Adaptation to pollination by fungus gnats underlies the evolution of pollination syndrome in the genus Euonymus.

BACKGROUND AND AIMS: Dipteran insects are known pollinators of many angiosperms, but knowledge on how flies affect floral evolution is relatively scarce. Some plants pollinated by fungus gnats share a unique set of floral characters (dark red display, flat shape and short stamens), which differs from any known pollination syndromes. We tested whether this set of floral characters is a pollination syndrome associated with pollination by fungus gnats, using the genus Euonymus as a model. METHODS: The pollinator and floral colour, morphology and scent profile were investigated for ten Euonymus species and Tripterygium regelii as an outgroup. The flower colour was evaluated using bee and fly colour vision models. The evolutionary association between fungus gnat pollination and each plant character was tested using a phylogenetically independent contrast. The ancestral state reconstruction was performed on flower colour, which is associated with fungus gnat pollination, to infer the evolution of pollination in the genus Euonymus. KEY RESULTS: The red-flowered Euonymus species were pollinated predominantly by fungus gnats, whereas the white-flowered species were pollinated by bees, beetles and brachyceran flies. The colour vision analysis suggested that red and white flowers are perceived as different colours by both bees and flies. The floral scents of the fungus gnat-pollinated species were characterized by acetoin, which made up >90 % of the total scent in three species. Phylogenetically independent contrast showed that the evolution of fungus gnat pollination is associated with acquisition of red flowers, short stamens and acetoin emission. CONCLUSIONS: Our results suggest that the observed combination of floral characters is a pollination syndrome associated with the parallel evolution of pollination by fungus gnats. Although the role of the red floral display and acetoin in pollinator attraction remains to be elucidated, our finding underscores the importance of fungus gnats as potential contributors to floral diversification.

Phylogeny of gracillariid leaf-mining moths: evolution of larval behaviour inferred from phylogenomic and Sanger data.

Gracillariidae is the most taxonomically diverse cosmopolitan leaf-mining moth family, consisting of nearly 2000 named species in 105 described genera, classified into eight extant subfamilies. The majority of gracillariid species are internal plant feeders as larvae, creating mines and galls in plant tissue. Despite their diversity and ecological adaptations, their phylogenetic relationships, especially among subfamilies, remain uncertain. Genomic data (83 taxa, 589 loci) were integrated with Sanger data (130 taxa, 22 loci), to reconstruct a phylogeny of Gracillariidae. Based on analyses of both datasets combined and analyzed separately, monophyly of Gracillariidae and all its subfamilies, monophyly of the clade "LAMPO" (subfamilies: Lithocolletinae, Acrocercopinae, Marmarinae, Phyllocnistinae, and Oecophyllembiinae) and relationships of its subclade "AMO" (subfamilies: Acrocercopinae, Marmarinae, and Oecophyllembiinae) were strongly supported. A sister-group relationship of Ornixolinae to the remainder of the family, and a monophyletic leaf roller lineage (Callicercops Vári + Parornichinae) + Gracillariinae, as sister to the "LAMPO" clade were supported by the most likely tree. Dating analyses indicate a mid-Cretaceous (105.3 Ma) origin of the family, followed by a rapid diversification into the nine subfamilies predating the Cretaceous-Palaeogene extinction. We hypothesize that advanced larval behaviours, such as making keeled or tentiform blotch mines, rolling leaves and galling, allowed gracillariids to better avoid larval parasitoids allowing them to further diversify. Finally, we stabilize the classification by formally re-establishing the subfamily ranks of Marmarinae stat.rev., Oecophyllembiinae stat.rev. and Parornichinae stat.rev., and erect a new subfamily, Callicercopinae Li, Ohshima and Kawahara to accommodate the enigmatic genus Callicercops.

Slippery flowers as a mechanism of defence against nectar-thieving ants.

