Interview with the authors: Genomic basis of Y‐linked dwarfism in cichlids pursuing alternative reproductive tactics

In a recent paper in Molecular Ecology, Singh et al. used genome sequencing, bioinformatics and population genetic analyses to gain insight into the genetics and evolution of a fascinating mating system. The species in question, Lamprologous callipterus, exhibits a mating system with two males morphs. Large “bourgeois” males carry empty snail shells that are inhabited and used as nests by the females. An alternate male morph, much smaller than the “bourgeois” males, also exists and inhabits shells along with the females. Previous genetic work had established that this mating system was Y-linked and that the male body size was a Mendelian trait, but the sex-determining locus had not been identified until this study.

We sent some questions to Pooja Singh, the author who led this work, to get more detail on this study.

Photo credit: Drawing by Pooja Singh, based on Barbara Taborsky’s original image.

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

The most novel aspect of this study is that we found an example of a young sex chromosome that may have evolved due to sexual antagonism over body size. While the sexual antagonism theory is considered the classical model of sex chromosome evolution, few empirical examples exist to support it. The other exciting finding was that the candidate body size/dwarfism gene that we propose for L. callipterus, GHRHR, is a well-known dwarfism gene in mammals. Fish and mammals shared a common ancestor over 440 million years ago, so the body size development pathway is genetically constrained through deep evolutionary time.

What difficulties did you run into along the way? 

The major challenge for me was that I knew little about sex chromosome evolution when I started this project, so I really had to do a lot of groundwork reading relevant literature and researching methods to be able to get things going. I had to start thinking beyond the classical XY and WZ old sex systems and familiarize myself with the workings of early stages of sex chromosome evolution.

When I read your paper, I had never heard of the fascinating mating system of these cichlids. They reminded me of the ruff, and the multiple inversions that seem to be involved in the different reproductive strategies in that system. You mention in the paper that you were not able to identify inversions based on the bioinformatic approaches you used. Is there a sense for how much chromosome evolution during the radiation? Could the use of the divergent reference genome have anything to do with the lack of a signal of inversions? 

To my knowledge the broad scale chromosomal structure of African cichlid species is similar. However, small scale structural variations (inversions, indels, translocations etc.) have not been investigated systematically. So yes, it is totally possible that our short read data and the divergent (and fragmented) reference genome assembly may have hindered our ability to detect inversions. The system really needs a long-read de novo genome assembly to resolve the inversion question.

In the Discussion, you talk about the possibility of different male Y-haplotypes. Is your data sufficiently high resolution that you could examine insertion/deletion polymorphisms in your dataset? 

Yes, we could technically identify small insertions/deletions in our data. Might certainly be something to investigate in the future, in combination with long-read Y assembly.

A recently proposed model of sex-chromosome evolution indicates that gene expression differences may predominate at the early stages of sex chromosome evolution (Lenormand and Roze 2022 Science – This is intriguing given that you didn’t find any smoking gun loci with signals of sexual antagonism. Do you have plans to look at patterns of gene expression across the different morphs?

While Lenormand and Roze’s theory is certainly exciting for the field of sex chromosome evolution, I think it is less plausible for the L. callipterus mating system because antagonistic body sizes in females and males are crucial shell-brooding success and fitness. And because we found the candidate sex-determining gene and body-size gene to be physically linked. I am certainly not a proponent of the ‘one classical theory explains all’ narrative and I really look forward to seeing what RNA-seq data reveal about sex chromosome evolution in this species. It would be especially interesting to see the landscape of cis/trans eQTLs of genes in our proposed L. callipterus sex chromosome and how much it reflects the expectations from Lenormand and Roze’s model. Beyond just this species, cichlid fishes are an excellent system to test the sexual antagonism vs gene regulation models of sex chromosome evolution.

Regarding the coverage analysis you used to identify the putative sex-linked locus. Given the hypothesis that the divergence of the sex chromosomes is recent, reads sampled from Y-linked regions may still map well to the orthologous region on the putative X-chromosome.  Did you tweak mapping quality filters at all?

I did run a less stringent mapping analysis, which overall had slightly higher mapping statistics, but the reduced coverage pattern on the L. callipterus sex chromosome was still significant.

