Fossil-Calibrated Molecular Phyiogenies Reveal That Leaf-Mining Moths Radiated Millions of Years After Their Host Plants

Home , Acer carpinifolium, Adenocarpus, Bucculatrix ulmella, Callisto denticulella, Caloptilia azaleella, Caloptilia populetorum

doi: 10.1111/j.1420-9101.2005.01070.x

Fossil-calibrated molecular phyiogenies reveal that leaf-mining moths radiated millions of years after their host plants

C. LOPEZ-VAAMONDE, N. WIKSTRÖr ,t C. LABANDEIRA4§ H. C. J. GODFRAY.t S. J. GOODMAN** & J. \/l. COOKU *INRA-Orkans. Laboratoire de Zoologie Forestière, Ardon, Olivet Cedex, France ^Department of Systematic Botany, Evolutionary Biology Centre, Uppsala University, Norbyvdgen, Sweden XDepartment of Paleobiology, Smithsonian Institution. National Museum of Natural History, Washington. DC, USA %Department of Entomology, University of Maryland, College Park, MD, USA ^Division of Biology and NERC Centre for Population Biology, Imperial College London. Silwood Park Campus, Ascot, Berkshire, UK **Institute of Integrative and Comparative Biology, University of Leeds, Leeds, UK

Keywords: Abstract coevolution; Coevolution has been hypothesized as the main driving force for the cospeciation; remarkable diversity of insect-plant associations. Dating of insect and plant divergence times; phyiogenies allows us to test coevolutionary hypotheses and distinguish evolutionary rates; between the contemporaneous radiation of interacting lineages vs. insect 'host fossil record; tracking' of previously diversified plants. Here, we used nuclear DNA to host shift; reconstruct a molecular phytogeny for 100 species of Phyllonorycter leaf-mining leaf-mining moth; moths and 36 outgroup taxa. Ages for nodes in the moth phylogeny were penalized likelihood; estimated using a combination of a penalized likelihood method and a plant-insect interactions; Bayesian approach, which takes into account phylogenetic uncertainty. To sequential evolution. convert the relative ages of the moths into dates, we used an absolute calibration point from the fossil record. The age estimates of (a selection of) moth clades were then compared with fossil-based age estimates of their host plants. Our results show that the principal radiation of Phyllonorycter leaf- mining moths occurred well after the main radiation of their host plants and may represent the dominant associational mode in the fossil record.

detect and understand macroevolutionary patterns in Introduction insect-plant associations (Farrell, 1998b, 2001; Sequeira The associations between vascular plants and insects or & Farrell, 2001; Percy et al, 2004; Kergoat et al, 2005). their early arthropodan relatives have a fossil record This research has received added impetus in recent years dating back to the first known terrestrial ecosystems of due to the increasing avaflability of molecular phyioge- the early Devonian, 415-400 Ma (Kevan et al, 1975; nies of speciose groups of plants and their herbivores. Labandeira, 2002). More recently, during the Cretaceous, There are now many estabfished examples of clades of from 145 to 65.5 Ma, flowering plants and their insect herbivores that have radiated on phylogenetically related herbivores have dominated terrestrial biomes and cur- groups of plants (e.g. FarreU, 1998a; Ronquist & Liljeblad, rently constitute a major fraction of the earth's biodiver- 2001; Weiblen & Bush, 2002). Less clear is the temporal sity, perhaps accounting for as much as 50% of all relationship between the plant and insect diversiflca- described species (excluding micro-organisms; Strong tions. At least three patterns are possible, but these et ai, 1984). Since the pioneering work of Ehrlich & should be seen as parts of a continuum rather than Raven (1964) on the coevolution of butterflies and their discrete categories. First, the insects might speciate host plants, there has been great interest in trying to contemporaneously with their host plants (cospeciation) so that their phyiogenies are congruent. Secondly, insect diversification may follow rapidly (in geochronological Correspondence: Dr Carlos Lopez-Vaamonde, INRA-Orleans, Laboratoire de Zoologie Forestière, BP 20619 Ardon, F- 45166 Olivet Cedex, France. terms) after plant diversification ('fast colonization hypo- Tel.: +33 2384 17861; lax: -^33 2384 17879; thesis'), but with the pattern of plant colonization e-mail: [email protected] influenced by some factors in addition to host-plant

phylogeny, resulting in an insect radiation that is slightly mid-level taxonomic ranks to extant taxa (Labandeira younger and incongruent with that of the plants. Thirdly, et al, 1994). This facOitates the temporal calibration of colonization of plants may require an evolutionary the insect phylogeny, which is more difficult in other innovation and occur only when this arises, some time insect groups. after plant diversification ('delayed colonization hypo- In a previous study, we found no evidence for thesis'). Again, an imperfect match between the phylo- cospeciation between Phyllonorycter and their host plants genies is predicted, but now we also predict a time lag (Lopez-Vaamonde et al, 2003). However, Phyllonorycter before colonization. species from the same host-plant genus frequently form a Testing the above alternative scenarios ideally requires monophyletic clade. Closely related moth species often phytogenies of both taxa, along with fossil data for both feed on closely related plants, which we interpret as due groups, to provide absolute time calibrations. Recently, to host switching (colonization followed by speciation) Farrell (1998a) studied the evolution of host-plant use being more likely to happen amongst phylogenetically among herbivorous Phytophaga beetles (weevils, long- related plants. horned and leaf beetles), using phylogenetic and fossil The aim of this paper was to test whether the main data. He showed two bursts of beetle speciation: one diversification of Phyllonorycter moths occurred near- during the mid-Cretaceous (90 Ma) and the other in the contemporaneously (on a geochronological time scale) later-Cainozoic (10 Ma). This bimodal distribution may with that of their host plants, or after a significant time be attributable to three events: (1) a major spurt of lag. To do this we generated a molecular phylogeny of herbivore diversification coinciding with the mid-Creta- 100 species of Phyllonorycter leaf-mining moths using 28S ceous ecological expansion of angiosperms, (2) extinction rDNA sequence data. We then used a penalized likeli- of many specialized associations at the K/T boundary and hood method with a Bayesian approach to estimate (3) a gradual rebound to late-Cretaceous species diversity divergence times, a method that takes into account levels during the earUer Cainozoic (Wilf et al, 2001; phylogenetic uncertainty. We converted the relative ages Labandeira et al, 2002). of the moths into absolute dates using a calibration point More recently, Percy et al. (2004) compared fossil- from the fossil record. The absolute ages of the moths dated molecular phylogenies of both jumping plant-fice were then compared with fossil-based estimates of the (Psylloidea) and their legume host plants. They found dates of diversification of their host plants. that there was significant topological congruence of the phylogenies, but that the psyllids radiated after their Material and methods legume hosts, except in the geologicafiy recent Canary Islands where psylUds and genistoid legumes radiated Insect sampling contemporaneously. So far, attempts to time the diversifications of insect Moths were reared and collected as described by Lopez- and plant groups have focused mainly on external Vaamonde et al (2003). We used several other genera of feeders. Here, we explore this issue in a speciose genus gracillariids (from the subfamilies Gracillariinae and Phyl- of leaf-mining moths, Phyllonorycter (Lepidoptera: Gracil- locnistinae as well as the Lithocolletinae to which Phyl- lariidae). Radiations of internal feeders, such as leaf lonorycter belongs), one species of Bucculatricidae (in the miners, may differ from those of external feeders. For same suborder) and one species of Micropterigidae (a basal example, the larvae of leaf-mining moths have very group of lepidopterans) as outgroups (see Appendix SI). intimate associations with their host plants and this may mean that there are more host-imposed constraints on Data generation and laboratory procedures their ecology and evolution compared with externally feeding herbivores. Several authors have suggested that We estimated phylogenies using sequence data from an internal feeders show both greater host-plant specificity approximately 1000 bp fragment of the 28S nuclear and greater phylogenetic conservatism in colonizing new ribosomal subunit gene. We added 21 new outgroup species of plants (e.g. Powefi, 1980; Mitter & Farrefi, species and 22 new Phyllonorycter species to the published 1991; Lopez-Vaamonde et al, 2003). 28S rDNA phylogeny of 77 Phyllonorycter species in There are also some particular advantages to studying Lopez-Vaamonde et al. (2003). All new sequences have these issues with leaf-mining microlepidoptera. First, the been deposited in GenBank (accession numbers: earliest fossil evidence for leaf-mining moth clades AY521484-AY521528; see Appendix SI). We used the extends to at least 99 Myr, almost to the start of the polymerase chain reaction and sequencing procedures major explosion of angiosperm diversity during the described by Lopez-Vaamonde et al (2001). 28S Cretaceous (130-90 Myr; Powell, 1980; Powefi et al, sequences showed substantial length variation and were 1999). Secondly, the fossO record preserves important afigned following the procedure of Lopez-Vaamonde host-plant associations of leaf-mining insects, often in et al. (2003). The DNA alignments are available from exquisite detail, and replete with larval behavioural and TREBBASE (http://www.treebase.Org/treebase/; study developmental characters that often are assignable at accession number = S1423).

Estimating phylogenies Table 1 Estimates of the age when particular plant groups were colonized by Phyllonorycter leaf-mining moths (column 3). We used Markov Chain Monte Carlo (MCMC) methods (Larget & Simon, 1999) within a Bayesian framework to Number Mode 90% 90% Probability estimate the posterior probabilities of the phylogenetic Taxen of node (Myr) LHPD UHPD (%) trees. By adopting this Bayesian approach, we take Fágales 2 37.8 27.3 50.8 32 account of phylogenetic uncertainty, both with respect Malpighiales 2 37.8 27.3 50.8 27 to topology and to branch lengths, in our estimates of Populus 3 29.9 21.1 42.5 76 divergence age. Ulmus 4 32.6 24.5 48.4 79 We used hierarchical likelihood ratio tests in the Acer 5 13 7 24.5 79 computer program MODELTEST 3.0B6 (Posada & Crandall, Fagaceae 6 24.4 14.6 34.1 51 1998) to select the best model of nucleotide substitution, Lyonia 7 18.8 10.9 27.2 68 which was a general time reversible (GTR) model, Malus 8 11.4 7.6 17.7 43 allowing for rate heterogeneity across sites assuming a AInus 9 25.7 18.1 36.2 97 Corylus 10 11.3 7.4 17.5 98 discrete y-distribution and for a proportion of sites to be Salicaceae 11 15.9 11.1 24.4 65 invariable (GTR + I + F). The phylogenies were then Prunus 12 9.6 6.2 16.2 98 estimated using MRBAYES V3.OB4 (Huelsenbeck & Acer 13 18 12.1 27.8 73 Ronquist, 2001) and the MCMC was run for 10'^ cycles, Carpinus 14 14.8 9.8 22.2 81 sampling one tree every 1000th cycle. After filtering the Quercus 15 15.8 11 24.4 47 trees for our defined nodes of interest, a total of 8368 trees remained for use in the age estimation analyses. Number of node is indicated on the cladogram (see Fig. 3). LHPD, 90% lower highest posterior density; UHPD, 90% upper highest posterior density; probability (%), the figures represent the Estimating divergence times probabifity (as averaged over the 100 randomly sefected trees from the posterior distribution) that a particular plant group is recon- We estimated node ages for the moths using a penalized structed for our node of interest and not reconstructed for the node likelihood approach in the computer program RSS v. 1.50 below (i.e. the colonization event has occurred along the stem (Sanderson, 2003; http://ginger.ucdavis.edu/r8s/). For lineage to our node of interest). each node of interest (see below) the posterior distribution of the divergence time, conditioned on both the phylogenetic model and the calibration point obtained (the 8368 trees sampled, filtered for our nodes of from the fossil record, was obtained by local density interest) using MESQUITE version 1.0 (buUd e58; http:// estimation using the program LOCFIT (Loader, 1999), mesquiteproject.org; see Table SI). We selected clades implemented in the 'R' statistical package (Ihaka & that grouped closely related moths feeding on closely Gentlemen, 1996 see script in Fig. SI). To summarize the related plants (same host-plant genus, family, order). On fitted distributions, we report their modes and 90% this basis, 15 nodes of interest, supported by posterior highest posterior density (HPD) limits (Table 1 and probabilities of at least 92%, were chosen for insect and Fig. SI). The mode represents the most fikely value for plant clade age comparisons (Tables 1 and 2). the divergence time of a particular node, and the HPD provides a confidence interval for this estimate. It is Crown-group and stem-group ages important to emphasize that we have inferred age from the entire posterior probability distribution of the 8368 The distinction between crown and stem-group ages is trees sampled rather than using a single topology, so our crucial when dealing with colonization events. Consider age estimates account for both branch length and a clade of insects that aU feed on the same host-plant topological uncertainties. taxon. A colonization event is inferred to have occurred The ages of the host-plant groups were obtained from somewhere along the stem lineage to this clade. How- the literature based on the fossil record (Table 2). ever, the date of this colonization event can be estimated either as the date of origin of the stem group or as the Character mapping and pliylogenetic uncertainty date of origin of the crown group (see Fig. 1). We have consistently used stem-group ages for both moth and We mapped the character 'host-plant taxa' onto the host-plant groups in our analyses. In terms of testing for a moth phylogeny using Bayesian methodology in order to significant delay between plant and insect radiations, incorporate phylogenetic and mapping uncertainty. insect stem-group ages are the conservative option as Unfortunately, current software (MRBAYES and SIMMAP, they provide the older date. For the host plants, crown- http://www.simmap.com) does not allow sufficient char- group ages (younger) would be more conservative for the acter states for this analysis. Consequently, we recon- hypothesis test. However, palaeobotanical reports of the structed the evolution of host use manuafiy on 100 trees first occurrence of particular plant groups rarely identify drawn randomly from the Bayesian posterior distribution the taxon as a stem-group lineage or part of the crown