BACKGROUND AND AIMS: The great diversity of floral characteristics among animal-pollinated plants is commonly understood to be the result of coevolutionary interactions between plants and pollinators. Floral antagonists, such as nectar thieves, also have the potential to exert an influence upon the selection of floral characteristics, but adaptation against floral antagonists has attracted comparatively little attention. We found that the corollas of hornet-pollinated Codonopsis lanceolata (Campanulaceae) and the tepals of bee-pollinated Fritillaria koidzumiana (Liliaceae) are slippery to nectar-thieving ants living in the plant's habitat; because the flowers of both species have exposed nectaries, slippery perianths may function as a defence against nectar-thieving ants. METHODS: We conducted a behavioural experiment and observed perianth surface microstructure by scanning electron microscopy to investigate the mechanism of slipperiness. Field experiments were conducted to test whether slippery perianths prevent floral entry by ants, and whether ant presence inside flowers affects pollination. KEY RESULTS: Scanning electron microscopy observations indicated that the slippery surfaces were coated with epicuticular wax crystals. The perianths lost their slipperiness when wiped with hexane. Artificial bridging of the slippery surfaces using non-slippery materials allowed ants to enter flowers more frequently. Experimental introduction of live ants to the Codonopsis flowers evicted hornet pollinators and shortened the duration of pollinator visits. However, no statistical differences were found in the fruit or seed sets of flowers with and without ants. CONCLUSIONS: Slippery perianths, most probably based on epicuticular wax crystals, prevent floral entry by ants that negatively affect pollinator behaviour. Experimental evidence of floral defence based on slippery surfaces is rare, but such a mode of defence may be widespread amongst flowering plants.

Active pollination drives selection for reduced pollen-ovule ratios.

PREMISE: Variation in pollen-ovule ratios is thought to reflect the degree of pollen transfer efficiency-the more efficient the process, the fewer pollen grains needed. Few studies have directly examined the relationship between pollen-ovule ratio and pollen transfer efficiency. For active pollination in the pollination brood mutualisms of yuccas and yucca moths, figs and fig wasps, senita and senita moths, and leafflowers and leafflower moths, pollinators purposefully collect pollen and place it directly on the stigmatic surface of conspecific flowers. The tight coupling of insect reproductive interests with pollination of the flowers in which larvae develop ensures that pollination is highly efficient. METHODS: We used the multiple evolutionary transitions between passive pollination and more efficient active pollination to test if increased pollen transfer efficiency leads to reduced pollen-ovule ratios. We collected pollen and ovule data from a suite of plant species from each of the pollination brood mutualisms and used phylogenetically controlled tests and sister-group comparisons to examine whether the shift to active pollination resulted in reduced pollen-ovule ratios. RESULTS: Across all transitions between passive and active pollination in plants, actively pollinated plants had significantly lower pollen-ovule ratios than closely related passively pollinated taxa. Phylogenetically corrected comparisons demonstrated that actively pollinated plant species had an average 76% reduction in the pollen-ovule ratio. CONCLUSIONS: The results for active pollination systems support the general utility of pollen-ovule ratios as indicators of pollination efficiency and the central importance of pollen transfer efficiency in the evolution of pollen-ovule ratio.

Pollination by fungus gnats and associated floral characteristics in five families of the Japanese flora.