Snail shell nest of L. callipterus with the nest owner in the left centre.
Photo credit: Koblmueller et al. 2007

Could you describe the significance of this research for the general scientific community in one sentence?

Sexual antagonism over body-size may have driven sex chromosome evolution in a shell-brooding cichlid fish where giant and dwarf male reproductive types have evolved.

Moving forward, what are the next steps in this area of research (unless otherwise covered)?

Our main priority right now is to keep the L. callipterus dwarf males alive and breeding. Our fish were recently moved from the University of Bern in Switzerland to the University of Graz in Austria, and it has proved difficult to get the dwarf males happy and breeding in the new facility. This is (probably) the only collection in the world of L. callipterus dwarf males outside Lake Tanganyika so they are very precious. Our next step is to write a convincing grant to get funding to build a long-read improved genome assembly, conduct RNA-seq, and sample and sequence more individuals from natural populations. I would like to use the RNA-seq data to map expression QTLs and investigate the regulatory interactions of candidate genes related to sex, morphology, physiology, and behaviour that we found in or around the L. callipterus sex region. It would also be interesting to study sex chromosomes in related lamprologine species, as our pre-liminary analysis in this manuscript suggests that the sex region may be shared across the lamprologine tribe

Interview with the authors: Whole-genome analysis of multiple wood ant population pairs supports similar speciation histories, but different degrees of gene flow, across their European ranges

In a recent paper in Molecular Ecology, Portinha et al. used population genomic data to analyse the speciation history of two closely related species of wood ants, Formica polyctena and F. aquilonia. Using a demographic modelling approach, the authors reconstruct the history of divergence for multiple heterospecific pairs of populations. In all cases, the authors found that there was evidence for divergence with gene flow. However, for a sympatric population pair sampled in Finland there was evidence for substantially elevated gene flow between the species. Their findings imply that population genomic analysis of speciation history may be geographically variable for particular species.

We sent some questions to Beatriz Portinha and Pierre Nouhaud, the corresponding authors of this work, to get more detail on this study.

Ant mound surface covered in ants. Photo credit: Jack Beresford

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

Knowledge on the demographic and speciation histories is essential for understanding
contemporary genomic patterns in natural populations, which is why we wanted to
reconstruct it for the emerging Formica model system. Our study species, Formica polyctena
and F. aquilonia, are known to hybridize naturally in Southern Finland, where their hybrids
have been studied for over 10 years (Kulmuni et al., 2010; Martin-Roy et al. 2021). We
wanted to test whether a similar divergence history was consistently inferred across the
European ranges of both species, or whether the Finnish populations would stand apart,
possibly because of gene flow mediated by hybrid populations in the area.

What difficulties did you run into along the way? 

Formica polyctena and F. aquilonia had a limited genomic toolbox when we started the
project, and we initially relied on a distant and non-contiguous reference assembly.
Meanwhile, our group assembled a high quality reference genome (Nouhaud et al., 2022),
which improved the quality of our inferences.

The demographic modelling software we used, fastsimcoal2, can simulate a large panel of
evolutionary scenarios. When planning this study, we wanted to design models that
considered alternative scenarios for the divergence of the species which would be as
biologically meaningful as possible, while keeping the number of models low enough that the
project 1) would not be a huge computational burden and 2) would be executable in the
available time frame (Beatriz’s MSc. project, funded by Erasmus+ and Societas pro Fauna et
Flora Fennica). This was an especially important aspect as we used four distinct population
pairs to reconstruct the history of the two species, so each model had to be run, at least, four
different times.

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

We found that there was already bidirectional gene flow occurring in Finland before the
hybridization events that led to the present-day hybrid populations. This was not suspected
before, as there is no evidence in the literature, and it suggests that F. polyctena in Finland
may be admixed, which is supported by the fact that we have not found non-admixed F.
individuals in Finland.

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

The divergence history we inferred between F. polyctena and F. aquilonia can be used to
run simulations about the evolution of the hybrid populations, which is what we did in a
subsequent work (Nouhaud et al. 2022). In the longer run, it would also be important to
extend this work by reconstructing the divergence history of the whole F. rufa species group,
which encompasses 5 species (including F. aquilonia and F. polyctena) and where gene flow
is prevalent (Seifert, 2021).

