Molecular Phylogenetics and Evolution 54 (2010) 194–210 Contents lists available at ScienceDirect Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev Evolutionary diversification of the gall midge genus Asteromyia (Cecidomyiidae) in a multitrophic ecological context John O. Stireman III a,*, Hilary Devlin a, Timothy G. Carr b, Patrick Abbot c a Department of Biological Sciences, Wright State University, 3640 Colonel Glenn Hwy., Dayton, OH 45435, USA b Department of Ecology and Evolutionary Biology, Cornell University, E145 Corson Hall, Ithaca, NY 14853, USA c Department of Biological Sciences, Vanderbilt University, Box 351634 Station B, Nashville, TN 37235, USA article info abstract Article history: Gall-forming insects provide ideal systems to analyze the evolution of host–parasite interactions and Received 3 April 2009 understand the ecological interactions that contribute to evolutionary diversification. Flies in the family Revised 17 August 2009 Cecidomyiidae represent the largest radiation of gall-forming insects and are characterized by complex Accepted 9 September 2009 trophic interactions with plants, fungal symbionts, and predators. We analyzed the phylogenetic history Available online 16 September 2009 and evolutionary associations of the North American cecidomyiid genus Asteromyia, which is engaged in a complex and perhaps co-evolving community of interactions with host-plants, fungi, and parasitoids. Keywords: Mitochondrial gene trees generally support current classifications, but reveal extensive cryptic diversity Adaptive diversification within the eight named species. Asteromyia likely radiated after their associated host-plants in the Aste- Fungal mutualism Insect-plant coevolution reae, but species groups exhibit strong associations with specific lineages of Astereae. Evolutionary asso- Cryptic species ciations with fungal mutualists are dynamic, however, and suggest rapid and perhaps coordinated Parasitoid changes across trophic levels. Asteraceae Ó 2009 Elsevier Inc. All rights reserved. Astereae Diptera 1. Introduction architecture’ underlying the similarities and differences in macro- evolutionary patterns (Price, 2005; Nyman et al., 2007). Recent phylogenetic and ecological analyses of endophytic in- Of all the major radiations of insects with endophytic lifestyles, sects are providing novel insights into the intricate ecological the Cecidomyiidae, or gall midges, are arguably the most poorly interactions between parasites and their hosts and the causes of understood. They represent the most extensive radiation of gall- evolutionary diversification (e.g., Eurosta gall flies, Craig et al., forming insects, with approximately 5400 extant described species 2001; Waring et al., 1990; Andricus gall wasps, Cook et al., 2002; (and many more undescribed species; Gagné, 2004), the overwhelm- Nematine sawflies; Nyman et al., 2006). These insects—gall-form- ing majority of which form galls on plants. The causes of this diver- ers, leaf-miners and stem borers—experience intimate interactions sity, and even its major features, remain largely mysterious with host-plant tissues, and possess traits that, when polymorphic, (Dorchin et al., 2004; Yukawa and Rohfritsch, 2005; Yukawa et al., can incur intense trade-offs in fitness among alternative hosts or 2005; Joy and Crespi, 2007). What is known is that, unlike many of host-plant modules (Yukawa, 2000; Karban and Agrawal, 2002; the other major gall-forming lineages of insects, which tend to be re- Harris et al., 2003; Wool, 2004), leading to ecologically-mediated stricted to one or a few plant families (e.g., Cynipidae [gall wasps] on genetic divergence and speciation (Craig et al., 1993, 1997; Fagaceae and Rosaceae), gall midges have radiated onto an enormous Stireman et al., 2005; Condon et al., 2008a,b). Moreover, endo- array of plant taxa from ferns to grasses (Gagné, 1989, 1994). In North phytic insects can be focal points for complex multi-trophic inter- America at least 89 plant families are known to host one or more actions which may strongly shape their anagenetic and species of gall midge (Gagné, 1989; Price, 2005), and in Japan at least cladogenetic evolution (Price et al., 1987; Stone and Schonrogge, 90 families are known to be used (Yukawa and Rohfritsch, 2005). 