The long proboscis of the aphid Stomaphis yanonis (Aphididae Lachninae) is advantageous for avoiding predation by tending ants.

Whether in ant-aphid mutualism the ants exert evolutionary selection pressure on aphid morphology has not yet been fully tested. Here, we tested whether the long proboscises of Stomaphis yanonis (Aphididae Lachninae) aphids confer an advantage in preventing predation by the tending ants. Specifically, we tested the hypothesis that aphids with a shorter proboscis would excrete less honeydew, making them more likely to be preyed upon by ants. Our results showed that aphid individuals with a shorter proboscis took up less phloem sap and excreted less honeydew than individuals with a longer proboscis. In addition, among aphids with a similar body size, those with a shorter proboscis were more susceptible to predation by ants than those with a longer proboscis. These results suggest that predation by tending ants, by exerting selection pressure on aphid proboscis morphology, has caused the aphids to evolve longer proboscises.

Sympatric co-existence of two ecotypes of Impatiens noli-tangere (Balsaminaceae) with different morphology and flowering phenology.

In angiosperms, intraspecific variation of flowering phenology may affect reproductive isolation and, consequently, speciation. This study focused on Impatiens noli-tangere (Balsaminaceae), which is distributed over broad latitudinal and altitudinal ranges in Japan. We aimed to reveal the phenotypic mixture of two ecotypes of I. noli-tangere with different flowering phenology and morphological traits in a narrow contact zone. Previous studies have shown that I. noli-tangere has early- and late-flowering types. The early-flowering type makes buds in June and is distributed at high-elevation sites. The late-flowering type makes buds in July and is distributed at low-elevation sites. In this study, we analyzed the flowering phenology of individuals at an intermediate elevation site where the early- and late-flowering types grow in sympatry (contact zone). We found no individuals showing intermediate flowering phenology at the contact zone, and early- and late-flowering types were clearly distinguishable. We also found that the differences in many other phenotypic traits between the early- and late-flowering types were maintained, including the number of flowers produced (total number of chasmogamous and cleistogamous flowers), leaf morphology (aspect ratio, number of serrations), seed traits (aspect ratio), and flower bud formation positions on the plant. This study showed that these two flowering ecotypes maintain many different traits in sympatry.

Evolutionary diversification of Japanese Stomaphis aphids (Aphididae, Lachninae) in relation to their host plant use and ant association.

Phytophagous insects are among the most diverse of the earth's organisms, and their diversification patterns and the driving forces behind these have attracted considerable research interest. Host shifting to closely related plant species is thought to play an important role in phytophagous insect diversification, but the extent to which other interactions such as mutualistic associations affect diversification is not yet known. In this study, we reconstructed the molecular phylogeny of Japanese Stomaphis aphids and determined whether host shifting or mutualistic association with different ant species could explain diversification in this aphid genus. We analyzed 12 species of Stomaphis and grouped them into ten well-supported DNA lineages. Species in each lineage used a single or a few host plant species, but were mutualistically associated with many ant species of the genus Lasius. This result suggests that Stomaphis evolutionarily diversified primarily through host plant shifts. Interestingly, the reconstructed phylogeny suggests that Stomaphis host shifts occasionally occurred between very distantly related host plant taxa (spanning up to five plant orders). The dependence of Stomaphis on long-lasting Lasius ant colonies situated in temperate deciduous forests where Lasius is the dominant ant genus may have led the aphids to shift to distantly related but spatially adjacent host tree species.

Cryptic Diversity in the Aphid-Parasitizing Wasp Protaphidius nawaii (Hymenoptera: Braconidae): Discovery of Two Attendant-Ant-Specific mtDNA Lineages.

The parasitoid wasp Protaphidius nawaii parasitizes the aphid Stomaphis japonica, which is obligatorily attended by several species of ants of genus Lasius. Subgenus Lasius or Dendrolasius ants use different defense strategies to protect the aphids that they attend (Lasius, shelter building; Dendrolasius, aggressive attack). We performed molecular phylogenetic analysis based on partial mitochondrial DNA sequences of P. nawaii and found that the parasitoid wasp consists of two highly differentiated genetic lineages. Although these two lineages distributed sympatrically, one tends to parasitize aphids attended by ants of subgenus Lasius, and the other parasitizes aphids attended by ants of subgenus Dendrolasius. The two lineages of P. nawaii appear to exhibit different oviposition behaviors adapted to the different aphid-protection strategies of the two ant subgenera.

