Interview with the authors: Parent and offspring genotypes influence gene expression in early life

Early life stress can often have long-term fitness effects on organisms, and the molecular mechanisms behind this have long been of interest to biologists. While much work has demonstrated that changes in DNA methylation patterns are involved, the transcriptional effects of early life stress are less well-understood, particularly at a genome-wide level. In a recent Molecular Ecology paper, Daniel J. Newhouse and colleagues investigate the transcriptional effects of different parental care strategies in white-throated sparrows. In white-throated sparrows, there are two morphs, and two associated mating pair types: tan male x white female (TxW) pairs, and white female x tan male (WxT) pairs. While TxW pairs provide biparental care, WxT pairs provide female-biased parental care. Newhouse and colleagues use RNA sequencing to assess the transcriptional effects of these differences in parental care strategies. They find evidence of an elevated stress response in offspring of WxT pairs, which provide female-biased parental care. For more information, read the full article, and see the in-depth interview with the authors below.

 A white morph female white-throated sparrow feeding her nestlings. Photo credit: Tiffany Deater.

What led to your interest in this topic / what was the motivation for this study? 
Early in graduate school, I participated in the white-throated sparrow genome sequencing project. That project was my crash course in white-throated sparrow biology, and the unique genetics and associated behaviors of the sparrows fascinated me. Most work on white-throated sparrows focuses on the adults, but nestlings are relatively understudied.  Depending on the adult pair type of the nest, nestlings will either receive biparental care (parents=tan morph male & white morph female) or female-biased parental care (parents=white morph male & tan morph female). Essentially, I wanted to see how this parental care variation impacted the nestlings.

What difficulties did you run into along the way? 
Finding white-throated sparrow nests was much harder than I ever imagined. Hiking through bogs while fighting off swarms of biting insects made it even more difficult. Thankfully, I have wonderful collaborators who are amazing at finding nests.
Also, when we designed this study, there weren’t many examples of RNA-seq from bird blood. White-throated sparrow nestlings are very small, so the amount of blood we can collect is quite small. RNA extractions proved more difficult than expected, but we managed to sequence a sufficient amount for the study.

What is the biggest or most surprising finding from this study? 
It was surprising to see a solid signature of morph-specific gene expression. As adults, there are many differences in the transcriptome between morphs and these correlate strongly with their behavior. White-morph and tan-morph nestlings look the same and do not exhibit any morph-specific behaviors like we see in adults. Despite this, we found that a large number of genes found within the chromosomal inversion are differentially regulated. Some of these genes have also been previously identified in the brain of adult white-throated sparrows. It was cool to see the same genes appear very early in life and in a much different tissue (blood).

Moving forward, what are the next steps for this research? 
From a genomics perspective, it would be great to identify the regulatory mechanisms underlying the gene expression signatures we identified here. Additionally, within a single nest, there are both white morph and tan morph nestlings. This allows us to look at nestling morph specific responses to variation in parental care. We identified some differences between the morphs within a nest, but were ultimately limited by sample size to discuss this in depth. I think this will be a really interesting topic to explore further.

What would your message be for students about to start their first research projects in this topic? 
I suggest pursuing integrative projects, like much of the work published in Molecular Ecology. Associated with that, I suggest networking and establishing collaborations early. We can’t all be experts in everything, so collaborating with research groups that complement your interests can be beneficial.
More generally, keep up with the literature as much as you can. The more you know about your system and anything related to it, the better. Don’t forget to read up on methods papers, too. Data analysis is very important so having a grasp on analytical concepts will really help.

What have you learned about science over the course of this project? 
There’s no universal way to analyze data. There are so many tools to process genomic data, so it can be overwhelming at times to keep track of everything. I also learned that data analysis takes much longer than you plan. Inevitably something won’t work, so keeping a positive attitude throughout is crucial.

Describe the significance of this research for the general scientific community in one sentence.
Parental genotype is correlated with a transcriptomic stress response in their offspring.

Describe the significance of this research for your scientific community in one sentence.
Half of all adult white-throated sparrow pairs provide female-biased parental care and this stable parental care strategy induces a transcriptomic stress response in their offspring.

Newhouse DJ, Barcelo‐Serra M, Tuttle EM, Gonser RA, Balakrishnan CN. Parent and offspring genotypes influence gene expression in early life. Mol Ecol. 2019;28:4166–4180. https://doi.org/10.1111/mec.15205.

Interview with the authors: Lack of gene flow: Narrow and dispersed differentiation islands in a triplet of Leptidea butterfly species

A diverse array of evolutionary processes contribute to diversity and divergence, and as large genomic datasets become more readily available our ability to parse apart these processes increases. In their recent Molecular Ecology publication, Talla et al. generate genomic data from six populations of wood white butterflies and use this data to try to tease apart the effects of introgression, recombination rate variation, selection, and genetic drift. In contrast to many previous genome-scan studies, they find no evidence of introgression or parallelism. Rather, they find support for genetic drift and directional selection as having shaped genomic divergence between species. Read the full article here: https://doi.org/10.1111/mec.15188, and learn more below with a behind-the-scenes interview with the authors.

What led to your interest in this topic / what was the motivation for this study? 
We have a general interest in understanding the contribution of different molecular mechanisms and evolutionary forces to genomic differentiation between diverging lineages. Previous research in this area has revealed a rather complex interaction between selection, genetic drift, recombination rate variation and introgression and we thought we had found an ideal study system to tell these factors apart. In addition, we believe that it will be key to describe the divergence landscapes in many different taxonomic groups to understand the relative importance of different molecular and evolutionary factors in lineages with different genetic/genomic features, demographic histories and life-history characteristics.