Table 2 Earliest fossil occurrences of plant taxa that currently are hosts for Phyllonoryder leaf-mining moths.

Earliest occurrence Host-plant faxen Epoch Stage Absolute age (Myr)* Type References

Acer Paleoceno Ttianefian 56.5 Leaves, fruit Crane ei a/. (1990) Alms Eocene [Middle] Lutetian 48.5 Leaves, fruits Crane (1989) Carpinus't Eocene [Middle] Lutetian 49.0 Fruit Wilde & Frankenfiäuser (1998) Corylus Eocene [Middle] Lutetian 48.5 Fruit Pigg ef al. (2003) Fagalesi Gallic Santonian 85.0 Pollen Doyle & Robbins (1977), Romero (1986), Hill & Dettmann (1996), Simsefa/. (1998), Herendeen et al. (1999) Fagaceae Gallic Upper Santonian 84 Staminate Simsefa/. (1998) inflorescences, flowers, cupules and fruits Lyonia Miocene [Middle] Langhian 15.5 Fruits, seeds Friis (1985) Malpighiales§ Late-Cretaceous Turonian 91.5 Flowers Crepet & Nixon (1998) Malus'i Oligocène [Early] Rupelian 31.0 Leaves Meyer & Manchester (1997) Populus Paleoceno [Late] Thanetian 55.0 Leaves S. R. Manchester, personal communication to CCL Prunus Eocene [Middle] Lutetian 48.0 Fruits Cevallos-Ferriz & Stockey (1991); Manchester (1994) Quercus Eocene [Middle] Lutetian 48.5 Acorns Manchester (1994) Salicaceae Middle-Eocene [Middle] Lutetian 45 Reproductive Manchester et al. (1986) foliage and leaves Ulmus Eocene [Middle] Lutetian 48.0 Leaves, fruit Manchester (1989, 1999)

*To nearest 0.5 Myr; stage-interval data are given as midpoints; some are radioisotopic age dates. Time scale is from Gradstein & Ogg (2004). tThe assignment is 'atf. Carpinus'; of which the same deposit has the very closely related Paleocarpinus, also a member ot the subfamily Coryleae. jFagales comprises the Betulaceae, Casuariniaceae, Fagaceae, Juglandaceae, Myricaceae, Nothofagaceae and Rhoipteleaceae (Judd et al, 1999). §MalpighiaIes comprises the Malpigiaceae, Clusiaceae, Euphorbiaceae, Chrysobalanaceae, Rhizophoraceae, Salicaceae, Passifloraceae, Violaceae and Flacourtiaceae (Judd et al. 1999). *|Leaves of Malus are difficult to distinguish from Pyrus. group of living species. Generating crown-group age Table S2). However, it is unclear whether they should be estimates would therefore require an extensive taxo- assigned to the stem or crown group as the adult nomic evaluation of both fossil and living plant taxa, morphology is not preserved, and therefore cannot be clearly beyond the scope of this study. Therefore, we use compared with extant species. Consequently, we applied stem groups wherever possible; one exception being the a 99 Myr calibration point to the Phyllocnistinae stem- estimate for the origin of Phyllonorycter. This taxon was group node (the time of divergence of the Phyllocnistinae used to establish a diversification rate estimate based only from its sister taxon). on numbers of extant species, so a crown-group estimate is more appropriate. Estimating diversification rates We estimate rates of diversification (r) for the genus Fossil record and calibration points Phyllonorycter as a whole by using Magallón & Sander- To convert the relative ages obtained through the son's (2001) eqn (7) for a crown-group age penalized likelihood analysis into dates, we used an absolute calibration point from the fossil record. Mines of fe =-[\oi[-n{\-e^) + 2e the gracillariid subfamily Phyllocnistinae have been recorded from magnoliid dicot leaves originally reported + -(1 -e)y^ñ(ñe^ •8e-l-2ne + K)]-log2], to be at least 97 Myr old (Labandeira et al, 1994; Fig. 2), but this date has now been revised to at least 99 Myr, where n is number of species, e is the extinction rate and t based on new evidence (Brenner et al, 2000; Wang, the crown-group age. 2002; Gradstein & Ogg, 2004). In addition, we estimate r using two extinction rate These mines represent the oldest fossil evidence for the values at opposite ends of the spectrum of possible family Gracillariidae (Kristensen & Skalski, 1999; see values: one corresponds to zero extinction (fo) and the

Stem group

Stem Sister clade representatives Crown group (on specific tiost)

Crown group age

Stem iineage (where coionization occurs)

Fig. 1 Distinction between crown-group and stem-group ages (based on Magallón, Stem group age 2004).

Other to a very high extinction rate (ro.9; for a similar whereas two other nodes were identifled as colonizations approach see: Strathmann & Slatkin, 1983; Magallón & of the plant famfly Fabaceae (results not shown). When Sanderson, 2001). We previously estimated the number phylogenetic uncertainty was taken into account, the of extant Phyllonorycter species as 400 (Lopez-Vaamonde robustness of these host-plant colonization events et al, 2003) and used this estimate for the age of the decreased. Thus, the mapping of host use on the 100 crown group obtained in this study. posterior trees showed node 2 as a colonization event of Fágales and Malpighiales (Table 1), but there was not a single tree out of the 100 sampled that supported the two Results nodes as colonization events of Fabaceae (see Table SI). Bayesian analysis This clearly indicates that using a single topology for reconstructing character evolution can yield misleading We obtained 28S rDNA sequences for 100 Phyllonorycter results. species and 36 outgroup taxa. The aligned Dl -1- D2 -1- D3 region sequences comprised 1130 bp. Results of the Comparing moth and plant age estimates Bayesian analysis are presented as a 50% majority rule consensus tree in Fig. 3. As in a previous study (Lopez- When we compare the age estimates of the 15 nodes Vaamonde et al, 2003), Cameraria is the sister group of (Table 1) with those of the associated host plants (Table 2), Phyllonorycter, and Phyllonorycter species are arranged in we observe, as expected, that the moths are younger than clades that reflect their host-plant relationships (Fig. 3). the plants. For example, we placed the origin of the genus The R8s analysis suggests that the genus Phyllonorycter Phyllonorycter in the Palaeocene (62.3 Myr), considerably originated during the early Palaeocene (mode = postdating the origin of both extant angiosperm clades 62.3 Myr, lower and higher 90% posterior density (Early Cretaceous, 130-110 Myr) and extant eudicot limits = 50.3 and 76.3 Myr). subclades (mid-Cretaceous, 110-90 Myr; Stuessey, 2004). Simflarly, the colonization of the order Fágales Character evolution and phylogenetic uncertainty (Fig. 3a, node 2), a major group of hosts for Phyllonorycter, occurred during the late-Eocene (37.8 Myr) whereas the The mapping of host-plant use onto the 100 randomly order Fágales originated during the late-Cretaceous chosen trees from the Bayesian posterior distribution (84 Myr; Herendeen et al., 1999; Table 2). From these (Fig. 3) enabled us to identify 15 host-plant colonization data, on average, moths are 32.3 ± 16.6 Myr younger events. Nodes, indicating where these events occurred, than their host plants and this difference is signiflcant are numbered from 1 to 15 on Fig. 3. (paired i-test: Í14 = -7.52, P < 0.0001). When host use was mapped onto Fig. 3, node 2 was The ages of the host plants were obtained from matched to a host shift onto the plant order Fágales the fossU record (Table 2) and compared with the

Fig. 2 Gracillariidae (Phyllocnistinae) leaf mine on host plant Rogersia parlatorii Fontaine, whose affinities is closest to extant Lauraceae within the order Laurales cf. Lauraceae. (a-c) Specimen UF7351, showing a camera lucida drawing of the entire leaf (a), terminal mine chamber (b) and mine loop adjacent to midrib at the leaf base (c). (d-g) Specimen UF4818, displaying a camera lucida drawing of the entire leaf (d), mine course near leaf apex (e), terminal chamber (f) and oviposition site with adjacent frass trail from early instars (g). Both specimens are from the Cenomanian-Albian boundary interval of the Dakota Formation and were found at the Braun's Ranch Locality, Cloud Co., Kansas, USA.

sequence-derived ages of the moths. An alternative would be to estimate the ages of the plants from Discussion molecular data as well (with fossil data providing minimum age constraints). However, it has been shown that Contemporary or sequential radiations? fossil-based ages are nearly always younger than the This is the first study to make a rigorous quantitative corresponding sequence-derived ages (Rodriguez-Trelles comparison of the timing of radiations by internally et al, 2002; Wikström et al, 2003) and hence this would feeding insects and their host plants. Our results provide simply increase the time span between host-plant origin strong evidence for sequential radiation of the insects and moth colonization. onto an already diversified group of plants. The analyses suggest that the principal Phyllonorycter radiation was at Rate of diversification least 27.3-50.8 Ma, when these moths colonized plants in the orders Fágales and/or Malpighiales (Fig. 3a, node Given that there are about 400 Phyllonorycter species extant 2). By contrast, the main radiation of Fágales occurred at today (Lopez-Vaamonde et al, 2003), we can use our least 84 Myr (Hill & Dettmann, 1996; Sims et al, 1998) estimate of the age of the crown group (62.3 Myr) to and that of Malpighiales at least 90 Ma (Crepet & Nixon, obtain a measure of the net diversification rate as a 1998). function of the relative extinction rate (e). The values we On average moths took 32.3 ± 16.6 Myr to colonize obtain are 0.04 net speciation events per mifiion years in new plant clades. However, this figure is an underesti- the absence of extinction (e = 0), and a minimum of 0.03 mate of the true figure because we have no information under the assumption of a high relative extinction rate about the number and age of suitable but as yet (e = 0.9). uncolonized plant clades. Moreover, fossil calibration