Background and aims: Pollination by fungus gnats (Mycetophilidae and Sciaridae) is uncommon, but is nevertheless known to occur in 20 genera among eight angiosperm families. Because many fungus gnat-pollinated plants possess a dark red floral display, we hypothesized that fungus gnat pollination is more widespread among plants with similar floral display than currently known. We thus studied the pollination biology of flowers with dark red pigmentation in five families, focusing particularly on plants having small, flat, actinomorphic flowers with exposed nectaries and short stamens, because these floral characteristics mirror those of a known fungus gnat-pollinated genus (Mitella). Methods: We observed daytime and night-time floral visitors for a total of 194.5 h in Aucuba japonica (Garryaceae), Euonymus spp. (Celastraceae), Disanthus cercidifolius (Hamamelidaceae), Micranthes fusca (Saxifragaceae) and Streptopus streptopoides (Liliaceae). Visitors were categorized into functional groups, and a pollination importance index (PII) was calculated for each functional group based on visitation frequency, pollen load and behaviour on flowers. Key results: Fungus gnats were dominant among the 1762 insects observed (36-92 % depending on the plant species) and were the most important pollinators among all plants studied (PII: 0.529-1). Fungus gnat visits occurred during the daytime and, more frequently, at dusk. Most often, pollen grains became clumped on the ventral side of the head and/or thorax as the short-proboscid fungus gnats foraged on nectar and came into contact with anthers located close to the flower base. Conclusions: Pollination by fungus gnats is probably more common than previously thought, especially in habitats similar to those of the plants studied (moist forest understorey, streamside or subalpine meadow) where fungus gnats are abundant year-round. Our results further suggest that there may be a previously unnoticed association between fungus gnat pollination and dark red coloration, and a shared overall floral architecture among the plants studied.

Patterns of temporal and enemy niche use by a community of leaf cone moths (Caloptilia) coexisting on maples (Acer) as revealed by metabarcoding.

The diversity of herbivorous insects is often considered a function of host plant diversity. However, recent research has uncovered many examples of closely related herbivores using the same host plant(s), suggesting that partitioning of host plants is not the only mechanism generating diversity. Herbivores sharing hosts may utilize different parts of the same plant, but such resource partitioning is often not apparent; hence, the factors that allow closely related herbivores to coexist are still largely undetermined. We examined whether partitioning of phenology or natural enemies may explain the coexistence of leaf cone moths (Caloptilia; Gracillariidae) associated with maples (Acer; Sapindaceae). Larval activity of 10 sympatric Caloptilia species found on nine maple species was monitored every 2-3 weeks for a total of 13 sampling events, and an exhaustive search for internal parasitoid wasps was conducted using high-throughput sequencing. Blocking primers were used to facilitate the detection of wasp larvae inside moth tissue. We found considerable phenological overlap among Caloptilia species, with two clear peaks in July and September-October. Coexisting Caloptilia species also had largely overlapping parasitoid communities; a total of 13 chalcid and ichneumon wasp species attacked Caloptilia in a nonspecific fashion at an overall parasitism rate of 46.4%. Although coexistence may be facilitated by factors not accounted for in this study, it appears that niche partitioning is not necessary for closely related herbivores to stably coexist on shared hosts. Co-occurrence without resource partitioning may provide an additional axis along which herbivorous insects attain increased species richness.

Pollinia transfer on moth legs in Hoya carnosa (Apocynaceae).

PREMISE OF THE STUDY: The genus Hoya (Apocynaceae; Asclepiadoideae) is characterized by a set of complex floral characters unique among the asclepiads, but their role in pollination is poorly understood. Here, we report a new mechanism of asclepiad pollination in the wax plant Hoya carnosa: the pollinaria are transferred on the legs of medium or large settling moths. METHODS: Floral visitors and their behavior were observed on Amami-Oshima Island during 2013-2015, and the efficacy of different pollinators was determined by counting the pollinarium loads on different flower visitors. The floral characters were studied to establish the process of pollination in relation to pollinator behavior on the flower. KEY RESULTS: Hoya carnosa was visited by various settling moths at night, but pollinia attachment was confirmed predominantly on the legs of the large moth Erebus ephesperis (Noctuidae) and less frequently on the legs of the medium-sized moths Bastilla arcuata (Crambidae) and Cleora injectaria (Geometridae). The moths walked around and searched for nectar on the inflorescence, and the corpusculum became clipped to the arolia (adhesive pads on moth tarsi) as they stepped on the pollinaria between the staminal corona. The downward spherical inflorescence of aggregated flowers with flat, velvety petals and a slippery corona provides restricted footholds for the visitors, which efficiently leads pollinator legs to the pollinaria. CONCLUSIONS: Although the pollination system of Hoya is largely unknown, pollination by insect legs may be a major pollination system in this genus because these basic floral characters are shared among many species.

