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Please focus on the results! Do not focus on the methods (skim these at best)

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Please focus on the results! Do not focus on the methods (skim these at best).

Paper #1: Farrell, B.D. 1998. “Inordinate fondness” explained: Why are there so many beetles? Science 281: 555-559.

Paper #2: Nakadai, R. and A. Kawakita. 2016. Phylogenetic of speciation by host shift in leaf cone moths (Caloptilia) feeding on maples (Acer). Ecology and Evolution 6: 4958-4970.

1. What do you think is the link between these two papers?

2. Provide two discussion questions. These questions should motivate some aspects of discussion. They do not need to be separated out by paper (indeed, integrative questions are often the best). Your questions should provide insight beyond simple rote explanations or definitions. Very good questions may have detailed explanations, examples, or justifications.REPORTS tions in all visual cortical areas could underlie a three-dimensional spatial code for addressing and binding of computations carried out in different cortical compartments. References and Notes 1. A. H. Holway and E. G. Boring, Am. J. Psychol. 54, 21 (1941); A. S. Gilinsky, ibid. 68, 173 (1955). 2. N. Humphrey and L. Weiskrantz, Q. J. Exp. Psychol. 21, 255 (1969); L. G. Ungerleider, L. Ganz, K. H. Pribram, Exp. Brain Res. 27, 251 (1977). 3. H. Sakata, H. Shibutani, K. Kawano, J. Neurophysiol. 43, 1654 (1980); R. A. Andersen and V. B. Mountcastle, J. Neurosci. 3, 532 (1983); J. W. Gnadt and L. F. Mays, J. Neurophysiol. 73, 280 (1995); C. Galletti and P. P. Battaglini, J. Neurosci. 9, 1112 (1989). 4. T. G. Weyand and J. G. Malpeli, J. Neurophysiol. 69, 2258 (1993). 5. Y. Trotter, S. Celebrini, B. Stricanne, S. Thorpe, M. Imbert, Science 257, 1279 (1992). , J. Neurophysiol. 76, 2872 (1996). 6. 7. S. P. Wise and R. Desimone, Science 242, 736 (1988); R. A. Andersen, L. Snyder, C.-S. Li, B. Stricanne, Curr. Opin. Neurobiol. 3, 171 (1993). 8. S. Petersen, J. Baker, J. Allman, Brain Res. 197, 507 (1980); R. Desimone and S. J. Schein, J. Neurophysiol. 57, 835 (1987). 9. Recording chambers were positioned to permit access to foveal and perifoveal V4 as well as V1 and V2. Two macaque monkeys were trained to reliably fixate a small spot on a computer monitor for a juice reward, and fixation was monitored monocularly with a noninvasive infrared video-based eye tracker [ J. Barbur, W. Thomson, P. Forsyth, Clin. Vision Sci. 2, 131 (1987)]. 10. The computer monitor was on a movable platform that could be set at 22.5, 45, 90, 180, or 360 cm from the monkey. Interleaved blocks of trials were obtained at three to five of the viewing distances with multiple blocks at each distance. Stimuli were presented in blocks consisting of randomly interleaved presentations of bars of varying size (aspect ratio, 4 :1 or 8 :1) and scaled with distance so that the bar size was of fixed retinal image size (lengths: 0.2, 0.4, 0.8, 1.6, and 3.2°). At 22.5 cm, the smallest bar (0.2°) was omitted, and at 360 cm, the largest bar (3.2°) was omitted because of physical limitations of the monitor and pixel size. Stimulus intensity was 160.96 6 3.4 cd z m–2. The bars were swept over the receptive field during fixation at the preferred orientation, direction, and color for the cell. Speed and length of excursion were scaled proportionally with distance to keep retinal speed and excursion constant. 11. Distance modulation and disparity modulation are distinct properties, therefore we use the terms “nearness” and “farness” to distinguish monotonic distance modulation from cells showing near and far binocular disparity-tuning as described by G. F. Poggio and B. Fischer [ J. Neurophysiol. 40, 1392 (1977)]. Classification of cells as monotonic (nearness or farness) is not completely certain, because a maximum or minimum could conceivably occur at an unsampled distance. A study of distance and disparity in V1 appears to show that for disparity-selective cells, farness cells are more common than nearness cells (6), but differences in stimuli, methods, and analysis preclude direct comparison with our results. 12. Tuning for absolute distance (at least for nearness) has been reported in the ventral intraparietal area of posterior parietal cortex [C. L. Colby, J. R. Duhamel, M. E. Goldberg, J. Neurophysiol. 69, 902 (1993)] and in ventral premotor cortex [M. Gentilucci et al., Exp. Brain Res. 50, 464 (1983); L. Fogassi et al., J. Neurophysiol. 76, 141 (1996)]. However, a study that manipulated viewing distance and binocular disparity in V1 did not find the systematic shifts in preferred disparity with viewing distance that absolute distance tuning would predict (5, 6). Moreover, a cell tuned to an intermediate absolute distance would not show monotonic response with viewing distance, as the majority of cells here do. Nonmonotonic cells could be tuned for absolute distance, but these cells made up only 13% of our sample. iiii 13. Cells were assigned to a visual cortical area based on receptive field position, size, and properties, and position relative to the lunate sulcus. Uncertainty about whether certain cells were in V1 or V2 led us to combine V1 and V2 for quantitative analysis. 14. H. Wallach and C. Zuckerman, Am. J. Psychol. 76, 404 (1963); H. W. Leibowitz and D. Moore, J. Opt. Soc. Am. 56, 1120 (1966); T. S. Collett, U. Schwarz, E. C. Sobel, Perception 20, 733 (1991). 15. K. Nakayama and S. Shimojo, Vision Res. 30, 1811 (1990); J. E. W. Mayhew and H. C. Longuet-Higgins, Nature 297, 376 (1982); B. J. Rogers and M. F. Bradshaw, ibid. 339, 253 (1993). 16. To ensure that ocular artifacts were not significant, a number of precautions were taken. Both monkeys were refracted by an optometrist using slit retinoscopy to establish that they were capable of accommodation over the range of distances used in the experiment (uncertainty ,0.25 diopters). During the experiments, the monitored eye varied its position with distance consistent with the appropriate change of vergence. Pupil radius was measured with the eye tracker and did not vary with distance in either monkey (2.33 6 0.01 mm; 1.73 6 0.02 mm). The monkeys were required to maintain fixation within a 0.25° square fixation window during the trial. 17. If viewing distance affected neural response, the measurements were repeated under either binocular or monocular restricted-field viewing conditions. Measurements were then repeated under the initial viewing conditions. The monkey viewed the stimuli through either monocular or binocular apertures (6.5° diameter). The remainder of the scene was masked such that only the monitor screen was visible to the monkey. 18. Because all receptive fields were in or close to the fovea (,2.5° eccentric in all cases), horizontal disparity of stimuli relative to the fixation point would be expected to be very close to zero at all distances. However, if the monkeys made vergence errors during fixation that varied systematically with distance, the responses of disparity-selective neurons could vary with viewing distance during binocular viewing. 19. 20. 21. 22. 23. 24. In the absence of binocular disparity, this argument does not apply, and 15 of 33 neurons maintained distance modulation under monocular restricted-field viewing, demonstrating that distance modulation cannot be attributed to fixation-induced disparity. An independent line of evidence on this point is provided by the modulation of spontaneous activity observed in the absence of a stimulus in half the neurons studied (88/178, P , 0.01). For this cell, manipulating the frame size had no effect (Fig. 2E; see figure legend for details), ruling out a center-surround artifact. Local image variations with viewing distance, such as slight changes in brightness or contrast, or changes in pixellation, are common to all the viewing conditions and cannot account for the difference between full-field and restricted-field responses. Nor can fixation disparityinduced horizontal disparity be responsible, because distance modulation is not dependent on binocular viewing. Therefore, local image variation with viewing distance cannot account for distance modulation. D. Zipser and R. A. Andersen, Nature 331, 679 (1988); A. Pouget and T. J. Sejnowski, Cereb. Cortex 4, 314 (1994). L. G. Ungerleider and M. Mishkin, in Analysis of Visual Behavior, D. J. Ingle, M. A. Goodale, R. J. W. Mansfield, Eds. (MIT Press, Cambridge, MA, 1982), pp. 549 –586. M. A. Goodale and A. D. Milner, Trends Neurosci. 15, 20 (1992); A. D. Milner and M. A. Goodale, The Visual Brain in Action (Oxford Univ. Press, Oxford, 1995). G. K. Aguirre and M. D’Esposito, J. Neurosci. 