CO Phyllocnistis sp. 2 c Phyllocnistis sp. 1 Phyllocnistis meliacella Phyllocnistis labyhnthella c Phyllocnistis saligna o Phyllocnistis citrella Lithocolletinae sp. Cameraria ohridella Cameraria hamadryadella CL Cameraria guttifinitella Cameraria sp. 2 P. grewiella P. quinqueguttella Figure 3B P. harrisella P. populifoliella P. sagitella Fágales P. comparella Populus P. pastorella P. pastorella P. bifurcata P. lonicerae P. schreberella o P. millierella c P. loxozana P. emberizaepennella 'S, P. sf). 4 P. trifasciella P. scabiosella P. tristrigella P. bicinctella Ulmus P. acaciella P. agüella Liocrobyla lobata Parectopa robiniella Metrichroa sp. 2 Metrichroa sp. 4 Dendrorycter marmaroides Marmara sp. Metrichroa sp. 3 Parornix carpinella Callista denticulella Bucculatrix ulmella Caloptilia falconipennella Caloptilia azaleella Caloptilia sp. Caloptilia populetorum Calybites auroguttella Fig. 3 Bayesian tree with posterior probab- Eucalybites aureola Gibbovalva quadrifasciata ility values above the branches. Nodes of Dialéctica sp. interest (where host-plant colonization Eucosmophora pithecellobiae Metriochroa sp. 1 events are inferred) are numbered from 1 to Acrocercops heptadeta Acrocercops brongniardella 15 and their stem-group age estimates are Acrocercops unistriata reported in Table 1.

• p. lantanella (b) • P. hilarella • P. cerasinella • P. genistella • P. medicaginella • P. maestingella • P. insignitella • P. geniculella ~r HI 00 • P. sylvella Acer • P. planatoidella j- • P. ulicicolella P. sp. 6 -10.54 P. sp. 7 _ • P. sçssilifolia • P. similis • P. distentella Fagaceae • P sp. 2 ^ -ioST • P. roboris -^ P. acutissimae -9 P /yon/ae I LyOA7/a -11.00 • P. japónica • P. japónica • P. junoniella • P. crataegella • P. sorbicolla • P. elmaella • P. blancardella • P. oxyacanthae Malus • P. mespilella -10.96 • P. pomonella • P. cydoniella • P. gerasimovii • P. pyrifoliella • P. platani • P. pygmaea HI .00 • P. /ss//f// -^ P. issikii -11.00 -iE) p. nicelii X Corylus • P. stettinensis • P. cavella Al nus Hi .00 HI .00 P. kleemanella • P. froelichiella • P. salicicolella • P. brevilineatella -(Do • P. dubitella Salicaceae • P. viminiella HO.62 • P. salictella • P. heringiella • P. saportella • P. heegeriella • P. cerasicolella ~r pmnnc -(Ell .00 • P spinicolella 1 '^'^'•^S • P. nigrescentella • P. onentalii T A^or -if) 1.00 P sp. 8 1 ^^^^ • P. strigulatella • P. ulmifoliella P. sp. 5 • P. rajella -rrpD" • P. anderidae • P. anderidae HO.58 • P. viminetorum • P. /ai/fe//a • P. corylifocrataegus • P. corylifoiiella ex Crataegus • P. corylifoiiella ex Sorbus • P. tenerella ~ ^ • P. quinnata • P. coryli Carplnus • P. aemula • P. sorb/ -11.00 • P. leucographeila • P. jovielia ~ Hi.00 • P. mesaniella • P. quercifolielia Hi .00 • P. sp. Ï • P. sp. Ï Quercus Hi 00 • P. sp. 3 • P. cretata Ho.90 P. muellerieila • P. iiicifoliella HI 00 • P. baidensis • P. scopariella • P. staintonielia Hi 00 • P. parvifoiiella Fig. 3 Continued • P. telonensis

dates are underestimates that are more likely to increase Based on recent studies of the three major types of rather than decrease with additional fossil data. insect herbivore radiation on new host-plant clades - Regardless, our findings clearly suggest substantial delays cospeciation, fast colonization and delayed or sequential (15-52 Myr) between the radiation of host-plant taxa radiation (see Labandeira, 2002) - it appears that the and their colonization by Phyllonorycter moths, rejecting sequential mode is the most common. This pattern is the hypothesis of 'fast colonization' and supporting the supported by several other studies focusing on extant hypothesis of 'delayed colonization' or sequential evolu- plant host and their insect herbivore clades (Schoon- tion (Jermy, 1976; Percy et al, 2004). Interestingly, the hoven et al, 1998) as well as by our own study. Detection moth phylogeny is poorly resolved around the time of of sequential evolution (Jermy, 1976) in the fossil record colonization of Fágales (internal branches are very short, requires geochronologicafly measurable time lags Lopez-Vaamonde et al, 2003) and this probably reflects between the origin of a plant clade and its subsequent rapid speciation following this key host shift. colonization by insects. In contrast, the rapid radiation of

angiosperms that occurred from the later-, early- and that uncertainty in the estimate of a phylogeny esti- mid-Cretaceous is normally thought to be associated with mate can lead to spurious mapping results. Mapping onto cospeciation or rapid insect diversification (Grimaldi, 100 trees sampled randomly from a posterior distribution 1999), based exclusively on insect fossil data. If further is similar to using nonparametric bootstrapping to studies support this conclusion then our data and the account for phylogenetic uncertainty (Ronquist & other studies discussed above indicate a different mode of Liljeblad, 2001). However, neither approach addresses colonization in subsequent colonization events. Conse- mapping uncertainty - i.e. the error associated with quently, we suggest that delayed sequential colonization reconstructing the evolution of a character on a given may represent the norm for insect herbivores during the tree - adequately (Ronquist, 2004). Only a fufi Bayesian overwhelmingly longer durations of geological time that approach can account simultaneously for both mapping are characterized by more typical levels of plant ciado- and phylogenetic uncertainty, but such a procedure has genesis. yet to be applied in any empirical study. We hope that Our data suggest a substantial delay between the future software development will permit analysis of radiation of host plants and their colonization by host- characters with more than 10 states, a constraint that specific Phyllonorycter moths. The moths, in turn, also prevented us from using the full Bayesian approach here. harbour host-plant-specific parasites, namely parasitoid wasps of the genus Achrysocharoides (Hymenoptera: lUlolecular dating and uncertainty Eulophidae; Lopez-Vaamonde et al, 2005). It is clear that these wasps have not cospeciated with either the It is well established that molecular rates vary across plants or the moths (Lopez-Vaamonde et al, 2005), but lineages, and that this makes it difficult to estimate there has not been a formal comparison of the ages of divergence dates accurately using DNA sequence data moth and parasitoid radiations. However, comparisons of (Arbogast et al, 2002). There are two major approaches both nuclear and mitochondrial DNA reveal greater to deafing with this problem (Welch & Bromham, 2005). sequence divergences between pairs of moth species The first, nonparametric rate smoothing (NPRS) was than matched pairs of their parasitoids, suggesting developed by Sanderson (1997) and allows different rates strongly that the parasitoids radiated across an already on different branches of the tree. It assumes that rates are diversified group of moth hosts (Lopez-Vaamonde et al, constant on individual branches and that branch length 2005). Consequently, this plant-leafminer-parasitoid sys- estimates are their true values. The penalized likefihood tem seems to be characterized by two waves of sequential method is a superior modification of NPRS that relies on colonization, which we might call 'bottom-up sequential a parameter-rich model to estimate rates on branches radiation'. and, like NPRS, employs a numerical penalty to avoid rapid changes between neighbouring branches. The relative importance of the model vs. the penalty function Dealing with uncertainty is determined by a smoothing parameter, the choice of Our analyses are subject to three potentially important which is based on the data using cross-vaUdation sources of error. These occur in the procedures of (Sanderson, 2002). phylogeny estimation, character mapping and node A second approach was developed by Thorne et al dating respectively. (1998), and uses Bayesian statistics to model explicitly Phylogeny estimation is difficult when based on a the rate of evolution in a fully parametric setting. This is a single genetic marker due to the stochastic nature of more sophisticated technique, but it would be impractical coalescence events (Hudson, 1990). Indeed, the use of a to apply it to our data for two reasons. First, it is too single locus to estimate divergence times can be prob- computationally intensive to apply this approach to the lematic (Edwards & Beerli, 2000; Arbogast et al, 2002). fufi set of >8000 trees. Secondly, although in theory we To address this issue, at least partly, we used a Bayesian could apply it to a suitable consensus tree, there is no framework for phylogenetic reconstruction. It is very satisfactory consensus tree for this data set, because of probable that obtaining data from one or more additional poor resolution (<50% bootstrap support) at many genes would decrease the error bounds of our age nodes. We thus chose to use the penafized likefihood estimates. However, our main result about the timing method for practical reasons, but also note that this does of speciation is sufficiently marked that it is very unlikely not necessarily perform less well than the alternative to change. approach (Welch & Bromham, 2005). Any phylogeny generated from data is only an Age estimates may be poor if the fossil calibration is estimate of the true phylogeny and may be little better inaccurate, or if there is substantial variation in the rate than some alternative estimates. Conclusions from map- of sequence evolution (Magallón, 2004). As we have ping exercises using a single tree can depend crucially on shown a significant delay between host-plant radiation a few influential (but not necessarOy wefi supported) and moth colonization, we are most concerned here with nodes. Our comparison between mapping on a single factors that might lead us to accept the hypothesis of topology and on a sample of 100 posterior trees shows sequential radiation erroneously, i.e. to underestimate