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

Genomes from individuals sampled thousands of kilometers apart tell the same ancient
history, while their most recent history may be different.

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

The divergence history between two species can be reliably and consistently inferred from a
small number of individuals sampled across the species’ ranges.

Portinha, B., Avril, A., Bernasconi, C., Helanterä, H., Monaghan, J., Seifert, B., Sousa, V. C., Kulmuni, J., & Nouhaud, P. (2022). Whole-genome analysis of multiple wood ant population pairs supports similar speciation histories, but different degrees of gene flow, across their European ranges. Molecular Ecology, 31, 3416– 3431.

Kulmuni, J., Seifert, B. & Pamilo, P. (2010). Segregation distortion causes large-scale
differences between male and female genomes in hybrid ants. Proceedings on the National
Academy of Sciences
, 107(16), 7371-7376.

Martin-Roy, R., Nygård, E., Nouhaud, P. & Kulmuni, J. (2021). Differences in thermal
tolerance between parental species could fuel thermal adaptation in hybrid wood ants.
American Naturalist, 198(2), 278-294.

Nouhaud, P., Beresford, J. & Kulmuni, J. (2022). Assembly of a hybrid Formica aquilonia× F.
ant genome from a haploid male. Journal of Heredity, esac019, 1-7.

Nouhaud, P., Martin, S. H., Portinha, B., Sousa, V. C. & Kulmuni, J. (2022). Rapid and
repeatable genome evolution across three hybrid ant populations. bioRxiv.

Seifert, B. (2021). A taxonomic revision of the Palaearctic members of the Formica rufa
group (Hymenoptera: Formicidae) – the famous mound-building red wood ants.
Myrmecological News, 31, 133-179.

Interview with the authors: Mega-fire in redwood tanoak forest reduces bacterial and fungal richness and selects for pyrophilous taxa that are phylogenetically conserved

In a recent paper in Molecular Ecology, Enright et al. examined how soil microbiomes are affected by extreme fires. The Soberanes mega-fire provided the authors with an opportunity to study how such extreme events, which are increasingly common with climate-change, can have lasting effects on ecology. By sampling the soil microbiome before and after the Soberanes mega-fire, Enright at al. demonstrated dramatically altered soil communities and a reduction in species richness associated with the mega-fire. There was a clear phylogenetic pattern to the particular microbes that increased or decreased abundance after the fire. Drawing from their results, Enright et al. propose a framework to predict the traits that post-fire microbial communities might exhibit.

We sent some questions to Sydney Glassman, one of the corresponding authors of this work, to get more detail on this new study.

Aerial view of the Soberanes mega-fire. Photo credit: Calfire

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

I had originally been interested in sampling the redwood tanoak forests of Big Sur because I was interested in what the cascading effects of sudden oak death (SOD) induced mortality would be on soil fungal communities during my PhD at UC Berkeley. Prof Dave Rizzo at UC Davis had a large plot network investigating the effects of SOD on plant mortality. I teamed up with him in 2011 to select a subset of plots to collect soils to investigate the impacts on the soil microbial community via amplicon sequencing. Then, in 2016, I learned that half my plots burned in the catastrophic Soberanes Megafire. It’s extremely rare to have pre- and post-fire samples from the same sampling locations before and after a mega-fire. I was really curious about what the impact of a mega-fire would be on soil microbial communities especially since they had never been studied in redwood tanoak forests before. These forests are endemic and charismatic megflora of Califronia that are facing multiple global change factors and it is really unclear how the soil microbial communities will respond to wildfires and how that will influence the recovery of the vegetation. I had already moved to southern California at this time to start a post-doc at UC Irvine, so I asked Kerri Frangioso, who lived in Big Sur, if she would be able to re-sample any of the plots that burned. Using GPS, she was able to collect soils from the exact same sampling locations that I had sampled in 2011 from 3 of the plots (2 burned and 1 unburned) within 30 days of the fire being declared over. She mailed these soils to me, I extracted the DNA, and froze everything until I was able to start my own lab at UC Riverside in 2018.

