Behavioural, ecological and genetic evidence ... - Wiley Online Library

doi:10.1111/j.1420-9101.2009.01696.x

Behavioural, ecological and genetic evidence confirm the occurrence of host-associated differentiation in goldenrod gall-midges N. DORCHIN,* E. R. SCOTT, ,à C. E. CLARKIN,* M. P. LUONGO,* S. JORDAN* & W. G. ABRAHAMSON* *Department of Biology, Bucknell University, Lewisburg, PA, USA Whitman College, Walla Walla, WA, USA

Keywords:

Abstract

assortative mating; Cecidomyiidae; enemy-reduced space; host races; reproductive isolation; speciation.

Host-associated differentiation (HAD) is considered a step towards ecological speciation and an important mechanism promoting diversification in phytophagous insects. Although the number of documented cases of HAD is increasing, these still represent only a small fraction of species and feeding guilds among phytophagous insects, and most reports are based on a single type of evidence. Here we employ a comprehensive approach to present behavioural, morphological, ecological and genetic evidence for the occurrence of HAD in the gall midge Dasineura folliculi (Diptera: Cecidomyiidae) on two sympatric species of goldenrods (Solidago rugosa and S. gigantea). Controlled experiments revealed assortative mating and strong oviposition fidelity for the natal-host species. Analysis of mitochondrial DNA showed an amount of genetic divergence between the two host-associated populations compatible with cryptic species rather than host races. Lower levels of within-host genetic divergence, gall development and natural-enemy attack in the S. gigantea population suggest this is the derived host.

Introduction One of the mechanisms thought to promote speciation in phytophagous insects is shifts to new hosts that lead to the establishment of new species via an intermediate step of host-race formation (Bush, 1969; Jaenike, 1981; Dre`s & Mallet, 2002). The role of host adaptation in speciation, especially in sympatry, has been one of the most debated topics among evolutionary ecologists over the last four decades (reviews in Tauber & Tauber, 1989; Via, 2001; Dre`s & Mallet, 2002), since Bush (1969) first argued the occurrence of sympatric host-race formation in the fruit fly Rhagoletis pomonella. Populations that experience hostassociated differentiation (HAD – speciation that is driven Correspondence: Netta Dorchin, Museum Koenig, Adenauerallee 160, Bonn 53113, Germany. Tel.: +49 228 9122 292; fax: +49 228 9122 212; e-mail: [email protected] àPresent address: E. R. Scott, School of Integrative Biology, University of Illinois, Urbana, IL 61801, USA.

by the adaptation to hosts) can be categorized as constituting a heterogeneous panmictic species, hostraces or established sister species based on the amount of gene flow between them (Dre`s & Mallet, 2002), with FST estimates for host-races, for example, typically ranging from 0.01 to 0.21 (e.g. McPheron et al., 1988; Via, 1999; Nason et al., 2002; Blair et al., 2005; Stireman et al., 2005). In order for HAD to occur, the host-associated populations must exhibit certain premating and postmating mechanisms that promote reproductive isolation and thus lead to genetic divergence. Possible premating mechanisms include assortative mating, wherein individuals show preference for mating within the same host-associated population (Berlocher & Feder, 2002; Rundle & Nosil, 2005), and phenological differences exist in emergence or activity times between host-associated populations (e.g. Bush, 1969; Smith, 1988; Craig et al., 1993; Nason et al., 2002; Thomas et al., 2003). A major post-mating isolating mechanism is host fidelity by ovipositing females, which has been

ª 2009 THE AUTHORS. J. EVOL. BIOL. 22 (2009) 729–739 JOURNAL COMPILATION ª 2009 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY

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documented in many case studies of phytophagous insects (summary in Dre`s & Mallet, 2002). Once a shift to a new host has occurred, escape from natural enemies may be a factor that offsets the cost of lower fitness resulting from physiological maladaptations to this host (Price et al., 1980; Jeffries & Lawton, 1984; Jaenike, 1990). Despite the accumulation in recent years of empirical evidence for HAD in herbivorous insects, the data currently available represent only a tiny fraction of potential cases within this extremely speciose and diverse group. Most documented case studies are based on a single type of evidence (usually genetic), and many involve man-made rather than natural scenarios [e.g. the fruit fly R. pomonella on hawthorn and apple (Bush, 1969), the European corn borer Ostrinia nubilalis on corn and mugwort (Bethenod et al., 2005; Malausa et al., 2005) and the aphid Acyrthosiphon pisum on alfalfa and red clover (Via, 1999; Via et al., 2000)]. Difficulty or reluctance to publish negative results, for cases in which HAD has not been found, further contribute to the difficulty of estimating the prevalence of this process. Many more examples are therefore needed to establish how prevalent HAD is under natural conditions, and under what circumstances it is likely to occur. In providing such empirical evidence it would be most informative to explore HAD in unrelated groups of herbivores that use the same set of hosts (Nason et al., 2002; Blair et al., 2005; Stireman et al., 2005). If HAD has indeed occurred independently in such cases, it would imply that this process constitutes an important source of biodiversity in nature. An exceptional model system in this context is the rich insect community associated with goldenrods (Solidago spp., Asteraceae) in North America, many members of which are highly specialized herbivores (McEvoy, 1988). Among nine cases of goldenrod-feeding insects in which HAD has been studied to date, four proved to have radiated on the sympatric S. altissima and S. gigantea (Abrahamson & Weis, 1997; Nason et al., 2002; Eubanks et al., 2003; Blair et al., 2005; Stireman et al., 2005). In the present study, we employ a multiple-evidence approach to obtain genetic, behavioural, morphological and ecological indications for HAD in the gall midge Dasineura folliculi (Diptera: Cecidomyiidae), which induces galls on Solidago rugosa and S. gigantea. We rely on multiple rather than on a single type of evidence as we consider it to be a more powerful approach for establishing the existence of HAD, while also inherently providing insight into premating and post-mating mechanisms that lead to and ⁄ or maintain HAD in a studied organism (see Via, 2001; Berlocher & Feder, 2002). Specifically we asked whether: (1) adults exhibit hostrelated morphological differences, (2) midges prefer mates that originate from the same host (is there assortative mating), (3) females prefer to oviposit on their natal host (is there host fidelity), (4) the hostassociated populations are genetically differentiated and

(5) one of the hosts offers enemy-reduced space to the population associated with it. We then discuss the significance of parallel host shifts in phylogenetically unrelated organisms as a means for estimating the prevalence of HAD in phytophagous insects.

Materials and methods The system The gall midge D. folliculi (Diptera: Cecidomyiidae) induces bud galls on the common and sympatric goldenrods S. rugosa and S. gigantea (Dorchin et al., 2007). Adults of this species live for 1–3 days, mate on or under the plants, and lay eggs between leaves that surround the apical buds. The galls do not have defined chambers, and the gregarious larvae develop among widened and thickened leaves that form loose clusters on growing shoot tips, with 5–80 larvae per gall. Larvae mature within 3–4 weeks and then leave the gall to pupate in the soil. Dasineura folliculi is multivoltine, completing at least four generations a year between early May and October (Dorchin et al., 2007). Collecting and rearing of insects We collected galls in 13 field sites within a 90-km radius of Lewisburg, Pennsylvania (N4057¢ W7653¢) between late April and October in 2005 and 2006. The collection sites included roadsides, natural areas and old fields that supported large, sympatric populations of S. rugosa and S. gigantea; although in some localities one of the plant species was much more abundant than the other. We collected all the galls we encountered that appeared mature (i.e. containing third-instar larvae) and dissected each of them under a stereomicroscope on the same or on the next day. The numbers of larvae of gall inducers and of all types of natural enemies were recorded for each gall, and mature larvae were transferred into 25-mL plastic cups filled with ProMix BXTM (Premier Horticulture, Dorval, QC, Canada) potting mix to allow pupation. Each soil cup contained all viable larvae originating from a single gall. The cups were individually covered with ventilated caps and kept moist in the laboratory, at room temperature, until the end of emergence (up to 4 weeks). The percentage of galls attacked by different types of natural enemies was compared between the two host plants via likelihood-ratio tests using J M P version 5.1.2 (SAS Institute, Cary, NC, USA). Morphology Emerging adults were preserved in 70% ethyl alcohol for morphological study, and 50–60 individuals from each host-associated population were later mounted on permanent microscope slides in euparal according to the method outlined in Gagne´ (1989). To detect possible