the age of insect clades or overestimate the age of plant Cretaceous, including leaf-rofling hispine beetles and clades. their ginger hosts (Wilf et al, 2000) as weU as water liUes The low estimate of moth diversification rate (see and their scarab beetle polfinators (Ervik & Knudsen, below) might indicate that our calibration point is too 2003) and seed-beetles and their legume hosts (Kergoat old, resulting in Phyllonorycter crown-group age being etal, 2005). overestimated. However, any younger age would further In conclusion, determining the precise timing of strengthen the result of sequential evolution, so this is phylogenies has become increasingly important to dis- not a great concern. To favour contemporaneous radi- entangle the evolution of interactions between insects ation, a considerably older Phyllonorycter origin would be and plants (Percy et al, 2004; Kergoat et al, 2005). Our necessary and there is no fossil evidence to support this. dated phylogeny supports the hypothesis that the diver- However, this might only reflect the general paucity of sification of Phyllonorycter leaf-mining moths occurred phyflocnistine leaf mines in the Cretaceous fossil record, well after the diversification of their host plants. This a general concern in paleobiology (Marshall, 1997) and it scenario of asynchronous radiations might apply to other has been shown recently that divergence dates based on predominantly monophagous insect-plant systems and single calibration points tend to be underestimates (Yoder has implications for the generation of biodiversity at & Yang, 2004). Unfortunately, no other calibration points geochronological time scales. are currently available, although further studies of plant- insect associations from the Dakota Formation and other Acknowledgments Cretaceous biotas may provide new calibration points in the future. Particularly promising are a Cenomanian The authors thank the colleagues fisted in the Appen- (approximately 97 Ma) deposit from Israel (Krassilov & dix SI for providing specimens for DNA sequencing. CLV Bacchia, 2000) and a younger Turonian (approximately is most grateful to Tosio Kumata, Ohshima Issei and 92 Ma) flora from Kyzl Zhar, Kazakhstan (Kozlov, 1988), Kazuhiro Sugisima for help during field coflections in both yielding dicot leaf mines, and possibly older strata Hokkaido. We also thank David Dilcher of the Florida such as the Barremian (approximately 125 Ma) Yixian Museum of Natural History for the loan and identifica- deposits from Hebei Province, China (Ren, 1995; Sun tion of the host-plant specimens (UF735I, UF4818) upon et al, 2001) and the Hauterivian (approximately 135 Ma) which one of our dates is based. The authors thank Baissa deposits from Transbaikalia, Russia (Zherikhin Elisabeth Herniou and Tim Barraclough for comments on etal, 1999). earlier versions of this manuscript, and Finnegan Marsh Additionally, variation in the rate of sequence evolu- for assistance with Fig. 2. This study has been supported tion can bias both relative and absolute age estimates in a by the National Environment Research Council Centre nonlinear way. The direction and magnitude of errors for Population Biology. This is contribution no. 121 of the can only be evaluated on an individual node basis by Evolution of Terrestrial Ecosystems consortium at the direct comparison with an independent age estimate, National Museum of Natural History. typically a fossil. A recent comparison between fossil- and sequence divergence-based age estimates of land-plant References lineages indicated that rate variation can impose large error bounds on age estimates (Soltis et al, 2002). Arbogast, B.S., Edwards, S.V., Wakeley, J., Beerli, P. & When taken overall, the fossil record supports the Slowinski, J.B. 2002. Estimating divergence times from hypothesis that the colonization of Fágales by Phyllono- molecular data on phylogenetic and population genetic rycter moths occurred significantly after the diversifica- timescales. Annu. Rev. Ecol. Syst. 33: 707-740. Brenner, R.L., Ludvigson, G.A., Witzke, B.J., Zawistoski, A.N., tion of major fagalean lineages (Table 2). Kvale, E.P., Ravn, R.L. & Joeckel, R.M. 2000. Late Albian Kiowa-Skull Creek marine transgression. Lower Dakota Interpreting diversification rates Formation, eastern margin of Western Interior Seaway, USA. J. Sediment. Res. 70: 868-878. The estimated rate of diversification (r) for Phyllonorycter Cevallos-Ferriz, S.R. & Stockey, R.A. 1991. 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Insect- Palaeoentomological Conference, Moscow (1998) (P. Vrsansky, Plant Biology: From Physiology to Evolution. 409 p. Chapman and ed.), pp. 185-191. AMBA Projects, Bratislava, Slovakia. Hall, London, UK. Sequeira, A.S. & Farrell, B.D. 2001. Evolutionary origins of Gondwanan interactions: how old are Araucaria beetle Supplementary Material herbivores? Biol. J. Linn. Soc. 74: 459-474. Sims, H., Herendeen, P.S. & Crane, P.R. 1998. New genus of The following supplementary material is available for this fossil Fagaceae from the Santonian (late Cretaceous) of central article online: Georgia, USA. Int. J. Plant Sei. 159: 391-404. Fig. SI Distributions of age estimates. Age: mode of the Soltis, P.S., Soltis, D.E., Savolainen, V., Crane, P.R. & Barra- distribution. It represents the most likely value for the clough, T.G. 2002. Rate heterogeneity among lineages of divergence time of a particular node; LHPD: 90% lower tracheophytes: integration of molecular and fossil data and evidence of molecular living fossils. Proc. Nati Acad. Sei. USA highest posterior density limit; UHPD: 90% upper highest 99: 4430-4435. posterior density limit. Nodes of interest are numbered Strathmann, R.R. & Slatkin, M. 1983. The improbability of from 1 to 15 and indicated on the cladogram (see animal phyla with few species. Paleobiology 9: 97-106. Fig. 3a,b; script for 'R' statistical package).

Table SI Probability (as averaged over the 100 Appendix SI. Specimens used in this study. They randomly selected trees from the posterior distribution) have been ordered according to the plant classification that a particular plant group is reconstructed for our (APG, 1998). node of interest and not reconstructed for the node This material is available as part of the online article from below. http://www.blackwell-synergy.com Table S2 Body and trace fossil record of the Gracilla- rioidea (Lepidoptera: Ditrysia, Gracillariidae and Buccu- Received 15 June 2005; revised 18 October 2005; accepted 17 November latricidae). 2005

Fossil-calibrated molecular phylogenies reveal that leaf-mining moths radiated several million years after their host plants

C. LOPEZ-VAAMONDE\ N. WIKSTRöM^ C. LABANDEIRA^ H. C. J. GODFRAY^

S.J. GOODMAN^ & J. M. COOK^

Institute of Zoology, Zoological Society of London, Regent's Park, London, NWl 4RY,

U.K.

Department of Systematic Botany, Evolutionary Biology Centre, Uppsala University,

Norbyvägen 18D, Sweden.

Smithsonian Institution, National Museum of Natural History, Department of

Paleobiology, Washington, DC 20013-7012 USA. and Department of Entomology,

University of Maryland, College Park, MD, 20741 USA.

Division of Biology andNERC Centre for Population Biology, Imperial College London,

Silwood Park Campus, Ascot, Berkshire, SL5 7PY, U.K.

Department of Biology, University of Leeds, L. C Miall Building, Clarendon Way,

LeedsLS2 9JT, UK

AUTHOR FOR CORRESPONDENCE:

Dr. Carlos Lopez-Vaamonde

D>íRA-Orleans, Laboratoire de Zoologie Forestière, BP 20619 Ardon, F- 45166 Olivet

Cedex, France (e-mail: carlos.lopez-vaamondeÇSiorleans.inra.fr)

39 Figure Caption

Figure 4. Distributions of age estimates. Age = mode of the distribution. It represents the

most likely value for the divergence time of a particular node. LHPD= 90% Lower

Highest Posterior Density limit. UHPD= 90% Upper Highest Posterior Density limit.

Nodes of interest are numbered from 1 to 15 and indicated on the cladogram (see

figure 3a,b).

Script for R' statistical pacliage

# The authors accept no liability for the accuracy, use/misuse of this script, or whether it is fit for purpose # This script 'automatically' calculates mode and 90%HPD intervals for Bayesian distributions given a matrix of values drawn from a posterior distribution. # The contributed package "Locfit" is required (see http://www.r- project.org) # A data file is required, consisting of a comma delimited ".csv" file, exported from a spreadsheet such as Excel. # The first row should be column headings with the name of each variable # The first column can be an index field, listing the iteration (or draw number) for the posterior distribution # Each column is the values for each variable and the rows are the values from a given iteration. # You will need to adjust the filename for the .csv file on the second line to match your own data file. # It is recommended that you perform some exploratory analysis with locfit first to ensure the default settings allow unbiased estimation of the distributions # You should plot the locfit objects to check the shape of the distributions, these should be smooth, not spiky! # This is not a 'blackbox' analysis and users must be aware of the need to check that it is appropriate for their datasets and to confirm their own results . library(locfit) age<-read.CSV("agesheet.CSV") #reads in the file "agesheet.csv", you'll need to change the file name to match your data file attach(age) #makes age the main data frame sum.stat<-summary(age) #summary stats for all columns print(sum.stat) lnam<-length(names(age))

40 res<-c(O,o,0) #sets up dummy variable for results table, use this if the first the column of values is an index field (i.e. contains the iteration number) #res<-c() #sets up dummy variable for results table, use this if there is instead of the above if there is no index field # in this case you also need to change the 'for loop' below to (s in 1 :Inam)

#Calculations begin for (s in 2 :Inam) {

max.node<-max(age[s], na.rm=TRUE) #gets max value for interval min.node<-min(age[s], na.rm=TRUE) #gets min value for interval interval<-c(min.node,max.node) x<-as.matrix(age[s]) If <- locfit(~x) #fits density distribution

mfd<-function(x,If){return(predict(If,x))} #defines function used to get predicted values # next line finds the mode of the distribution

mmaxd<-optimise(mfd, interval=c(min.node,max.node), lower=min(interval), upper=max(interval), maximum=TRUE, tol=0.001. If)

f<-fitted(lf) #next few lines find 90% HPD values fi<-order(f) j<-length(f) jl<-0.1*j k<-fi[jl:j] r<-range(x[k]) # r #this is the 90% HPD range ml<-as.numeric(mmaxd[1]) #converts mode value to numeric object res<-append(res,ml) #appends to results vector res<-append(res,r) #appends to results vector

###Process results vector for output cn<-c("Name","Mode","90% LHPD","90% UHPD") #column names for results table res.mat<-t(matrix(res,3, (length(res)/3))) #turns the results vector in to a matrix # res.tab<-data.frame(res.mat) #makes the results matrix in to a table for ouput res.tab<-data.frame(cbind(names(age),res.mat)) #does the same as above but adds names of original data columns colnames(res.tab)<-cn #assigns column names

41 res.tab

# write . table(res.tab,file="results.txt") #writes a table to file, you should set your own file name write.table(res.tab,file="results.txt",quote=FALSE,sep="\t") #does the same but without quotes and tab delimited, more convenient for import into other programmes.

### End of script.

42 Table 3. Probability (as averaged over the 100 randomly selected trees from the posterior distribution) that a particular plant group is

reconstructed for our node of interest and not reconstructed for the node below.

Tree Node 2 Node 2 Node 3 Node 4 Nodes Node 6 Node? Nodes Node 9 Node 10 Node 11 Node 12 Node 13 Node 14 Node 15 Nr. Fágales Malpighiales Populus Ulmus Acer Fagaceae Lyonia Malus Alnus Corylus Salicaceae Prunus Acer Carpinus Quercus 1 0.4 0.4 1.0 0.5 0.7 0.0 0.7 0.8 0.4 0.4 0.4 1.0 0.5 0.75 0.5 2 0.4 0.4 0.5 0.9 0.7 1.0 0.7 0.2 1.0 0.4 0.4 0.5 0.9 0.75 0 3 0.4 0.4 1.0 0.9 0.0 1.0 0.7 0.7 1.0 0.4 0.4 1.0 0.9 0.667 0.67 4 0.4 0.4 0.5 0.9 0.7 0.8 0.9 0.1 0.8 0.4 0.4 0.5 0.9 0.75 0.5 5 0.4 0.4 1.0 0.9 0.0 0.5 0.7 0.4 1.0 0.4 0.4 1.0 0.9 0.667 0.67 6 0.4 0.4 1.0 0.9 1.0 0.8 0.8 0.7 1.0 0.4 0.4 1.0 0.9 0.75 0 7 0.4 0.4 0.3 0.8 1.0 1.0 0.7 0.7 0.0 0.4 0.4 0.3 0.8 0.667 0.67 8 0.4 0.4 1.0 0.9 0.5 0.5 0.7 0.2 0.7 0.4 0.4 1.0 0.9 0.75 0.5 9 0.4 0.4 1.0 0.5 1.0 0.5 0.7 0.1 0.0 0.4 0.4 1.0 0.5 0.75 0.5 10 0.3 0.3 1.0 0.9 0.8 0.7 0.7 0.7 1.0 0.3 0.3 1.0 0.9 1 0 11 0.4 0.4 0.3 0.9 0.7 0.0 0.7 0.8 1.0 0.4 0.4 0.3 0.9 0.75 0.5 12 0.3 0.3 1.0 0.8 1.0 1.0 0.7 0.2 0.5 0.3 0.3 1.0 0.8 0.75 0.5 13 0.0 0.0 0.4 1.0 1.0 0.8 0.8 0.4 0.5 0.0 0.0 0.4 1.0 1 0 14 0.0 0.0 0.4 0.9 0.0 0.5 0.7 0.7 1.0 0.0 0.0 0.4 0.9 0.75 0.5 15 0.8 0.0 0.4 0.9 0.5 0.5 0.7 0.8 1.0 0.8 0.0 0.4 0.9 0.75 0.5 16 0.4 0.4 1.0 0.9 0.7 0.5 0.7 0.4 0.8 0.4 0.4 1.0 0.9 0.75 0.5 17 0.0 0.0 0.4 0.9 0.0 0.5 0.7 0.7 1.0 0.0 0.0 0.4 0.9 1 0.5 18 0.0 0.0 0.4 1.0 0.7 0.0 0.7 0.2 1.0 0.0 0.0 0.4 1.0 0.75 0.5 19 0.4 0.4 1.0 0.9 0.7 1.0 0.7 0.4 1.0 0.4 0.4 1.0 0.9 0.857 0 20 0.0 0.0 0.4 0.7 1.0 0.0 0.7 0.7 1.0 0.0 0.0 0.4 0.7 0.667 0.667 21 0.4 0.4 1.0 1.0 1.0 0.8 0.7 0.8 0.0 0.4 0.4 1.0 1.0 0.75 0.5 22 0.3 0.3 0.3 0.9 0.7 0.0 0.7 0.0 1.0 0.3 0.3 0.3 0.9 1 1 23 0.4 0.4 1.0 0.9 0.7 0.5 0.7 0.7 1.0 0.4 0.4 1.0 0.9 0.75 0.5