Limiting the cost of mutualism: the defensive role of elongated gynophore in the leafflower-moth mutualism.

Mutualisms are interactions from which both partners benefit but may collapse if mutualists' costs and benefits are not aligned. Host sanctions are one mechanism whereby hosts selectively allocate resources to the more cooperative partners and thereby reduce the fitness of overexploiters; however, many mutualisms lack apparent means of host sanctions. In mutualisms between plants and pollinating seed parasites, such as those between leafflowers and leafflower moths, pollinators consume subsets of the seeds as larval food in return for their pollination service. Plants may select against overexploiters by selectively aborting flowers with a heavy egg load, but in many leafflower species, seeds are fully eaten in some fruits, suggesting that such a mechanism is not present in all species. Instead, the fruits of Breynia vitis-idaea have stalk-like structures (gynophore) through which early-instar moth larvae must bore to reach seeds. Examination of moth mortality in fruits with different gynophore lengths suggested that fruits with longer gynophore had higher moth mortality and, therefore, less seed damage. Most moth mortality occurred at the egg stage or as early larval instar before moths reached the seeds, consistent with the view that gynophore functions to prevent moth access to seeds. Gynophore length was unaffected by plant size, extent of moth oviposition, or geography; thus, it is most likely genetically controlled. Because gynophores do not elongate in related species whose pollinators oviposit directly into the ovary, the gynophore in B. vitis-idaea may have evolved as a defense to limit the cost of the mutualism.

Pollination system and the effect of inflorescence size on fruit set in the deceptive orchid Cephalanthera falcata.

Larger inflorescences in reward-producing plants can benefit plants by increasing both pollinator attraction and the duration of visits by individual pollinators. However, ultimately, inflorescence size is determined by the balance between the benefits of large inflorescences and the increased cost of geitonogamy. At present, little is known about the relationship between inflorescence size and fecundity in deceptive plants. Given that pollinators are likely to leave inflorescences lacking rewards quickly, it seems unlikely that longer pollinator visits and the risk of geitonogamy would be strong selective pressures in these species, which indicates that pollinator attraction might be the most important factor influencing their inflorescence size. Here we examined the pollination ecology of the deceptive orchid Cephalanthera falcata in order to clarify the effects of inflorescence size on the fruit set of this non-rewarding species. Field observations of the floral visitors showed that C. falcata is pollinated by the andrenid bee Andrena aburana, whilst pollination experiments demonstrated that this orchid species is neither autogamous nor apogamous, but is strongly pollinator dependent. Three consecutive years of field observations revealed that fruit set was positively correlated with the number of flowers per inflorescence. These results provide strong evidence that the nectarless orchid C. falcata benefits from producing larger inflorescences that attract a greater number of innate pollinators. Large inflorescences may have a greater positive effect on fruit set in deceptive plants because a growing number of studies suggest that fruit set in reward-producing plants is usually unaffected by display size.

Active pollination favours sexual dimorphism in floral scent.

Zoophilous flowers often transmit olfactory signals to attract pollinators. In plants with unisexual flowers, such signals are usually similar between the sexes because attraction of the same animal to both male and female flowers is essential for conspecific pollen transfer. Here, we present a remarkable example of sexual dimorphism in floral signal observed in reproductively highly specialized clades of the tribe Phyllantheae (Phyllanthaceae). These plants are pollinated by species-specific, seed-parasitic Epicephala moths (Gracillariidae) that actively collect pollen from male flowers and pollinate the female flowers in which they oviposit; by doing so, they ensure seeds for their offspring. We found that Epicephala-pollinated Phyllanthaceae plants consistently exhibit major qualitative differences in scent between male and female flowers, often involving compounds derived from different biosynthetic pathways. In a choice test, mated female Epicephala moths preferred the scent of male flowers over that of female flowers, suggesting that male floral scent elicits pollen-collecting behaviour. Epicephala pollination evolved multiple times in Phyllantheae, at least thrice accompanied by transition from sexual monomorphism to dimorphism in floral scent. This is the first example in which sexually dimorphic floral scent has evolved to signal an alternative reward provided by each sex, provoking the pollinator's legitimate altruistic behaviour.