Host-associated speciation in goldenrod gall-midges

morphological differences between adults from the two host-associated populations, we measured wing length (which reflects body size) and the length of tergite 8 relative to that of tergite 6 of the abdomen in both males and females. The relative lengths of the distal abdominal tergites are one of the few morphological traits that may vary among species in the otherwise rather morphologically uniform adults in the genus Dasineura (Gagne´, 1989). The wing and tergite measurements were analysed with a two-way A N O V A (with host plant and sex as main effects). Tergite measurements were analysed after )X )0.5 transformation to better meet the distributional assumptions of A N O V A . Analyses were carried out in J M P version 5.1.2 (SAS Institute). Mate-choice experiments Adults of both sexes reared from field-collected galls as described above were paired in small glass vials in the four possible host-associated combinations (gigantea female with rugosa male, gigantea female with gigantea male, rugosa female with gigantea male and rugosa female with rugosa male). Because D. folliculi galls yield mostly single sex progeny (Dorchin et al., 2007), all females used in the experiments were known to be virgins. In order for mating to occur, females had to exhibit ‘calling behaviour’, during which they stood still and extended and slightly waved their ovipositor, probably emitting pheromones. Mating never occurred when females were not calling. We conducted the mating experiments in a nochoice design, using a single male and a single female in a vial at a time. Each couple was given 5 min to mate as long as the female was calling. If a female stopped calling during the observation, we stopped the clock and resumed the time measurement once she started calling again. The great majority of mating events occurred within 10–120 s from the time a male was introduced into the vial. Once a female had mated she retracted her ovipositor and did not call or mate again. Overall, we conducted 220 mating trials between individuals reared from the same host (controls), and 139 mating trials between individuals from different hosts (crosses). The individuals used in these trials originated from at least 50 different galls collected at different localities and times. Mating frequencies were compared with likelihood-ratio tests using J M P version 5.1.2 (SAS Institute). Oviposition-choice and performance experiments We tested female oviposition preference in a choice experiment in which females were offered both host plants in a greenhouse setting. We introduced individual females immediately after they had mated with males from the same host plant into ca. 0.5-L mesh cages covering 20-cm standard pots that each contained one S. rugosa and one S. gigantea ramet that were propagated from rhizomes. Rhizomes for these experiments were

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collected from numerous plants in two field sites and, although we did not verify their genetic identity, each of the host species must have been represented by at least six different genets. This design assumed that variation among the plant species would be of greater significance to the gall midges than variation between individual plants within the same species. The ramets were 4– 10 weeks old at the time of the experiment and were matched for age and height in each pot. Females remained in the cage until death (1–3 days later), after which the cage was removed and the ramets were inspected for eggs and then daily for gall development. Like other gall midges, D. folliculi adults are very shortlived [males live for up to 24 h, females for up to 3 days (Dorchin et al., 2007)], and therefore females were expected to engage in host searching and oviposition soon after they had mated. Because Dasinuera females do not insert their eggs into the plant tissues, the eggs are visible on the shoot tips following oviposition. Overall, we tested 137 females reared from S. gigantea and 87 females reared from S. rugosa. The differences in host choice (i.e. presence of eggs on the shoot tip) and progeny performance (i.e. gall induction and development of midges to maturity) between females from the two host-associated populations were compared via goodness of fit tests using J M P version 5.1.2 (SAS Institute). Genetic methods We extracted genomic DNA from 35 whole adult midges with the DNeasy Blood & Tissue extraction kit (QIAGEN Inc. Valencia, CA, USA) and PCR-amplified a fragment of ca. 650 bp of the mitochondrial cytochrome oxidase subunit I (COI) for individuals from each host-associated population across our collecting localities. PCR reactions contained 1–2 lL genomic DNA, 2.5 lL (10 mM ) 10· PCR Buffer, 2 lL (10 mM ) DNTP solution, 2.5 lL (1 mM ) MgCl2, 2.5 lL (10 lM ) of forward and reverse primers, 0.2 lL (5 U lL)1) AmpliTaq Gold polymerase (Applied Biosystems, Foster City, CA, USA), and dH2O to 25 lL. The primers we used were LCO1490 (F) and HCO2198 (R) (Folmer et al., 1994). The PCR conditions consisted of an initial 10-min denaturation at 95 C, 35 cycles of 95 C for 30 s, 50 C for 1 min, 72 C for 1 min and a final 72 C elongation phase of 4 min. PCR products were purified using the MinElute PCR Purification Kit (QIAGEN Inc.). Sequencing was conducted on an automated ABI Hitachi 3730XL DNA Analyzer (Applied Biosystems) at the Pennsylvania State University nucleic acid facility. Sequences were initially aligned using CodonCode Aligner version 1.5.2 (CondonCode Corporation, Dedham, MA, USA). GenBank accession numbers are given in Appendix 1 (http://www.ncbi.nlm.nih.gov/). We carried out a maximum-likelihood (ML) phylogenetic analysis of 21 unique ingroup and four unique