Seasonal Migration in the Aphid Genus Stomaphis (Hemiptera: Aphididae): Discovery of Host Alternation Between Woody Plants in Subfamily Lachninae.

About 10% of aphid species show host alternation. These aphids migrate between primary and secondary host plant species in spring and autumn. Host alternation has not been observed in subfamily Lachninae, although it has been suggested on the basis of circumstantial evidence that Stomaphis japonica (Takahashi) may alternate its host between Quercus serrata (Murray) and Quercus acutissima (Carruth). However, a molecular phylogenetic study has indicated that the Stomaphis individuals feeding on these two plant species belong to two different lineages and aphids feeding on Q. acutissima and Pinus densiflora (Sieb. & Zucc.) belong to the same lineage. Here, we examined host alternation in Stomaphis species by comparing molecular phylogenetic identities, morphological features, and life cycles. The molecular analysis and morphological examination showed that aphids feeding on Q. acutissima were the same as those feeding on P. densiflora, whereas aphids feeding on Q. serrata were different from those feeding on Q. acutissima or on P. densiflora. Furthermore, winged aphids were observed on both Q. acutissima and P. densiflora in autumn, but we did not observe winged aphids on Q. serrata. These results indicate that Stomaphis (Walker) individuals feeding on Q. serrata and Q. acutissima belong to two species, one that feeds year-round on Q. serrata, and another, heteroecious species that feeds on P. densiflora as a primary host and on Q. acutissima as a secondary host. This study documents host alternation in subfamily Lachninae for the first time and discusses the acquisition of host alternation by Stomaphis from evolutionary and ecological perspectives.

Positive geographic correlation between soldiers' weapon size and defensive prowess in a eusocial aphid, Ceratovacuna japonica.

Some aphid species produce a soldier caste with enlarged forelegs and horns (weapons). It has been hypothesised that the evolution of morphological specialization by soldiers in social aphids is accelerated by high predation pressure, but this possibility has not been tested. Here, we investigated the relationship between local predator abundance and soldiers' weapon size and aggressiveness in a prey-predator system comprising a eusocial aphid, Ceratovacuna japonica, and its predators (larvae of the butterfly Taraka hamada and of the moth Atkinsonia ignipicta) in two populations with different predator abundances. We found that the soldiers in the predator-abundant population had larger weapons and were more aggressive than those in the population with lower predator abundance. Furthermore, the soldiers' defensive prowess (evaluated as the survival of aphids in the presence of predators) was greater in the predator-abundant population. These results provide the first evidence that a population of eusocial aphids experiencing high predation pressure has soldiers with pronounced defensive traits and defensive prowess.

Intraspecific independent evolution of floral spur length in response to local flower visitor size in Japanese Aquilegia in different mountain regions.

Geographic differences in floral traits may reflect geographic differences in effective pollinator assemblages. Independent local adaptation to pollinator assemblages in multiple regions would be expected to cause parallel floral trait evolution, although sufficient evidence for this is still lacking. Knowing the intraspecific evolutionary history of floral traits will reveal events that occur in the early stages of trait diversification. In this study, we investigated the relationship between flower spur length and pollinator size in 16 populations of Aquilegia buergeriana var. buergeriana distributed in four mountain regions in the Japanese Alps. We also examined the genetic relationship between yellow- and red-flowered individuals, to see if color differences caused genetic differentiation by pollinator isolation. Genetic relationships among 16 populations were analyzed based on genome-wide single-nucleotide polymorphisms. Even among populations within the same mountain region, pollinator size varied widely, and the average spur length of A. buergeriana var. buergeriana in each population was strongly related to the average visitor size of that population. Genetic relatedness between populations was not related to the similarity of spur length between populations; rather, it was related to the geographic proximity of populations in each mountain region. Our results indicate that spur length in each population evolved independently of the population genetic structure but in parallel in response to local flower visitor size in different mountain regions. Further, yellow- and red-flowered individuals of A. buergeriana var. buergeriana were not genetically differentiated. Unlike other Aquilegia species in Europe and America visited by hummingbirds and hawkmoths, the Japanese Aquilegia species is consistently visited by bumblebees. As a result, genetic isolation by flower color may not have occurred.

