In the fall of 2008, I was wrapping up my research as a M.Sc. student in the Population and Conservation program at Texas State University. I had spent several months narrowing down potential Ph.D. advisors. I was looking for a lab that studied plant-insect interactions and insect speciation. After sending out scores of emails, I had narrowed my search down to five prospective labs.
In November of that same year, I was presenting my research at the Entomological Society of America’s annual meeting in Reno, Nevada. Luckily, four of my five potential Ph.D. advisors were also attending the meeting. As a self-promotional tool, I sent out emails inviting the four potential advisors to attend my talk. My goal was simple—give a talk so great that I would be offered a Ph.D. position on the spot. Unfortunately, I was scheduled to present at 8:15 am on the final day of the conference, which practically ensured low attendance in general and no-shows from all four candidates. In my experience, the night before the last day of a conference is the most social and goes well into the night. My talk about differences in body size and fecundity between the alternating generations of cyclically parthenogenic gall wasps was surely not going to get tired bodies out of bed.As 8:00 am approached the morning of my talk, a few people filed into the room (i.e., my current advisor, the moderator, and a few early-morning speakers). A few minutes before 8:00, the moderator informed me that the first talk had been cancelled. Great, I thought, even the 8:00 am speaker couldn’t show up for his own talk! I walked out into the hall realizing my plan to make a positive impression on potential advisors had failed. Then I heard someone say, “Glen Hood?” I looked up to see Jeff Feder, one of the potential Ph.D. advisors, surrounded by two oversized luggage bags and one crammed backpack. Of the four labs I had contacted, this was the one I was most interested in, as Jeff studied ecological speciation in Rhagoletis fruit flies. He explained that he was currently on sabbatical in Germany, was briefly in town to give an invited talk, and had to catch a flight in less than two hours. Despite his busy schedule, Jeff said he wanted to make sure we were able to chat before he left. To this day, I still do not know if Jeff was able to catch his originally scheduled flight to Germany. However, in the 10 minutes before I was scheduled to present, Jeff introduced me to the topic that was to consume the next six years of my life.
When I joined Jeff’s lab in the summer of 2009, the groundwork was already laid for what would result in a paper we recently published in PNAS (Hood et al. 2015). That same year, Andrew Forbes, a former graduate student in the Feder lab (now in the faculty at the University of Iowa), was wrapping up his dissertation research. The central theme of Andrew’s research was simple: a major cause of biodiversity may be biodiversity itself. The process, referred to as “sequential” or “cascading” speciation or divergence, has been proposed to help explain a number of diverse patterns including radiations following mass extinctions, and species diversity in the tropics. However, sequential speciation could perhaps be most important for understanding the incredible diversity of plant feeding insects and their parasitoids. The idea is that when plant-feeding insects diversify by adapting to new host plants, they create a new habitat for their insect parasites (parasitoids) to exploit and adapt to. If a parasitoid shifts to the new habitat, it can encounter the selection pressures as its insect host, which could result in the parallel divergence of insect host and parasitoid. However, there were relatively few empirical examples of sequential speciation within insect communities (Stireman et al. 2006, Abrahamson & Blair 2008, Feder & Forbes 2010). The major issue is that analyses of sequential speciation across trophic levels can be complicated by a lack of information about the natural history and geographic context of host shifting. What Andrew and Jeff needed to test the sequential speciation hypothesis was a well-defined system with a well-resolved natural history to directly test whether ecological adaptation can sequentially amplify diversity.
It just so happens that Jeff had spent his entire career working in the perfect system to address these issues. Fruit flies in the Rhagoletis pomonella species complex are a model for ecological speciation via host-plant shifting. In particular, the recent host shift of the apple maggot fly, R. pomonella, from its ancestral host plant hawthorn to introduced, domesticated apples in the last ~160 years, is an example of incipient speciation (i.e., host race formation) in action. To test for sequential divergence, Andrew and Jeff used the Rhagoletis-specific parasitoid wasp, Diachasma alloeum that lays it eggs into the larvae of the fly. Their study showed that populations of D. alloeum attacking hawthorn and apple host races of R. pomonella as well as sister species R. mendax (host: blueberry) and R. zephyria (host: snowberry) had indeed formed genetically distinct host races as a result of specializing on diversifying fly hosts. In addition, the same ecological traits that differentially adapt R. pomonella to their respective host plants and reduce gene flow between diverging populations (host-related differences in the timing of adult eclosion, and host fruit odor discrimination behaviors) are the same barriers that reproductively isolate D. alloeum to their respective fly hosts. Andrew, Jeff, and colleagues published the results in a paper in Science (Forbes et al. 2009).
Fig. 1. (A) a single sequential divergence event and (B) sequential divergence with multiplicative amplification of biodiversity.