17, 2512 (1997). We thank A. Leonardo for contributions to the experiments; E. Dobbins, M. Lewicki, J. Mazer, and D. Rosenbluth for reviewing the manuscript; T. Annau, M. Lewicki, and J. Mazer for assistance with software tools; R. Desimone for providing data collection software; T. Joe for optometric assistance; and H. Weld and J. Baer for veterinary care. All methods of animal care conform to the guidelines of the Caltech Institutional Animal Care and Use Committee and the NIH. 19 February 1998; accepted 8 June 1998 “Inordinate Fondness” Explained: Why Are There So Many Beetles? Brian D. Farrell The phylogeny of the Phytophaga, the largest and oldest radiation of herbivorous beetles, was reconstructed from 115 complete DNA sequences for the 18S nuclear ribosomal subunit and from 212 morphological characters. The results of these analyses were used to interpret the role of angiosperms in beetle diversification. Jurassic fossils represent basal lineages that are still associated with conifers and cycads. Repeated origins of angiosperm-feeding beetle lineages are associated with enhanced rates of beetle diversification, indicating a series of adaptive radiations. Collectively, these radiations represent nearly half of the species in the order Coleoptera and a similar proportion of herbivorous insect species. When the British biologist J. B. S. Haldane was asked by a group of theologians what one could conclude as to the nature of the Creator from a study of His creation, Haldane is said to have answered, “An inordinate fondness for beetles” (1). Haldane’s remark reflects the Museum of Comparative Zoology, Harvard University, Cambridge, MA 02138, USA. E-mail: bfarrell@oeb. harvard.edu numerical domination of described species by the insect order Coleoptera (2), the diversity of which exceeds that of any other known animal or plant group. Because over half of all beetles are herbivorous and because the diversity of the remainder is comparable to that of other large, young, and nonherbivorous insect orders (3), a reconstruction of the phylogenesis of beetle herbivory would contribute substantially to an understanding of www.sciencemag.org SCIENCE VOL 281 24 JULY 1998 555 REPORTS possible reasons for the apparent success of the Coleoptera. Most phytophagous beetles feed on angiosperms, which are the most diverse group of vascular plants. Although the diversity of insects and angiosperms has been thought to result from the interaction of these two groups (3), the impact that the rise of flowering plants had on insect diversification has been recently challenged (4) by evidence that the appearance rate of insect families did not increase with angiosperm radiation during the Cretaceous. Indeed, most insect families that contain present-day associates of flowering plants were in place by the Jurassic (5), with the origins of actual angiosperm associations following later. The most direct test of the influence of flowering plant diversity on insect diversity must evaluate insect diversification rates before and after the origins of associations with angiosperms and must examine diversity within insect families. Phytophagous beetles are critical subjects for these tests, not only because they represent much of the diversity that must be explained, but also because several lineages of phytophagous beetles have colonized angiosperms independently. Plant feeding arose early in beetle history, about 50 million years after the origin of the Coleoptera in the Permian (5). Herbivorous species doubled beetle diversity by the midJurassic and overshadowed the nonherbivorous taxa by the beginning of the Tertiary; this interval coincided with the rise of angiosperms (Fig. 1). The most successful insectangiosperm associations involve the beetle sister superfamilies Chrysomeloidea and Cur- Fig. 1. The number of beetle genera of each of three trophic levels (34) per geological period (Permian to Tertiary) and epoch (Recent) (5, 35). Permian fossils are entirely of the saprophagous Archostemmata (5), and the first Adephaga and Polyphaga (the curculionoid Obrienidae) appear in the Triassic (9). Low diversity in the Cretaceous likely reflects the paucity of studied strata. The proportions of fossil genera in each beetle series (defined by Crowson) in the Tertiary and Recent are significantly correlated (P 5 0.001). The disproportionate rise in the diversity of the post-Cretaceous phytophagous beetles likely reflects the exponential rise in angiosperm diversity, particularly of herbaceous taxa. 556 culionoidea. These comprise the Phytophaga clade and likely exceed 135,000 species (6) [;80% of herbivorous beetles and ;50% of herbivorous insects (3)]. The Curculionoidea superfamily consists of six relatively depauperate families (Nemonychidae, Anthribidae, Attelabidae, Belidae, Brentidae, and Rhynchophoridae) and the considerably more diverse Curculionidae, whereas the Chrysomeloidea superfamily consists of the species-rich Cerambycidae and Chrysomelidae families. This assemblage of families contains different lineages, which are associated with cycads or conifers or with monocots or dicots (7, 8). The ancestor of the Phytophaga existed ;230 million years ago in the Triassic, as evidenced by the fossils of the now-extinct curculionoid family Obrienidae (9). However, the most important Mesozoic strata for fossil weevils and chrysomelids are the Jurassic Karatau beds in Kazakhstan (10). These beds contain no angiosperms but are rich in remains of Pteridophyta, Ginkgoales, Gnetales, Coniferales (that is, Araucariaceae and Podocarpaceae), Cycadales, and nowextinct Bennettitales (10). The angiosperm and phytophagous beetle fossil records are richest in the post-Jurassic Period, with most of the currently dominant subgroups of monocots and dicots and their herbivores proliferating in the early Tertiary. Because the diversification of seed plants and beetle herbivores has been at least broadly contemporaneous, it is plausible that this history has determined, at least in part, present-day beetle associations and diversity. To resolve the diversification history of these beetles, DNA sequences for the entire 18S ribosomal subunit gene were produced for samples of 115 species, which were drawn from all beetle subfamilies, representing the major variations in host-plant affiliations (11). These data were complemented by the addition of a matrix of 212 morphological characters compiled from recent reviews (12, 13). The most parsimonious trees (14) (Fig. 2) showed basal conifer- and cycad-feeding beetle lineages in the Chrysomeloidea and Curculionoidea branches. The Chilean Araucaria-feeding nemonychid subfamily Rhinorhynchinae [represented by Mecomacer (15)] is at the base of the Curculionoidea (Fig. 2A), whereas the Araucaria-feeding Palophaginae [represented by Palophagoides (16)] subtends the basal branch in the Chrysomeloidea (Fig. 2B). Immediately following these first branches of the Curculionoidea and Chrysomelidae are branches leading to the Araucariaceae-associated Oxycoryninae [Oxycraspedus (17)] and Orsodacninae [Orsodacne (18)] and their respective cycad-feeding sister groups Allocoryninae [Rhopalotria (19)] and Aulacoscelidinae [Aulacoscelis (20)]. Similarly, within the Cerambycidae family, the conifer-affiliated Aseminae (Asemum) and Spondylinae (Spondylis) (Fig. 2B) are the most basal live-plant feeders. All described larvae of these taxa feed on internal host tissues; the feeding of these chrysomelid and curculionoid larvae on the male pollenbearing strobili of conifers and cycads suggests that attack on these nutrient-rich reproductive structures preceded foliage feeding. The current affiliations of these oldest beetle lineages with pre-angiosperm seed plants support the hypothesis that these lineages retain affiliations that were formed early in the Mesozoic, before the diversification of flowering plants. Also supportive of early Mesozoic origins are the south temperate distributions of the basal curculionoids and chrysomelids, which are relictual and represent a broader previous distribution on Gondwanaland, before the late Mesozoic breakup (21). Thus, the evidence from phylogenetic position and biogeography points to the con- Table 1. Five independent contrasts of groups associated with gymnospermous seed plants versus angiosperms. All five contrasts yield a positive difference in favor of the hypothesis that angiosperm feeding is associated with enhanced diversity (one-tailed sign test, P 5 0.03). Addition of the remaining (mostly weevil) subfamilies, not yet sequenced, will bring the total number of species to 135,000. For two comparisons, alternative topologies are three to four steps (combined changes in nucleotides and morphological characters) away (comparisons 3 and 5), but these alternatives yield the same conclusion of ancestral beetle associations with gymnosperms. Thus, for comparison 3 (the Cerambycidae), the closest alternative grouping (within four steps) is of the Spondylinae as sister to the angiospermassociated clade, with Aseminae as sister to this assemblage. For comparison 5, the closest alternative (within three steps) is of Orsodacninae as sister to the angiosperm feeders. Comparison Primitively gymnosperm-associated taxon Diversity 1 Nemonychidae 85 2 OxycoryninaeAllocoryninae Aseminae-Spondylinae Palophaginae OrsodacninaeAulacoscelidinae 3 4 5 Primitively angiosperm-associated taxon Diversity 44,002 30 Attelabinae-Rhynchitinae, Apioninae, and Curculionidae-Rhynchophoridae Belinae 78 3 26 Lepturinae and Lamiinae-Cerambycinae Megalopodinae-Zeugophorinae Remaining Chrysomelidae 25,000 400 33,400 24 JULY 1998 VOL 281 SCIENCE www.sciencemag.org 150 REPORTS clusion that these associations of beetles with conifers and cycads are nearly 200 million years old and are therefore the oldest extant insect-plant interactions known. The phylogenetic ordering of beetle-plant associations is borne out by the concordant stratigraphic distributions of taxa in the