2003). Because there have been repeated and often dramatic Despite the great range of host-plants used by the family as a whole, radiations of endophytic insects in terrestrial ecosystems (Price, most individual species tend to be highly specific, feeding on only 2005), they offer excellent opportunities to compare the ‘ecological one or a few related host-plant species (Gagné, 1989), and often on particular plant organs (e.g., leaf, stem, flower; Jones et al., 1983; Waring and Price, 1989; Joy and Crespi, 2007). * Corresponding author. Fax: +1 937 775 3320. Few studies have analyzed the evolution of gall midge-host- E-mail address: [email protected] (J.O. Stireman III). plant associations with modern phylogenetic and comparative 1055-7903/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2009.09.010 J.O. Stireman III et al. / Molecular Phylogenetics and Evolution 54 (2010) 194–210 195 methods. Here, we seek to provide some insight into the evolution 1.1.3. Fungal associations of these associations by reconstructing the phylogeny and the In addition to the developing midge larvae, the galls of most evolution of key traits and associations for members of one small Asteromyia species house fungi. Such associations are not uncom- cecidomyiid genus, Asteromyia. We employ DNA sequence-based mon among galling Cecidomyiidae, as two major clades are known phylogenetic reconstructions and data on ecological associations to have fungal associations, the tribe Asphondyliini (Supertribe to examine the evolutionary history of host-plant use and its Cecidomyiidi) and the (likely sister) tribes Alycauliini and Lasiopte- consequences for diversification. We take a community perspec- rini (Supertribe Lasiopteridi; Gagné, 1989; Roskam, 2005; Yukawa tive in examining the evolutionary history of this genus, consider- and Rohfritsch, 2005). Like Scolytinae and Platypodinae ‘‘ambrosia” ing not only the associations with particular host-plant taxa and beetles, which have nutritional symbioses with fungi, these cecid- host-plant structures, but also associations with fungal symbionts omyiids are sometimes referred to as the ambrosia gall midges that may mediate interactions with the host-plant, and with (e.g., (Bisset and Borkent, 1988). The symbiosis with fungi by Aster- natural enemies that may encourage ecological diversification. omyia midges is particularly unusual. The galls cause only minor We explore how each of these interacting partners may have deformation of plant tissue itself. The bulk of the gall structure is influenced the evolutionary diversification of Asteromyia gall mid- formed by an associated hemibiotrophic fungus in the genus Bot- ges, utilizing this system as a model to assess and generate ryosphaeria (Camp, 1981; Janson pers. comm.). The function of hypotheses and predictions concerning the evolution of insect- the symbiosis with fungi is not entirely clear, in Asteromyia or in plant interactions. cecidomyiids in general. There has been debate about whether Asteromyia is a plant or fungus feeder (Gagné, 1968, 1989; Bisset and Borkent, 1988), although recent data support the latter 1.1. The genus Asteromyia (Janson, pers. comm.). While most of the ambrosia gall midges are likewise thought to be fungus-feeders (Bisset and Borkent, 1.1.1. Taxonomy 1988), the debate is fueled by the degree of dietary variation be- The genus Asteromyia currently consists of eight recognized tween closely-related species. A. modesta, for example, is appar- species, primarily occurring in North America, but with one spe- ently herbivorous: fungal mycelia are sparse or absent in the cies, A. modesta, extending into South America (Table 1;(Gagné, simple inconspicuous ‘‘pocket” like blister galls (Fig. 1), which 1968, 1969, 1989, 1994). Thirty-three species of Asteromyia were resemble circular ‘‘blotch” mines of some leaf-mining insects. originally described in the early 20th century (primarily by Felt, How this variation is maintained is uncertain, because the basal e.g., Felt, 1910), however due to the lack of consistent morpholog- lineages of the Cecidomyiidae, subfamilies Porricondylinae and ical differences, this multitude of species was distilled down to se- Lestremiinae, are not associated with plants at all, but rather feed ven morphologically distinguishable species by Gagné (1968) in his exclusively on fungus, suggesting that the fungal associations in careful revision of the genus, with an additional species recognized the ambrosia gall midges may reflect ancestral associations (Bisset one year later (Gagné, 1969). In his recent catalogue of Cecidomyii- and Borkent, 1988). At the moment, there have been no rigorous dae, (Gagné, 2004) lists an additional species, A. grindeliae, which phylogenetic analyses of the evolution of fungal associations in he had previously synonymized with A. modesta (Gagné, 1968); cecidomyiids (though see Roskam, 2005 for an overview of the however, we could find no published justification or official rein- alternative arguments). statement of this species. Several additional species occurring on Baccharis were recognized by Gagné and Boldt (1995); these taxa 1.1.4. Natural enemies were not described, however, due to lack of