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outgroup haplotypes. First, we identified an appropriate model of molecular evolution using P A U P * (Swofford, 1998) and the methods of Frati et al. (1997) and Buckley et al. (2002). Both likelihood-ratio tests and the Akaike information criterion supported the GTR+ G model, and it was used in all phylogenetic analyses. A heuristic search was then carried out using 10 random addition sequence replicates and Tree Bisection-Reconnection (TBR) branch swapping. ML model parameters were initially estimated using a neighbour-joining topology and fixed for the first heuristic search. Model parameters were then re-estimated using the resulting ML tree, and fixed for a second heuristic search to confirm the results. In order to assess nodal support, we performed 1000 bootstrap pseudoreplicates using model parameters estimated from the ML tree, one addition sequence replicate, retaining one tree per replicate and TBR branch swapping. The mean genetic distance for the ingroup sequences, mean genetic distances within each of the host-plant groups and net mean genetic distances between the groups were calculated using M E G A 4.0 (Tamura et al., 2007) and the Tamura-Nei + G model, with alpha fixed at 0.25 based on estimates from the phylogenetic analysis (Tamura & Nei, 1993). M E G A does not include the GTR model, so we chose a model of similar complexity. Standard errors of these values were calculated using 1000 bootstrap pseudoreplicates. We then calculated nucleotide and haplotype (gene) diversity using AR L E Q U I N 2.0 (Schneider et al., 2000). Finally, we used AR L E Q U I N 2.0 to infer the population genetic structure with an analysis of molecular variance (A M O V A ). Here, we defined two populations corresponding to the hostassociated populations. We used the Tamura and Nei model with a gamma correction, alpha = 0.25 (Tamura & Nei, 1993) and significance tests based on 1023 permutations.

Results Morphology We found significant morphological differences between adult gall midges reared from S. rugosa and S. gigantea (Table 1). Based on wing-length measurements, individ-

Table 1 Wing length (mm, mean ± 1 SE) and the length ratio of abdominal tergites 8 to 6 in males and females of Dasineura folliculi from Solidago rugosa and S. gigantea. Individuals from the two hostassociated populations differed significantly (P < 0.01) in all measured characters.

Male Female

Wing length Tergite ratio Wing length Tergite ratio

S. rugosa

S. gigantea

2.77 0.47 2.29 1.70

2.58 0.41 2.19 1.47

± 0.03 (N = 31) (N = 14) ± 0.04 (N = 27) (N = 25)

± 0.04 (N = 28) (N = 19) ± 0.04 (N = 24) (N = 21)

uals reared from S. rugosa were larger than individuals from S. gigantea, (F1,106 = 16.0, P = 0.0001), and males were larger than females regardless of host and locality (F1,106 = 135.7, P < 0.0001). The interactions between host and sex and between host and locality were not significant (P = 0.28 and 0.33 respectively). The ratio in length of tergite 8 to tergite 6 of the abdomen was allometrically higher in both males and females from S. rugosa than in those from S. gigantea (females: F1,44 = 19.3, P < 0.001; males: F1,31 = 7.4, P = 0.01), hence male and female post-abdomens are longer in the population associated with S. rugosa. Because segment 8 of the female abdomen constitutes the basal part of the ovipositor, this observation means that females associated with S. rugosa have relatively longer ovipositors. Mate-choice experiments The percentage of mating events within the same hostassociated populations was significantly higher than between host-associated populations (Table 2), indicating assortative mating in D. folliculi based on host association. Because individuals reared from S. gigantea mated more readily than those from S. rugosa, we also found significant differences in mating percentages between the control groups (i.e. within host-plant species) (v2 = 14.78, P = 0.0001). No difference was found between the two combinations that represented crosses between individuals from different hosts. Oviposition-choice and offspring survival experiments Forty-five of 87 (52%) mated females reared from S. rugosa (‘rugosa females’) and 42 of 137 (31%) mated females reared from S. gigantea (‘gigantea females’) oviposited on potted plants. All of the rugosa females that oviposited and 38 of the 42 gigantea females (90%) chose their natal host species for oviposition, thus indicating significant preference of females for oviposition on their natal host (v2 = 71.7, P < 0.0001, Fig. 1). Larvae had higher performance on S. rugosa than on S. gigantea (v2 = 51.1, P < 0.0001, Fig. 1). Oviposition by

Table 2 Percentages of matings out of total number of trials (N) in the four possible crosses between Dasineura folliculi individuals from the two host-associated populations. Cross

N

% Mating

gigantea $ · gigantea # rugosa $ · rugosa # gigantea $ · rugosa # rugosa $ · gigantea #

129 91 71 68

87.6a 65.9b 31.0c 38.2c

Different letters indicate significant statistical differences (P < 0.05) according to likelihood-ratio tests.