two groups. The nemonychid subfamily Rhinorhynchinae (22), the belid subfamily Oxycoryninae (23, 24 ), and the chrysomelid subfamily Palophaginae (25), all of which attack the male strobili of Araucaria, contain members that are found in Kazakhstan in the Jurassic Karatau Formation, in which Araucaria fossils are prominent. The Araucariaceae show remarkable continuity between Mesozoic and extant forms, because Jurassic fossil cones and leaves are attributable to extant sections of Araucaria (26, 27 ). Indeed, the investment of fossil and extant Araucaria reproductive parts with defensive resin canals supports an argument for the early and con- tinued vulnerability of Araucaria to herbivorous insects (28). The discovery of extremely well preserved Araucaria strobili (some with apparent beetle damage) and foliage in the Jurassic fossils of Argentina suggests that these Argentine beetles may have been continuously associated with their hosts in a single place. Such continuity in insect associations therefore extends the morphological continuity of the Araucariaceae to include ecological interactions with herbivores. Some present-day cycad associates predate the rise of angiosperms. The phylogeny estimate predicts the early appearance of the cycad-feeding beetle subfamilies Allocoryninae and Aulacoscelinae, insects that are found in the Jurassic Karatau beds (29, 30). The pairing of the cycad-feeding taxa with associates of Araucariaceae in both the Chrysomeloidea and Curculionoidea apparently reflects the codominance of these Late Jurassic flora members and also reflects, perhaps, the nutritional similarity of their relatively large male strobili (31). Although the fidelity of the oldest beetlehost associations might reflect features of conifers and cycads (or features of these particular beetles) that promote their stability, many angiosperm-affiliated beetle subfamilies or tribes are restricted to taxonomic groups of monocots or dicots as well (Fig. 2). The persistent affiliations of beetle clades with plants that represent the range of potential host groups that formed throughout the latter half of the Phanerozoic Eon clearly impose a strong imprint of evolutionary history on the structure of modern insect-plant communities and thereby bear implications for their relative diversity. The phylogeny estimate permits a test of the hypothesis that proposes that the angiospermfeeding origins in the beetles are associated with enhanced diversity. To apply this estimate, the diversity of each group for which angio- B A Fig. 2. Estimate of the phylogeny of host associations in the Phytophaga, on the basis of simultaneous analyses of DNA sequences and morphological characters for (A) Curculionoidea, (B) Chrysomeloidea, and outgroups. The strict consensus tree for the two superfamilies, minus outgroups, is presented in two parts for legibility, with numbers indicating the number of synapomorphies/only those bootstrap values that exceed 50% (length, 2086; consistency index, 0.5; rescaled consistency index, 0.4; retention index, 0.83). Individual numbers also represent the number of synapomorphies. The Phytophaga, Chrysomeloidea, and Curculionoidea are all monophyletic, and the erotylid and melyrid sequences form the sister group to the Phytophaga, with Tenebrio outside these. Common groups between separate analyses of DNA sequences and morphological characters are represented by bold lines (DNA sequences are the sole source of resolution below the subfamily level in the Chrysomeloidea and below the family level in the Curculionoidea). Colors indicate the major host group attributable to the common ancestor of each group (green, Coniferae; brown, Cycadales; red, dicotyledonous angiosperms; blue, monocotyledonous angiosperms; black, subfamilies that do not feed on living plants). Approximate ages of Mesozoic and early Tertiary fossils only are indicated where known, because almost all subfamily groups are known from the mid-Tertiary fossil record. www.sciencemag.org SCIENCE VOL 281 24 JULY 1998 557 REPORTS Fig. 3. The phylogeny of the families and subfamilies of Phytophaga represented by genera in Fig. 2, with estimates of the number of current species in parentheses (36). Branches are colored by major host-plant group as in Fig. 2, but with purple indicating the collective use of angiosperms. The approximate age of each clade (estimated from the beetle fossil record) is indicated by the depth of the branches, with dotted lines superimposed for each period. The five origins of associations with angiosperms are numbered. In the Curculionoidea, an equally parsimonious interpretation would be an origin of angiosperm association at 1 followed by a reversal to cycad-Araucaria association at 2. However, this interpretation seems less plausible than two separate origins in the Cretaceous, because angiosperms were not developed in the Jurassic (37). sperm association was clearly the ancestral habit was contrasted with the diversity of the respective sister group for which cycad feeding or conifer feeding was clearly ancestral (Fig. 3). This analysis identified five such contrasts (Table 1), all of which show an increased diversity (of several orders of magnitude) in the angiosperm-associated group (one-tailed sign test, P 5 0.03). The total increase in beetle diversity is ;100,000 species, which is directly attributable to a series of adaptive radiations onto angiosperms. The diversification of the phytophagous beetles is consistent with the coevolutionary model of Ehrlich and Raven (32), who ascribe differences in the present diversity of insect and plant groups to evolutionary changes in characters (which affect their ecological interactions) and who predict that older plants should harbor older herbivores. Combined evidence from the phylogeny estimates presented here and from the fossil record shows a pronounced conservatism in the evolution of beetle-plant associations, which is important for the implication that plants might escape herbivory via key innovations (28, 32). Correlated with angiosperm feeding is the proliferation of life-history traits in the curculionids and chrysomelids. In contrast with the strobilus feeding of conifer- and cycad-associated ancestors, diversification of the subfamilies that attack flowering plants has been accompanied by larval folivory, leaf mining, and seed and root feeding, which exempli- 558 fy the concept of adaptive radiation. Although Haldane’s remark reflected a common and understandable emphasis on explaining the diversity of a particular taxon, explanations may be more readily found through comparative investigations of ecological breakthroughs that have evolved sufficiently often to permit multiple comparisons to be made (33). The success of the order Coleoptera thus seems to have been enabled by the rise of flowering plants. 11. 12. 13. 14. 15. References and Notes 1. G. E. Hutchinson, Am. Nat. 93, 145 (1959). Haldane himself often repeated this quip, although the circumstances and precise wording of the original remark have been controversial [see the summary of recent exchanges by S. J. Gould, Nat. Hist. 1, 4 (1993)]. 2. N. Stork, Biol. J. Linn. Soc. 35, 321 (1988). 3. D. R. Strong, J. H. Lawton, T. R. E. Southwood, Insects on Plants (Harvard Univ. Press, Cambridge, MA, 1984). 4. C. C. Labandeira and J. J. Sepkoski Jr., Science 261, 310 (1993). 5. F. M. Carpenter, Treatise on Invertebrate Paleontology, Part R of Arthropoda, vol. 4 of Superclass Hexapoda (Geological Society of America, Boulder, CO, 1992). 6. J. F. Lawrence, in Synopsis and Classification of Living Organisms, S. P. Parker, Ed. (McGraw-Hill, New York, 1982), vol. 2, pp. 482–553. 7. R. T. Thompson, J. Nat. Hist. 26, 835 (1992). 8. P. Jolivet and T. J. Hawkeswood, Host-Plants of Chrysomelidae of the World (Backhuys, Leiden, Netherlands, 1995). 9. V. G. Gratshev and V. V. Zherikhin, Paleontol. J. 29, 112 (1995). 10. L. V. Arnoldi’i, V. V. Zherikhin, L. M. Nikritin, A. G. 16. 17. 18. Ponomorenko, Mesozoic Coleoptera (Oxonian Press, New Delhi, India, 1991). Beetle groups that are restricted to particular higher plant taxa were scored for the least inclusive plant taxon that contained their hosts. The cerambycid subfamilies Prioninae and Parandrinae were left unscored for host, as these do not feed on live plant tissues but on dead or decaying wood [E. G. Linsley, Univ. Calif. Berkeley Publ. Entomol. 