Issei Ohshima

Ecological speciation in phytophagous insects

Divergent natural selection has been shown to promote speciation in a wide range of taxa. For example, adaptation to different ecological environments, via divergent selection, can result in the evolution of reproductive incompatibility between populations. Phytophagous insects have been at the forefront of these investigations of ‘ecological speciation’ and it is clear that adaptation to different host plants can promote insect speciation. However, much remains unknown. For example, there is abundant variability in the extent to which divergent selection promotes speciation, the sources of divergent selection, the types of reproductive barriers involved, and the genetic basis of divergent adaptation. We review these factors here. Several findings emerge, including the observation that although numerous different sources of divergent selection and reproductive isolation can be involved in insect speciation, their order of evolution and relative importance are poorly understood. Another finding is that the genetic basis of host preference and performance can involve loci of major effect and opposing dominance, factors which might facilitate speciation in the face of gene flow. In addition, we raise a number of other recent issues relating to phytophagous insect speciation, such as alternatives to ecological speciation, the geography of speciation, and the molecular signatures of speciation. Throughout, we aim to both synthesize what is known, as well as highlight areas where future work is especially needed.

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

BackgroundResearchers 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.ResultsPartial 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.Conclusions1) 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.

DNA barcoding reveals a largely unknown fauna of Gracillariidae leaf-mining moths in the Neotropics.

Higher taxa often show increasing species richness towards tropical low latitudes, a pattern known as the latitudinal biodiversity gradient (LBG). A rare reverse LBG (with greater richness towards temperate high latitudes) is exhibited by Gracillariidae leaf‐mining moths, in which most described species occur in northern temperate areas. We carried out the first assessment of gracillariid species diversity in two Neotropical regions to test whether the relatively low tropical species diversity of this family is genuine or caused by insufficient sampling and a strong taxonomic impediment. Field surveys in six French Guianan and one Ecuadorian site produced 516 gracillariid specimens that were DNA barcoded to facilitate identification and to match larvae inside leaf mines with adults. Species delineation from sequence data was approximated using Automatic Barcode Gap Discovery and Refined Single Linkage Analysis through the Barcode Index Number system, and the proportion of described/undescribed species was estimated after comparison with types of 83% of described species. Locally, alpha‐diversity far exceeds that of any known temperate fauna, with as many as 108 candidate species (59.3% as singletons) collected at one site, and with an estimated species richness lower bound of 240 species. Strikingly, at least 85% of the species collected as adults were found to be undescribed. Our sampling represents the most thorough survey of gracillariid species diversity in the Neotropics to date and the results from both our molecular and morphological analyses indicate that the current reverse LBG seen in this group is an artefact of insufficient sampling and a strong description deficit in the Neotropics.

A molecular phylogeny and revised higher-level classification for the leaf-mining moth family Gracillariidae and its implications for larval host-use evolution

Gracillariidae are one of the most diverse families of internally feeding insects, and many species are economically important. Study of this family has been hampered by lack of a robust and comprehensive phylogeny. In the present paper, we sequenced up to 22 genes in 96 gracillariid species, representing all previously recognized subfamilies and genus groups, plus 20 outgroups representing other families and superfamilies. Following objective identification and removal of two rogue taxa, two datasets were constructed: dataset 1, which included 12 loci totalling 9927 bp for 94 taxa, and dataset 2, which supplemented dataset 1 with 10 additional loci for 10 taxa, for a total of 22 loci and 16 167 bp. Maximum likelihood analyses strongly supported the monophyly of Gracillariidae and most previously recognized subfamilies and genus groups. On this basis, we propose a new classification consisting of eight subfamilies, four of which are newly recognized or resurrected: Acrocercopinae Kawahara & Ohshima subfam. n.; Gracillariinae Stainton; Lithocolletinae Stainton; Marmarinae Kawahara & Ohshima subfam. n.; Oecophyllembiinae Réal & Balachowsky; Parornichinae Kawahara & Ohshima subfam. n.; Ornixolinae Kuznetzov & Baryshnikova stat. rev.; and Phyllocnistinae Zeller. The subfamily Gracillariinae is restricted to the monophyletic group comprising Gracillaria Haworth and closely related genera. We also formally transfer Acrocercops scriptulata Meyrick to Ornixolinae and use the name Diphtheroptila Vári, creating Diphtheroptila scriptulata comb. n. An exploratory mapping of larval host‐use traits on the phylogeny shows strong conservation of modes of leaf mining but much higher lability of associations with host plant orders and families, suggesting that host shifts could play a significant role in gracillariid diversification.

