ORIGINAL ARTICLE doi:10.1111/evo.13683 Cascading reproductive isolation: Plant phenology drives temporal isolation among populations of a host-specific herbivore Glen R. Hood,1,2,3 Linyi Zhang,1 Elaine G. Hu,1 James R. Ott,4 and Scott P. Egan1 1Department of Biosciences, Anderson Biological Laboratories, Rice University, Houston, Texas 77005 2Department of Biological Sciences, Wayne State University, Detroit, Michigan 48202 3E-mail: [email protected] 4Population and Conservation Biology Program, Department of Biology, Texas State University, San Marcos, Texas 78666 Received November 5, 2018 Accepted January 7, 2019 All organisms exist within a complex network of interacting species, thus evolutionary change may have reciprocal effects on multiple taxa. Here, we demonstrate “cascading reproductive isolation,” whereby ecological differences that reduce gene flow between populations at one trophic level affect reproductive isolation (RI) among interacting species at the next trophic level. Using a combination of field, laboratory and common-garden studies and long-term herbaria records, we estimate and evaluate the relative contribution of temporal RI to overall prezygotic RI between populations of Belonocnema treatae, a specialist gall- forming wasp adapted to sister species of live oak (Quercus virginiana and Q. geminata). We link strong temporal RI between host-associated insect populations to differences between host plant budbreak phenology. Budbreak initiates flowering and the production of new leaves, which are an ephemeral resource critical to insect reproduction. As flowering time is implicated in RI between plant species, budbreak acts as a “multitrophic multi-effect trait,” whereby differences in budbreak phenology contribute to RI in plants and insects. These sister oak species share a diverse community of host-specific gall-formers and insect natural enemies similarly dependent on ephemeral plant tissues. Thus, our results set the stage for testing for parallelism in a role of plant phenology in driving temporal cascading RI across multiple species and trophic levels. KEY WORDS: Belonocnema treatae, live oak, multitrophic multieffect trait, Quercus, reproductive isolation. Ecology plays an essential role in the speciation process when (Talley et al. 2001). However, a synthetic view of how ecologically barriers to gene flow evolve between populations as a result of driven RI arises requires understanding (1) if and how individual ecologically based divergent natural selection (Rundle and Nosil phenotypes are affected by divergent selection between popula- 2005). Thus, knowledge of how such barriers arise is necessary tions experiencing different environments, (2) if those effected for understanding how divergence among populations is initiated phenotypes reduce gene flow between diverging populations, and and maintained. In the last 35 years, the role that ecology plays (3) how multiple barriers accumulate to contribute to RI. during population divergence and the evolution of reproductive To date, a small but growing number of studies have esti- isolation (RI) has been the subject of intensified research (Nosil mated the combined effect of multiple barriers to RI (e.g., Ramsey 2012). Consequently, the study of ecologically based RI has in- et al. 2003; Martin and Willis 2007; Matsubayashi and Katakura creased our understanding of how barriers to gene flow evolve in a 2009; Dopman et al. 2010; Sanchez-Guillen et al. 2012; Lackey diversity of taxa including plants (Richards and Ortiz-Barrientos and Boughman 2017; Paudel et al. 2018; Sambatti et al. 2012). 2016), fishes (Rundle 2002), insects (Feder et al. 1994; Egan However, even for well-studied systems, biologists frequently and Funk 2009), birds (Huber et al. 2007), amphibians (Twomey lack a detailed understanding of the relative contributions of the et al. 2014), reptiles (Rosenblum et al. 2010), and mammals individual components of RI to total RI and/or the chronological C 2019 The Author(s). Evolution C 2019 The Society for the Study of Evolution. 