43 24 0.3 0.3 1.0 0.8 1.0 0.8 0.7 0.8 1.0 0.3 0.3 1.0 0.8 0.75 0.5 25 0.0 0.0 0.4 0.7 1.0 0.5 0.7 0.1 1.0 0.0 0.0 0.4 0.7 1 0.5 26 0.4 0.4 0.3 0.9 1.0 0.8 0.8 0.2 1.0 0.4 0.4 0.3 0.9 0.75 0.5 27 0.0 0.0 0.4 1.0 0.8 0.8 0.7 0.7 0.4 0.0 0.0 0.4 1.0 0.75 0 28 0.4 0.4 1.0 0.9 1.0 0.5 0.7 0.1 1.0 0.4 0.4 1.0 0.9 1 0.5 29 0.0 0.0 0.4 0.8 1.0 0.0 0.7 0.8 0.0 0.0 0.0 0.4 0.8 0.75 0.5 30 0.4 0.4 1.0 0.9 0.8 0.5 0.7 0.4 1.0 0.4 0.4 1.0 0.9 1 0 31 0.0 0.0 0.4 0.8 0.5 0.5 0.7 0.1 1.0 0.0 0.0 0.4 0.8 0.667 0.667 32 0.3 0.2 0.0 0.7 1.0 0.0 0.7 0.1 0.0 0.3 0.2 0.0 0.7 1 0.5 33 0.4 0.4 1.0 0.8 0.0 1.0 0.7 0.8 1.0 0.4 0.4 1.0 0.8 0.75 0 34 0.0 0.0 0.4 0.8 0.5 0.5 0.7 0.1 0.5 0.0 0.0 0.4 0.8 0.75 0.5 35 0.4 0.4 1.0 0.5 1.0 0.5 0.7 0.8 0.0 0.4 0.4 1.0 0.5 0.75 0.5 36 0.4 0.4 0.3 0.9 1.0 0.0 0.7 0.2 1.0 0.4 0.4 0.3 0.9 0.667 0.5 37 0.4 0.4 1.0 0.7 0.5 1.0 0.7 0.8 1.0 0.4 0.4 1.0 0.7 0.667 0.667 38 0.3 0.3 0.3 0.8 1.0 0.8 0.8 0.2 1.0 0.3 0.3 0.3 0.8 0.667 0.667 39 0.0 0.0 0.4 0.9 1.0 0.5 0.7 0.2 1.0 0.0 0.0 0.4 0.9 1 0 40 0.3 0.3 1.0 0.8 0.8 0.8 0.8 0.8 0.4 0.3 0.3 1.0 0.8 1 0 41 0.4 0.4 1.0 0.9 1.0 0.8 0.7 0.0 1.0 0.4 0.4 1.0 0.9 1 0.5 42 0.4 0.4 1.0 0.5 1.0 0.8 0.7 0.4 1.0 0.4 0.4 1.0 0.5 0.75 0.5 43 0.4 0.4 0.3 0.9 1.0 0.0 0.7 0.7 0.7 0.4 0.4 0.3 0.9 0.667 0.667 44 0.4 0.4 1.0 0.5 0.8 0.5 0.7 0.1 0.5 0.4 0.4 1.0 0.5 0.667 0.667 45 0.3 0.2 0.4 0.8 0.7 0.5 0.7 0.7 1.0 0.3 0.2 0.4 0.8 0.75 0.5 46 0.4 0.4 1.0 0.9 1.0 1.0 0.7 0.4 1.0 0.4 0.4 1.0 0.9 0.75 0.5 47 0.4 0.4 1.0 0.9 1.0 0.5 0.7 0.2 1.0 0.4 0.4 1.0 0.9 1 0.5 48 0.3 0.3 1.0 0.5 0.0 0.5 0.7 0.8 1.0 0.3 0.3 1.0 0.5 0.75 0.5 49 0.4 0.4 1.0 0.5 0.0 0.8 0.7 0.7 1.0 0.4 0.4 1.0 0.5 0.667 0.667 50 0.4 0.4 1.0 0.9 1.0 0.8 0.7 0.4 1.0 0.4 0.4 1.0 0.9 1 0.5 51 0.4 0.4 1.0 0.7 1.0 0.5 0.7 0.7 1.0 0.4 0.4 1.0 0.7 1 0 52 0.4 0.4 1.0 0.9 1.0 0.5 0.7 0.2 1.0 0.4 0.4 1.0 0.9 0.75 0.5

44 53 0.0 0.0 0.4 0.9 0.7 0.5 0.7 0.2 1.0 0.0 0.0 0.4 0.9 1 0.5 54 0.4 0.4 1.0 0.9 1.0 0.0 0.7 0.4 1.0 0.4 0.4 1.0 0.9 0.75 0.5 55 0.0 0.0 0.4 0.8 0.5 0.0 0.7 0.1 1.0 0.0 0.0 0.4 0.8 1 0.5 56 0.0 0.0 0.3 0.9 1.0 0.0 0.7 0.2 1.0 0.0 0.0 0.3 0.9 1 0.5 57 0.0 0.0 0.4 0.9 1.0 0.0 0.7 0.8 1.0 0.0 0.0 0.4 0.9 0.75 0.5 58 0.3 0.3 1.0 0.9 1.0 0.5 0.7 0.7 0.0 0.3 0.3 1.0 0.9 0.667 0 59 0.4 0.4 1.0 0.9 1.0 0.8 0.7 0.2 1.0 0.4 0.4 1.0 0.9 1 0.5 60 0.4 0.4 1.0 1.0 1.0 1.0 0.7 0.2 1.0 0.4 0.4 1.0 1.0 1 0 61 0.4 0.4 1.0 0.8 1.0 0.5 0.7 0.7 0.4 0.4 0.4 1.0 0.8 0.75 0.5 62 0.4 0.4 1.0 0.9 1.0 0.8 0.7 0.2 1.0 0.4 0.4 1.0 0.9 0.75 0.5 63 0.3 0.3 1.0 0.8 1.0 1.0 0.7 0.8 0.5 0.3 0.3 1.0 0.8 0.667 0.667 64 0.3 0.3 1.0 0.9 0.8 1.0 0.7 0.4 0.5 0.3 0.3 1.0 0.9 0.75 0.5 65 0.8 0.0 0.8 0.8 1.0 0.0 0.7 0.7 1.0 0.8 0.0 0.8 0.8 0.667 0.667 66 0.4 0.4 1.0 1.0 1.0 0.0 0.7 0.8 0.0 0.4 0.4 1.0 1.0 1 0.5 67 0.4 0.4 1.0 0.0 0.7 1.0 0.7 0.2 0.5 0.4 0.4 1.0 0.0 0.75 0 68 0.4 0.4 1.0 0.5 0.7 1.0 0.7 0.8 0.5 0.4 0.4 1.0 0.5 0.667 0.667 69 0.4 0.4 1.0 0.9 1.0 0.8 0.7 0.2 1.0 0.4 0.4 1.0 0.9 0.75 0.5 70 0.4 0.4 1.0 1.0 0.8 0.8 0.7 0.8 0.7 0.4 0.4 1.0 1.0 1 0.5 71 0.4 0.4 0.3 0.9 1.0 1.0 0.7 0.7 1.0 0.4 0.4 0.3 0.9 0.75 0.5 72 0.4 0.4 1.0 0.8 1.0 0.5 0.7 0.7 0.0 0.4 0.4 1.0 0.8 0.75 0.5 73 0.3 0.3 1.0 0.7 1.0 0.5 0.7 0.2 1.0 0.3 0.3 1.0 0.7 0.667 0.667 74 0.4 0.4 1.0 0.5 1.0 1.0 0.7 0.2 0.5 0.4 0.4 1.0 0.5 0.75 0.5 75 0.4 0.4 1.0 0.8 1.0 0.0 0.7 0.2 0.0 0.4 0.4 1.0 0.8 0.667 0.667 76 0.4 0.4 0.3 1.0 0.8 1.0 0.8 0.7 0.4 0.4 0.4 0.3 1.0 0.75 0.5 77 0.0 0.0 0.4 0.7 0.5 0.0 0.7 0.2 0.7 0.0 0.0 0.4 0.7 0.667 0.667 78 0.4 0.4 0.3 0.9 0.7 0.0 0.7 0.2 0.7 0.4 0.4 0.3 0.9 0.75 0.5 79 0.4 0.4 1.0 0.8 0.0 1.0 0.7 0.0 1.0 0.4 0.4 1.0 0.8 0.667 0.667 80 0.0 0.0 0.4 0.8 0.8 0.5 0.7 0.8 1.0 0.0 0.0 0.4 0.8 1 0 81 0.4 0.4 1.0 0.9 0.5 0.0 0.7 0.7 1.0 0.4 0.4 1.0 0.9 0.667 0.667

45 82 0.0 0.0 0.4 0.9 1.0 0.0 0.7 0.4 0.0 0.0 0.0 0.4 0.9 0.667 0.667 83 0.4 0.4 1.0 0.8 0.5 0.8 0.7 0.2 1.0 0.4 0.4 1.0 0.8 0.667 0.667 84 0.4 0.4 1.0 1.0 1.0 0.5 0.7 0.2 0.0 0.4 0.4 1.0 1.0 1 0.5 85 0.3 0.3 1.0 0.9 1.0 0.0 0.7 0.8 1.0 0.3 0.3 1.0 0.9 0.75 0.5 86 0.4 0.4 1.0 1.0 0.7 1.0 0.7 0.4 1.0 0.4 0.4 1.0 1.0 0.75 0.5 87 0.8 0.0 0.4 0.8 0.7 0.0 0.7 0.4 1.0 0.8 0.0 0.4 0.8 1 0.5 88 0.3 0.3 1.0 0.8 1.0 0.5 0.7 0.7 1.0 0.3 0.3 1.0 0.8 1 0.5 89 0.4 0.4 1.0 0.8 1.0 0.5 0.7 0.7 0.0 0.4 0.4 1.0 0.8 0.75 0.5 90 1.0 0.0 1.0 0.8 1.0 0.8 0.8 0.0 1.0 1.0 0.0 1.0 0.8 1 1 91 0.0 0.0 0.5 0.9 1.0 0.5 0.7 0.4 1.0 0.0 0.0 0.5 0.9 0.75 0.5 92 0.0 0.0 0.4 0.5 0.5 0.5 0.7 0.2 1.0 0.0 0.0 0.4 0.5 0.75 0.5 93 1.0 0.0 1.0 0.7 1.0 0.0 0.7 0.4 1.0 1.0 0.0 1.0 0.7 1 0.5 94 0.0 0.0 0.4 0.9 1.0 0.0 0.7 0.0 1.0 0.0 0.0 0.4 0.9 0.667 0.667 95 0.4 0.4 0.3 0.9 0.5 0.0 0.7 0.8 1.0 0.4 0.4 0.3 0.9 0.75 0.5 96 0.8 0.0 0.4 0.5 1.0 0.0 0.7 0.8 1.0 0.8 0.0 0.4 0.5 0.8 0.8 97 0.3 0.3 1.0 0.7 1.0 0.5 0.7 0.7 1.0 0.3 0.3 1.0 0.7 1 0 98 0.4 0.4 1.0 0.8 1.0 0.0 0.7 0.2 1.0 0.4 0.4 1.0 0.8 1 0.5 99 0.4 0.4 1.0 0.8 0.7 1.0 0.7 0.2 1.0 0.4 0.4 1.0 0.8 1 0.5 100 0.4 0.4 1.0 0.9 1.0 0.0 0.7 0.4 0.7 0.4 0.4 1.0 0.9 0.75 0.5 Total 32.2 26.8 76.1 78.7 78.8 50.7 67.9 43.1 77.1 32.2 26.8 76.1 78.7 80.748 46.816

46 Table 4. Body and trace-fossil record of the Gracillarioidea (Lepidoptera: Ditrysia,

Gracillariidae and Bucculatricidae).