18, 1 (1961)]. The only higher beetle taxa for which DNA sequences were not obtainable were the Sagrinae and Anthribidae. G. Kuschel, Mem. Entomol. Soc. Wash. 14, 5 (1995). C. A. M. Reid, in Biology and Classification of Coleoptera: Papers Celebrating the 80th Birthday of Roy A. Crowson, J. Pakaluk and S. A. Slipinski, Eds. (Muzeum I Instytut Zoologi PAN, Warsaw, 1995), pp. 559 – 631. Sequences were obtained for the complete 18S ribosomal subunit gene from 115 of these beetle taxa and from the outgroup species from the Tenebrionidae (Tenebrio molitor, GenBank accession number 70810), Melyridae (Collops quadrimaculatus), and Erotylidae (Cypherotylus boisduvali) with methods that were given by M. F. Whiting, J. C. Carpenter, Q. D. Wheeler, W. C. Wheeler, Syst. Biol. 46:, 1 (1997). These sequences were aligned using Sequencher 3.0 (Gene Codes Corporation Ann Arbor, MI, 1995), producing a matrix of 2117 positions. Three ;40 – base pair (bp), hypervariable regions could not be unambiguously aligned and were excluded from the analyses, as were the two 50-bp ends of the gene, to avoid excessive missing data in parts of the matrix. The remaining 1874 positions yielded 355 potentially informative characters. These characters were analyzed separately and together with the morphological matrix compiled from Kuschel and Reid (12, 13). Analyses using the program PAUP* 4.0 version d59 included 100 initial heuristic searches using random taxon addition sequences and tree bisection-reconnection (TBR) branch swapping, setting MAX TREES (maximum number of trees held in memory) to 200, and keeping two trees per replicate search. This set of 200 trees was then subjected to TBR branch swapping with MAX TREES set to 10,000. Bootstrap analysis used 1000 random taxon addition sequences, with branch swapping limited to 100 trees per replicate. Tests of incongruence (using simple addition sequences and limiting MAXTREES to 100) between morphological and molecular data sets were not significant (incongruence length difference, P . 0.5). The Rhinorhynchinae subfamily includes the most morphologically plesiomorphic nemonychids, and they currently consist of 14 genera associated with strobili of Araucariaceae or Podocarpaceae in Chile, Argentina, and Australia plus a single species living on Pinaceae in Colorado [G. Kuschel, Rev. Chil. Hist. Nat. 54, 97 (1954)]. The closely related Holarctic Doydirhynchinae comprise 19 species living on Pinaceae. Crowson removed the nominate genus Nemonyx to the Anthribidae [R. A. Crowson, Entomol. Mon. Mag. 121, 144 (1985)]. The Palophaginae consist of three species in two genera, which develop in the male strobili of Araucariaceae in Chile, Argentina, Australia, and New Zealand [G. Kuschel and B. M. May, N. Z. Entomol. 19, 1 (1996)]. The most plesiomorphic oxycorynine belid genus Oxycraspedus attacks Araucaria strobili in Chile and Argentina [G. Kuschel, Invest. Zool. Chil. 5, 229 (1959)]. Crowson also suggested that Oxycraspedus and Rhopalotria are sister taxa but did not place the morphologically disparate oxycorynine genera reported from the Hydnoraceae and Balanophoraceae, which are families of tree parasites [R. A. Crowson, in Advances in Coleopterology, M. Zunino, X. Belles, M. Blas, Eds. (European Association of Coleopterology, Barcelona, 1991), pp. 13–28]. The belid tribe Pachyurini comprises 13 genera associated with Araucaria and Agathis in Australia and New Zealand and a single genus associated with Podocarpaceae and Cupressaceae in Brazil. The Orsodacninae comprise the Australian genus Cucujopsis, which is associated with the male strobili of the araucariaceous genus Agathis and the Holarctic 24 JULY 1998 VOL 281 SCIENCE www.sciencemag.org REPORTS 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. genus Orsodacne [ J. S. Mann and R. A. Crowson, J. Nat. Hist. 15, 727 (1981)]. Although the larval affiliations of Orsodacne are still unconfirmed, these are probably in the male strobili of Pinaceae (with which all eight species co-occur), a resource available during the early spring flights of the pollen-feeding adults. The belid subfamily Allocoryninae comprises .20 species in the Neotropical genus Rhopalotria, which attack the male strobili of Zamia and Dioon. The chrysomelid subfamily Aulacoscelidinae comprises 18 species in two Neotropical genera restricted to the Cycadaceae. L. Brundin, Evolution 19, 496 (1965). G. Kuschel, in Australian Weevils, E. Zimmerman, Ed. [Commonwealth Scientific and Industrial Research Organization (CSIRO), Melbourne, Australia, 1994], p. 569. Other nemonychids in the Karatau Formation apparently belong to the now-extinct subfamily Brenthorrhininae (9). The Nemonychidae are also represented by Libanorhinus succinus in Lower Cretaceous amber derived from Araucariaceae resins [G. Kuschel and G. O. Poinar, Entomol. Scand. (Group 2) 24, 143 (1993)] and by the Lower Cretaceous Slonik in the central Asian trans-Baikal deposits [G. Kuschel, GeoJournal 7, 499 (1983)]. The oxycorynine Archeorrhynchus paradoxopus (Belidae) is found in the Karatau Formation [G. Kuschel, in Australian Weevils, E. Zimmerman, Ed. (CSIRO, Melbourne, Australia, 1994), p. 244]. Oxycoryninae are also represented in the Lower Cretaceous Santana Formation of Brazil [D. A. Grimaldi, Ed., Bull. Am. Mus. Nat. Hist. 195, 8 (1990)]. Additional Karatau belids include the extinct subfamily Eobelinae [V. V. Zherikhin and V. G. Gratshev, in Biology and Classification of Coleoptera: Papers Celebrating the 80th Birthday of Roy A. Crowson, J. Pakaluk and S. A. Slipinski, Eds. (Muzeum I Instytut Zoologi PAN, Warsaw, 1995), p. 646]. The belid subfamily Carinae, which attacks strobili of the coniferous Cupressaceae, occurs in the Jurassic Karatau beds, as represented by Eccoptarthrus and Emanrhynchus [V. V. Zherikhin and V. G. Gratshev, in Biology and Classification of Coleoptera: Papers Celebrating the 80th Birthday of Roy A. Crowson, J. Pakaluk and S. A. Slipinski, Eds. (Muzeum I Instytut Zoologi PAN, Warsaw, 1995), pp. 634 –777]. The Carinae also appear in the Lower Cretaceous transBaikal beds (Cretonanophyes and Baissorhynchus); the Carinae presently contains Car, which is found in Australia and Tasmania, and Chilecar and Caenominurus, which are found in Chile and Argentina [E. Zimmerman, Ed., Australian Weevils (CSIRO, Melbourne, Australia, 1994), p. 504. The chrysomelid Cerambyomima longicornis, attributed to the Aulacoscelinae [G. Kuschel and B. M. May, Invertebr. Taxon. 3, 697 (1993)], resembles the orsodacnine Cucujopsis in the grooved frons and may be an intermediate form. Jurassic fossil cones of Araucaria mirabilis from Argentina closely resemble A. bidwellii and show damage similar to that caused by weevil larvae [see R. A. Stockey, Paleontographica 166, 1 (1978)]. A. bidwellii is host to extant species in both the Nemonychidae and Palophaginae. R. A. Stockey, J. Plant Res. 107, 493 (1994). B. D. Farrell, D. Dussourd, C. Mitter, Am. Nat. 138, 881 (1991). The allocorynine Scelocamptus curvipes is found in the Karatau beds (10). The aulacosceline genera Protoscelis, Protosceloides, and Pseudomegamerus are found in the Karatau beds (5). T. N. Taylor and E. L. Taylor, The Biology and Evolution of Fossil Plants (Prentice-Hall, Englewood Cliffs, NJ, 1993). P. R. Ehrlich and P. H. Raven, Evolution 18, 586 (1964); B. D. Farrell and C. Mitter, Biol. J. Linn. Soc. 68, 533 (1998). J. Jernvall, J. P. Hunter, M. Fortelius, Science 274, 1489 (1996). Assignments of feeding habits and numbers of recent genera are from Lawrence (6). The number of genera was extracted from the totals per beetle family in Lawrence (6). Estimates of diversity are from the following sources: Curculionoidea (7); Chrysomelidae [P. Jo- livet, E. Petitpierre, T. H. Hsiao, Eds., Biology of Chrysomelidae (Kluwer Academic, Dordrecht, Netherlands, 1988)]; Cerambycidae [S. Bily and O. Mehl, Longhorn Beetles (Coleoptera, Cerambycidae) of Fennoscandia and Denmark, vol. 22 of Fauna Entomologica of Scandinavica (Brill, Leiden, Netherlands, 1989)]. 37. For a discussion of the use of fossils to assign character optimizations, see J. M. Doyle and M. J. Donoghue, Rev. Palaeobot. Palynol. 50, 63 (1987). 38. For supplying specimens or identifications of key or austral taxa, I especially thank F. Andrews, J. Chemsak, L. Diego-Gomez, J. Donaldson, C. Duckett, T. Erwin, W. Flowers, D. Furth, C. D. Johnson, J. King- solver, G. Kuschel, J. Lawrence, A. Newton, K. Norstog, R. Oberprieler, C. O’Brien, and E. G. Riley, among many others. I also thank A. Salmore, M. Blair, and L. Morrissey for technical lab support; A. Berry, M. Donoghue, D. Futuyma, A. Knoll, D. Lewontin, E. Mayr, C. Mitter, N. Moran, B. Normark, S. Palumbi, N. Pierce, and E. O. Wilson for helpful discussions; and A. Knoll, C. Labandeira, and D. Maddison for detailed comments on a late draft. This research was supported by NSF, USDA, and the Putnam Expedition Fund of the Museum of Comparative Zoology. 