Non-congruent colonizations and diversification in a coevolving pollination mutualism on oceanic islands.

A challenge for coevolutionary theory is how different types of interaction influence the diversification of coevolving clades. Reciprocal specialization is characteristic of certain coevolving, mutualistic interactions, but whether this specialization seen in ecological time constrains changes in patterns of interaction over evolutionary time remains unclear. Here, we examine the co-radiation of Glochidion trees (Phyllanthaceae: Phyllanthus s. l.) and pollinating, seed-predatory Epicephala moths (Lepidoptera: Gracillariidae) on young (mostly later than 5 Ma) oceanic islands in southeastern Polynesia. Epicephala are the sole known pollinators of Glochidion trees, and show extreme reciprocal specialization in continental Asia. We find that Glochidion and Epicephala diversified across these islands through repeated, non-congruent colonizations, and that one recently colonizing Epicephala lineage has spread across 12 host species in three archipelagos in less than 1 Myr. These results indicate that reciprocal specialization and coadaptation do not prevent dramatic changes in associations between intimately associated taxa over short evolutionary time scales. Not only are these host associations more dynamic than previously recognized, but these changes in patterns of interaction may play an important role in the diversification of coevolving taxa.

Modified leaves with disk-shaped nectaries of Macaranga sinensis (Euphorbiaceae) provide reward for pollinators.

PREMISE OF THE STUDY: Nectar is the most common reward provided by animal-pollinated flowers. Diversity in position and structure of floral nectaries suggests that floral nectar production evolved repeatedly, but the evolutionary origins are not well known. Flowers of the genus Macaranga (Euphorbiaceae) are apetalous and lack floral nectar. Nevertheless, many Macaranga species possess disk-shaped nectaries on their leaves, sought by ants that defend plants from herbivory. In some Macaranga species, similar glands also occur on the bracteoles-modified leaves subtending the flowers. We investigated whether these glands on the bracteoles of M. sinensis are involved in pollination. • METHODS: Flower visitors were captured, and body pollen was examined. The behavior of flower visitors on inflorescences was also observed. Sugar composition of the nectar from bracteoles and leaves was collected and analyzed using high-performance liquid chromatography. • KEY RESULTS: Various bees and flies with body pollen visited both male and female inflorescences, feeding on nectar from the bracteoles and touching anthers and stigmas in the process. Sugar composition of nectar from the bracteoles and the leaves did not differ. • CONCLUSIONS: Macaranga sinensis was pollinated by insects foraging on the disk-shaped nectaries on bracteoles. The similar appearance, position, and sugar composition of nectar suggest that disk-shaped nectaries on bracteoles and leaves are homologous and that nectaries on leaves were recruited to inflorescences to serve floral function in M. sinensis. Having protective mutualism with ants has likely opened an unusual route for the evolution of floral nectar in otherwise non-nectar-producing flowers of M. sinensis.

Variation in the strength of association among pollination systems and floral traits: evolutionary changes in the floral traits of Bornean gingers (Zingiberaceae).