Leaf Miner Host plant Age (epoch, stage) Formation References

and locality

cf. Bucculatrix Tilia sp. late Pliocene, Willershaus (Strauss, 1977)

thoracella (Málvales: (Piacenzian; -2.7 en.

Thunburg Tiliaceae) m.y.) Germany

cf. Caloptilia Fagus cf. late Pliocene, Willershaus (Strauss, 1977)

alchimiella occidentalis (Piacenzian; -2.7 en.

S.C. (Fágales: m.y.) Germany

Fagaceae)

cf. Caloptilia Juglans late Pliocene, Willershaus (Strauss, 1977)

roscipennis (Fágales: (Piacenzian; -2.7 en.

Juglandaceae) m.y.) Germany

cf. Corsicium Magnolia sp. late Pliocene, Willershaus (Strauss, 1977)

sp. (Magnoliales: (Piacenzian; -2.7 en.

Magnoliaceae) m.y.) Germany

, or Syringia

sp. (Lamíales:

Oleaceae)

Lithocolletis unknown host late Pliocene, Willershaus (Strauss, 1977)

maestingella (Piacenzian; ~2.7 en.

47 Zetterstedt m.y.) Germany

cf. Parornix Amelanchier late Pliocene, Willershaus (Strauss, 1977)

sp. sp. (Rosales: (Piacenzian; -2.7 en,

Rosaceae) m.y.) Germany

cf. Caloptilia Fágales: early Pliocene Chuzbaia, (Givulescu, 1984)

roscipenella Betulaceae (Zanclian; ~4.4 Romania

m.y.)

Caloptilia sp. 1 Quer cus Miocene Latah (Lewis, 1969)

(Fágales: [undifferentiated] Formation;

Fagaceae) eastern

Acrocercops Quercus late Miocene Trout (Opler, 1973)

sp. consimilis (Messinian; ~6.2 Creek,

Newberry m.y.) Oregon,

(Fágales: USA

Fagaceae)

"Lithocolletis" Populus late Miocene Stewart (Opler, 1973)

sp. trichocarpa (Messinian; ~6.2 Valley,

var. ingrata m.y.) Nevada,

(Malpighiales: USA

Salicaceae)

Bucculatrix sp. Quercus late Miocene Buffalo (Opler, 1973)

hanibalii Dorf (Messinian; ~6.2 Canyon,

(Fágales: m.y.) Nevada,

48 Fagaceae) USA

Cameraria sp. Quercus early to late Thorn (Opler, 1973)

simulata Miocene Creek,

Knowlton (Tortonian/(Serravv Idaho, USA

(Fágales: allian; -13 m.y.)

Fagaceae)

"Lithocolletis" Quercus late middle Upper (Opler, 1973)

hanibalii Dorf Miocene, Goldyke,

(Fágales: (Serravallian; ~13 Cedar

Fagaceae) m.y.)

Gracillariidae early Miocene, La Toca (Poinareia/., 1991)

(body fossil) (Aquitanian; ~22 Formation

m.y.) amber;

Phyllonorycterl Symplocos sp. early Oligocène Tremembé (Martins-Neto, 1989)

Olive irae (Ericales: (Rupelian; ~31 m.y.) Formation;

Martins-Neto Symplocaceae) Taubaté, Sao

Paulo, Brazil

Bucculatrix sp. "Ze/ztova" late Eocene Florissant (Opler, 1982;

drymeja (Priabonian; -34.5 Formation; Kozlov, 1988)

(Rosales: m.y.) Park,

Ulmaceae) County,

Colorado,

USA

49 Graduar Ute s late Eocene, Bembridge (Jarzembowski,

sp. (body (Priabonian; ~35 Marls, 1980; Kozlov, 1987)

fossil) m.y.) Gunnard

Bay, Isle of

Wight,

U.K.

"Lithocolletis" unknown dicot late Eocene, White Lake (Freeman, 1965;

sp. (Priabonian; ~35 Basin, Kozlov, 1988)

m.y.) British

Columbia,

Canada

Graduar Ute s middle Eocene, Baltic (Kozlov, 1987)

mixtus (Lutetian; -44.1 amber

Kozlov (body m.y.)

fossil)

GradllarUtes middle Eocene, Baltic (Kozlov, 1987)

Uthuani- (Lutetian; -44.1 amber

Kozlov (body m.y.)

fossil)

Gracillariid unknown dicot middle Eocene, (?Barto Bransome (Langeia/., 1995)

[recent analog: Sand

Lyonettia Formation; prunifoliella Hübr Bournemouth,

50 Gracillariid unknown dicot middle Eocene, Bransome (Stevenson, 1992;

(recent analog: (Bartonian) Sand hang et al, 1995)

Lithocolletis Formation;

rajella Bournemouth,

on Alnus

Gracillariid unknown dicot middle Eocene, Bransome (Stevenson, 1992;

(recent analog: (Bartonian) Sand Stevenson & Scott,

Phyllocnistis Formation; 1992; Lang ei a/., 1995

sujfusella Zeller Bournemouth,

on Populus

(Salicaceae)

Gracillariid unknown dicot middle Eocene, Republic, (Labandeira, 2002)

(Lutetian; -48 m.y.) Ferry County,

Washington, U

Phyllocnistis Cedrela sp. early Eocene, Wind River (Hickey & Hodges,

sp. (Sapindales: (Ypresian; ~51 Formation; 1975)

Meliaceae) m.y.) Dubois,

Wyoming,

USA

Gracillariid Marmarthia Late Cretaceous, Hell Creek (Lang, 1996;

(recent analog: pearsoni (late Maastrichtian; Formation; Labandeira, 2002;

Bucculatrix (Laurales) ~ 91 m.y.) Willis ton Labandeira et al.,

albella Sft. on Basin, 2002)

51 Paliurus North

(Rhamnaceae) Dakota,

USA

IPhyllocnistis ''Staphyleal Late Cretaceous Frontier (Knowlton, 1917)

fremontii" [an (Turonian; ~91 Formation;

unknown m.y.) Lincoln

dicot] County,

Wyoming,

USA

Bucculatrix Platanus sp. Late Cretaceous Kizyl-Zhar, (Kozlov, 1988) platani Kozlov (Proteales: (Turonian; -91 Kazakstán

Platanaceae) m.y.)

Phyllocnistis sp. Rogersia Early Cretaceous (late Dakota (Labandeiraei a/..

parlatorii Albian; -99 m.y.) Formation; 1994; Labandeira,

Laurales: cf. Kansas and 1998; Kristensen &

Lauraceae); Nebraska, Skalski, 1999)

Densinevum sp. USA

(cf. Chlor

anthaceae);

Pabiana

variloba

(Laurales)

1. Systematic placement of plant hosts follows Judd (1999)

52 2. Geochronologic dates are given as midpoints of stages (Gradstein & Ogg,

1996), unless specific isotopic dates are provided.

Freeman, T.N. 1965. A lepidopterous leaf-mine from the Tertiary Period. Can. Entomol.

97: 1069-1070.

Givulescu, R. 1984. Pathological elements on fossil leaves from Chiuzbaia (galls, mines

and other insect traces). Däri Seama Sedin. Inst. Geol. Geofiz. Paleont. 68: 123-

133.

Gradstein, F.M. & Ogg, J.G. 1996. A Phanerozoic time scale. Episodes 19: 1-3.

Hickey, L.J. & Hodges, R.W. 1975. Lepidopteran leaf mine from the Early Eocene Wind

River Formation of northwestern Wyoming. Science 189: 718-720.

Jarzembowski, E.A. 1980. Fossil insects from the Bembridge Marls, Palaeogene, of the

Isle of Wight, southern England. Bull. Brit. Mus. Nat. Hist. (Geol.) 33: 237-293.

Judd, W.S., Campbell, C.S., Kellogg, E.A. & Stevens, P.F. 1999. Plant Systematics: A

Phylogenetic Approach Sinauer, Sunderland, Massachusetts.

Knowlton, F.H. 1917. A fossil flora from the Frontier Formation of southwestern

Wyoming. U. S. Geol. Surv. Prof. Paper 108: 73-95.

Kozlov, M.V. 1987. New Ditrysan lepidopterans from Baltic amber. Palentol. J. 4: 59-

67.

53 Kozlov, M.V. 1988. Paleontology of lepidopterans and problems of the phylogeny of the

order Papilionida. In: The Mesozoic-Cenozoic Crisis in the Evolution of Insects

(ed.A.G. Ponomarenko, eds), pp. 16-69. Academy of Sciences, Moscow.

Kristensen, N.P. & Skalski, A.W. 1999. Phylogeny and palaeontology. In: Lepidoptera,

Moths and Butterflies•Volume 1 : Evolution, Systematics, and Biogeography

(ed.N.P. Kristensen, eds), pp. 7-25. De Gruyter, Berlin.

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ecosystems. In: Lower and Middle Cretaceous Terrestrial Ecosystems. (S.G.

Lucas, J.I. Kirkland, J.W. Estep, eds), pp. 105-124.

Labandeira, C.C. 2002. Paleobiology of middle Eocene plant-insect associations from the

Pacific Northwest: a preliminary report. Rocky Mountain Geology 37: 31-59.

Labandeira, C.C, Dilcher, D.L., Davis, D.R. & Wagner, D.L. 1994. Ninety-seven

millions years of angiosperm-insect association: paleobiological insights into the

meaning of coevolution. Proc. Nati. Acad. Sei. USA 91: 12278-12282.

Labandeira, C.C, Johnson, K.R. & Wilf, P. 2002. Impact of the terminal Cretaceous

event on plant-insect associatons. Proc. Nati. Acad. Sei. USA 99: 2061-2066.

Lang, P.J. 1996. Fossil Evidence for Patterns of Leaf-Feeding from the Late Cretaceous

and Early Tertiary Ph.D., University of London.

Lang, P.J., Scott, A.C. & Stephenson, J. 1995. Evidence of plant-arthropod interactions

from the Eocene Branksome Sand Formation, Bournemouth, England:

Introduction and description of leaf mines. Tert. Res. 15: 145-174.