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Genetic results

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Gall-midge populations associated with S. rugosa and S. gigantea were genetically divergent. We identified 22 unique ingroup haplotypes from 35 individuals, and the alignment of 646 bp was unambiguous and contained no indels. The overall mean mtDNA-corrected genetic distance was 0.148 ± 0.024 and the net mean genetic distance between the S. gigantea and S. rugosa groups was 0.188 ± 0.041. ML phylogenetic analysis found genetic differentiation between the host-associated populations with strong bootstrap support (Fig. 2). Withinhost mtDNA distances showed a higher level of variation in the population associated with S. rugosa than in the population associated with S. gigantea [0.020 ± 0.003 (N = 14) and 0.015 ± 0.004 (N = 21) respectively]. Similarly, both haplotype (gene) and nucleotide diversity were higher among S. rugosa individuals (0.96 ± 0.036 and 0.042 ± 0.022 respectively) than among S. gigantea individuals (0.89 ± 0.057 and 0.0129 ± 0.007). The A M O V A showed that 78.9% of the genetic variation was between host-associated populations. The corresponding FST value of 0.789 was found to be highly significant (P < 0.0001), suggesting low levels of gene flow between host-associated populations, although increased sample sizes would be needed to confirm this.

40 Number of females

733

35 30 25 20 15 10 5 0 Eggs on rugosa

Gall on rugosa

Eggs on gigantea

Gall on gigantea

Fig. 1 Oviposition choice and larval performance on Solidago rugosa and S. gigantea plants in a greenhouse experiment in which individual Dasineura folliculi females were offered simultaneously their natal host and the alternative host for oviposition. Oviposition choice is represented by the presence of eggs. Larval performance is represented by the development of galls. Filled bars represent females reared from S. rugosa; open bars represent females reared from S. gigantea.

all 45 (100%) rugosa females resulted in gall development, whereas only 10 of the 38 (26%) oviposition events among gigantea females resulted in gall development, one of which was on an S. rugosa plant. All but four of the galls produced adults (93%).

Occurrence of natural enemies Natural enemies found in D. folliculi galls included parasitic wasps from the families Pteromalidae and

88 54 55

gigMa, rugV gigH2 gigH1 gigLair1, gigLair2 gigFR1, gigFR2 gigSt8 gigNA267 gigNA216, gigCr, gigNA98, gig2541, gigM1, gigM2

99

gigLB137,rugNA1 74 gigV1, gigV2 gig2542 rugNA197, rugH2 rugLair2, rugH1 rugNA2, rugNA3, rugRick rugLB1

100

Fig. 2 A phylogenetic tree inferred by maximum likelihood for Dasineura folliculi based on 646 bp of the mtCOI gene. Numbers indicate bootstrap support. The epithets ‘rug’ and ‘gig’ refer to Solidago rugosa and S. gigantea, respectively, as the host from which individuals were reared. The letters and numbers following the host refer to collecting sites (details in Appendix 1). Individuals that yielded identical haplotypes are listed on the same terminal branch.

54

92 61 100

rugM1 rugSN1 rugLB3 rugM2 rugSN2 rugLair226

Dasineura carbonaria 100

0.1


Dasineura n.sp.1(Syd) Dasineura n.sp.2(Vic) Mayetiola destructor

Galls attacked (% )

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50 45 40 35 30 25 20 15 10 5 0 Parasitoids

Inquilines

Others

Overall

Fig. 3 Percentages of Dasineura folliculi galls on Solidago rugosa and S. gigantea that were attacked by different types of natural enemies. The term ‘others’ refers to inquilinous caterpillars and predatory gall midges. Filled bars represent galls on S. rugosa (n = 645); open bars represent galls on S. gigantea (n = 518).

Torymidae, the inquilinous gall midge Macrolabis americana and larvae of an unidentified lepidopteran that fed on the gall tissue (Dorchin et al., 2007). Each of these natural enemies can have a devastating effect on larvae of the gall inducer. For instance, a single parasitoid larva usually killed many of the gall-midge larvae, and if more than one parasitoid was present, the majority of the gall midges were killed. Larvae of the inquiline M. americana were often found in large numbers in a gall, where they caused indirect mortality of the gall inducer, most probably due to competition for gall resources. Feeding by a single lepidopteran caterpillar typically consisted of tunnelling through the centre of the gall and physically destroying it. The percentage of galls attacked by each of the types of natural enemies was significantly higher on S. rugosa than on S. gigantea (wasps: v2 = 9.2, P = 0.0024; inquilines: v2 = 50.1, P < 0.001; others: v2 = 22.7, P < 0.001), and overall attack by natural enemies was about three times higher in galls on S. rugosa (v2 = 60.1, P < 0.001, Fig. 3).

Discussion Evidence for host-associated differentiation The data we present provide multiple types of evidence showing D. folliculi has differentiated into two distinct populations on its two hosts, S. rugosa and S. gigantea, which represent cryptic species or well-established host races. This is one of a handful of cases for which a full array of genetic, behavioural, morphological and ecological evidence for HAD is available (e.g. R. pomonella, Eurosta solidaginis, Zeiraphera diniana). Dasineura folliculi exhibited assortative mating and host choice for oviposition (i.e. host fidelity), and mtDNA sequence data showed the host-associated populations are genetically divergent.