PREMISE OF THE STUDY: Diversification of floral traits in angiosperms is often attributed to have been driven by adaptations to pollinators. Nevertheless, phylogenetic studies on the relationships among evolutionary changes in floral traits and pollination systems are still limited. We examined the relationships between floral trait changes and pollinator shifts in Bornean gingers (Zingiberaceae). These plants have strongly zygomorphic flowers pollinated by spiderhunter birds, bees of the genus Amegilla, and halictid bees. • METHODS: We identified pollination systems through field observations and recorded petal color, quantity of floral rewards, and seven measures of flower morphology in 28 ginger species. Phylogenetic trees were constructed from nucleotide sequences of the matK and ITS regions. We examined the correlations between the evolution of pollination systems and floral traits using phylogenetically independent contrasts. • KEY RESULTS: Significant association was found between pink color and spiderhunter pollination, orange and Amegilla pollination, and yellow and white and halictid pollination. Sugar production was higher in spiderhunter-pollinated species and lower in halictid-pollinated. Meanwhile, there was a significant association only for a subset of the floral morphological characters measured. Floral tube length, which is often thought to evolve to match the lengths of pollinator probing apparatuses, did not show any correlation. • CONCLUSIONS: There is considerable variation in the strength of association among pollination systems and floral traits. Lack of significant correlation in some traits could partly be explained by floral functions other than pollination, such as adaptations to prevent herbivore damage to the ovules. Further studies on these factors may improve understanding of plant-pollinator interactions.

Diversity and larval leaf-mining habits of Japanese jewel beetles of the tribe Tracheini (Coleoptera, Buprestidae).

From the Japanese Archipelago, 12 Habroloma and 20 Trachys species (Buprestidae: Tracheini) have been recorded. Two new Habroloma species were found, which are associated with Elaeocarpaceae and Loranthaceae, also new host plant families/orders for Tracheini. The two new species are described as Habrolomaelaeocarpusisp. nov. and Habrolomataxillusisp. nov., and the latter is the first Tracheini species shown to be associated with epiphytes. Leaf mines of 31 Tracheini species are also reported in this work, including new records of leaf mines for 16 Tracheini species. The larvae of all these recorded species are full-depth linear-blotch mesophyll miners of mature leaves and pupate within their mines. The mining habits of Habroloma species associated with Symplocos (Symplocaceae) are unique: the young larvae bore into midribs and petioles and cause leaf fall, and the larvae then mine the fallen leaves.

Erratum: Corrigendum: Kato M, Kawakita A (2023) Diversity and larval leaf-mining habits of Japanese jewel beetles of the tribe Tracheini (Coleoptera, Buprestidae). ZooKeys 1156: 133-158. doi: 10.3897/zookeys.1156.97768.

[This corrects the article DOI: 10.3897/zookeys.1156.97768.].

Molecular phylogeny of the bivalve superfamily Galeommatoidea (Heterodonta, Veneroida) reveals dynamic evolution of symbiotic lifestyle and interphylum host switching.

BACKGROUND: Galeommatoidea is a superfamily of bivalves that exhibits remarkably diverse lifestyles. Many members of this group live attached to the body surface or inside the burrows of other marine invertebrates, including crustaceans, holothurians, echinoids, cnidarians, sipunculans and echiurans. These symbiotic species exhibit high host specificity, commensal interactions with hosts, and extreme morphological and behavioral adaptations to symbiotic life. Host specialization to various animal groups has likely played an important role in the evolution and diversification of this bivalve group. However, the evolutionary pathway that led to their ecological diversity is not well understood, in part because of their reduced and/or highly modified morphologies that have confounded traditional taxonomy. This study elucidates the taxonomy of the Galeommatoidea and their evolutionary history of symbiotic lifestyle based on a molecular phylogenic analysis of 33 galeommatoidean and five putative galeommatoidean species belonging to 27 genera and three families using two nuclear ribosomal genes (18S and 28S ribosomal DNA) and a nuclear (histone H3) and mitochondrial (cytochrome oxidase subunit I) protein-coding genes. RESULTS: Molecular phylogeny recovered six well-supported major clades within Galeommatoidea. Symbiotic species were found in all major clades, whereas free-living species were grouped into two major clades. Species symbiotic with crustaceans, holothurians, sipunculans, and echiurans were each found in multiple major clades, suggesting that host specialization to these animal groups occurred repeatedly in Galeommatoidea. CONCLUSIONS: Our results suggest that the evolutionary history of host association in Galeommatoidea has been remarkably dynamic, involving frequent host switches between different animal phyla. Such an unusual pattern of dynamic host switching is considered to have resulted from their commensalistic lifestyle, in which they maintain filter-feeding habits even in symbiotic habitats. The results of the molecular phylogenetic analysis did not correspond with the current taxonomic circumscription. Galeommatidae and Lasaeidae were polyphyletic, and Basterotia, which is traditionally assigned to Cyamioidea, formed a monophyletic clade within Galeommatoidea.

