Discovery of the primary aphid (Hemiptera: Aphidomorpha) and scale insect (Hemiptera: Coccomorpha) type specimens from the collection of Theodor Hartig (18051880).

Theodor Hartigs aphid and scale insect type specimens have been presumed lost or destroyed for the last 140 years. Here we document their discovery at the Bavarian State Collection of Zoology (Zoologische Staatssammlung Mnchen, ZSM), in Munich, Germany. These specimens include primary types for 24 aphid, three adelgid, and two armored scale insect species named by Hartig between 1834 and 1851, as well as other specimens of unknown importance.

Geographic isolation drives speciation in Nearctic aphids.

Across herbivorous insect clades, species richness and host-use diversity tend to positively covary. This could be because host-use divergence drives speciation, or because it raises the ecological limits on species richness. To evaluate these hypotheses, we performed phylogenetic path model analyses of the species diversity of Nearctic aphids. Here, we show that variation in the species richness of aphid clades is caused mainly by host-use divergence, whereas variation in speciation rates is caused more by divergence in non-host-related niche variables. Aphid speciation is affected by both the evolution of host and non-host-related niche components, but the former is largely caused by the latter. Thus, our analyses suggest that host-use divergence can both raise the ecological limits on species richness and drive speciation, although in the latter case, host-use divergence tends to be a step along the causal path leading from non-host-related niche evolution to speciation.

Molecular phylogeny and evolution of Calaphidinae (Hemiptera: Aphididae).

Calaphidinae is the second-largest subfamily in the family Aphididae. Despite their species diversity and some taxonomic controversy, no phylogenetic studies have been conducted on them thus far. Herein, we report the first molecular phylogeny of Calaphidinae and two related lineages, Phyllaphidinae and Saltusaphidinae, based on five genes (3418 bp) for 126 taxa. Maximum parsimony, maximum-likelihood and Bayesian inference phylogenetic analyses were performed on the multilocus dataset. Divergence time estimation, biogeographical reconstruction, ancestral host plant reconstruction and PhyloType analyses were performed to identify evolutionary trends in Calaphidinae. Our phylogenetic results lead to several conclusions: Phyllaphidinae is a sister group to Calaphidinae s.l.; Calaphidinae is paraphyletic with respect to the former "Saltusaphidinae"; the ingroup clade was subdivided into nine newly recognized lineages; and three subtribes of Calaphidinae (Monaphidina, Calaphdina and Panaphidina) and many genera were not recovered as monophyletic. A new classification is proposed with eight tribal divisions that reflect our phylogenetic results, including three new tribes (Pterocallidini trib.n., Pseudochromaphidini trib.n. and Shivaphidini trib.n.) and three new statuses (Saltusaphidini stat.n., Therioaphidini stat.n. and Myzocallidini stat.n.). The ancestral reconstruction results imply that the ingroup taxa's common ancestor originated in the Eastern Palaearctic and might have fed on Fagaceae in the Late Cretaceous. Later, multiple host shifts and an expanding geographical distribution led to the current species diversity of Calaphidinae. Our reconstructions suggest that species diversification cannot solely be explained by speciation via host shifts and that geographical isolation probably also played a key role. Our results provide new insight into the natural classification and history of the host plant associations and biogeography of Calaphidinae s.l.

Hemiptera of Canada.

The Canadian Hemiptera (Sternorrhyncha, Auchenorrhyncha, and Heteroptera) fauna is reviewed, which currently comprises 4011 species, including 405 non-native species. DNA barcodes available for Canadian specimens are represented by 3275 BINs. The analysis was based on the most recent checklist of Hemiptera in Canada (Maw et al. 2000) and subsequent collection records, literature records and compilation of DNA barcode data. It is estimated that almost 600 additional species remain to be discovered among Canadian Hemiptera.

Thysanoptera of Canada.

The known Canadian Thysanoptera fauna currently consists of 147 species, including 28 non-native species, and there are five additional species found only indoors. DNA barcoding data, presence of species in adjacent regions, and preliminary evidence of the presence of host-associated cryptic species suggest that there may be as many as 255 additional species awaiting discovery or description in Canada.

Dynamic Acquisition and Loss of Dual-Obligate Symbionts in the Plant-Sap-Feeding Adelgidae (Hemiptera: Sternorrhyncha: Aphidoidea).