19 January 1998; accepted 8 June 1998 Activity-Dependent Cortical Target Selection by Thalamic Axons Susan M. Catalano* and Carla J. Shatz† Connections in the developing nervous system are thought to be formed initially by an activity-independent process of axon pathfinding and target selection and subsequently refined by neural activity. Blockade of sodium action potentials by intracranial infusion of tetrodotoxin in cats during the early period when axons from the lateral geniculate nucleus (LGN) were in the process of selecting visual cortex as their target altered the pattern and precision of this thalamocortical projection. The majority of LGN neurons, rather than projecting to visual cortex, elaborated a significant projection within the subplate of cortical areas normally bypassed. Those axons that did project to their correct target were topographically disorganized. Thus, neural activity is required for initial targeting decisions made by thalamic axons as they traverse the subplate. During the wiring of connections between the thalamus and cortex in mammals, there is an intermediate step in which thalamic axons grow and interact with a special population of neurons—subplate neurons— before they contact their ultimate target neurons within the cortical plate (1, 2). For example, LGN axons en route to visual cortex emit transient side branches that extend into the subplate under both target and nontarget cortical areas (3) and form functional synaptic contacts with subplate neurons (4). During this period of development, spontaneous action potential activity generated in the retina and relayed through the LGN likely drives these subplate synapses in vivo (5). Thus, synaptic relations within the subplate could support activitydependent interactions during the process of thalamocortical axon target selection. To examine if activity is needed for thalamic axons to form connections with their appropriate cortical target area, we infused Howard Hughes Medical Institute and Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720 –3200, USA. *Present address: Division of Biology, 216-76, California Institute of Technology, Pasadena, CA 91125, USA. E-mail: scatalan@cco.caltech.edu †To whom correspondence should be addressed. Email: cshatz@socrates.berkeley.edu tetrodotoxin (TTX, a sodium channel antagonist that blocks action potentials) or vehicle through osmotic minipumps (6) into the brain of cat fetuses between E42 (E42 5 42 days of gestation) and E56. At E42, the first LGN axons have just reached the subplate underneath visual cortex but still have side branches along their trajectory. Between E42 and E50, the majority of LGN axons have arrived in the visual subplate; by E56, many have departed the subplate and reached their ultimate target, layer 4 of the cortical plate (3). To assess the consequences of the treatments on the thalamocortical projection, we injected carbocyanine dyes at E56 to label retrogradely LGN neurons (7) and subsequently counted the numbers of neurons sending axons to the subplate or cortical plate of either visual (the correct target) or auditory (an incorrect target) cortex. The number of LGN neurons projecting to visual cortex was decreased in TTX-infused animals (Fig. 1), both within the subplate [Fig. 1C; an average of 69 6 5% SEM fewer neurons than vehicle controls, n 5 8 animals; 4 littermate pairs treated with TTX or vehicle and matched for similar 1,19-dioctadecyl-3,3,39,39tetramethylindocarbocyanine perchlorate (DiI) injection sizes] and within the cortical plate (Fig. 1C; 94 6 0.5% SEM, n 5 8 animals; 4 www.sciencemag.org SCIENCE VOL 281 24 JULY 1998 559 Phylogenetic test of speciation by host shift in leaf cone moths (Caloptilia) feeding on maples (Acer) Ryosuke Nakadai & Atsushi Kawakita Center for Ecological Research, Kyoto University, Hirano 2-509-3, Otsu, Shiga 520-2113, Japan Keywords Diversification, herbivorous insect, host plant, host shift, speciation. Correspondence Ryosuke Nakadai, Center for Ecological Research, Kyoto University, Hirano 2-509-3, Otsu, Shiga 520-2113, Japan. Tel: +81-77-549-8018; Fax: +81-77-549-8201; E-mail: r.nakadai@ecology.kyoto-u.ac.jp Funding Information Japan Society for the Promotion of Science (grant/award number: 15H04421, 15J00601, 26650165). Received: 12 May 2016; Revised: 26 May 2016; Accepted: 27 May 2016 Ecology and Evolution 2016; 6(14): 4958– 4970 Abstract The traditional explanation for the exceptional diversity of herbivorous insects emphasizes host shift as the major driver of speciation. However, phylogenetic studies have often demonstrated widespread host plant conservatism by insect herbivores, calling into question the prevalence of speciation by host shift to distantly related plants. A limitation of previous phylogenetic studies is that host plants were defined at the family or genus level; thus, it was unclear whether host shifts predominate at a finer taxonomic scale. The lack of a statistical approach to test the hypothesis of host-shift-driven speciation also hindered studies at the species level. Here, we analyze the radiation of leaf cone moths (Caloptilia) associated with maples (Acer) using a newly developed, phylogeny-based method that tests the role of host shift in speciation. This method has the advantage of not requiring complete taxon sampling from an entire radiation. Based on 254 host plant records for 14 Caloptilia species collected at 73 sites in Japan, we show that major dietary changes are more concentrated toward the root of the phylogeny, with host shift playing a minor role in recent speciation. We suggest that there may be other roles for host shift in promoting herbivorous insect diversification rather than facilitating speciation per se. doi: 10.1002/ece3.2266 Introduction Herbivorous insects comprise one of the major components of earth’s biodiversity. Because the diversity of herbivorous insects is often correlated with host plant diversity (Lawton and Schroeder 1977; Wiegmann et al. 2002; Janz et al. 2006; Joy and Crespi 2012; Ferrer-Paris and Sanchez-Mercado 2013; Isaka and Sato 2015; Lin et al. 2015), the cycle of host plant adaptation and host plant shift is commonly invoked as the major process generating high diversity (Mitter and Brooks 1983; Craig et al. 2001; Wheat et al. 2007; Futuyma and Agrawal 2009; Bennett and O’Grady 2012). For example, a classical study by Farrell (1998) showed that herbivorous insects using angiosperms as hosts are more species rich than those using gymnosperms among the Phytophaga beetles, suggesting that the diversity of angiosperms has facilitated speciation by host shift in the beetles that feed on them. Studies of host races in herbivorous insects showed that specialization to a novel host plant sometimes results in reproductive isolation between insects using different 4958 hosts (Feder et al. 1988; Groman and Pellmyr 2000; Hawthorne and Via 2001; Nosil et al. 2002; Thomas et al. 2003; Malausa et al. 2005; Ohshima 2012; Xue et al. 2014), providing a mechanistic explanation of how host shifts may promote speciation. Understanding the role of host plant shifts in generating diversity is thus a current focus in the study of herbivorous insect diversification (Marvaldi et al. 2002; Stireman et al. 2005; Wheat et al. 2007; Winkler et al. 2009; Fordyce 2010; Funk 2010; Matsubayashi et al. 2010; Nyman 2010; Soria-Carrasco et al. 2014). However, phylogenetic analyses of herbivorous insect radiation have often demonstrated conservatism in host plant use by herbivorous insects (Crespi et al. 1998; Lopez-Vaamonde et al. 2003; Wahlberg 2007; Winkler and Mitter 2008; Nyman et al. 2010; Jousselin et al. 2013; Doorenweerd et al. 2015). For example, Nyman et al. (2010) showed that only 20% of the speciation events in nematine sawflies were accompanied by shifts between host plant families, and Doorenweerd et al. (2015) showed that host use was generally conserved at the plant ª 2016 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. R. Nakadai & A. Kawakita Phylogenetic Test of Speciation by Host Shift family level, with biogeographic processes playing a greater role in the recent speciation of nepticulid moths. Extreme cases of host plant conservatism are found in gall wasps feeding on oaks (Stone et al. 2009) or micropterigid moths that have radiated on a single liverwort species (Imada et al. 2011). However, many phylogenetic studies that tested for host conservatism defined host plants at the plant family or genus level (Lopez-Vaamonde et al. 2003; Wahlberg 2007; Nyman et al. 2010; Jousselin et al. 2013; Doorenweerd et al. 2015). The relative importance of host shifts in herbivorous insect speciation should ideally be assessed using species-level phylogenies with data on all known host associations. Two major obstacles hamper analysis at the species level. First, because most radiations of herbivorous insect groups occur at the continental scale, it is usually difficult to achieve complete taxon sampling while having host association data for each species. It is therefore not surprising that some of the best-sampled phylogenies are those for less mobile herbivorous insect groups (e.g., Imada et al. 2011). Second, an appropriate method of analyzing host plant shifts along phylogenies has been lacking. Coding host plant associations at the family or genus level would simplify analysis because methods such as ancestral character state reconstructions are then applicable. However, many herbivorous insects use several closely related plant species (i.e., polyphagy) with varying levels of preference (Smiley 1978; Roininen and Tahvanainen 1989; Thompson 1998; Scheirs et al. 2000; D’Costa et al. 2013; Nakadai and Murakami 2015), which complicates analysis of the ancestral state regarding host use. In addition, individual host plant species cannot be considered as discrete character states because they are phylogenetically nonindependent (Pearse and Altermatt 2013). Ideally, the dissimilarity of host use between a pair of herbivorous insect species should be weighed by the phylogenetic disparity of the host plants. In this study, we assess the importance of host shifts in the speciation process of herbivorous insects by developing a new method that overcomes these issues. This method focuses on whether host plant shifts are concentrated toward the roots or the tips of the insect phylogenetic tree, while taking into account host plant phylogeny in the calculation of host use dissimilarity between a pair of herbivorous insect species. If most speciation events are associated with host shifts, the level of disparity in host use between a pair of herbivorous insect species will on average be greater for