Development of nine markers and characterization of the microsatellite loci in the endangered Gymnogobius isaza (Gobiidae).

Gymnogobius isaza is a freshwater goby endemic to Lake Biwa, Japan. They experienced a drastic demographic bottleneck in the 1950s and 1980s and slightly recovered thereafter, but the population size is still very small. To reveal dynamics of genetic diversity of G. isaza, we developed nine microsatellite markers based on the sequence data of a related goby Chaenogobius annularis. Nine SSR (Simple Sequence Repeats) markers were successfully amplified for raw and formalin-fixed fish samples. The number of alleles and expected heterozygosities ranged from one to 10 and from 0.06 to 0.84, respectively, for the current samples, while one to 12 and 0.09 to 0.83 for historical samples. The markers described here will be useful for investigating the genetic diversity and gene flow and for conservation of G. isaza.

Increased gene sampling strengthens support for higher-level groups within leaf-mining moths and relatives (Lepidoptera: Gracillariidae).

BACKGROUND: Researchers conducting molecular phylogenetic studies are frequently faced with the decision of what to do when weak branch support is obtained for key nodes of importance. As one solution, the researcher may choose to sequence additional orthologous genes of appropriate evolutionary rate for the taxa in the study. However, generating large, complete data matrices can become increasingly difficult as the number of characters increases. A few empirical studies have shown that augmenting genes even for a subset of taxa can improve branch support. However, because each study differs in the number of characters and taxa, there is still a need for additional studies that examine whether incomplete sampling designs are likely to aid at increasing deep node resolution. We target Gracillariidae, a Cretaceous-age (~100 Ma) group of leaf-mining moths to test whether the strategy of adding genes for a subset of taxa can improve branch support for deep nodes. We initially sequenced ten genes (8,418 bp) for 57 taxa that represent the major lineages of Gracillariidae plus outgroups. After finding that many deep divergences remained weakly supported, we sequenced eleven additional genes (6,375 bp) for a 27-taxon subset. We then compared results from different data sets to assess whether one sampling design can be favored over another. The concatenated data set comprising all genes and all taxa and three other data sets of different taxon and gene sub-sampling design were analyzed with maximum likelihood. Each data set was subject to five different models and partitioning schemes of non-synonymous and synonymous changes. Statistical significance of non-monophyly was examined with the Approximately Unbiased (AU) test. RESULTS: Partial augmentation of genes led to high support for deep divergences, especially when non-synonymous changes were analyzed alone. Increasing the number of taxa without an increase in number of characters led to lower bootstrap support; increasing the number of characters without increasing the number of taxa generally increased bootstrap support. More than three-quarters of nodes were supported with bootstrap values greater than 80% when all taxa and genes were combined. Gracillariidae, Lithocolletinae + Leucanthiza, and Acrocercops and Parectopa groups were strongly supported in nearly every analysis. Gracillaria group was well supported in some analyses, but less so in others. We find strong evidence for the exclusion of Douglasiidae from Gracillarioidea sensu Davis and Robinson (1998). Our results strongly support the monophyly of a G.B.R.Y. clade, a group comprised of Gracillariidae + Bucculatricidae + Roeslerstammiidae + Yponomeutidae, when analyzed with non-synonymous changes only, but this group was frequently split when synonymous and non-synonymous substitutions were analyzed together. CONCLUSIONS: 1) Partially or fully augmenting a data set with more characters increased bootstrap support for particular deep nodes, and this increase was dramatic when non-synonymous changes were analyzed alone. Thus, the addition of sites that have low levels of saturation and compositional heterogeneity can greatly improve results. 2) Gracillarioidea, as defined by Davis and Robinson (1998), clearly do not include Douglasiidae, and changes to current classification will be required. 3) Gracillariidae were monophyletic in all analyses conducted, and nearly all species can be placed into one of six strongly supported clades though relationships among these remain unclear. 4) The difficulty in determining the phylogenetic placement of Bucculatricidae is probably attributable to compositional heterogeneity at the third codon position. From our tests for compositional heterogeneity and strong bootstrap values obtained when synonymous changes are excluded, we tentatively conclude that Bucculatricidae is closely related to Gracillariidae + Roeslerstammiidae + Yponomeutidae.

