hence it is unclear if the same factors drive the diversity of large and small organisms.

Hyalosphenia papilio from Le Cachot Bog, Swiss Jura Mountains. Picture by Prof. Daniel Lahr.

Using molecular tools, we studied the distribution and diversity of a species complex of the testate (shell-producing) amoeba species Hyalosphenia papilio, a microorganism restricted to Sphagnum peatland of Eurasia and North America. H. papilio is a complex of 14 distinct molecular lineages. Based on the DNA sequences, we inferred how, where and when this diversity evolved.

Our results suggest that H. papilio evolved in western North America and subsequently colonized other regions of Eurasia and North America during interglacial periods. Colonization of Eurasia occurred most recently, possibly after the last glaciation.

The patterns we observed for H. papilio are consistent with those commonly observed for macroscopic plants and animals. This in turn suggests that microbial diversity may be much higher than currently thought and may include “relict” taxa with restricted distributions, as commonly found among macroscopic plants and animals.

Read the full article: Singer D, Mitchell EAD, Payne RJ, etal. Dispersal limitations and historical factors determine thebiogeography of specialized terrestrial protists. Mol Ecol. 2019;28:3089–3100.

Interview with the Author: Conservation of old individual trees and small populations is integral to maintain species’ genetic diversity of a historically fragmented woody perennial

What is the unit of conservation? Is it similar for different types of plants? How does the reproductive biology of the organism can inform the best practices in conserving threatened species? In her Doctoral research, Nicole Bezemer is studying Eucaliptus species from South Western Australia to better understand population dynamics in long-lived organisms and how this can lead to better management of their populations. Surprisingly, many of the small and fragmented populations of the two subspecies of E. caesia she studied are genetically differentiated at a fine spatial scale, and high levels of heterozygosity persists even in populations with a dozen of individuals. Nicole and colleagues suggest the clonal and perennial nature of E. caesia might contribute to these unusual patterns of genetic diversity and divergence, and suggest that traditional conservation genetic approaches might be detrimental for naturally fragmented species with these life-history characteristics. Read here about her experience in developing this research.

A multi-stemmed genet of Eucalyptus caesia at Mocardy Hill, Western Australia. Photo by NB.

What led to your interest in this topic / what was the motivation for this study? 
Eucalyptus caesia is an intriguing study species, given the combination of a distribution on scattered granite outcrops, a long history of geographic and genetic insularity, a capacity for individual longevity via lignotuber re-sprouting, a lack of recent recruitment in most known stands, and adaptation for pollination by nectarivorous birds. After completing my Honours research at the Boyagin stand of E. caesia, I was hooked. The present study came into fruition upon discovering that one of my PhD experiments, involving 6 months of controlled cross-pollinations, was killed by a series of frosts. I had already genotyped two large stands of E. caesia and I was curious about what patterns of genetic structure might exist in other stands, and across the species’ landscape distribution. 

What difficulties did you run into along the way? 
Some stands of E. caesia are located on immense granite outcrops, often hidden in hard-to-access gullies or behind thick barricades of vegetation. The first challenging aspect of the project was to find the sub-populations of E. caesia at each new location. For many populations, I did so by embarking on a Google Earth tour led by my supervisor, Steve Hopper, who has worked on the granite outcrop flora of south-west Australia and on E. caesia for nearly four decades. Nonetheless, I spent many hours traversing granite outcrops, sometimes in circles, which occasionally led to finding additional plants or, in the case of the E. caesia at Old Muntadgin, a previously undocumented population of several hundred plants.

What is the biggest or most surprising innovation highlighted in this study? 
I was surprised by the apparent lack of genetic interconnection between some stands over relatively small spatial scales. Given the long history of population fragmentation and reproductive biology of E. caesia (multiple modes of reproduction and gravity-dispersed seed), I anticipated that high levels of genetic differentiation would feature. Regardless, it was surprising to find that, in some instances, the level of genetic differentiation within stands exceeded that among stands. Another interesting result revealed by comprehensive genotyping were some very small census population sizes. Seven stands were comprised of fewer than ten unique multi-locus genotypes, and three locations had only one or two genotypes. Localised clonal reproduction is clearly of paramount importance to the persistence of these stands.