Even in the absence of host plants, the gall midges showed preference to mate within the same host-associated population, a phenomenon that was not found in E. solidaginis, for example, where no assortative mating was observed in the absence of hosts (Craig et al., 1993). Difficulties stemming from the tiny size, ephemeral adult longevity and other aspects of the biology of gall midges precluded experiments in the presence of hosts. We assumed that differences in mate choice and host fidelity would characterize the host-associated populations in general despite possible variations among different field sites; hence we pooled all individuals from the same host from all field sites for the analyses. Although this set-up precludes a statistical estimation of inter-population variation, our data suggest very limited variation at least with regard to mate choice (N. Dorchin, unpublished data). The ML tree based on our mtDNA data placed two individuals that were reared from S. rugosa in the clade otherwise representing the population associated with S. gigantea. The occurrence of these exceptions, together with the trend found in the mating experiments, implies that the host-associated populations of D. folliculi experience a certain amount of gene flow. This amount is considerably lower than that found between host races of E. solidaginis (Itami et al., 1998) and of the gall moth Gnorimoschema gallaesolidaginis (Stireman et al., 2005), and is similar to the amount of gene flow between the sister gall-midge species Rhopalomyia solidaginis and R. capitata (Stireman et al., 2005). However, it is noteworthy that in cases of rapid divergence, an incomplete lineage sorting may occur, thereby sorting of genes into monophyletic trees lags behind a speciation event that is already manifested by adaptive traits (Forister et al., 2008). Allochronic emergence or activity times of adults from different host-associated populations were proposed as important premating factors that prevent gene flow in some species (e.g. Bush, 1969; Smith, 1988; Craig et al., 1993; Nason et al., 2002; Thomas et al., 2003). However, these do not seem to play a major role in the case of D. folliculi, because we did not find appreciable differences in larval development and adult emergence times between the hosts (Dorchin et al., 2007). Although peaks of gall development and adult emergence on S. rugosa usually lag about a week behind those on S. gigantea (N. Dorchin, personal observation), there is considerable overlap in adult emergence between the populations, which means midges from different hosts could potentially interbreed. A primary post-mating cause for reproductive isolation in D. folliculi appears to be host choice by ovipositing females. If adults prefer to mate on the same host species in which they developed, then an oviposition choice made by a female predisposes her offspring to choose the same host for mating. Coupled with assortative mating, host fidelity by ovipositing females can result in reproductive isolation leading to genetic differentiation



between host-associated populations. In D. folliculi, galls developing in the greenhouse contained much larger numbers of larvae than field-collected galls (Dorchin et al., 2007), suggesting that, under field conditions, females spread their eggs among several plants. However, in our greenhouse experiments, females deposited their eggs in a single plant despite the availability of an individual of the alternate host species. This behaviour indicates that females not only choose their natal host species for oviposition but also specifically avoid nonnatal hosts (see Forbes et al., 2005). A second potential source of post-mating isolation is reduced fitness in hybrids (Craig et al., 1997; Feder & Filchak, 1999; Via et al., 2000). Although hybrids between the S. rugosa and the S. gigantea populations of D. folliculi are viable (N. Dorchin, unpublished data), we currently do not know whether their fitness differs from that of the pure populations. We found both strong host fidelity and morphological differences between host-associated populations in D. folliculi. The differences in body size between the populations could result from variable environmental factors, but may be genetically based, given that these differences were consistent in time and space. Morphological differences between host-associated populations have been documented in several systems (Wood, 1980; Rossi et al., 1999; Nosil et al., 2002; Pappers et al., 2002; Emelianov et al., 2003; Ikonnen et al., 2003; Tabuchi & Amano, 2003; Tokuda et al., 2004; Blair et al., 2005), and when directly associated with mate attraction (Brown & Cooper, 2006) or host use (Carroll et al., 1997; Diegisser et al., 2003), they contribute to the establishment of genetic differentiation. In D. folliculi, we found that females from S. rugosa are larger and have longer ovipositors than those from S. gigantea. A possible explanation is that a longer ovipositor allows better manoeuvrability and thus better access to the pubescent S. rugosa buds, whereas the smooth surface of S. gigantea plants does not require such an adaptation. Indeed, longer ovipositors among Dasineura species are correlated with less accessible oviposition sites, and when accompanied by a divided tergite 8, as in the case of D. folliculi, they are assumed to permit more flexibility of the post-abdomen (Sylveń & Tasta´s-Duque, 1993). Despite the overall morphometric differences, there may be some overlap between the host-associated populations, especially among males; hence we are reluctant to use these differences alone for distinguishing between the populations. Direction of the shift At least four lines of evidence suggest that the direction of the shift in D. folliculi was from S. rugosa to S. gigantea. First, our genetic data show that the average within-host mtDNA distance and nucleotide and gene diversity are lower in the S. gigantea population than in the S. rugosa population. Lower genetic diversity can be expected in a