54 Lewis, S.E. 1969. Lepidopterous larval-mining of an oak (?) leaf from the Latah

Formation (Miocene) of eastern Washington. Ann. Entomol. Soc. Am. 62: 1210-

1211.

Martins-Neto, R.G. 1989. Novos insetos Terciarios do Estado de Sao Paulo. Rev. Bras.

Geociênc. 19: 375-386.

Opler, P.A. 1973. Fossil lepidopterous leaf mines demonstrate the age of some insect-

plant relationships. Science 179: 1321-1323.

Opler, P.A. 1982. Fossil leaf-mines of Bucculatrix (Lyonetiidae) on Zelkova (Ulmaceae)

from Florissant, Colorado. /. Lepid. Soc. 36: 145-147.

Poinar, CO., Treat, A.E. & Southcott, R.V. 1991. Mite parasitism of moths: examples of

paleosymbioisis in Dominican amber. Experientia 47: 210-212.

Stevenson, J. 1992. Evidence of PlantIInsect Interactions in the Late Cretaceous and

Early Tertiary, University of London.

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Terr. Nov. 4: 542-552.

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Harz. Verhandlungen des Botanischen Vereins der Provinz Brandenburg 113: 43-

80.

55 Appendix 1. Specimens used in this study. They have been ordered according to the plant classification (APG, 1998)

Host Plant Species name Collection site Voucher CoUec GenBank accesión number tor number 28S rDNA

ORDER Proteales Family Platanaceae Platanus sp. Phyllonorycter platani Osteriey Park, Middlesex, 49(91) CLV AF477565 (Staudinger, 1870) UK ASTERIDS ORDER Ericales Family Ericaceae Vaccinium vitis-idaea Phyllonorycter junoniella Loch Garten, Inverness- 67(150) MS AF477555 (Zeller, 1846) shire, Scotland Lyonia ovalifolia Phyllonorycter lyoniae Asahi-Kogen, Honsyu, 266 KS AY521508 Kumata, 1963 Japan Euasterid II ORDER Dipsacales Family Caprifoliaceae Lonicera sp. Phyllonorycter lonicerae Oguni-Zinaya, Honsyu, 268 KS AY521517 Kumata, 1963 Japan Lonicera sp. Phyllonorycter trifasciella Hanwell, London, UK 13,14(142) CLV AF477580 (Haworth, 1828) Symphoricarpos sp. Phyllonorycter sp.4 Santa Rosa, Guanajuato, 242 CLV AY230759 Mexico Symphoricarpos rivularis Phyllonorycter Pett's Wood, Kent, UK 104(182) DO AF477526 emberizaepennella (Bouche, 1834) Family Valerianaceae Viburnumx carlcephalum Phyllonorycter lantanella Kew Gardens, UK 158 CLV AF477502 (Schrank, 1802) Family Dipsacaceae Scabiosa sp. Phyllonorycter scabiosella Addington, Kent, UK 92(172) DO AF477518 (Douglas, 1853) ROSIDS Eurosid II ORDER Málvales Family Malvaceae Grewia sp. Phyllonorycter grewiella Bontebok, South Africa 248 CLV AY230753 (Van, 1961) Tilia platyphylla Phyllonorycter issikii Bialowiezca, Poland 246 EO AY230754 (Kumata, 1963)

56 Tilia japónica Ishikari, Hokkaido, Japan 321 HK AY521521 Dombeya tórrida Phyllonorycter loxozana Kenya: Zuni 126 DA AF477550 (Meyrick, 1936) ORDER Sapindales Family Sapindaceae Acer pseudoplatanus Phyllonorycter geniculella Silwood Park, Ascot, 26(110) CLV AF477575 (Ragonot, 1874) Berkshire, UK Acer platanoides Phyllonorycter platanoidella Silwood Park, Ascot, 41(128) CLV AF477563 (Joannis, 1920) Berkshire, UK Acer campestre Phyllonorycter sylvella Lepe, New Forest, UK 28(137) CLV AF477582 (Haworth, 1828) Acer carpinifolium Phyllonorycter orientalis Honsyu, Simasima-dani, JP16 KS AY521519 Kumata, 1963 Azumi Vill. Nagano Pref., Japan Acer mono Phyllonorycter sp. 8 Kannon-Zawa, Sapporo JP15 KS AY521518 City, Hokkaido, Japan

Eurosid I ORDER Malpighiales Family Salicaceae Salix repens Phyllonorycter La Chaume, Vendee, 91(167) CLV AF477532 quinquéguttella (Stainton, France 1851) Salix caprea Phyllonorycter viminiella Lepe, Hampshire, UK 29(138) CLV AF477571 (Sircom, 1848) Silwood Park, Ascot, 42(114) CLV AF477571 Berkshire, UK Salix cinérea. Phyllonorycter salicicolella Kristianstad, Sweden 177 SV AF477522 (Sircom, 1848) Salix sp. Phyllonorycter dubitella Silwood Park, Ascot, 40(129) CLV AF477572 (Herrich-Schaffer, 1855) Berkshire, UK Salix fragilis. Phyllonorycter salictella Kristianstad, Sweden 179 SV AF477505 (Zeller, 1846) Salix aurita Phyllonorycter brevilineatella Jamshog, Sweden 178 SV AF477538 (Benander, 1946) Salix viminalis Phyllonorycter viminetorum Woolhampton, Reading, 159 IS AF477530 (Stainton, 1854) UK Not reared Phyllonorycter hilarella TwoUuvaara, Sweden 227 SV AF477534 (Zetterstedt, 1839) Salix sp. Phyllonorycter heringiella Bjalbo, Marstad, Sweden 181 SV AF477542 Gronlien, 1932 Salix sp. Phyllonorycter pastorella Trudovec, Bulgary 272 RT AF477535

57 (Zeller, 1846) Salix miyabeana Sapporo, Hokkaido, Japan 323 HK AF477509 Populas canescens Phyllonorycter comparella Andreze, Maine Loire, 86(164) CLV AF477539 (Duponchel, 1843) Populus trémula Phyllonorycter sagitella Worcestershire, UK 209 MH AF477532 (Bjerkander, 1790) Populas sp. Phyllonorycter populifolilella Uglich, Russia 80 EO AF477571 (Treitschke, 1833) ORDER Fabales Family Fabaceae

Medicago sp. Phyllonorycter medicaginella Butan, Bulgary 275 RT AY521515 (Gerasimov, 1930) Vicia sp. Phyllonorycter nigrescentella Darenth Wood, Kent, UK 98(176) DO AF477515 (Logan, 1851) Trifolium sp. Phyllonorycter insignitella La Gachere, Brem sur 85(165) CLV AF477540 (Zeller, 1846) Mer, France Sarothamnus scoparias Phyllonorycter scopariella Mandagout, Cevennes, 71(153) CLV AF477552 (Zeller, 1846) France Ulex europaeas Phyllonorycter ulicicolella Silwood Park, Ascot, 69(151) CLV AF477516 (Stainton, 1851) Berkshire, UK Genista pilosa Phyllonorycter staintoniella Perranporth, Cornwall, UK 154 FHNS AF477540 (Nicelli, 1853) Genista radiata Phyllonorycter baldensis Corna Piaña,, Monte 192 GD AF477504 Deschka, 1986 Valdo, Verona, Italy Genista florida Phyllonorycter genistella Miraflores de la Sierra, 258 EN AY521512 (Rebel, 1900) Madrid, Spain Adenocarpus complicatas Phyllonorycter parvifoUella Branza, Arzua, Galicia, 175(218) CLV AF477510 (Ragonot, 1875) Spain Chamaespartiam Phyllonorycter cerasinella Alajar, Sierra de Aracena, 255 EN AY521509 tridentatum (Reutti, 1852) Huelva, Spain Stauracanthus genistoides Phyllonorycter sp.6 Magazon, Huelva, Spain 256 EN AY521510 Genista polyantha Phyllonorycter sp.7 El Garrobo, Sevilla, Spain 257 EN AY521511 Adenocarpus telonensis Phyllonorycter parvifoUella Miraflores de la Sierra, 259 EN AY521513 (Ragonot, 1875) Madrid, Spain ORDER Fágales Family Betulaceae Alnus sp. Phyllonorycter sp.5 Santa Rosa, Guanajuato, 244 CLV AY230760 Mexico Alnus glutinosa Phyllonorycter stettinensis Silwood Park, Ascot, 43(112) CLV AF477545 (Nicelli, 1852) Berkshire, UK Virginia Water, Surrey, 5(6.2) CLV AF477545

58 UK " " Silwood Park, Ascot, 111(189) CLV AF477545 Berkshire, UK Alnus glutinosa Phyllonorycter froelichiella Silwood Park, Ascot, 6(6.3) CLV AF477586 (Zeller, 1839) Berkshire, UK Alnus glutinosa Phyllonorycter rajella Silwood Park, Ascot, 55(113) CLV AF477568 (Linnaeus, 1758) Berkshire, UK Alnus incana Phyllonorycter strigulatella Bookham, Surrey, UK 99(158) CLV AF477511 (Lienig & Zeller, 1846) " " Medmenham, Berks, UK 162 IS AF477511 Alnus glutinosa Phyllonorycter strigulatella Blacknest, Brimpton 161 IS AF477511 (Lienig & Zeller, 1846) Common, Berks, UK " Phyllonorycter kleemannella Rostock, Sweden 184 SV AF477549 (Fabricius, 1781) Betula péndula Phyllonorycter corylifoliella Val d'Aran, Pirineos, 94 EO AF477503 (Hubner, 1796) Spain Betula sp. Phyllonorycter ulmifoliella Osterley Park, Middlesex, 38(101) CLV AF477508 (Hubner, 1817) UK " " Osterley Park, Middlesex, 56(101) CLV AF477508 UK " ÍÍ Chateauneuf sur Loire, 90(88) CLV AF477508 Loiret, France Betula pubescens Phyllonorycter anderidae (W. Dagsmosse, Sweden 163 SV AF477519 Fletcher, 1875) Betula sp. " Medmenham, Berks, UK 232 IS AY521504 Betula pubescens Phyllonorycter cavella Dagsmosse, Sweden 228 SV AF477512 (Zeller, 1846) Corylus avellana Phyllonorycter nicellii Silwood Park, Ascot, 9(11.1) CLV AF477562 (Stainton, 1851) Berkshire, UK Not reared Virginia water, Surrey, UK 66(149) CLV AF477562 flying Corylus avellana Phyllonorycter coryli (Nicelli, Silwood Park, Ascot, 110(188) CLV AF477507 1851) Berkshire, UK Ostrya carpinifolia Phyllonorycter aemula Nago, Trento, Italy 193 GD AF477559 Triberti, Deschka & Huemer, 1997 Carpinus betulus Phyllonorycter tenerella Chateauneuf sur Loire, 89(86) CLV AF477546 (Joannis, 1915) Loiret, France