What difficulties did you run into along the way? 

The terrain in Big Sur is notoriously challenging to traverse. It is extremely steep, lots of windy dirt roads, and there is a lot of poison oak. There is no cell reception in any of our plots and most are at least an hour from the nearest town.  Collecting the soil even before the fire was challenging enough. However, after fires, it is really challenging to access sites because roads are closed, landslides are common, and dead or dying trees are extremely hazardous especially in the case of wind. We were very lucky to be able to re-sample even 3 of our plots so fast after the fire.

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

I was really surprised that many of the same pyrophilous “fire loving” microbes that have been found to increase in frequency after pine forest fires also increased in frequency after redwood tanoak fires. That indicates that soil microbes are selected for by slightly different pressures than plants because the plants that regenerate post-fire in pine forests vs redwood tanoak forests are very different. It seems more likely that microbes instead survive via temperature thresholds and if fire is high severity enough, similar groups of microbes will respond. We collaborated with Kazuo Isobe to implement the CONSENTRAIT analysis and identified that microbial response to fire was indeed phylogenetically conserved, and it seemed that related groups of bacteria and fungi did indeed positively or negatively respond to fires. This will greatly enhance our ability to predict which microbes will respond to fire in any ecosystem since certain lineages seem evolutionarily adapted to survive fires. We also found that a basidiomycete yeast Basidioascus, dominated the fungal sequences at 30 days post-fire, and that had never been found before, probably because most post-fire sampling historically has been based on fruiting bodies.

Morphological diversity of soil microbes. Photo credit: Jenna Maddox

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

I was able to leverage some of these results and results from my work sampling wildfires in Southern California chaparral to help me acquire a USDA grant from their Agricultural Microbiomes program (described here). The purpose of this grant is to characterize the traits of pyrophilous microbes and begin to get our knowledge of fire adaptation in microbes to that of plants. We understand a lot of the traits that enable plants to survive wildfires (like thick bark, vegetative resprouting, serotinous cones, etc) but we don’t have similar understanding of those traits in microbes. In order to understand these traits, Dylan Enright has begun performing biophysical trait assays on these microbes to determine their traits based on a large culture collection of pyrophilous microbes that I have been developing since I started my lab in July 2018. Over the last four years, 2 lab managers, one PhD student (Dylan Enright), 13 UCR undergraduates, and one part time laboratory technician have been involved in developing this culture collection of over 400 isolates of bacteria and fungi from burned soils from wildfires. Our goal is to characterize their traits with biophysical assays and eventually with genomics.

Have you gone back (or have you any plans to go back) to sample soils in the post-fire period? How long lasting do you think the effects of fire on microbial communities would be? 

Unfortunately, I have not been able to get this particular project funded (despite several attempts) and everything I did for this paper was completely unfunded. So I have not been able to return to these plots to sample again. I would be interested in returning to them eventually. I would predict the effects of the fire on the microbial communities could last decades if not longer, depending on if the plants themselves have been able to recover. Most of the literature on pyrophilous microbes suggests that high severity fire can have long term impacts on soil microbes that can last at least a decade or more. Given that the richness of both bacteria and fungi was reduced by up to 70% in one of our plots, I would predict it will take a long time to recover.

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

Megafires have long lasting impacts on both plants and soil microbes alike, and it is important to understand the impacts on soil microbes since they drive plant and soil regeneration. 

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

The pyrophilous microbes that respond to a mega-fire in redwood tanoak forests are similar to those that respond to high severity wildfires in better studied pine forest systems, and the fact that they are phylogenetically conserved indicates that we will be able to predict what microbes will respond to wildfires in any system. Further, we are beginning to identify conserved trait responses that enable wildfire response that are analogous to plants and will help us bin and better understand fire adaptation traits in microbes.

Enright, D. J., Frangioso, K. M., Isobe, K., Rizzo, D. M., & Glassman, S. I. (2022). Mega-fire in redwood tanoak forest reduces bacterial and fungal richness and selects for pyrophilous taxa that are phylogenetically conserved. Molecular Ecology, 31, 2475– 2493.

Nominations for Molecular Ecology Prize 2022

We are soliciting nominations for the annual Molecular Ecology Prize.