Intraspecific convergence of floral size correlates with pollinator size on different mountains: a case study of a bumblebee-pollinated Lamium (Lamiaceae) flowers in Japan.

BACKGROUND: Geographic differences in floral size sometimes reflect geographic differences in pollinator size. However, we know little about whether this floral size specialization to the regional pollinator size occurred independently at many places or occurred once and then spread across the distribution range of the plant species. RESULTS: We investigated the relationship between the local floral size of flowers and local pollinator size in 12 populations of Lamium album var. barbatum on two different mountains in the Japan Alps. Then, using 10 microsatellite markers, we analyzed genetic differentiation among the 12 populations. The results showed that local floral size was correlated with the average size of relevant morphological traits of the local pollinators: floral size was greater in populations visited frequently by the largest flower visitors, Bombus consobrinus queens, than it was in other populations. We also found that the degree of genetic similarity between populations more closely reflected interpopulation geographic proximity than interpopulation similarity in floral size. CONCLUSIONS: Although genetic similarity of populations was highly associated with geographic proximity, floral size varied independently of geographic proximity and was associated with local pollinator size. These results suggest that in L. album var. barbatum, large floral size evolved independently in populations on different mountains as a convergent adaptation to locally abundant large bumblebee species.

An ancient tripartite symbiosis of plants, ants and scale insects.

In the Asian tropics, a conspicuous radiation of Macaranga plants is inhabited by obligately associated Crematogaster ants tending Coccus (Coccidae) scale insects, forming a tripartite symbiosis. Recent phylogenetic studies have shown that the plants and the ants have been codiversifying over the past 16-20 million years (Myr). The prevalence of coccoids in ant-plant mutualisms suggest that they play an important role in the evolution of ant-plant symbioses. To determine whether the scale insects were involved in the evolutionary origin of the mutualism between Macaranga and Crematogaster, we constructed a cytochrome oxidase I (COI) gene phylogeny of the scale insects collected from myrmecophytic Macaranga and estimated their time of origin based on a COI molecular clock. The minimum age of the associated Coccus was estimated to be half that of the ants, at 7-9Myr, suggesting that they were latecomers in the evolutionary history of the symbiosis. Crematogaster mitochondrial DNA (mtDNA) lineages did not exhibit specificity towards Coccus mtDNA lineages, and the latter was not found to be specific towards Macaranga taxa, suggesting that patterns of associations in the scale insects are dictated by opportunity rather than by specialized adaptations to host plant traits.

Host-ant specificity of endangered large blue butterflies (Phengaris spp., Lepidoptera: Lycaenidae) in Japan.

Large blue butterflies, Phengaris (Maculinea), are an important focus of endangered-species conservation in Eurasia. Later-instar Phengaris caterpillars live in Myrmica ant nests and exploit the ant colony's resources, and they are specialized to specific host-ant species. For example, local extinction of P. arion in the U. K. is thought to have been due to the replacement of its host-ant species with a less-suitable congener, as a result of changes in habitat. In Japan, Myrmica kotokui hosts P. teleius and P. arionides caterpillars. We recently showed, however, that the morphological species M. kotokui actually comprises four genetic clades. Therefore, to determine to which group of ants the hosts of these two Japanese Phengaris species belong, we used mitochondrial COI-barcoding of M. kotokui specimens from colonies in the habitats of P. teleius and P. arionides to identify the ant clade actually parasitized by the caterpillars of each species. We found that these two butterfly species parasitize different ant clades within M. kotokui.

Demonstration of pollinator-mediated competition between two native Impatiens species, Impatiens noli-tangere and I. textori (Balsaminaceae).