Sap-sucking insects typically engage in obligate relationships with symbiotic bacteria that play nutritional roles in synthesizing nutrients unavailable or in scarce supply from the plant-sap diets of their hosts. Adelgids are sap-sucking insects with complex life cycles that involve alternation between conifer tree species. While all adelgid species feed on spruce during the sexual phase of their life cycle, each adelgid species belongs to a major lineage that feeds on a distinct genus of conifers as their alternate host. Previous work on adelgid symbionts had discovered pairs of symbionts within each host species, and unusual diversity across the insect family, but left several open questions regarding the status of bacterial associates. Here, we explored the consistency of symbionts within and across adelgid lineages, and sought evidence for facultative vs. obligate symbiont status. Representative species were surveyed for symbionts using 16S ribosomal DNA gene sequencing, confirming that different symbiont pairs were consistently present within each major adelgid lineage. Several approaches were used to establish whether symbionts exhibited characteristics of long-term, obligate mutualists. Patterns of symbiont presence across adelgid species and diversification with host insects suggested obligate relationships. Fluorescent in situ hybridization and electron microscopy localized symbionts to bacteriocyte cells within the bacteriome of each species (with one previously known exception), and detection of symbionts in eggs indicated their vertical transmission. Common characteristics of long-term obligate symbionts, such as nucleotide compositional bias and pleomorphic symbiont cell shape were also observed. Superimposing microbial symbionts on the adelgid phylogeny revealed a dynamic pattern of symbiont gains and losses over a relatively short period of time compared to other symbionts associated with sap-sucking insects, with each adelgid species possessing an older, "senior" symbiont and a younger "junior" symbiont. A hypothesis relating adelgid life cycles to relaxed constraints on symbionts is proposed, with the degradation of senior symbionts and repeated acquisition of more junior symbionts creating opportunities for repeated colonization of new alternate-conifer hosts by adelgids.

Cryptic diversity of the subfamily Calaphidinae (Hemiptera: Aphididae) revealed by comprehensive DNA barcoding.

Aphids are a species rich group comprising many important pests. However, species identification can be very difficult for aphids due to their morphological ambiguity. DNA barcoding has been widely adopted for rapid and reliable species identification as well as cryptic species detection. In this study, we investigated cryptic diversity in the subfamily Calaphidinae (Hemiptera: Aphididae) based on 899 sequences of cytochrome c oxidase I (COI) for 115 morphospecies (78 species collected in this study and sequences of 73 species downloaded from Genbank). Among these 115 morphospecies, DNA barcoding results of 90 (78.3%) species were identical to results of morphological identification. However, 25 (21.7%) morphospecies showed discrepancies between DNA barcoding and traditional taxonomy. Among these 25 discordances, a total of 15 cryptic species were identified from 12 morphospecies. We also found three morphologically distinct species pairs that sharing DNA barcoding. Based on molecular operational taxonomic unit (MOTU) estimation, we discussed on species delimitation threshold value for these taxa. Our findings confirm that Calaphidinae has high cryptic diversity even though aphids are relatively well-studied.

Ancient and modern colonization of North America by hemlock woolly adelgid, Adelges tsugae (Hemiptera: Adelgidae), an invasive insect from East Asia.

Hemlock woolly adelgid, Adelges tsugae, is an invasive pest of hemlock trees (Tsuga) in eastern North America. We used 14 microsatellites and mitochondrial COI sequences to assess its worldwide genetic structure and reconstruct its colonization history. The resulting information about its life cycle, biogeography and host specialization could help predict invasion by insect herbivores. We identified eight endemic lineages of hemlock adelgids in central China, western China, Ulleung Island (South Korea), western North America, and two each in Taiwan and Japan, with the Japanese lineages specializing on different Tsuga species. Adelgid life cycles varied at local and continental scales with different sexual, obligately asexual and facultatively asexual lineages. Adelgids in western North America exhibited very high microsatellite heterozygosity, which suggests ancient asexuality. The earliest lineages diverged in Asia during Pleistocene glacial periods, as estimated using approximate Bayesian computation. Colonization of western North America was estimated to have occurred prior to the last glacial period by adelgids directly ancestral to those in southern Japan, perhaps carried by birds. The modern invasion from southern Japan to eastern North America caused an extreme genetic bottleneck with just two closely related clones detected throughout the introduced range. Both colonization events to North America involved host shifts to unrelated hemlock species. These results suggest that genetic diversity, host specialization and host phylogeny are not predictive of adelgid invasion. Monitoring non-native sentinel host trees and focusing on invasion pathways might be more effective methods of preventing invasion than making predictions using species traits or evolutionary history.