Differential introgression causes genealogical discordance in host races of Acrocercops transecta (Insecta: Lepidoptera).

Recently diverged populations often exhibit incomplete reproductive isolation, with a low level of gene flow continuing between populations. Previous studies have shown that, even under a low level of gene flow, genetic divergence between populations can proceed at the loci governing local adaptation and reproductive isolation but not at other neutral loci. A leaf‐mining moth, Acrocercops transecta, consists of Juglans‐ and Lyonia‐associated host races. The two host races differ in host preferences of ovipositing females and in larval adaptation to host plants but mate readily in the laboratory, producing fertile hybrids. The Juglans and Lyonia races are often sympatric in the wild, implying that gene introgression could occur in nature between the two host races. We tested this hypothesis by combining phylogenetic analyses with coalescent simulations, focusing on mitochondrial genes (COI and ND5) and the nuclear Tpi, Per and Ldh genes located on the Z‐chromosome. The mitochondrial genes clearly distinguished the Lyonia race from the Juglnas race, whereas the Tpi, Per and Ldh genealogies did not reflect the two host races. Coalescent simulations indicated gene flow at the three Z‐linked genes in both directions, whereas there was no introgression in the mitochondrial genes. The lack of introgression in mitochondrial genes suggests that female host preference is the primary force leading to the bifurcation of maternally inherited loci. Thus, the results show that a low level of gene flow coupled with the inflexible female host preference differentiates histories of divergence between maternally and biparentally inherited genes in this host race system.

Phylogeny, biogeography, and host-plant association in the subfamily Apaturinae (Insecta: Lepidoptera: Nymphalidae) inferred from eight nuclear and seven mitochondrial genes.

The subfamily Apaturinae consists of 20 genera and shows disjunct distributions and unique host-plant associations. Most genera of this subfamily are distributed in Eurasia South-East Asia and Africa, whereas the genera Doxocopa and Asterocampa are distributed mainly in South America and North America, respectively. Although the Apaturinae larvae mainly feed on the Cannabaceae, those of the genus Apatura are associated with Salix and Populus (Salicaceae), which are distantly related to the Cannabaceae. Here, we infer the phylogeny of Apaturinae and reconstruct the history of host shifting and of colonization in the New World. We analyzed 9761 bp of nuclear and mitochondrial DNA sequence data, including the genes encoding EF1a, Wg, ArgK, CAD, GAPDH, IDH, MDH, RpS5, COI, COII, ATPase8, ATPase6, COIII, ND3, and ND5 for 12 apaturine genera. We also inferred the phylogeny with six additional genera using mitochondrial sequence data alone. Within the Apaturinae, two major clades are recovered in all the datasets. These clades separate the New World genera, Doxocopa and Asterocampa, indicating that dispersal to the New World occurred at least twice. According to our divergence time estimates, these genera originated during the Early Oligocene to the Early Miocene, implying that they migrated across the Bering Land Bridge rather than the Atlantic Land Bridge. The temporal estimates also show that host shifting to Salix or Populus in Apatura occurred more than 15 million years after the divergence of their host plants. Our phylogenetic results are inconsistent with the previously accepted apaturine genus groups and indicate that their higher classification should be reconsidered.

Techniques for continuous rearing and assessing host preference of a multivoltine leaf‐mining moth, Acrocercops transecta (Lepidoptera: Gracillariidae)

Techniques for rearing the leaf‐mining moth Acrocercops transecta successively over several generations are described. Based on continuous rearing, some life historical parameters in A. transecta were determined. Because of its short generation time, successive rearing makes the moth suitable for mating experiments and a model organism to elucidate the mechanism of host‐associated speciation.

