Friday, March 31, 2017

Dissecting phases of speciation in stick-insects

I have worked on many different study systems over the years, including killer whales, livebearing fishes (family Poeciliidae) and Timema stick insects (for more detail please see my homepage). Originally, I started my research career with a Diploma in Biology in Germany (equivalent to a Masters in other countries), with my thesis work focusing on whistle communication in diverging killer whale populations around Vancouver Island in British Columbia, Canada. However, I soon realized that there are only so many questions you can ask using a study system that largely precludes running controlled experiments. Thus, for my PhD thesis and subsequent postdoctoral work, I focused on population divergence and speciation in livebearing fishes (Poeciliidae) living along various environmental gradients (e.g., gradients of predation, toxicity, and access to light). It was during this time that I realized that my main interest was not so much in one particular study system, but rather in discovering the mechanisms that create, maintain, and sometimes constrain, biodiversity. That interest eventually led me to add yet another study system to the mix in 2012: the system of Timema stick insects I am writing about here. More specifically, I wanted to use that particular system to study the potential role chemical communication might have on population divergence and speciation.

As the above paragraph suggests, each of my study systems has their own system-specific peculiarities [e.g., cultural differences seem to play a prominent role in driving population divergence in killer whales (Riesch et al. 2012), but not in stick insects or livebearing fishes]. However, they also have a lot in common. For example, selection from predation is integral to both, the livebearing-fish and stick-insect systems I study, while foraging specialization plays a prominent role in population divergence of both, stick insects and killer whales. Thus, the three systems simply constitute different examples of how ecologically-based divergent selection drives population divergence and ultimately (ecological) speciation.
A rain shower moving through the chaparral near Santa Barbara, California. Timema are often found in this biome of dense thickets and thorny bushes.
The idea that speciation can be thought of as a continuum is yet another concept that dates back at least to Charles Darwin’s world-changing On the origin of species. The concept in its modern form posits that pairs of populations move along a continuum between panmixis on one extreme end and complete reproductive isolation on the other. Progress can be towards speciation or towards collapse, the latter showcased by studies on speciation reversal in European whitefish Coregonus spp. (Vonlanthen et al. 2012), three-spined stickleback Gasterosteus aculeatus (Tayler et al. 2006), and cichlid fishes (Seehausen et al. 1997).

This concept of a speciation continuum has gained traction again in recent years (e.g., Hendry et al. 2009). Consequently, studies across closely related taxa at different phases of speciation are beginning to illuminate the processes and genetic changes underlying the formation of new species (Seehausen et al. 2014). It is well-known, of course, that speciation involves genetic differentiation, and that, in the absence of gene flow, genome-wide differentiation can readily build up by selection and drift. If speciation is to happen in the face of gene flow, however, the picture gets more complex. According to the genic model of speciation (Wu 2001), speciation is initiated by a few genetic regions that become resistent to gene flow before others. This results in a localized pattern of genetic differentiation, which becomes more genome-wide as speciation progresses.

In a recent study just published in the April issue of Nature Ecology and Evolution (http://www.nature.com/articles/s41559-017-0082), we took a closer look at the transitions between phases of genomic differentiation during speciation of Timema stick insects. Like other studies on the speciation continuum (including my other study systems), we were faced with a key problem: speciation is often slow enough that we cannot simply follow a single lineage through time to see in real-time how the process unfolds. The solution then is to take as many different snapshots of the process from different pairs of natural populations as possible, and to then start to reconstruct a bigger picture of what might be happening across different moments in time. This is exactly what we did using data from >100 populations of 11 species of Timema stick insects. Our work suggests that speciation can be initiated by few genetic changes associated with natural selection on few loci, but the overall process is multi-faceted and involves mate choice and genome-wide differentiation.


A male Timema cristinae on one of its host plants (genus Ceanothus).
Photo: Moritz Muschick
This study is the culmination of almost 30 years of research into this system, and consists of data collected between 1996 and 2014, including >1000 re-sequenced whole genomes. In fact, research in this system began when two of our coauthors, Cristina Sandoval (University of California in Santa Barbara, USA) and Bernie Crespi (Simon Fraser University, Canada), recognized this group harbours variation in phases of speciation. Patrik Nosil then entered the system in 2000 and eventually wrote his PhD on it, using experiments to estimate reproductive isolation. An important component of the current paper was the chemical ecology of stick insects. This part of the project was born ~2009, emerging out of initial discussions between Patrik and I, with additional input from Bernie Crespi and Gerhard Gries at Simon Fraser University. Fast-forward to the year 2012, where the alignment of different projects getting funded finally enabled us to team up at the University of Sheffield in the UK (key components were a European Research Council Grant to study the genomics of speciation to Patrik, a Human Frontier Science Program Postdoctoral Fellowship to study the role of chemical communication in speciation to myself, and a burgeoning collaboration with Zach Gompert, a statistical population geneticist from Utah State University).

The emphasis of the chemical ecological aspect of the project was on cuticular hydrocarbons (CHCs), the oily/waxy chemicals on the cuticle of insects that can function to prevent desiccation and physical injury (Drijfhout et al. 2013 in Behavioral and Chemical Ecology, pp. 91-114), but that have also been repeatedly implicated as integral to mate choice (e.g., Blows and Allan 1998; Chung et al. 2014). For stick insects, we found that populations that differed more strongly in their CHC profiles also had higher degrees of sexual isolation and stronger genome-wide differentiation. We confirmed the causal role of CHCs in mate choice by means of a perfuming experiment.

Evaporating hexane samples in Santa Barbara, California, as part of the perfuming experiment on Timema mate choice.
Preparing another CHC-hexane sample for analysis with the gas chromatograph in the Gries lab at Simon Fraser University in 2014. Photo: Sean McCann

When combining this CHC-data with other phenotypic data and genomic analyses, we uncovered that, consistent with early phases of genic speciation, colour-pattern loci that confer camouflage to particular host plants reside in localised genetic regions of accentuated differentiation between populations experiencing gene flow. Transitions to genome-wide differentiation are also observed with gene flow, but appear to have little to do directly with differentiation in color. Rather, genome-wide differentiation is associated with divergence in CHCs, which we show to be polygenic, modestly heritable traits. Thus, intermediate phases of speciation are not associated with growth of a few peaks or ‘islands’ in the genome. Finally, we show that complete reproductive isolation was associated with a conspicuous increase in the overall degree of genomic differentiation. Thus, although speciation is perhaps continuous, this does not mean it always proceeds in a strictly uniform fashion (this component was led by our collaborators Moritz Muschick, now a postdoctoral fellow at EAWAG in Switzerland, and Victor Soria-Carrasco, who is now a Leverhulme Early Career Fellow at the University of Sheffield). Overall, the results suggest that substantial progress towards speciation may involve the alignment of multi-faceted aspects of differentiation. We suspect similar conclusions may apply to other systems where strong reproductive isolation involves many traits and evolves in a polygenic fashion.

A female Timema bartmani, cryptic against the needles of white fir. Photo: Moritz Muschick.

In conclusion, although many questions remain unanswered, it seems clear that speciation in this group involves more than divergence in cryptic coloration, and the results point to mating isolation and other reproductive barriers, as well as geographic separation, as being important. Thus, the striking example of crypsis in Timema that has been the focus of many previous studies (e.g., Sandoval 1994; Nosil and Crespi 2006; Comeault et al. 2015) represents only one aspect of the multi-faceted speciation process.

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