Aphid Transmission of the Ontario Isolate of Plum Pox Virus.

Utilization of timed virus acquisition access probes in studies of plum pox virus (PPV) transmission by aphids demonstrated that endemic species transmitted the virus readily from plum, Prunus domestica (L.) Batsch; peach, P. persica (L.); or dwarf flowering almond, P. glandulosa Thunberg., to peach seedlings. The green peach aphid, Myzus persicae (Sulzer), was shown to be the most efficient vector. Acquisition of virus by green peach aphids from infected peach leaves resulted in 18-28% infected peach seedlings, while aphids previously fed on infected leaves of plum transferred virus to 36% of peach seedlings. Although the spirea aphid, Aphis spiraecola (Patch), was a less efficient vector than M. persicae it is perhaps more important for the spread of PPV due to its greater abundance and occurrence earlier in the season when peach trees are thought to be more susceptible to infection. Virus transmission rates varied depending on the virus source and healthy test plant species. In contrast to many previous studies, aphid inoculation of the experimental host Nicotiana benthamiana Domin occurred at a low rate, never exceeding 4%. Acquisition of PPV by M. persicae from infected peach fruit was greatly reduced compared with acquisition from leaves. The results of this research indicate that the Ontario isolate of PPV-D is readily transmissible by aphids to peach and natural spread of the virus needs to be considered in future management or eradication programs.

The hemiptera (insecta) of Canada: constructing a reference library of DNA barcodes.

DNA barcode reference libraries linked to voucher specimens create new opportunities for high-throughput identification and taxonomic re-evaluations. This study provides a DNA barcode library for about 45% of the recognized species of Canadian Hemiptera, and the publically available R workflow used for its generation. The current library is based on the analysis of 20,851 specimens including 1849 species belonging to 628 genera and 64 families. These individuals were assigned to 1867 Barcode Index Numbers (BINs), sequence clusters that often coincide with species recognized through prior taxonomy. Museum collections were a key source for identified specimens, but we also employed high-throughput collection methods that generated large numbers of unidentified specimens. Many of these specimens represented novel BINs that were subsequently identified by taxonomists, adding barcode coverage for additional species. Our analyses based on both approaches includes 94 species not listed in the most recent Canadian checklist, representing a potential 3% increase in the fauna. We discuss the development of our workflow in the context of prior DNA barcode library construction projects, emphasizing the importance of delineating a set of reference specimens to aid investigations in cases of nomenclatural and DNA barcode discordance. The identification for each specimen in the reference set can be annotated on the Barcode of Life Data System (BOLD), allowing experts to highlight questionable identifications; annotations can be added by any registered user of BOLD, and instructions for this are provided.

DNA barcodes for Nearctic Auchenorrhyncha (Insecta: Hemiptera).

BACKGROUND: Many studies have shown the suitability of sequence variation in the 5' region of the mitochondrial cytochrome c oxidase I (COI) gene as a DNA barcode for the identification of species in a wide range of animal groups. We examined 471 species in 147 genera of Hemiptera: Auchenorrhyncha drawn from specimens in the Canadian National Collection of Insects to assess the effectiveness of DNA barcoding in this group. METHODOLOGY/PRINCIPAL FINDINGS: Analysis of the COI gene revealed less than 2% intra-specific divergence in 93% of the taxa examined, while minimum interspecific distances exceeded 2% in 70% of congeneric species pairs. Although most species are characterized by a distinct sequence cluster, sequences for members of many groups of closely related species either shared sequences or showed close similarity, with 25% of species separated from their nearest neighbor by less than 1%. CONCLUSIONS/SIGNIFICANCE: This study, although preliminary, provides DNA barcodes for about 8% of the species of this hemipteran suborder found in North America north of Mexico. Barcodes can enable the identification of many species of Auchenorrhyncha, but members of some species groups cannot be discriminated. Future use of DNA barcodes in regulatory, pest management, and environmental applications will be possible as the barcode library for Auchenorrhyncha expands to include more species and broader geographic coverage.