The field of molecular ecology is young and inherently interdisciplinary. As a consequence, research in molecular ecology is not currently represented by a single scientific society, so there is no body that actively promotes the discipline or recognizes its pioneers. The editorial board of the journal Molecular Ecology therefore created the Molecular Ecology Prize in order to fill this void, and recognize significant contributions to this area of research. The prize selection committee is independent of the journal and its editorial board.

The prize will go to an outstanding scientist who has made significant contributions to molecular ecology.  These contributions would mostly be scientific, but the door is open for other kinds of contributions that were crucial to the development of the field.  The previous winners are: Godfrey Hewitt, John Avise, Pierre Taberlet, Harry Smith, Terry Burke, Josephine Pemberton, Deborah Charlesworth, Craig Moritz, Laurent Excoffier, Johanna Schmitt, Fred Allendorf, Louis Bernatchez, Nancy Moran, Robin Waples, Scott Edwards, Victoria Sork, and Fuwen Wei.

Please send your nomination with a short supporting statement (no more than 250 words; longer submissions will not be accepted) and the candidate’s CV directly to Anne Yoder ( by Monday, June 6, 2022.  Organized campaigns to submit multiple nominations for the same person are not necessary and can be counterproductive.  Also, note that nominations from previous years do not roll over.

With thanks on behalf of the Molecular Ecology Prize Selection Committee

Interview with the authors: Associations between MHC class II variation and phenotypic traits in a free-living sheep population

In a recent paper in Molecular Ecology, Huang, Dicks and colleagues analysed variation in the major histocompatibility complex (MHC) and phenotypic traits in an unmanaged population of sheep living on an island off the coast of Scotland. This population of sheep has been studied closely for more around 70 years, providing a very rare level of insight and statistical power to evolutionary genetic studies. The MHC is among the most variable parts of mammalian genomes and has long been known to be encode proteins central to the adaptive immune system. Through their analyses, Huang, Dicks and colleagues found associations with levels of circulating antibodies and variation at MHC loci.

We sent some questions to the corresponding author of this work, Wei Huang, to get more detail on this new study.

Rams in St Kilda. Photo credit, Martin Adam Stoffel.

Can you describe the significance of this research for the general scientific community in one sentence?

This study demonstrated the direct link between immune genes and antibody levels in wild populations.

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

The major histocompatibility complex (MHC) contains a number of genes linked with immune defence in vertebrates. Associations between MHC variation and phenotypic traits or pathogens have been identified in many species. Also, selection on MHC genes has also been demonstrated in some studies. However, many previous studies only examined associations between MHC variation and a limited number of phenotypic traits or pathogens. Few of them have examined both MHC-fitness associations and MHC-trait associations. The longitudinal study of Soay sheep in St Kilda is a great system to study the associations between MHC variation and phenotypic traits and how the associations are linked with selection on MHC genes. Using three representative phenotypic traits monitored in thousands of sheep over decades, we are able to provide a full picture of MHC-trait associations in wild populations.

Can you describe the significance of this research for your scientific community in one sentence?

This study suggests associations between MHC and phenotypic traits are more likely to be found for traits more closely associated with pathogen defence than integrative traits and highlights the association between MHC variation and antibodies in wild populations.

What difficulties did you run into along the way? 

It is extremely hard to monitor populations and collect longitudinal data over decades. Thanks to our great field assistants and volunteers, the Soay sheep data has provided a good foundation. In terms of the specific study, the first difficulty is to genotype MHC in a large number of sheep. We used two steps to genotype the MHC genes. We first used genotype-by-sequencing to genotype hundreds of sheep. Then, benefiting from the high-density sheep SNP chip, we were able to use 13 SNPs to genotype MHC in the other thousands of sheep successfully.

Additionally, it is hard to choose the appropriate model. Some of our traits are not normally distributed and are also not closed to other common error structures. We instead used Bayesian statistical methods to run the analysis.