phylogenetically more closely related pairs (Fig. 2A). Alternatively, if most host shifting events occurred during the initial stage of the radiation and more recent speciation events were independent of host shifts, the level of difference in host use would be larger toward the root of the phylogenetic tree (Fig. 2B). We focused on the interaction between a group of leaf cone moths (Caloptilia, Gracillariidae) and their maple hosts (Acer, Sapindaceae). The Caloptilia–Acer interaction is appropriate for testing host-shift-driven speciation at fine taxonomic scales because a previous study demonstrated large variation in the pattern of host use among Caloptilia species (Nakadai and Murakami 2015). The genus Acer is one of the most taxonomically diverse groups of trees in the Northern Hemisphere, particularly in the temperate regions of East Asia, eastern North America, and Europe (van Gelderen et al. 1994). The genus comprises 124 species in the Northern Hemisphere, 81% of which are distributed in China, Korea, and Japan (Renner et al. 2007). A previous taxonomic study of Caloptilia identified 11 species associated with Acer in Japan alone, which have high morphological affinity to each other (Kumata 1982). Based on extensive geographic sampling, we establish full host plant records for these 11 species and three newly found ones, and analyze them using the above method to assess the relative importance of host shift in the speciation of Caloptilia moths feeding on Acer trees. ª 2016 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. 4959 Materials and Methods Study material The genus Caloptilia is globally distributed and includes nearly 300 described species, of which 27 feed on maples (De Prins and De Prins 2015). In Japan, 51 species are described feeding on 21 host plant families, and 11 of them use Acer, which is the most common host plant genus of Japanese Caloptilia (Kumata et al. 2013). The feeding habits of the larvae change dramatically between the early and late developmental stages. Upon hatching, larvae mine the surface layer of the leaf (i.e., leaf miners) until the third instar, then exit the mine, and form the edge of the leaf into a roll within which they feed externally until the final instar (hence the name leaf cone moth) (Kumata et al. 2013). Some species are leaf gallers or blotch miners at the final instar and do not roll leaves. Each species is usually associated with a single plant genus. Sampling, DNA sequencing, and phylogenetic analyses We sampled Caloptilia moths that use Acer trees at 73 sites covering a wide geographic range in Japan (Figs. 1, S2) during May–October of 2011–2015. Moths were sampled by searching for larvae in leaf rolls (fourth or fifth instar) or pupae on maple leaves. In total, 254 specimens were obtained, used to delimit species and to establish the Phylogenetic Test of Speciation by Host Shift R. Nakadai & A. Kawakita Continental Asia Mainland Japan 500 km Figure 1. Sampling localities of Caloptilia moths collected from Acer trees in Japan. Sampling information for each species shown in Figure S2. host range for each species. Delimitation of species was based on sequences of the mitochondrial cytochrome oxidase subunit I (COI) gene; major divergences in COI sequences clearly corresponded with differences in wing pattern and genital morphology. Species were morphologically identified following Kumata (1982). To further determine whether the Caloptilia species feeding on maples resulted from a single radiation, we additionally sampled 44 Caloptilia species that use nonmaple hosts and six species in closely related genera (Gracillaria, Calybites, and Eucalybites; for details, see Table S1) and reconstructed a species-level phylogeny of Caloptilia. For the species-level phylogeny, one representative specimen of each Caloptilia species feeding on maple was included in the analysis. All moth specimens were kept in ethanol prior to DNA extraction. We extracted genomic DNA using the NucleoSpin Tissue Kit (Macherey-Nagel, D€ uren, Germany). The head capsule of the larva or the head, wings, and abdomen of the adult were stored as vouchers. The COI gene was sequenced for all of the 254 moths collected from maples. For the species-level phylogenetic analysis, we sequenced four genomic regions: COI and the nuclear arginine kinase (ArgK), carbamoyl-phosphate synthetase 2 (CAD), and elongation factor 1-alpha (EF-1a) genes. We designed new primer sets for ArgK, CAD, and EF-1a (Table S3) based on sequences available for other species of Gracillariidae in the database. The information on existing primer sets for CO1 and EF-1a is also provided in Table S3. Polymerase chain reaction (PCR) amplifications were carried out under the following conditions: initial denaturation step at 94°C for 5 min; 30 cycles of 94°C for 30 sec, 50°C for 30 sec, and 72°C for 1 min; and a final extension at 72°C for 7 min. Products were sequenced on an ABI 3100 automated sequencer using BigDye chain termination chemistry (Applied Biosystems, Foster City, CA), and obvious sequence errors were manually corrected using MEGA 6.06 (Tamura et al. 2013). Obtained sequences were aligned using Mafft ver. 6.901 (Katoh and Toh 2008) under the default settings. The resulting dataset contained 658, 573, 614, and 541 base pairs of COI, ArgK, CAD, and EF-1a, respectively. Species-level phylogenetic trees were constructed using two datasets: (1) an all-genes dataset (COI + ArgK + CAD + EF-1a) and (2) a nuclear-only dataset (ArgK + CAD + EF-1a). The latter was created because a previous phylogenetic study of Gracillariidae suggested that nuclear genes provide strong phylogenetic signals at the genus and species levels (Kawahara et al. 2011). We reconstructed phylogenetic trees by maximum-likelihood and Bayesian methods for each dataset. The maximum-likelihood analysis was performed using RAxML ver. 8.0 (Stamatakis 2014). We 4960 ª 2016 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. R. Nakadai & A. Kawakita Phylogenetic Test of Speciation by Host Shift conducted 100 replicates of shotgun search for the likelihood ratchet and assessed nodal support using bootstrap analyses with 1000 replications. We also conducted Bayesian phylogenetic analysis using MrBayes5D (Tanabe 2008), a modified version of MrBayes3.1.2 (Ronquist and Huelsenbeck 2003). We used the following settings for the Bayesian analysis: number of Markov chain Monte Carlo generations, five million; sampling frequency, 100; and burn-in, 5001. The burn-in size was determined by checking the convergence of log likelihood (ln L) plotted against generation time. In both methods, we used Kakusan4 (Tanabe 2011) to determine appropriate models of sequence evolution under the BIC4 criterion. Hypothesis and randomization tests for validation To test the relative importance of host shift in the speciation process from phylogeny, we assumed two contrasting scenarios (Fig. 2). If most speciation events are associated with host shifts, the dissimilarity in host use will on average be larger for phylogenetically more closely related pairs of Caloptilia moths (Fig. 2A). Conversely, if most speciation events occur during the initial stage of the radiation and more recent speciation events are independent of host shifts, host use dissimilarity will be larger for phylogenetically more distantly related pairs of Caloptilia moths (Fig. 2B). A similar framework was proposed by Nyman et al. (2010), but their method cannot be applied to species-level analysis. Following Barraclough et al. (1999), we used randomizations to compare the observed pattern of host use to that expected under a null model of no association with cladogenesis. Our null model hypothesized that changes occurred at random and independently across the tree. The statistic used to test the association between phylogenetic distance and the degree of difference in host use is expressed as the sum across all nodes of phylogenetic distance Xi multiplied by the degree of host use dissimilarity Hi (see the next section for detailed calculation of dissimilarity), i¼m X Xi Hi : i¼1 If differences in host use are greater between closely related species, the above statistic is expected to be smaller than that under the null model and vice versa. Thus, Host use dissimilarity Herbivorous insect phylogeny (A) Host use dissimilarity Figure 2. Phylogenetic distributions of host use arising from different speciation modes in herbivorous insects. (A) Distribution of host use on the phylogeny of a hypothetical insect group in which speciation is mainly associated with host shifts. (B) Distribution of host taxa when speciation mainly involves other processes without host shifts. Herbivorous insect phylogeny (B) Phylogenetic distance Host plant phylogeny ª 2016 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. between herbivorous insects 4961 Phylogenetic Test of Speciation by Host Shift R. Nakadai & A. Kawakita we tested for a significant concentration of changes toward either the tips or the root of the tree. A positive sign indicates the concentration of changes toward the tips, whereas a negative sign indicates that more changes occurred toward the root. The null distribution was obtained by randomly shuffling observed changes among branches of the tree and calculating the above statistic in each null trial. The two-tailed