Is ecological speciation a major trend in aphids? Insights from a molecular phylogeny of the conifer-feeding genus Cinara.

INTRODUCTION: In the past decade ecological speciation has been recognized as having an important role in the diversification of plant-feeding insects. Aphids are host-specialised phytophagous insects that mate on their host plants and, as such, they are prone to experience reproductive isolation linked with host plant association that could ultimately lead to species formation. The generality of such a scenario remains to be tested through macroevolutionary studies. To explore the prevalence of host-driven speciation in the diversification of the aphid genus Cinara and to investigate alternative modes of speciation, we reconstructed a phylogeny of this genus based on mitochondrial, nuclear and Buchnera aphidicola DNA sequence fragments and applied a DNA-based method of species delimitation. Using a recent software (PhyloType), we explored evolutionary transitions in host-plant genera, feeding sites and geographic distributions in the diversification of Cinara and investigated how transitions in these characters have accompanied speciation events. RESULTS: The diversification of Cinara has been constrained by host fidelity to conifer genera sometimes followed by sequential colonization onto different host species and by feeding-site specialisation. Nevertheless, our analyses suggest that, at the most, only half of the speciation events were accompanied by ecological niche shifts. The contribution of geographical isolation in the speciation process is clearly apparent in the occurrence of species from two continents in the same clades in relatively terminal positions in our phylogeny. Furthermore, in agreement with predictions from scenarios in which geographic isolation accounts for speciation events, geographic overlap between species increased significantly with time elapsed since their separation. CONCLUSIONS: The history of Cinara offers a different perspective on the mode of speciation of aphids than that provided by classic models such as the pea aphid. In this genus of aphids, the role of climate and landscape history has probably been as important as host-plant specialisation in having shaped present-day diversity.

Reconstructing the phylogeny of aphids (Hemiptera: Aphididae) using DNA of the obligate symbiont Buchnera aphidicola.

Reliable phylogenetic reconstruction, as a framework for evolutionary inference, may be difficult to achieve in some groups of organisms. Particularly for lineages that experienced rapid diversification, lack of sufficient information may lead to inconsistent and unstable results and a low degree of resolution. Coincidentally, such rapidly diversifying taxa are often among the biologically most interesting groups. Aphids provide such an example. Due to rapid adaptive diversification, they feature variability in many interesting biological traits, but consequently they are also a challenging group in which to resolve phylogeny. Particularly within the family Aphididae, many interesting evolutionary questions remain unanswered due to phylogenetic uncertainties. In this study, we show that molecular data derived from the symbiotic bacteria of the genus Buchnera can provide a more powerful tool than the aphid-derived sequences. We analyze 255 Buchnera gene sequences from 70 host aphid species and compare the resulting trees to the phylogenies previously retrieved from aphid sequences, only. We find that the host and symbiont data do not conflict for any major phylogenetic conclusions. Also, we demonstrate that the symbiont-derived phylogenies support some previously questionable relationships and provide new insights into aphid phylogeny and evolution.

Barcoding aphids (Hemiptera: Aphididae) of the Korean Peninsula: updating the global data set.

DNA barcode (mitochondrial COI) sequences are provided for species identification of aphids from the Korean Peninsula. Most (98%) of the 154 species had distinct COI sequences (average 0.05% intraspecific pairwise divergence) relative to the degree of sequence divergence among species (average value 5.84%). For species in common with other regions, barcodes for Korean samples fell near or within known levels of variation. Based on these results, we conclude that DNA barcodes can provide an effective tool for identifying aphid species in such applications as pest management, monitoring and plant quarantine.

Relationship of insecticide tolerance to esterase enzyme activity in Aphis pomi and Aphis spiraecola (Hemiptera: Aphididae).