What difficulties did you run into along the way? 
Our expectations were not really met regarding the study system. First, earlier observations suggested that hybridization occurs between species pairs in the Leptidea group when they occur in sympatry, indicating that introgression might differ between sympatric and allopatric species pairs, but this turned out to be wrong. Second, butterflies lack centromeres and this could indicate a more even recombination landscape than what is generally observed in taxa with centromeres, but this we could not address with our data. Third, we expected that the divergence time between lineages was short which was again not right. Finally, the three species are characterized by large differences in karyotype, and we wanted to investigate if chromosomal rearrangements could underlie reproductive isolation, but this goal was actually out of reach with our data.

What is the biggest or most surprising finding from this study? 
It was surprising to us that there was no evidence for interspecific gene flow since hybrids have been observed. We were also very surprised by the deep divergence times between these virtually identical species. Besides that, we do not think the results are really surprising, but they do give some novel insight into the patterns of genomic divergence when there is no introgression and when chromosomes lack centromeres. One observation that we found interesting was that regions with high genetic differentiation (FST) had higher genetic divergence (DXY) than the genomic average. This may sound intuitive, but many previous ‘genome-scan’ studies have in fact found a negative relationship between differentiation and divergence, most likely as a consequence of reduced recombination in some regions leading to reduced diversity already before lineages started to diverge.

Moving forward, what are the next steps for this research? 
We are developing more resources to generate genome assemblies of multiple species in the study system and we are also working on establishing high-density linkage maps for multiple populations with different karyotypes. These tools will help us pinpoint chromosome rearrangements and investigate if these have played a role in the divergence process. The data will also be used to quantify the effects of fissions and fusions on the recombination landscape. We are also delving into other approaches to understand how ecological and behavioral differences between species leave footprints in the DNA sequences or epigenetic marks (and vice versa). Given the deep divergence times between species and the apparent lack of gene flow, we will mainly focus on intraspecific comparisons where we observe some incompatibilities between some populations with distinct karyotypes.

What would your message be for students about to start their first research projects in this topic? 
We would suggest to read up on the previous literature in detail. We also encourage students to contact leading researchers in the field to discuss potential questions. Most people are really helpful and interested in knowing about other research efforts within their field. Discussing directly with experienced researchers also gives a hint on the key questions that should be addressed to extend the knowledge in the field. Given the copious amount of data we generate these days and the integrative nature of the questions we ask, it is also crucial to develop some skills in bioinformatics and scripting and to have a network of collaborators/colleagues that can provide help and support in both theory, experimental studies and data analyses.

What have you learned about science over the course of this project? 
That science is an extremely time-consuming and dynamic process and that the first glimpse on the data not necessarily reflects the final results. Moreover, that project plans need to be worked over regularly to accommodate for that the initial strategies did not really work out as they were outlined. We also acknowledge the importance of establishing a network of colleagues with expertise in different areas of the field – we experience that most research projects within our field are getting more and more integrative and it will be increasingly difficult to conduct advanced research without collaboration.

Describe the significance of this research for the general scientific community in one sentence.
We verify that genomic differentiation between diverging lineages is affected by a complex interaction between molecular mechanisms and evolutionary forces and stress the importance of studying organisms with different genomic features, demographic histories and life-history characteristics.

Describe the significance of this research for your scientific community in one sentence.
In contrast to much of the previous work on patterns of genomic diversity and differentiation, our study provides insight into divergence processes when the effects of gene flow and/or a shared and highly variable recombination landscape are absent.

Full article: Talla V, Johansson A, Dincă V, et al. Lack of gene flow: Narrow and dispersed differentiation islands in a triplet of Leptidea butterfly species. Mol Ecol. 2019;28:3756–3770. https://doi.org/10.1111/mec.15188.

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

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

Photo credit: Benjamin Titus.

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

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

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

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

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

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

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

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

Summary from the authors: Boomeranging around Australia: Historical biogeography and population genomics of the anti-equatorial fish Microcanthus strigatus (Teleostei: Microcanthidae)

Photo credit: Shigeru Harazaki.

The study of species and where they live is of particular interest to biologists, because it not only allows us to gain insight into genetic diversity, but also into how different populations interact. Animals with widespread distributions are often assumed to be of least concern. This can be misleading, as it does not take into account the possibility of fragmentation and population disjunction. The Stripey fish Microcanthus strigatus is one example, as it is listed as being of least concern on the IUCN Red List. Although it spans a wide distribution across the western Pacific and eastern Indian Oceans, our study suggests that populations in Western Australia, the southwest Pacific (including eastern Australia), Hawaii and East Asia are very genetically divergent. Several of these populations have been isolated since the last glacial cycle in the Pleistocene epoch, and are currently so fragmented that no contemporary genetic exchange occurs. This is of significant conservation concern as a once widespread population is revealed to consist of four cryptic groups, especially in light of evidence suggesting that the Hawaiian population is currently in decline and that the southwest Pacific population is distinct enough to warrant recognition as a different species. 

Read the full article:
Tea Y‐K, Van Der Wal C, Ludt WB, Gill AC, Lo N, Ho SYW. Boomeranging around Australia: Historical biogeography and population genomics of the anti‐equatorial fish Microcanthus strigatus (Teleostei: Microcanthidae). Mol Ecol. 2019;28:3771–3785. https://doi.org/10.1111/mec.15172

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

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

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

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

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

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

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

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

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

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

Full article:

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

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. https://doi.org/10.1111/mec.15146

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. https://doi.org/10.1111/mec.15117