probability of the observed value was calculated based on 10,000 randomizations. A similar randomization method was used by Barraclough et al. (1999) and Sauer and Hausdorf (2009) to study adaptive character evolution in tiger beetles and land snails, respectively. In addition, we calculated the standardized effect size (SES) as the observed test statistic minus the mean of the null distribution, divided by the standard deviation of the null distribution. This null model approach is commonly used for expressing biological differences regardless of the units of the indices (McCabe et al. 2012). Indices of dissimilarity in host use We used both Jaccard (Jaccard 1912; Koleff et al. 2003) and Unifrac (Lozupone and Knight 2005) indices to quantify the degree of difference in host use between a pair of Caloptilia moths feeding on Acer trees. Both indices are commonly used in community ecology for assessing the degree of dissimilarity between two communities (Cavender-Bares et al. 2009). The Unifrac index is analogous to the Jaccard dissimilarity index, but takes into account phylogenetic information (Lozupone and Knight 2005), which in the present case is the plant phylogeny. The Unifrac index has an advantage over the Jaccard index especially when there (B) Results Extensive sampling of Caloptilia moths throughout Japan identified 14 species feeding on maples (Figs. 4, S1), of which three were newly discovered in this study. This represents ca. 40% of the Caloptilia species known to feed on maples (De Prins and De Prins 2015). Most species were widely distributed throughout the range, although Low Turnover/High Nestedness Host plant phylogeny (C) High Turnover/Low Nestedness Herbivorous insect phylogeny Herbivorous insect phylogeny Low Turnover/Low Nestedness Herbivorous insect phylogeny (A) is missing information on host association; the latter index assumes an equal weight for all host plant species, whereas the former weighs host plants according to their phylogenetic relatedness and is thus less sensitive to missing data. In this study, we used the phylogeny of 30 Japanese Acer species published by Nakadai et al. (2014). In addition, both Jaccard and Unifrac indices can be partitioned into two components of dissimilarity: turnover and nestedness (Baselga 2010; Leprieur et al. 2012). In community ecology, the turnover of a species assemblage refers to the replacement of some species by others as a consequence of historical events, such as geographic barrier formation or environmental sorting (Baselga 2010). In contrast, the nestedness of a species assemblage occurs when the species composition of sites with a smaller number of the species is a subset of that of species-rich sites, which reflects a spatial pattern of species loss resulting from dispersal limitation or environmental filtering (Hirao et al. 2015). In our study, the turnover component indicates the degree of nonoverlapping host use, and the nestedness component represents the difference in the degree of specialization between insect species with shared host plants (Fig. 3). All indices were calculated using the “betapart” package (Baselga and Orme 2012) in R ver. 3.2.2 (R Core Team 2015). Host plant phylogeny Host plant phylogeny Figure 3. Possible patterns of plant–herbivore association. (A) Low turnover/low nestedness, (B) low turnover/high nestedness, and (C) high turnover/low nestedness. Both Jaccard and Unifrac indices perform similarly in (A) and (B), whereas in (C), the nestedness component of the Unifrac index between a pair of closely related herbivores will be lower than that of the Jaccard index. This is because host use is similar when host phylogeny is taken into account but maximally dissimilar in the absence of host phylogenetic information. 4962 ª 2016 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. R. Nakadai & A. Kawakita Phylogenetic Test of Speciation by Host Shift Figure 4. Phylogeny of Caloptilia moths and their related groups. The phylogeny was constructed by maximum-likelihood method using four genomic regions (COI, ArgK, CAD, and EF-1a) of 71 species. ª 2016 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. 4963 Phylogenetic Test of Speciation by Host Shift R. Nakadai & A. Kawakita some were only found at a limited number of sites (Fig. S2). Some species were apparently specialists on single Acer species (e.g., Caloptilia hidakensis, Caloptilia kurokoi), whereas others were collected from many hosts. Overall, each species uses 1–11 Acer species, with a mean of 3.0 3.0 (Fig. 5). Species-level phylogenetic analyses based on 2386 bp of the combined COI, ArgK, CAD, and EF-1a dataset produced a well-resolved phylogeny (Fig. 4). All of the Caloptilia species feeding on Acer were closely related, although they were not monophyletic. One species, Caloptilia gloriosa, was positioned outside of the clade consisting mainly of Acer-feeding Caloptilia (Fig. 4), and another species, Caloptilia aurifasciata, feeding on Toxicodendron (Anacardiaceae), was embedded within this clade (Fig. 4). We thus focused on the clade containing C. aurifasciata and the 13 species feeding on Acer for the analysis of host shifts. We conducted randomization tests separately for datasets with and without C. aurifasciata. Because information on the phylogenetic distance between Acer and Toxicodendron (the host of C. aurifasciata) was not available, we assumed the maximum turnover (1) and minimum nestedness (0) for the calculation of dissimilarity indices between C. aurifasciata and Acer-feeding Caloptilia. The results of randomization tests indicated that the turnover components and the combined turnover and nestedness components of both Jaccard and Unifrac indices are greater between distantly related species than expected under the null model (positive signs in Table 1), (A) C. sp. 1 C. kisoensis C. sp. 2 C. sp. 3 C. aceris C. acericola C. wakayamensis C. hidakensis C. sp. cf. yasudai C. sp. cf. heringi C. semifasciella C. monticola 0.04 0.06 0.08 0.10 0.12 4964 A. japonicum A. amoenum A. shirasawanum A. sieboldianum A. palmatum 0.14 Phylogenetic distance between Caloptilia species A. tenuifolium A. ukurunduense A. tschonoskii A. micranthum A. rufinerve A. capillipes A. pictum A. crataegifolium A. miyabei A. argutum A. ginnala A. cissifolium A. maximowiczianum A. nipponicum 1.0 0.6 0.4 0.0 0.2 Host use dissimilarity 0.8 (B) A. pycnanthum C. kurokoi Figure 5. The results of Acer–Caloptilia interactions obtained from wide range sampling in Japan. (A) Phylogram of 13 species of Caloptilia pruned from a phylogeny of this genus and related groups (Table S3) and a phylogram of 20 species of Acer trees pruned from a phylogeny of this genus in Japan (Nakadai et al. 2014). The complete phylogeny of Acer trees was the 50% majority-rule consensus of trees sampled from the stationary distribution of a Bayesian analysis of four chloroplast DNA loci sampled from 30 species, including some varieties. (B) The plot of phylogenetic distance between Caloptilia moths (all-genes dataset) versus host use dissimilarity (turnover and nestedness components of the Unifrac index). ª 2016 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. R. Nakadai & A. Kawakita Phylogenetic Test of Speciation by Host Shift Table 1. Relationships between differences of host use and phylogenetic distance between Caloptilia species feeding on Acer according to randomization tests. Dataset All-genes dataset Nuclear-only dataset Jaccard index Unifrac index Jaccard index Unifrac index Turnover + nestedness Turnover Sign SES Sign SES + + + + 1.66 2.17 1.90 2.72 + + + + 1.95 2.16 1.95 2.40 n.s. * n.s. ** Nestedness * * n.s. * Sign SES – – – – 1.26 0.85 1.10 0.60 n.s. n.s. n.s. n.s. Positive signs of differences in host use with phylogenetic distance suggest that changes are concentrated toward the root and negative signs suggest that changes occur near the tips. Significance level: n.s., P ≥ 0.05; *P < 0.05; **P < 0.01. although the trend was not significant for the Jaccard index except for the turnover component of the all-genes dataset. The nestedness component showed negative signs but was not statistically significant (Table 1). These results support the hypothesis of phylogenetic conservatism in host use (Fig. 2B). Inclusion of C. aurifasciata, which feeds on Toxicodendron, did not change the overall pattern but slightly strengthened the trend, with tests using both Jaccard and Unifrac indices becoming significant (Table S4). The SES values provide a quantitative measure of the strength of association between host use dissimilarity and phylogenetic distance (Table 1). Overall, the values for the turnover component and the combined turnover + nestedness component were greater when host plant phylogeny was taken into account (Unifrac index) than when it was not (Jaccard index). In this article, we describe a new method for testing the role of host shift in herbivorous insect speciation. We identified three beneficial features of this method. First, it is less sensitive to incomplete species sampling. It is usually difficult to sample every species for the entire radiation (Lopez-Vaamonde et al. 2003; Nyman et al. 2006; Agrawal and Fishbein 2008; Stone et al. 2009; Doorenweerd et al. 2015), and conventional methods of analyzing the effects of host shifts on phylogeny (e.g., ancestral character state