Moving forward, what are the next steps in this area of research?
The next step is to further test the genetic integrity of the two subspecies, E. caesia subsp. caesia and E. caesia subsp. magna, by genotyping plants from additional stands. Walyamoning and Yanneymooning are geographical outliers to other stands of subsp. caesia and occur within relatively close proximity to the group of subsp. magna populations located in the north-east of the species distribution. We propose to genotype a sample of individuals from the two outlier populations of subsp. caesia stands, and at three additional locations of subsp. magna, to test whether the two subspecies are genetically distinct even when populations are sympatric, and to determine if hybridisation has occurred.

What would your message be for students about to start developing or using novel techniques in Molecular Ecology? 
My message to other young or early-career researchers is to have a clear research outcome in mind before exploring the application of novel techniques. Avoid putting yourself in the position of having to come up with a hypothesis after the fact.

What have you learned about methods and resources development over the course of this project? 
Comprehensive genotyping at multiple spatial scales may provide a more complete picture of spatial genetic structure compared to studies where sampling efforts are focused on few individuals from many populations, or on many individuals from few populations. There is still much to be gained from population genetic studies, especially in understudied, biodiverse, endemism hotspots such as granite outcrops, and in understudied systems such as small, historically fragmented populations of long-lived trees.

Describe the significance of this research for the general scientific community in one sentence.
Anciently fragmented plant populations may be adept at persisting as small populations with low genetic diversity and limited genetic interconnection, and therefore attempts to connect such populations may be ineffective or even harmful.

Describe the significance of this research for your scientific community in one sentence.
Small populations of long-lived woody perennial plants, even those comprising a handful of individuals, may contain unique genotypes that contribute to overall species genetic diversity, and are worthy of conservation.

Enjoying the afternoon light from my field base camp underneath Eucalyptus caesia at Boyagin Rock. Photo by NB.

Summary from the authors: Genome‐scale sampling suggests cryptic epigenetic structuring and insular divergence in Canada lynx

Wild populations are often genetically structured in complex ways due to migration, selection, and drift. In highly mobile species such as the Canada lynx (Lynx canadensis), these complexities are exacerbated due to high levels of gene flow, which can make population delimitation challenging. Previously, Canada lynx populations appeared largely undifferentiated across continental North America at neutral genetic markers, with only small fine-scale differences across the landscape being correlated with climatic gradients. This climatic structuring aroused our interest in potential epigenetic differences between Canada lynx across their range, as environmentally-induced modifications to DNA could explain geographical or morphological differences that are not apparent in neutral DNA.

A lynx stalks its prey in the Northern forests of the Canadian Yukon, bordering Alaska. Photo credit to Dr. Melanie Boudreau.

To test this hypothesis, we examined neutral genetic differences and patterns of DNA methylation between 95 Canada lynx across 4 geographical regions (Alaska, Manitoba, Québec, and an insular population on Newfoundland). We found that Newfoundland lynx were the most distinct at both genetic and epigenetic markers, consistent with what we would expect for an island population. However, despite low neutral genetic differentiation between all mainland populations, we detected stark epigenetic differences between Alaska lynx and the remaining mainland lynx. Further analyses indicated that these differences might correlate with increased energetic demands, consistent with Alaskan lynx being the morphologically largest of all in their range. Our study exemplifies the utility of epigenetic markers for assessing population structure, even in non-model systems characterized by extreme levels of gene flow. 

Summary of neutral genetic structure with SNPs (left) and patterns of DNA methylation (right) between Canada lynx, where each circle represents an individual lynx colored by geographic region. Alaskan lynx (purple) are largely undifferentiated at neutral genetic markers compared to other mainland lynx, in contrast to their epigenetic profiles. 

Read the full article: Meröndun J, Murray DL, Shafer ABA. Genome-scale sampling suggests cryptic epigenetic structuring and insular divergence in Canada lynx. Mol Ecol. 2019;28:3186–3196.

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.

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:

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.