59 Carpinus betulus Phyllonorycter esperella Osterley Park, Middlesex, 32(89) CLV AF477569 (Goeze, 1783)= quinnata UK (Geoffroy, 1785) Carpinus laxiflora Phyllonorycter japónica Miyakonojo, Mizakazi, 262 YS AY521524 Kumata, 1963 Japan Carpinus cordata " Japan, ? 327 HK AY521525 Family Fagaceae Quercus robur Phyllonorycter messaniella Silwood Park, Ascot, 1(2.1) CLV AF477584 (Zeller, 1846) Berkshire, UK Quercus robur Phyllonorycter distentella Thorden Wood, East Kent, 101(179) DO AF477541 (Zeller, 1846) UK Quercus robur Phyllonorycter harrisella Richmond Park, 51(125.3) CLV AF477567 (Linnaeus, 1761) Middlesex, UK " " Richmond Park, 52(125.1) CLV AF477567 Middlesex, UK Quercus robur Phyllonorycter lautella Pett's Wood, Kent, UK 214 DO AF477579 (Zeller, 1846) Quercus robur Phyllonorycter quercifoliella Richmond Park, 57(125.1) CLV AF477561 (Zeller, 1839) Middlesex, UK Quercus robur Phyllonorycter saportella South Lopham, UK 210(191) CLV AF477543 (Duponchel, 1840) Quercus robur Phyllonorycter muelleriella Cyrencester park, Hatcled 157 IS AF477506 (Zeller, 1839) Quercus robur Phyllonorycter roboris Queen's wood, Dymock, 211(196) CLV AF477560 (Zeller, 1839) Worcestershire, UK Not reared Phyllonorycter heegeriella Torringe, Sweden 229 SV AF477531 (Zeller, 1846) Quercus Phyllonorycter ilicifoliella Val Lourian, Rougiens, 118 JN AF477529 (Duponchel, 1843) Var, France Quercus ilex Phyllonorycter joviella Montseny, Barcelona, 84 EO AF477523 (Constant, 1890) Spain Quercus crispula Phyllonorycter cretata Ishikari, Japan 260/264 IT AY521505 Kumata, 1963 Quercus acutissima Phyllonorycter acutissimae Nara, Japan 263 SH AY521522 Kumata, 1963 Quercus mongólica var. " Ishikari, Hokkaido, Japan 320 HK AY521522 grosseserrata Quercus mongólica var. Phyllonorycter similis Ishikari, Hokkaido, Japan 324 HK AY521528 grosseserrata (Kumata) Quercus sessilifolia Phyllonorycter similis Guangdong, Shao-guan 331 FH AY521527 (Kumata) City, China Quercus dentata Phyllonorycter pygmaea Kagoshima, Japan 265 YS AY521507

60 Kumata, 1963 Fagus sylvatica Phyllonorycter maestingella Kew Gardens, Middlesex, 73(156) CLV AF477520 (Müller, 1764) UK Quercus robur Phyllonorycter sp. 1 0 Carballal, Bueu, Spain 188 CLV AF477537 " " 0 Carballal, Bueu, Spain 189 CLV AF477551 Quercus pyrenaica Phyllonorycter sp 2 Serra do Xures, Lovios, 216 CLV AF477553 Spain Quercus suber Phyllonorycter sp.3 Serra do Xures, Lovios, 190 CLV AY230761 Spain ORDER Rosales Family Rosaceae Pyrus sp. Phyllonorycter cydoniella Virginia water, Surrey, UK 54(115) CLV AF477573 (Denis & Schiffermuller, 1775) Sorbus torminalis Phyllonorycter mespilella Bookham, Surrey, UK 75(162) CLV AF477548 (Hubner, 1805) Sorbus torminalis Phyllonorycter corylifoliella Bookham, Surrey, UK 76(161) CLV AF477536 (Hubner, 1796) Sorbus aucuparia Phyllonorycter sorbí (Frey, Silwood Park, Ascot, 7(8.1) CLV AF477585 1855) Berkshire, UK Sorbus commixta Phyllonorycter sorbicola Ishikari, Hokkaido, Japan 322 HK AY521523 (Kumata, 1963) Crataegus monogyna Phyllonorycter oxyacanthae Silwood Park, Ascot, 112(190) CLV AF477570 (Frey, 1856) Berkshire, UK Crataegus monogyna Phyllonorycter corylifoliella Silwood Park, Ascot, 106 CLV AF477514 (Hubner, 1796) Berkshire, UK Prunus capuli Phyllonorycter crataegella Santa Rosa, Guanajuato, 241 CLV AY230762 (Clemens, 1859) Mexico Prunus spinosa Phyllonorycter spinicolella Brent river park, Hanwell, 33(134) CLV AF477554 (Zeller, 1846) London, UK Prunus sp. Phyllonorycter cerasicolella Molins rei, Barcelona, 81 EO AF477528 (Peyerimhoff, 1872) Spain " " Brent river park, Hanwell, 105(183) CLV AF477528 London, UK Pyracantha sp. Phyllonorycter Hanwell, London, UK 60(143) CLV AF477556 leucographella (Zeller, 1850) Malus sp. Phyllonorycter blancardella Lepe, Hampshire, UK 34(135) CLV AF477564 (Fabricius, 1781) Malus sp Phyllonorycter elmaella USA 31 TU AF477558 Doganlar & Mutuurua, 1980 Malus sp. Phyllonorycter pomonella Emilia-Romagna, Spain 47 EO AF477578 Zeller, 1846 nee. auct.

61 Malus sp Phyllonorycter gerasimovi (M. Bucharest, Romania 119 EO AF477524 Hering, 1930) Malus sp Phyllonorycter pyrifoliella Silistra, Bulgary 271 RT AY521514 (Gerasimov, 1933) Family Ulmaceae Ulmus glabra Phyllonorycter schreberella Silwood Park, Ascot, 35(100) CLV AF477566 (Fabricius, 1781) Berkshire, UK Ulmus glabra Phyllonorycter tristrigella Virginia water, Surrey, UK 27(111) CLV AF477581 (Haworth, 1828) Ulmus davidiana var. Phyllonorycter bicinctella Ishikari, Hokkaido, Japan 328 HK AY521526 japónica (Matsumura) Ulmus sp. Phyllonorycter acaciella Sofia, Bulgary 277 RT AY521516 (Duponchel, 1843) Ulmus sp. Phyllonorycter agüella Sofia, Bulgary 279 RT AY521520 (Zeller, 1846) Family Celtidaceae Celtis australis Phyllonorycter millierella Barcelona, Spain 95 EO AF477517 (Staudinger, 1871) Celtis sinensis Phyllonorycter bifurcata Korimoto, Kyusyu, Japan 261/267 YS AY521506 Kumata, 1967 OUTGROUPS Superfamily Family Micropterigidae Micropteryx thunbergella Burnham Beeches, Slough, 54 IS AY521484 (Fabricius, 1794) England Superfamily Gracillarioidea Family Bucculatricidae Quercus robur Bucculatrix ulmella Zeller, Richmond Park, 53(125) CLV AF477574 1848 Middlesex, UK Family Gracillariidae Subfamily Gracillariinae Malus spp. Callisto denticulella Medmenham, Berks, UK 167 IS AF477525 (Thunberg, 1794) Hypericum perforatum Calybites auroguttella Bix Bottom, Oxon, UK 166 IS AF477521 (Stephens, 1835) Hypericum sp. Eucalybites aureola Kumata, Okusawa-suigenti, Otaru JPIO KS AY521500 1982 City, Hokkaido, Japan Margaritaria nobilis Caloptilia sp. Chiquibul Forest, Belize 150 OL AY521501 Betula sp. Caloptilia populetorum Chigwell Row, Essex, UK 175 IS AF477513 (Zeller, 1839) Rhododendron Caloptilia azaleella (Brants, Virginia water, Surrey, UK 65(148) CLV AF477577 1913)

62 Alnus glutinosa Caloptilia falconipennella Medmenham, Marlow, 170 IS AF477533 (Hubner, 1813) Bucks, UK Carpinus betulus Parornix carpinella (Frey, Silwood Park, Ascot, 37(90) CLV AF477583 1863) Berkshire, UK Tournefortia hirsutissima Dialéctica sp. Chiquibul Forest, Belize 141 OL AF477527 Mallotusphilippensis Acrocercops heptadeta le-rindo, Okinawa-honto, JP17 KS AY521489 Meyrick, 1936 Ryukyu, Japan Quercus ilex Acrocercops brongniardella La Gachere, Brem sur 127(171) CLV AF477501 (Fabricius, 1798) Mer, France " " " 128(171) CLV AF477501 Quercus robur " Syderstone, Norfolk 168 IS AF477501 Quercus serrata Acrocercops unistriata Oguni-zinya, Mori Town, JP14 KS AY521488 Decheng, 1986 Honsyu, Japan Malvaviscus arbóreas Eucosmophora pithecellobiae Chiquibul Forest, Belize 140-142 OL AY521495 Malachra fasciata nov.sp. Pisonia aculeata Marmara sp. Chiquibul Forest, Belize 153 OL AY521485 Coccoloba belizensis Metrichroa sp. 1 Chiquibul Forest, Belize 151 OL AY521498 Piper pseudofuligineum Metrichroa sp.2 Chiquibul Forest, Belize 145 OL AY521496 Psichotria nervosa Metrichroa sp.3 Chiquibul Forest, Belize 139 OL AY521499 Turpinia occidentalis Metrichroa sp.4 Chiquibul Forest, Belize 147 OL AY521497 Alnus hirsuta Dendrorycter marmaroides Eniwa-keikoku, Bniwa JPU KS AY521486 Kumata, 1978 City, Hokkaido, Japan Pueraría lobata Liocrobyla lobata Kuroko, Ogasa-yama, Daito Town, JP12 KS AY521501 1960 Honsyu, Japan Machilus thunbergi Gibbovalva quadrifasciata Hunaura, Uehara, JP13 KS AY521487 (Stainton, 1863) Iriomote-zima, Ryukyu, Japan Robinia Parectopa robiniella Primorsko, Bulgary 278 RT AY521502 Clemens, 1863 Family Gracillariidae Subfamily Phyllocnistinae Citrus limon Phyllocnistis citrella Stainton, California, USA 68 JMA AF477557 1856 Phyllocnistis saligna (Zeller, Schinnen, Holland 281/284 EN AY521491 1839) Phyllocnistis labyrinthella Zwannenwater, Holland 283 EN AY521494 (Bjerkander, 1790) Stigmaphyllon Phyllocnistis sp. 1 Chiquibul Forest, Belize 133 OL AY521490 lindenianum Swietenia macrophylla Phyllocnistis meliacella Chiquibul Forest, Belize 134 OL AY521492 Nectandra cf. Phyllocnistis sp.2 Chiquibul Forest, Belize 132-138 OL AY521493 longicaudata

63 Licaria peckii Family Gracillariidae Subfamily Lithocolletinae Aesculus flavax * pavia Cameraria sp 1 Virginia, USA 254 MW AY230758 Quercus sp. Cameraria sp 2 Santa Rosa, Guanajuato, 243 CLV AY230756 Mexico Cameraria guttifinitella USA 247 DD AY230755 (Clemens, 1859) Quercus sp. Cameraria hamadryadella Virginia, USA 250 MW AY230757 (Clemens, 1859) Aesculus hippocastanum Cameraria ohridella Deschka Austria 83 EO AF477544 & Dimic, 1986 unidentified leguminosous Lithocolletinae sp. Bourbon Hotel park, Foz 282 EN AY521503 tree do Iguaçu, Parana, Brasil

New sequences reported in this study are highlighted in bold.

Note. CLV: Carlos Lopez Vaamonde; EO: Ehsenda Olivella; DO: Dennis O'Keeffe; MS: Mark Shaw; TU: Tom, Uhru; S: Sruogal; JN: Jacques Nel; FHNS: Frank Smith; FH: Frank Hsu; OL: Owen Lewis; DA: David Agassiz.; IS: Ian Simms; SV: Ingvar Svensson; GD: Gerfi-ied Deschka; MH: Michael Harper; DD: Don Davis; MW: Mike Wise; EN: Erik J. Van Nieukerken; RT: Rumen Tomov; IT: Ishida T.; YS: Y. Sakamaki; SH: Sato H.; KS: K. Sugisima; JMA: Juan Manuel Alvarez; HK: Hodeki Kagata; MK: Marc Kenis; TK: Tosio Kumata.