reconstruction) are sensitive to species sampling. However, because our analysis focuses on whether host use changes are concentrated toward either the root or the tips of the phylogenetic tree, complete sampling is not required as long as species sampling is not biased (e.g., toward species feeding only on a particular species of host). Second, the method permits analysis of speciation by host shift at a broader geographic scale. In many cases, herbivorous insect species have broader distributions than individual host plant species, so sister herbivore species occurring in allopatry should always use different hosts, even if diet shift was not the major cause of speciation. The use of a dissimilarity index controlling for host phylogeny partly remedies this problem (Pearse and Altermatt 2013; Pearse et al. 2013) because related plant species are generally similar in their traits associated with susceptibility to herbivores (Rasmann and Agrawal 2011; D’Costa et al. 2014; Nakadai and Murakami 2015), and thus host use dissimilarity will consistently be low if no major diet shift has occurred during speciation. Caution is needed in cases where the group of herbivores being studied has extremely high or low host specificity because, in both cases, the method may overestimate host use conservatism. Finally, calculation of SES allows comparison of trends among different studies (McCabe et al. 2012) because SES is independent of differences in the number of herbivore species included in the dataset. Previous phylogenetic studies assessed the percentage of host shifts between host plant families in each taxonomic group (Lopez-Vaamonde et al. 2003; Nyman et al. 2010; Doorenweerd et al. 2015), but quantitative comparisons among studies were difficult due to the lack of a standardized measure for comparison. We note that our method has a link to those developed previously to test the degree of cospeciation between a pair of host and parasite. However, because they are designed to test for cospeciation, they either assume that each parasite is associated with only one host at any given time (Page 1994; Ronquist 1995; Charleston and Robertson 2002; Merkle and Middendorf 2005; Conow et al. 2010) or that host and parasite speciation events are simultaneous in time (Legendre et al. 2002), which are not realistic for many plant–herbivore associations. Recently, Rafferty and Ives (2013) and Hadfield et al. (2014) developed methods that do not require such assumptions and uses GLMM to test for interaction effect of two phylogenies, but the methods are not designed to test the polarity of trait divergence occurring either toward the tips or the root of the phylogeny as in our method. ª 2016 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. 4965 Discussion Application of randomization test in the study of herbivorous insect speciation Phylogenetic Test of Speciation by Host Shift One weakness of our analysis is that we treated host association based on presence/absence, but in reality, preference levels are not equal for all of the host plant species observed. We could not quantify host preference in this study because it is necessary to standardize both sampling effort and host abundance to obtain a comparative measure of host preference, which was difficult to accomplish at all sampling sites. However, the above-described method can easily incorporate host preference when such data are available, as dissimilarity measures (Unifrac and Jaccard indices) are also designed for quantitative data. The newly developed method is presently intended for testing host-shift-driven speciation in herbivorous insects, but the overall framework is applicable, in principle, to studies of other types of ecological speciation. The source code for running the analysis in R is provided as Data S4. The source code and datasets for running the analysis in R is provided as Data S1–4. Alternative hypothesis on the speciation process of leaf cone moths feeding on maples Application of the present method to the 13 species of maple-feeding leaf cone moths suggested that major dietary changes are concentrated toward the root of the herbivore phylogenetic tree (Table 1). Because the Unifrac index takes into account plant phylogeny whereas the Jaccard index does not, significant positive sign for the Unifrac index and lack of significance for the Jaccard index indicate that the trend exists only when host plant phylogeny is taken into account in the calculation of dissimilarity. Thus, the results indicate that major dietary shifts play a minor role in recent speciation events, but shifts between very closely related hosts may have took place during recent Caloptilia speciation. The addition of C. aurifasciata generally strengthened the trend for both Jaccard and Unifrac indices because C. aurifasciata diverged from all other species toward the root of the tree and has a completely different diet. The Jaccard test, which was only marginally insignificant in the absence of C. aurifasciata, became significant after the inclusion of this species (Table S4). Although our test indicated that speciation assisted by host shift may be relatively minor in this group, we do not deny the importance of major dietary changes as such events occur in some of the earliest speciation events. Nevertheless, host-shift-driven speciation may not be as important as commonly thought in generating the current diversity of Caloptilia. Because our analysis only tests for patterns, the alternative process that drives speciation in Caloptilia cannot be inferred from our data. However, previous studies proposed several possible processes by 4966 R. Nakadai & A. Kawakita which herbivorous insects speciate without changing their diet (Imada et al. 2011; Bennett and O’Grady 2012; Yamamoto and Sota 2012; Hamm and Fordyce 2015). For some phytophagous insect groups, allopatric speciation without host shift may be a major factor causing radiation (Nyman et al. 2010; Imada et al. 2011), but in the case of Japanese leaf cone moths, the pattern is unclear based on visual inspection of the current geographic distribution (Fig. S1). Ecological shift within a host plant is also a significant process (Condon and Steck 1997; Cook et al. 2002; Joy and Crespi 2007; Althoff 2014; Mishima et al. 2014). For example, Zhang et al. (2015) demonstrated divergence induced by host plant ages in sympatric sister beetles (Pyrrhalta maculicollis and Pyrrhalta aenescens) feeding on elm. There is clearly a need to sample from a broader geographic area and to collect additional information on microniche divergence among leaf cone moths to fully understand the process underlying their diversification. Adding timeline to the divergence events of both herbivores and host plants should also facilitate the understanding of the role of host shift in herbivore radiation. Revealing the role of host shifts in herbivorous insect diversification Our study proposed a method for assessing the relative importance of host shifts in herbivorous insect speciation. This method allows quantitative analysis at a fine taxonomic scale, but because we only applied it to one herbivorous insect group, the application of this method to various herbivorous insect groups will facilitate a more general discussion on herbivorous insect diversification. If host-shift-driven speciation turns out to be relatively minor in recent speciation, there may be another role for host shifts in promoting herbivorous insect diversification rather than facilitating speciation per se, such as facilitating the entry into novel niche spaces (Janzen 1968) and the coexistence of already diverged species (Rabosky 2009). Information on the phylogenetic pattern of host use is clearly increasing rapidly, and a standardized method would link studies using different systems and facilitate our understanding of the effects of host shift on herbivorous insect diversity. Acknowledgments We thank K. Mochizuki, T. Hirano, S. Furukawa, W. Toki, I. Ohshima, O. Kishida, K. Hashimoto, K. Watanabe, M. Kobayashi, T. Iwasaki, and M. Tsujimoto for sampling and field assistance; staff members of Teshio Experimental Forest, Uryu Experimental Forest, Tomakomai Experimental Forest, Wakayama Experimental Forest, ª 2016 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. R. Nakadai & A. Kawakita Ashiu Forest Research Station, and Shiiba Research Forest. We also thank two anonymous reviewers and the associated editors for comments that improved the manuscript. 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Phylogeny of Caloptilia moths feeding on Acer based on mitochondrial COI with information on sampling site. Figure S2. Distributions of 14 Caloptilia moth species feeding on Acer. Table S1. Specimen information. Table S2. DDBJ accession numbers. Table S3. Primers used in this study. Table S4. The results of randomization tests including C. aurifasciata that feeds on Toxicodendron. Data S1. Newick format data containing phylogenetic information of 13 Caloptilia moths feeding on Acer trees. Data S2. Newick format data containing phylogenetic information of 20 Acer species. Data S3. CSV format file containing host use information. Data S4. Text file containing the command for running the randomization analysis in R using Data S1-3. ª 2016 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.