My job as a new Feder lab graduate student was to attack the next, obvious question: How common is the sequential speciation phenomenon in a broader context? The plan was simple: follow the road map set by Forbes et al. (2009) in the Science paper to determine if sequential speciation could not just linearly (one fly to one parasitoid), but multiplicatively (one fly to many parasitoids) amplify biodiversity across the entire community of parasitoid wasps attacking R. pomonella group flies. Jeff, Andrew and I formed a team of evolutionary biologists including then Feder lab graduate student Tom Powell (currently a post-doc at University of Florida), Notre Dame research assistant professor Scott Egan (now faculty at Rice University), a graduate student of Forbes’, Gabriela Hamerlinck (currently a post-doc at the University of Wisconsin), and Jim Smith (faculty at Michigan State) to contribute to the cause.
Fig. 2. The apple maggot fly, Rhagoletis pomonella, on its native host hawthorn, Crataegus mollis. Photo credit: Hannes Schuler.
We first outlined a series of conditions modified from Dres & Mallet (2002) and Abrahamson & Blair (2008) that must be met to support the sequential divergence hypothesis. In addition these criteria helped guide our experimental approach. These conditions are as follows:
(1) Shift to a new host resource and multiple host associations occur in close geographic proximity
(2) Host-associated populations form distinct genetic clusters (spatially replicable), but experience gene flow at appreciable rates
(3) Females and potentially males display host preferences and discriminate among alternate hosts
(4) Host choice is linked to mate choice facilitating assortative mating resulting in prezygotic habitat isolation
(5) Host selection and fidelity are under some degree of genetic control and not due solely to maternal, learning or environmental effects
(6) Differences in insect phenologies track differences in host phenologies, resulting in temporal isolation
(7) Insect phenology is under some degree of genetic control, not due solely to maternal or environmental effects
(8) Fitness tradeoffs exist between host-associated populations resulting in migrants and hybrids having reduced fitness
To experimentally address if the conditions of sequential divergence were met in the remaining members of the parasitoid wasps community, Diachasmimorpha mellea and Utetes canaliculatus, we first sampled populations attacking multiple Rhagoletis hosts occurring in sympatry (populations attacking apple, hawthorn, flowering dogwood, snowberry, blueberry and black cherry flies). Given that members of the wasp community are specific to Rhagoletis, this fulfilled condition 1.
First, to test for genetic evidence of sequential divergence we genotyped populations of D. mellea and haplotype C U. canaliculatus for 20 and 21 microsatellite loci respectively. Similar to the pattern documented for D. alloeum by Forbes et al. (2009), both D. mellea and U. canaliculatus showed consistent allele frequency differences between host-associated populations. In addition, in genetic distance networks, populations clustered by host-association, not by geography. This result supports sequential divergence condition 2.
Fig. 3. Host fruit odor discrimination of (A) Diachasma alloeum, (B) Utetes canaliculatus, and (C) Diachasmimorpha mellea. Positive values represent preference for host fruit odor and negative values represent avoidance of host fruit odors in behavioral assays.
In their Science paper, Forbes et al. (2009) concluded that the origin of D. alloeum attacking the apple was not from a host shift from the hawthorn fly but from the blueberry fly. While our results for D. mellea and U. canaliculatus were not as conclusive, our study implies that Rhagoletis and its parasitoids may not always co-speciate in a strict 1:1 follow-the-leader fashion. Wasps attacking different flies in the community appear to be taking advantage of the new niche opportunity provided by Rhagoletis host shifts, not necessarily just the parasitoid infesting ancestral fly hosts. Thus, adaptive starbursts of sequential divergence may be the result of biodiversity radiating from several different origins within the community.
Key features of the fly and wasp life cycles and biology mirror each other, suggesting that the same host-plant related ecological adaptations that reproductively isolate the flies may also isolate wasps. For example, both flies and wasps use the volatiles emitted from the surface locate host plants, and both Rhagoletis and D. alloeum use the fruit as the site for courtship and mating. To test for host plant-related assortative mating caused by habitat isolation for D. mellea and U. canaliculatus, we coupled field observations of mating behavior with tests of host odor discrimination. By making observations of wasps at sympatric sites, we found that, similar to R. pomonella and D. alloeum, both D. mellea and U. canaliculatus mate on or near their host fruit. In addition, in tests of host fruit odor discrimination, wasps prefer the odors emitted from the surface of natal fruit and avoid non-natal odors. We estimated that fruit odor discrimination reproductively isolates D. alloeum, D. mellea and U. canaliculatus attacking different fly hosts by as much as 79%, 88% and 89% respectively, fulfilling sequential divergence conditions 3 and 4.
Fig. 4. The parasitoid wasp, Utetes canaliculatus, searching for its Rhagoletis fly host on a snowberry fruit. Photo credit: Hannes Schuler.