From east to west across the Palearctic: Phylogeography of the invasive lime leaf miner Phyllonorycter issikii (Lepidoptera: Gracillariidae) and discovery of a putative new cryptic species in East Asia

Knowing the phylogeographic structure of invasive species is important for understanding the underlying processes of invasion. The micromoth Phyllonorycter issikii, whose larvae damage leaves of lime trees Tilia spp., was only known from East Asia. In the last three decades, it has been recorded in most of Europe, Western Russia and Siberia. We used the mitochondrial cytochrome c oxidase subunit I (COI) gene region to compare the genetic variability of P. issikii populations between these different regions. Additionally, we sequenced two nuclear genes (28S rRNA and Histone 3) and run morphometric analysis of male genitalia to probe for the existence of cryptic species. The analysis of COI data of 377 insect specimens collected in 16 countries across the Palearctic revealed the presence of two different lineages: P. issikii and a putative new cryptic Phyllonorycter species distributed in the Russian Far East and Japan. In P. issikii, we identified 31 haplotypes among which 23 were detected in the invaded area (Europe) and 10 were found in its putative native range in East Asia (Russian Far East, Japan, South Korea and China), with only two common haplotypes. The high number of haplotypes found in the invaded area suggest a possible scenario of multiple introductions. One haplotype H1 was dominant (119 individuals, 67.2%), not only throughout its expanding range in Europe and Siberia but, intriguingly, also in 96% of individuals originating from Japan. We detected eight unique haplotypes of P. issikii in East Asia. Five of them were exclusively found in the Russian Far East representing 95% of individuals from that area. The putative new cryptic Phyllonorycter species showed differences from P. issikii for the three studied genes. However, both species are morphologically undistinguishable. They occur in sympatry on the same host plants in Japan (Sendai) and the Russian Far East (Primorsky krai) without evidence of admixture.

Host-associated pre-mating reproductive isolation between host races of Acrocercops transecta: mating site preferences and effect of host presence on mating.

1. A correlation between habitat and mating site preference is crucial for reproductive isolation between populations with different ecological requirements in the absence of any other barriers to gene flow. In phytophagous insects, host‐plant preference of ovipositing females has been postulated to directly influence the extent of assortative mating. 2. Although this pleiotropic effect of host preference is commonly postulated in both theoretical and empirical studies in phytophagous insects, few studies have assessed the mating site preferences of both sexes. In herbivorous insects, it is crucial whether males have the same host preference as ovipositing females. 3. This study examines whether males and females have preferences for mating sites and assesses the effects of host presence on mating activity in a leaf‐mining moth, Acrocercops transecta Meyrick (Gracillariidae). This species consists of two host races, which are associated with Juglans ailanthifolia Carr. (Juglandaceae) and Lyonia ovalifolia (Wall.) Drude (Ericaceae). The two host races clearly differ in host preferences of ovipositing females. 4. In an experiment of mating site selection, Lyonia‐associated host race females significantly preferred Lyonia, while males of the Lyonia‐associated host race and both sexes of the Juglans‐associated host race did not show a preference for either plant. The mating activity of the Lyonia‐associated host race was not reduced in the absence of host plants. In the Juglans‐associated host race, however, the proportion of successful matings was significantly reduced when the host was absent. 5. These laboratory experiments provide empirical evidence that the different host associations in A. transecta do not directly lead to host‐induced assortative mating between the two host races as a pleiotropic effect. However, the present results suggest that the combined effects of the mating‐site preference of Lyonia‐associated females and the mating propensity of Juglans‐associated females could contribute to reproductive isolation between the two host races.

Estimation of Time Lag Between Right-Turning Vehicles and Pedestrians Approaching from the Right Side

In Japan, where vehicles travel on the left side of the road (i.e., right turns correspond to left turns in the United States), right turns can cause difficulties for a driver at an intersection, because the driver has to judge the headway in oncoming traffic while avoiding pedestrians in the crosswalk. However, few investigations have addressed the drivers avoidance of conflicts with pedestrians in the crosswalk. This study investigates conflicts between the right-turning vehicle and the pedestrian coming from the right in the crosswalk. A field experiment was conducted to measure a drivers avoidance behavior according to the time lag, defined as the interval between the time that the right-turning vehicle reaches the conflict point and the time that the pedestrian coming from the right side reaches the conflict point. This study found that the time lag is constantly reevaluated during the right turn by the driver, and the driver slows and enters the crosswalk behind the pedestrian, if the time lag at the conflict point is less than 2 s. Also, the braking location of drivers who braked to avoid conflict with the pedestrian after starting was 10.3 m before the conflict point.