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derived lineage due to founder effects. Second, ‘oviposition mistakes’ were observed only among gigantea females in our greenhouse oviposition trials, whereas all 45 females from S. rugosa oviposited on their natal host. The weaker host fidelity exhibited by gigantea females could result from the fact that the development of host preference and recognition mechanisms occurred more recently in this population. Third, populations shifting to a new host are expected to be less adapted to utilizing it and will therefore have reduced fitness on this host compared with populations on the original host (Prokopy et al., 1988; Jaenike, 1990; Brown et al., 1995). In our greenhouse experiments, only 9 of 38 oviposition events (24%) by S. gigantea females on S. gigantea resulted in successful gall induction and development, compared to a success rate of 100% among S. rugosa females on S. rugosa. In cases where oviposition by S. gigantea females did not lead to gall formation, larvae hatched from the eggs and fed on the buds for several days, but died before moulting into second instars. This observation suggests that S. gigantea gall midges are less adapted to their host than those from S. rugosa. A fourth type of evidence is the level of natural-enemy attack on galls of the two host-associated populations. Enemy-reduced space on a derived host has been suggested as a factor facilitating host shifts or maintaining isolation between conspecific populations in several systems (e.g. Abrahamson et al., 1994; Brown et al., 1995; Feder, 1995; Thomas et al., 2003; Blair et al., 2005). Our results show that for D. folliculi, S. gigantea offers enemy-reduced space with regard to the two main enemy taxa we documented (parasitic wasps and gallmidge inquilines). Although the percentage of galls affected by parasitic wasps and by inquilines varied among study sites (N. Dorchin & G. Lee, unpublished) and generations (Dorchin et al., 2007), it was almost always higher on S. rugosa regardless of the relative local abundance of the two hosts. Mitochondrial DNA analyses showed that S. gigantea was the derived host in three other case studies of HAD among goldenrod insects, namely, E. solidaginis, G. gallaesolidaginis and R. solidaginis + R. capitata, all of which shifted to S. gigantea from S. altissima (Waring et al., 1990; Brown et al., 1996; Stireman et al., 2005). In all three cases the gigantea population exhibited less genetic variation than the altissima population. This parallel direction of the shift may reflect the fact that S. gigantea is simply less abundant than the other two species due to its narrower ecological requirements (Abrahamson et al., 2005). Alternatively, it is possible that S. gigantea is a younger species, or that it has expanded its distribution into habitats where S. altissima and S. rugosa were already established, thus offering an underused niche to the insects that were associated with these hosts (see Rice, 1987; Berlocher & Feder, 2002). Given the complex and yet unresolved classification of the Solidago species in this group (Zhang, 1996), and the lack of knowledge on their


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historic distribution in North America (Marks, 1983), these explanations remain speculative. However, it is noteworthy that a shift to S. gigantea has now been documented in four different systems that show varying amounts of gene flow. All three cases of goldenrod gall-inducing insects mentioned above, as well as the host races of the beetle Mordellistena convicta (Mordellidae) that attack E. solidaginis galls (Blair et al., 2005), involved populations only from S. altissima and S. gigantea, although each of these insects is also found occasionally on S. rugosa. Several other highly specialized herbivores that are shared by the three largely sympatric host plants include the gallinducing fruit fly Procecidochares atra (Tephritidae) and at least five additional species of gall midges (N. Dorchin, unpublished). These observations imply some involvement of S. rugosa in host shifts and speciation processes in these insects. It appears that despite their presumed distant phylogenetic relations (Zhang, 1996), S. rugosa and the S. altissima species complex (subsection Triplinerviae of Semple & Cook, 2006) are at least physiologically similar with respect to the conditions they offer for the development of gall inducers.

Conclusion Dasineura folliculi joins three species of gall inducers and one inquiline species from goldenrods that show HAD, thus further establishing the goldenrod insect fauna as a model system for the study of ecological diversification in phytophagous insects. Gall inducers in this community provide an excellent system for investigating possible factors that promote HAD, which is particularly true for gall midges, being by far the most numerous and biologically diverse group among goldenrod gallers. Until further molecular study, we cannot say whether the host-associated populations of D. folliculi represent wellestablished host races or whether they are cryptic species. The high genetic differentiation is indicative of cryptic species, but interspecific copulation, production of hybrids and possible oviposition mistakes suggest a lower degree of speciation. Major reproductive isolation factors found in this study are assortative mating and host fidelity by ovipositing females. We also found that S. gigantea provided enemy-reduced space to the gall midges, thus possibly counterbalancing their poor performance on this host.