Allopatric distribution and diversification without niche shift in a bryophyte-feeding basal moth lineage (Lepidoptera: Micropterigidae).

The Lepidoptera represent one of the most successful radiations of plant-feeding insects, which predominantly took place within angiosperms beginning in the Cretaceous period. Angiosperm colonization is thought to underlie the evolutionary success of the Lepidoptera because angiosperms provide an enormous range of niches for ecological speciation to take place. By contrast, the basal lepidopteran lineage, Micropterigidae, remained unassociated with angiosperms since Jurassic times but nevertheless achieved a modest diversity in the Japanese Archipelago. We explored the causes and processes of diversification of the Japanese micropterigid moths by performing molecular phylogenetic analysis and extensive ecological surveying. Phylogenetic analysis recovered a monophyletic group of approximately 25 East Asian endemic species that feed exclusively on the liverwort Conocephalum conicum, suggesting that niche shifts hardly played a role in their diversification. Consistent with the low flying ability of micropterigid moths, the distributions of the Conocephalum specialists are each localized and allopatric, indicating that speciation by geographical isolation has been the major process shaping the diversity of Japanese Micropterigidae. To our knowledge, this is the largest radiation of herbivorous insects that does not accompany any apparent niche differentiation. We suggest that the significance of non-ecological speciation during the diversification of the Lepidoptera is commonly underestimated.

Three new species of Ametrodiplosis (Diptera: Cecidomyiidae) from Japan, with a key to the Japanese species and a molecular phylogenetic analysis.

Ametrodiplosis Rübsaamen (Diptera: Cecidomyiidae: Clinodiplosini) is a mostly Holarctic gall midge genus whose species are associated with a wide range of seed plant families, either as gall-inducers or inquilines. In this study, we describe three species of Ametrodiplosis from Japan: A. adetos n. sp. feeding in the flowers of Tylophora aristolochioides Miq. (Apocynaceae); A. aeroradicis n. sp. inducing aerial root galls on Trachelospermum asiaticum (Sieb. et Zucc.) Nakai and T. gracilipes var. liukiuense (Apocynaceae); and A. stellariae n. sp. forming leaf bud galls on Stellaria uliginosa Murray var. undulata (Thunb.) Ohwi (Caryophyllaceae). A molecular phylogenetic analysis using mitochondrial COI and ribosomal 16S genes and nuclear ribosomal 28S gene were conducted for the three new Ametrodiplisis species and other clinodiplosine taxa sequences available in GenBank. The analysis supported the monophyly of Ametrodiplosis despite the variable life history of the three species. In addition, it indicated very low intraspecific genetic divergence among the individuals from different localities and/or host plants. A taxonomic key to the three new Japanese species of Ametrodiplosis is provided.