Plant-plant interspecific competition via pollinators occurs when the flowering seasons of two or more plant species overlap and the pollinator fauna is shared. Negative sexual interactions between species (reproductive interference) through improper heterospecific pollen transfer have recently been reported between native and invasive species demonstrating pollination-driven competition. We focused on two native Impatiens species (I. noli-tangere and I. textori) found in Japan and examined whether pollinator-mediated plant competition occurs between them. We demonstrate that I. noli-tangere and I. textori share the same pollination niche (i.e., flowering season, pollinator fauna, and position of pollen on the pollinator's body). In addition, heterospecific pollen grains were deposited on most stigmas of both I. noli-tangere and I. textori flowers that were situated within 2 m of flowers of the other species resulting in depressed fruit set. Further, by hand-pollination experiments, we show that when as few as 10% of the pollen grains are heterospecific, fruit set is decreased to less than half in both species. These results show that intensive pollinator-mediated competition occurs between I. noli-tangere and I. textori. This study suggests that intensive pollinator-mediated competition occurs in the wild even when interacting species are both native and not invasive.

Congruence of microsatellite and mitochondrial DNA variation in acrobat ants (Crematogaster subgenus Decacrema, Formicidae: Myrmicinae) inhabiting Macaranga (Euphorbiaceae) myrmecophytes.

A previously reported mitochondrial DNA (mtDNA) phylogeny of Crematogaster (subgenus Decacrema) ants inhabiting Macaranga myrmecophytes indicated that the partners diversified synchronously and their specific association has been maintained for 20 million years. However, the mtDNA clades did not exactly match morphological species, probably owing to introgressive hybridization among younger species. In this study, we determined the congruence between nuclear simple sequence repeat (SSR, also called microsatellite) genotyping and mtDNA phylogeny to confirm the suitability of the mtDNA phylogeny for inferring the evolutionary history of Decacrema ants. Analyses of ant samples from Lambir Hills National park, northeastern Borneo, showed overall congruence between the SSR and mtDNA groupings, indicating that mtDNA markers are useful for delimiting species, at least at the local level. We also found overall high host-plant specificity of the SSR genotypes of Decacrema ants, consistent with the specificity based on the mtDNA phylogeny. Further, we detected cryptic genetic assemblages exhibiting high specificity toward particular plant species within a single mtDNA clade. This finding, which may be evidence for rapid ecological and genetic differentiation following a host shift, is a new insight into the previously suggested long-term codiversification of Decacrema ants and Macaranga plants.

Codiversification in an ant-plant mutualism: stem texture and the evolution of host use in Crematogaster (Formicidae: Myrmicinae) inhabitants of Macaranga (Euphorbiaceae).

We investigate the evolution of host association in a cryptic complex of mutualistic Crematogaster (Decacrema) ants that inhabits and defends Macaranga trees in Southeast Asia. Previous phylogenetic studies based on limited samplings of Decacrema present conflicting reconstructions of the evolutionary history of the association, inferring both cospeciation and the predominance of host shifts. We use cytochrome oxidase I (COI) to reconstruct phylogenetic relationships in a comprehensive sampling of the Decacrema inhabitants of Macaranga. Using a published Macaranga phylogeny, we test whether the ants and plants have cospeciated. The COI phylogeny reveals 10 well-supported lineages and an absence of cospeciation. Host shifts, however, have been constrained by stem traits that are themselves correlated with Macaranga phylogeny. Earlier lineages of Decacrema exclusively inhabit waxy stems, a basal state in the Pachystemon clade within Macaranga, whereas younger species of Pachystemon, characterized by nonwaxy stems, are inhabited only by younger lineages of Decacrema. Despite the absence of cospeciation, the correlated succession of stem texture in both phylogenies suggests that Decacrema and Pachystemon have diversified in association, or codiversified. Subsequent to the colonization of the Pachystemon clade, Decacrema expanded onto a second clade within Macaranga, inducing the development of myrmecophytism in the Pruinosae group. Confinement to the aseasonal wet climate zone of western Malesia suggests myrmecophytic Macaranga are no older than the wet forest community in Southeast Asia, estimated to be about 20 million years old (early Miocene). Our calculation of COI divergence rates from several published arthropod studies that relied on tenable calibrations indicates a generally conserved rate of approximately 1.5% per million years. Applying this rate to a rate-smoothed Bayesian chronogram of the ants, the Decacrema from Macaranga are inferred to be at least 12 million years old (mid-Miocene). However, using the extremes of rate variation in COI produces an age as recent as 6 million years. Our inferred timeline based on 1.5% per million years concurs with independent biogeographical events in the region reconstructed from palynological data, thus suggesting that the evolutionary histories of Decacrema and their Pachystemon hosts have been contemporaneous since the mid-Miocene. The evolution of myrmecophytism enabled Macaranga to radiate into enemy-free space, while the ants' diversification has been shaped by stem traits, host specialization, and geographic factors. We discuss the possibility that the ancient and exclusive association between Decacrema and Macaranga was facilitated by an impoverished diversity of myrmecophytes and phytoecious (obligately plant inhabiting) ants in the region.