A common criticism of Forbes’ Science paper was that, unlike the work in Rhagoletis, there was no direct support for a genetic basis for host fruit odor discrimination. To address this issue, we reared D. alloeum originating from blueberry and hawthorn flies in non-natal apple fly and apple host plant environments. We then compared their response to the odors emitted from the surface of their parental host and their novel apple host. As predicted, both hawthorn- and blueberry-origin D. alloeum retained preferences for their respective parental host fruit odors, while avoiding non-natal apple volatiles. While not definitive, the rearing studies support condition 5, a genetic basis for behavioral differences in host fruit odor discrimination during sequential divergence.
The host plants of Rhagoletis fruit at different times of the year. For example, apples ripen 3–4 weeks before native hawthorns in sympatry. Thus, flies must eclose to coincide with the availability of ripe fruit to find mates and oviposition sites. Rhagoletis are univoltine, and live for 1 month as adults. Differences in eclosion timing therefore result in temporal mating isolation. The life cycle of the wasps mirrors that of their fly hosts. Wasps are also univoltine, and live 1–2 weeks. To assess the degree of temporal isolation due to variation in host phenology, we compared the timing of adult eclosion of U. canaliculatus and D. mellea attacking different fly populations in sympatry. We found that eclosion curves differed between sympatric populations of wasps attacking different Rhagoletis, tracking the eclosion times of fly hosts and the fruiting times of their host plants. Coupling the differences in eclosion times with calculations of adult longevity, we estimated that populations of D. alloeum, D. mellea, and U. canaliculatus are temporally reproductively isolated by as much as 75%, 55% and 96% respectively. This supports sequential divergence condition 6.
Fig. 5. Mean eclosion times averaged across collection sites of adult Rhagoletis attacking different host plants and Utetes canaliculatus, Diachasmimorpha mellea, and Diachasma alloeum attacking each fly host.
To link host-associated genetic differentiation to divergence in life history timing, we tested for associations between microsatellite genotypes and the timing of eclosion for U. canaliculatus and D. mellea. Similar to Rhagoletis and D. alloeum, we found 7 and 12 loci, for D. mellea and U. canaliculatus respectively, that displayed significant genotype or genotype × host effects with eclosion timing. This satisfied condition 7.
Finally, although host-associated fitness tradeoffs have been inferred for several species of Rhagoletis feeding in natal versus non-natal fruit, difficulty in reciprocally transplanting wasps in the lab made it difficult to directly experimentally test condition 8. However, hybrid wasps may display intermediate phenotypes for eclosion phenology and host odor discrimination that suffer reduced fitness for both parental host plant.
In conclusion, we found that sequential divergence can rapidly and multiplicatively amplify biodiversity of entire guilds or communities, as the same host-related ecological adaptations associated with host choice and life history timing cascade from host plant to fly to parasitoid. When combined with the results from Forbes et al. (2009), our study supports seven of the eight conditions we identified as necessary for sequential divergence in D. alloeum, D. mellea, and U. canaliculatus. Our results thus prompt the question: just how taxonomically widespread is sequential speciation and how often does it really contribute to the formation of biodiversity? For organisms such as insects and their parasites that experience and partition resources on a fine scale, the effects of new niche construction may cascade through ecosystems and have an important effect on biodiversity. I hope that our study motivates others to look for patterns of sequential divergence in their own systems.
To this day, I am still not sure what surprises me more – that sequential divergence can multiplicatively amplify biodiversity, or that Jeff risked missing an international flight to talk to a prospective graduate student!
Fig. 6. Notre Dame graduate student Glen Hood (left; big beard), and professor Jeffrey Feder (right; small beard) rearing Rhagoletis from rotting, infested apples.
References
Abrahamson WG, Blair CP (2008) Sequential radiation through host-race formation: herbivore diversity leads to diversity in natural enemies. Specialization, Speciation, and Radiation: The Evolutionary Biology of Herbivorous Insects, eds Tilmon KJ (University of California Press) pp 188–202.
Drès M, Mallet J (2002) Host races in plant-feeding insects and their importance in sympatric speciation. Philos Trans R Soc Lond B Biol Sci 357:471–492.
Feder JL, Forbes AA (2010) Sequential divergence and the diversity of insects. Ecological Entomology 35:67–76.
Forbes AA, Powell THQ, Stelinski LL, Smith JJ, Feder JL. 2009. Sequential sympatric speciation across trophic levels. Science 323:776–779.
Hood GR, Forbes AA, Powell THQ, Egan SP, Hamerlinck G, Smith JJ, Feder JL. 2015. Sequential divergence and the multiplicative origin of community diversity. PNAS 112:E5980–5989.
Stireman JO, Nason JD, Heard SB, Seehawer JM (2006) Cascading host-associated genetic differentiation in parasitoids of phytophagous insects. Proc Biol Sci 273:523–530.
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