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

We used three representative traits to examine the associations between MHC variation and phenotypic traits. The traits included a fitness-related integrative trait, body weight, a measure of gastrointestinal parasites, faecal egg count, and level of three antibodies. All of the three traits are related to fitness. We only found associations between MHC variation and antibodies. Such results reflect the important role of MHC in immune defence in wild populations. Our study is one of the first studies to examine associations between MHC variation and multiple phenotypic traits. 

How do you think your results generalize to other systems?
Our study is based on the longitudinal study of Soay sheep. The large sample size provides great statistical power. Therefore, our results are reliable and solid. Also, we investigated phenotypic traits that have different links with immune defence. Therefore, our results can reflect the general pattern of MHC-trait associations.  

You conclude from your study that MHC variation is more likely to be associated with immune traits. How would you validate your findings for species with less rich data?

First, it is possible to use experiments to test the associations. In terms of wild populations, future studies can investigate multiple populations or multiple traits in a single population if they are restricted by the study length.

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

Our study demonstrates that it is important to study MHC-antibody associations. Future studies should focus on immune traits rather than only examine MHC-pathogen associations. Also, previous studies are often restrained by small sample size. It would be nice if future studies could increase their sample size to strength the statistical power.

Huang, W.*, Dicks, K. L.*, Ballingall, K. T., Johnston, S. E., Sparks, A. M., Watt, K., Pilkington, J. G., & Pemberton, J. M. (2022). Associations between MHC class II variation and phenotypic traits in a free-living sheep population. Molecular Ecology, 31, 902– 915. 

*These authors contributed equally to this work

Interview with the authors

A holobiont view of island biogeography: Unravelling patterns driving the nascent diversification of a Hawaiian spider and its microbial associates

In their recent paper in Molecular Ecology, Armstrong and Perez-Lamarque et al investigated the evolution of the holobiont. The holobiont is the assemblage of species associated with a particular host organism. In the case of this study, the holobiont refers to the stick spider (Ariamnes), its microbiome and its endosymbionts. Taking advantage of the successive colonization of islands in a volcanic archipelego, Armstrong and Perez-Lamarque et al contrasted the evolutionary history of the host species to the different components of the holobiont on different islands in Hawai’i.

We sent some questions to the authors of this work and here’s what Benoît Perez-Lamarque, Rosemary Gillespie and Henrik Krehenwinkel had to say.

Ariames waikula (on the island of Hawaii). Photo credit: George Roderick

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

Gut microbiota play multiple roles in the functioning of animal organisms. In addition, host-associated microbiota composition can be relatively conserved over time and the concept of the “holobiont” has been proposed to describe the ecological unit formed by the host and its associated microbial communities. Yet, it remains unclear how the different components of the holobiont (the hosts and the microbial communities) evolve. This is what spurred our interest. Taking advantages of the chronologically arranged series of volcanic mountains of the Hawaiian archipelago, we were able to tackle this question and could investigate how the different components of the holobiont have changed as the host spiders colonized new locations.   

Can you describe the significance of this research for the general scientific community in one sentence?

The evolution of Hawaiian spider hosts and their associated microbes are differently impacted by the dynamic environment of the volcanic archipelago.
Can you describe the significance of this research for your scientific community in one sentence.

The host and its associated microbiota may not act as a single and homogeneous unit of selection over evolutionary timescales.

Ariames waikula (on the island of Hawaii). Photo credit: George Roderick

What difficulties did you run into along the way? 

All the different components of the holobiont are not as easy to study. For instance, for the host spiders, we used double digest RAD sequencing (ddRAD) to obtain genome-wide single nucleotide polymorphism data. With such data, we could precisely reconstruct the evolutionary histories of the different spider populations in the last couple of million years and tracked the finest changes in their genetic diversity. In contrast, characterizing the composition of the microbial components is much more challenging. We used metabarcoding of a short region of the 16S rRNA gene to identify the bacteria present. However, over such short evolutionary timescales, this DNA region is too conserved to accumulate many differences between isolated populations. Therefore, we had high-resolution data for the spider hosts but comparably low-resolution data for the bacterial communities. To ensure that the observed patterns were not artefactually driven by such differences of resolutions, we complemented our analyses with a range of simulations to assess the robustness of our findings.