Carbon and nitrogen contents of food bodies in three myrmecophytic species of Macaranga: implications for antiherbivore defense mechanisms.

In Macaranga myrmecophytes, differences in the production of the food bodies (FBs), on which symbiont ants feed, may relate to the intensity of antiherbivore defense by the ants. Interspecific comparisons among Macaranga species on such a mutualistic cost give important information on their strategies and evolution of antiherbivore defense. In this study, the carbon and nitrogen contents of FBs as well as the production rate of FBs were measured in three Macaranga species, M. winkleri, M. trachyphylla, and M. beccariana. There were significant differences in the production rates of FBs among species; the investment in FBs was greater in the Macaranga species in which ant defenses were more intensive. The carbon and nitrogen contents of FBs were significantly different among the three species, although they did not match the intensity of ant defense; the nitrogen content, especially, was greatest in the species of least intensive ant defense. It is suggested that Macaranga plants may have differentiated in the dependence on ant defense by controlling the total amount of nitrogen of FBs, not simply by nitrogen content.

Moth caterpillar solicits for homopteran honeydew.

A life-history in which an organism depends on ants is called myrmecophily. Among Lepidoptera (moths and butterflies), many species of lycaenid butterflies are known to show myrmecophily at the larval stage. Descriptions of myrmecophily among moth species, however, are very few and fragmentary. Here, we report the ant-associated behaviour of the tiny Japanese arctiid moth, Nudina artaxidia. Field observations revealed that the moth larvae associate with the jet black ant, Lasius (Dendrolasius) spp. The larvae, which we observed only near ant trails, showed an ability to follow the trails. Further, they solicit honeydew from ant-attended scale insects, without suffering attacks by the ants protecting the scale insects. These suggest that N. artaxidia is a myrmecophilous moth wholly dependent on ants and ant-attended homopterans. Considering the overwhelmingly plant-feeding habits of moth caterpillars, this discovery ranks in novelty with the discovery of the Hawaiian carnivorous moth larvae that stalk snails.

Changes in pollinator fauna affect altitudinal variation of floral size in a bumblebee-pollinated herb.

Geographic trait variations are often caused by locally different selection regimes. As a steep environmental cline along altitude strongly influences adaptive traits, mountain ecosystems are ideal for exploring adaptive differentiation over short distances. We investigated altitudinal floral size variation of Campanula punctata var. hondoensis in 12 populations in three mountain regions of central Japan to test whether the altitudinal floral size variation was correlated with the size of the local bumblebee pollinator and to assess whether floral size was selected for by pollinator size. We found apparent geographic variations in pollinator assemblages along altitude, which consequently produced a geographic change in pollinator size. Similarly, we found altitudinal changes in floral size, which proved to be correlated with the local pollinator size, but not with altitude itself. Furthermore, pollen removal from flower styles onto bees (plant's male fitness) was strongly influenced by the size match between flower style length and pollinator mouthpart length. These results strongly suggest that C. punctata floral size is under pollinator-mediated selection and that a geographic mosaic of locally adapted C. punctata exists at fine spatial scale.