Acknowledgments We thank Alan Snyder, Brian Lucas, Catherine Blair, Anna Latimer, Patti Scarff and Jeff Williams for help with field and laboratory work, Michael J. Wise for assistance in statistical analyses, and Catherine Blair and Michael J. Wise for discussions and comments on earlier versions of the manuscript. This study was supported by Bucknell’s David Burpee Plant Genetics Chair endowment and by

National Science Foundation Grant DEB-0343633 to W.G.A. and Jason T. Irwin.

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Appendix 1 Samples used for mtDNA analysis with host plants, collecting sites, dates, and GenBank accession numbers. All specimens were collected in Pennsylvania, USA unless otherwise noted. Sample name

Host plant

Site

Date

Accession no.

gig2541 gig2542 gigLB137 gigCr gigFR1 gigFR2 gigH1 gigH2 gigLair1 gigLair2 gigM1 gigM2 gigMa gigNA216 gigNA67 gigNA98 gigSt8 gigV1 gigV2 rugLB1 rugLB3 rugH1 rugH2 rugLair2 rugLair226 rugM1 rugM2 rugNA1 rugNA197 rugNA2 rugNA3 rugRick rugSN1

Solidago Solidago Solidago Solidago Solidago Solidago Solidago Solidago Solidago Solidago Solidago Solidago Solidago Solidago Solidago Solidago Solidago Solidago Solidago Solidago Solidago Solidago Solidago Solidago Solidago Solidago Solidago Solidago Solidago Solidago Solidago Solidago Solidago

Turbot Turbot Stein Ln. Lewisburg Pottsgrove Furnace Rd, Lewisburg Furnace Rd, Lewisburg Hughesville Hughesville Lairdsville Lairdsville Montour Preserve Montour Preserve Mauses Creek Bucknell University Chillisquaque Bucknell University Chillisquaque Bucknell University Chillisquaque Stein Ln, Lewisburg Mauses Creek Mauses Creek Lewisburg Lewisburg Hughesville Hughesville Lairdsville Lairdsville Montour Preserve Montour Preserve Bucknell University Chillisquaque Bucknell University Chillisquaque Bucknell University Chillisquaque Bucknell University Chillisquaque Ricketts Glen Selinsgrove

1 July 2006 1 July 2006 13 July 2005 9 August 2005 30 May 2006 30 May 2006 8 June 2006 8 June 2006 22 June 2005 22 June 2005 1 July 2006 1 July 2006 19 July 2005 21 June 2005 26 July 2005 9 September 2005 8 August 2005 10 July 2006 10 July 2006 20 June 2005 20 June 2205 8 June 2006 8 June 2006 22 June 2005 22 June 2005 4 June 2006 4 June 2006 27 June 2005 19 July 2005 27 June 2005 27 June 2005 29 June 2006 14 June 2006

EU375690 EU375683 EU375666 EU375667 EU375682 EU375677 EU375688 EU375689 EU375668 EU375669 EU375695 EU375696 EU375681 EU375665 EU375679 EU375684 EU375664 EU375691 EU375678 EU375670 EU375674 EU375699 EU375700 EU375685 EU375686 EU375675 EU375692 EU375671 EU375680 EU375672 EU375673 EU375701 EU375698

gigantea gigantea gigantea gigantea gigantea gigantea gigantea gigantea gigantea gigantea gigantea gigantea gigantea gigantea gigantea gigantea gigantea gigantea gigantea rugosa rugosa rugosa rugosa rugosa rugosa rugosa rugosa rugosa rugosa rugosa rugosa rugosa rugosa

Creek Natural Area Creek Natural Area Creek Natural Area

Creek Creek Creek Creek

Natural Natural Natural Natural

Area Area Area Area



739

Appendix 1 (Continued) Sample name

Host plant

Site

Date

Accession no.

rugSN2 rugV Dasineura carbonaria Dasineura n.sp.1(Syd) Dasineura n.sp.2(Vic) Mayetiola destructor

Solidago rugosa Solidago rugosa Euthamia graminifolia Leptospermum laevigatum Leptospermum laevigatum Triticum

Selinsgrove Mauses Creek Bucknell University Chillisquaque Creek Natural Area Sydney, NSW, Australia Pearcedale, Victoria, Australia Mishmar Hanegev, Israel

14 17 24 21 23 23

EU375702 EU375693 EU375703 EU375687 EU375694 EU375697

Received 30 June 2008; accepted 30 December 2008


June 2006 July 2006 May 2006 August 2004 September 2004 February 2005