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

We find that the different components of the holobiont (the host spiders, the intracellular endosymbionts, and gut microbial communities) respond in distinct ways to the dynamic environment of the Hawaiian archipelago. While the host spiders have experienced sequential colonizations from older to younger volcanoes, resulting in a strong (phylo)genetic structuring across the archipelago’s chronosequence, the gut microbiota was largely conserved in all populations irrespectively of the archipelago’s chronosequence. More intermediately, we found different endosymbiont genera colonizing the spiders on each island. This suggests that this holobiont does not necessarily evolve as a single unit over long timescales.

In the conclusion to your study, you point out how different components of the holobiont likely contribute differently to selection/colonization history in this system. If you had unlimited resources, what would you do to strengthen this conclusion? 

We indeed suspect that the different components of the holobiont probably did not act as a single and homogeneous unit of selection during the colonization of the Hawaiian archipelago. First, it would be ideal to perform an even broader sampling, targeting more Ariamnes populations and species from older islands, to better characterize the long-term changes of the different holobiont components. Using sequencing technics with better resolution (as detailed below) would also improve our characterization of the microbial component(s) of the holobiont. Second, to properly test for selection, we should perform transplant experiments of the bacterial communities between spider populations/species and measure whether or not it impacts holobiont fitness. We would expect to find a significant impact of the transplant for the endosymbionts, but no or low impact for the gut bacterial communities of these spiders.

The geological history of Hawai’i provides a powerful system to build understanding of the evolution of holobiont. Are you aware of other systems where similar studies could be performed? (I appreciate that this is related to the previous question!).

Many other archipelagos, with similar island chronosequences, like the Canary Islands or the Society Islands, are also ideal for testing hypotheses on the evolution of holobionts. Within the Hawaiian archipelago again, we could replicate our work on other holobiont systems. For instance, among arthropods, plant feeders might rely more importantly on their microbiota for their nutrition, and this might likely translate into different patterns of holobiont evolution.

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

As previously said, one main limitation is the low resolution of the 16S rRNA metabarcoding. This prevented us to look at the evolutionary history of the individual bacterial lineages. Using a new model, we have recently tackled this issue of low resolution ( and we reported little evidence of microbial vertical transmission in these holobionts. Yet, the next step would be to move from classical metabarcoding to metabarcoding with longer sequencing reads (e.g. the whole 16S rRNA gene) or even metagenomics. It would provide more resolution for looking at bacterial evolution and would also bring more information on the functioning of these bacterial communities (e.g. are gut microbiota contributing to the digestion of these Hawaiian spiders in natural environments?).

Armstrong, E. E.*, Perez-Lamarque, B.*, Bi, K., Chen, C., Becking, L. E., Lim, J. Y., Linderoth, T., Krehenwinkel, H., & Gillespie, R. G. (2022). A holobiont view of island biogeography: Unravelling patterns driving the nascent diversification of a Hawaiian spider and its microbial associates. Molecular Ecology, 31, 1299– 1316. 

*Authors contributed equally

Interview with the authors: Transposable elements mark a repeat-rich region associated with migratory phenotypes of willow warblers

In a recent paper in Molecular Ecology, Caballero-López and colleagues investigated the genetics of migratory behaviour in a two subspecies of willow warbler (Phylloscopus trochilus trochilus and Phylloscopus trochilus acredula). Previous work had identified several genetic markers associated with migratory behaviour in this species, but a particularly important candidate marker was unable to be mapped to previous genome assemblies. This suggested to Caballero-López et al, that the important marker may lie in a highly repetitive, and thus difficult to assemble, genomic region. Leveraging a recent genome assembly based on long-read technology and a quantitative PCR approach, Caballero-López et al found that the elusive migration marker is located in a genomic region rich in remnants of transposable elements.

We sent some questions to the primary author of this work, Violeta Caballero-López, to get some more insight and details about this exciting study.

Willow warbler male, 2017. Photo credit: Harald Ris.

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

My research aims to shed some light on our understanding of the genetics underpinning bird migration, which is currently very poor. Passerine birds migrate alone, and they follow the same routes to wintering grounds as their parents, fully relying on genetic mechanisms.