554 Evolution 73-3: 554–568 TEMPORAL ISOLATION IN GALL WASP POPULATIONS order in which components of RI evolve. Consequently, the host plants and/or the probability that immigrants successfully forms of RI most common during ecological speciation remain reproduce. Thus, for short-lived insect species, temporal RI may unclear (see “Twenty-Five Major, Yet Unresolved, Questions in represent an ecological adaptation of a population to its environ- Ecological Speciation,” Nosil 2012). ment that can also function as a critical barrier to gene flow and Here, we investigate an understudied driver of RI, which promote population divergence. we describe as “cascading RI,” whereby ecological differences in In their comprehensive review of the role of allochrony dur- traits that reduce gene flow between populations at one tropic level ing speciation, Taylor and Friesen (2017) conclude that (1) dif- “cascade” upward to similarly affect interacting organisms at the ferences in the timing of key life-cycle events can contribute to next trophic level. To test for cascading temporal RI in a special- RI, (2) the effect of allochrony can substantially reduce gene flow ized insect herbivore, we first document phenological differences when it disrupts the overlap of breeding times between popula- in the trait budbreak, a hypothesized driver of RI between sister tions, and (3) allochrony may often be the initial or key driver plant taxa occupying different microhabitats. Second, we test the of speciation. The last point is important owing to the sequen- role of ecology (differences in the timing of leaf availability) in tial nature of RI—those barriers that act earliest in the life cycle generating temporal RI between host-plant-associated insect pop- contribute more to total RI when multiple barriers are present ulations dependent on this ephemeral resource. Third, we combine (Hood et al. 2012; Ramsey et al. 2003). Thus, the accumulation estimates of temporal RI with two established prezygotic barri- of barriers to gene flow can act as sieves, with the earliest acting ers to estimate total RI among host-associated insect populations. barriers allowing fewer individuals to pass genes to the next gen- Cascading RI differs from “cascading divergence” whereby both eration. Despite the orthodoxy of the perceived role of allochrony ecological and genetic divergence at one trophic level results in in insect speciation, Forbes et al. (2017) found that allochronic concordant divergence of species at the next trophic level that isolation initiated speciation via host plant shifting in only 13 adapt to the newly created niche. For example, the shift and sub- out of 85 (15%) herbivorous insect systems, while Taylor and sequent divergence of Rhagoletis pomonella from native plant Friesen (2017) documented only nine cases of allochrony driv- hawthorn to apple resulted in the formation of a new niche that ing ecological speciation (five of which were in insect systems) was colonized by multiple parasitoid species that have diverged and an additional 56 systems in which “further study is needed.” in tandem with R. pomenella (Hood et al. 2015). Moreover, while Thus, the assertion that “allochronic isolation is common . for cascading RI is a necessary component of cascading divergence, phytophagous insects” (Berlocher and Feder 2002) may be less RI at both trophic levels must be under (partial) genetic control. substantiated by available data than models and theory alone Cascading RI, as defined herein, need not necessarily be con- suggest. trolled genetically. Herein, we postulate that the ecological driver (divergence Herbivorous insects and their host plants are excellent sys- in the timing of the trait budbreak) hypothesized to generate tems to investigate temporal RI as many species are highly spe- RI between sister species of live oaks Quercus virginiana and cialized on one or a few closely related plant species and depen- Q. geminata (Cavender-Bares and Pahlich 2009), cascades dent on specific tissues as sites for courtship, mating, feeding, across trophic levels to generate temporal RI between host- oviposition, and development (Bernays 1998; Funk et al. 2002). plant-associated populations of the gall-former, Belonocnema Host plant specialization often has a temporal component as plant treatae. Here, we combine field and laboratory observations, tissues can be used for only short windows of time. Thus, the tim- common-garden experiments and long-term herbaria records to ing of insect life-cycle events becomes critical when reproductive link phenological differences in host plant flowering and leaf success is intrinsically tied to the acquisition of ephemeral re- flush to temporal RI among associated gall-former populations. sources. As a result, host-tissue-specific populations of insects Importantly, two additional phenotypes, adult longevity and adapting to different plant environments that vary in the produc- breeding time, interact to affect temporal RI. If populations tion of a critical plant tissue may experience a degree of “tempo- emerge at different times on alternative host plants, but earlier ral” or “allochronic” RI as a consequence of ecologically based emerging individuals survive long enough to partially/completely selection (Mopper 2005). Evidence for allochrony among host- overlap with the later emerging population during reproduction, plant-associated insect populations exists for a number of taxa, for RI will be reduced/eliminated.