The motivation for this specific study was to try to characterize a region in the genome which varies between two subspecies of willow warbler that present differential migration to Africa. Until now, this region was only identified as an AFLP-derived marker which failed to be mapped to the genome. However, with the use of molecular techniques such as qPCR in combination with a good quality genome assembly, we could understand the nature of this element better.

AFLP: Amplified Fragment Length Polymorphism

qPCR: Quantitative Polymerase Chain Reaction

Can you describe the significance of this research for the general scientific community in one sentence?

Repeat-rich regions which are often considered “junk DNA” might have a larger role on phenotypes and function than previously thought.

Can you describe the significance of this research for your scientific community in one sentence?

It is important to revise the role of repeat DNA on the determination of a complex trait such as the determination of bird migratory routes.

Willow warbler singing in Siberia, 2017. Photo credit: Harald Ris.

What difficulties did you run into along the way? 

For more than 20 years “WW2” has been an elusive AFLP marker, observed to be fixed in the “northern” subspecies P. t. acredula. It could only be amplified in PCR as a 154 bp fragment and then sequenced, but its nature was totally unknown. The identification and curation of this sequence as a transposon (TE) was challenging because it is an old, degraded “LTR portion” of the full element. This required a willow warbler genome built with long read sequencing techniques that provided regions of the genome rich in repeat DNA. Locating the ends of this transposon was also complicated. Alignment “breaks” serve as a detection method for the target site duplications that mark the edge of these elements. However, they could not be used in our system because these TEs appear consistently embedded within a larger block of repeats. This interfered with our estimation of age and theories about the origin of the repeat.

LTR: Long terminal repeat

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

The most surprising finding here is the presence of a large repeat-rich region (>12 mb) that segregates in both willow warbler subspecies. This region is characterized by several copies of the WW2 derived variant, which turned out to be part of a transposable element belonging to the endogenous retrovirus family. Furthermore, we provide solid evidence of its independence from the other polymorphic regions in chromosomes 1 and 5. As this TE seems to be inactive, and no clear functional genes have been detected on its surroundings, it remains puzzling why this region correlates with migration in the willow warbler so strongly.

You end your paper describing how it’s premature to think that the association of the WW2 derived variant has a causal role on the trait. Based on your knowledge of the warbler genome, would you care to speculate as to the actual causal basis of the phenotype?

The most supported hypothesis is that migration is a complex trait influenced by gene packages. In the case of the willow warblers, I would speculate that the repeat rich region, and not necessarily the WW2 derived variant itself, could affect migration indirectly through 1) the formation of a structural variant in a chromosome that affects gene expression 2) the trans-regulation from this region of some gene(s) elsewhere in the genome 3) the presence of an adjacent gene outside this region that we have not been able to detect in the current genome assemblies so far 4) a missed single copy gene within the repeat rich region. However, the last one is the least likely given that areas with such a repeat density rarely contain functional genes.

Have you got any ideas of how you might test the hypothesis that chromosomal rearrangements were facilitated by the presence of TEs

The most exciting possibility is to visually confirm if these rearrangements have taken place. A way to test this empirically would be to obtain a karyotype of each subspecies and combine it with fluorescence in situ hybridization (FISH). First, a probe labelling the WW2 derived variants would signal the location of the repeat-rich region. Once the location of this region is resolved, it is possible to design several fluorescent probes outside of it to determine if the chromosomal arrangement around it is maintained both in the genome of P. t. acredula and its orthologue region in P. t. trochilus.

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

The biggest mystery within this study is the location in the genome of this repeat-rich region that contains several copies of the WW2 derived variant. One of the biggest challenges of genome assemblies is the mapping and correct location of repeat-dense sequences, and therefore future effort should be focused on targeting empirical evidence of the location of this region. Then we could get a better hint on if and/or how this region affects migration. Is it downstream or upstream of any gene complex? Is it silenced? how does its orthologue look in P. t. trochilus?

Typical working setup for the willow warbler team, 2021. Photo credit: Harald Ris.

Caballero-López, V., Lundberg, M., Sokolovskis, K., & Bensch, S. (2022). Transposable elements mark a repeat-rich region associated with migratory phenotypes of willow warblers (Phylloscopus trochilus). Molecular Ecology, 31, 1128– 1141.