Green apple aphid, Aphis pomi De Geer, and Aphis spiraecola Patch (both Hemiptera: Aphididae), are sympatric aphid species that are pests of apples (Malus spp.) and other crops. A. spiraecola has been shown to be significantly more tolerant to several insecticides compared with A. pomi. To establish the mechanisms contributing to this difference in insecticide response, clones of both species were collected from British Columbia, Canada, and Washington state. Dose-response bioassays were conducted to determine relative tolerances to the insecticides pirimicarb, dimethoate, and imidacloprid; these results have been reported previously. Samples of adult aphids from each clone were assayed for the activity of esterase enzymes often involved in the detoxification of insecticides. A. spiraecola had higher esterase activity compared with A. pomi; this was apparent for two model substrates, alpha-naphthyl acetate (alpha-NA) and alpha-naphthyl butyrate (alpha-NB). Aphid clones of both species collected from Washington had higher esterase activity than clones collected from British Columbia. Clones from both species and locations hydrolyzed alpha-NA to a greater extent than alpha-NB. Esterase activity measured with both substrates was significantly positively correlated with the relative response to pirimicarb and dimethoate; a significant positive correlation also was found for hydrolysis of alpha-NB and imidacloprid. The apparent involvement of esterases in the differential response of A. pomi and A. spiraecola to insecticides indicated that the choice of control chemicals for A. spiraecola should not involve chemistries that are metabolized predominantly by esterases.

Evolution of host specialization in the Adelgidae (Insecta: Hemiptera) inferred from molecular phylogenetics.

The Adelgidae form a small group of insects in the Aphidoidea. They are cyclically parthenogenetic with host alternating, multiple-generation complex life cycles and are restricted to certain host genera in the Pinaceae. Species that host alternate always have Picea as the primary host where sexual reproduction and gall formation occur, and another genus in the Pinaceae as the secondary host where a series of parthenogenetic generations are produced. Other species that do not host alternate complete their entire life cycle on one host and only reproduce parthenogenetically. We studied relationships within Adelgidae using DNA sequences from the mitochondrial COI, COII, and cytb genes, and the nuclear EF1alpha gene. Analysis of the combined data resulted in a well-resolved phylogeny in which the major adelgid clades correspond neatly to their association with secondary host genera. Specialization on each secondary host genus occurred only once and was followed by diversification on the host genus. Molecular dating of divergence times in the Adelgidae suggest that diversification among host genera occurred in the Late Cretaceous and Early Tertiary when the Pinaceae genera were diverging. It is not clear, however, whether the Adelgidae and Pinaceae co-diversified because the relationships among the Pinaceae genera are not fully resolved. We discuss implications for adelgid taxonomy, life cycle evolution, and evolution of the interaction between adelgids and their host plants.

Biology and evolution of adelgidae.

The Adelgidae form a small clade of insects within the Aphidoidea (Hemiptera) that includes some of the most destructive introduced pest species threatening North American forest ecosystems. Despite their importance, little is known about their evolutionary history and their taxonomy remains unresolved. Adelgids are cyclically parthenogenetic and exhibit multigeneration complex life cycles. They can be holocyclic, with a sexual generation and host alternation, or anholocyclic, entirely asexual and without host alternation. We discuss adelgid behavior and ecology, emphasizing plant-insect interactions, and we explore ways that the biogeographic history of their host plants may have affected adelgid phylogeny and evolution of adelgid life cycles. Finally, we highlight several areas in which additional research into speciation, population genetics, multitrophic interactions, and life-history evolution would improve our understanding of adelgid biology and evolution.

Susceptibilities of apple aphid and spirea aphid collected from apple in the Pacific Northwest to selected insecticides.

Laboratory bioassays using leaf disks of apple dipped in test solutions of insecticides demonstrated that the apple aphid, Aphis pomi De Geer, and the spirea aphid, Aphis spiraecola Patch, differed significantly in susceptibility to a number of insecticides registered for control of aphids on apple (Malus spp.). Compared with A. pomi, A. spiraecola was approximately four- and three-fold less susceptible to pirimicarb and lambda-cyhalothrin, respectively, whereas there was little difference in response to dimethoate. Pymetrozine is thought to act on aphids primarily as a feeding inhibitor. Exposure of aphids to this material generated data that fit the probit model for only half the tested clones. However, the LC50 value for one clone of A. spiraecola was nearly 1,000 times higher than the value for one clone of A. pomi. Although the results from these trials did not indicate that either species had developed significant levels of resistance to the test materials, differences in LC50 levels of > 10-fold suggest insecticide tolerances and the possibility of control failures in the future. The demonstrated differences in susceptibility to insecticides between these two morphologically similar species also should be considered during the evaluation of new products for use on apple.

Baseline susceptibilities to imidacloprid for green apple aphid and spirea aphid (Homoptera: Aphididae) collected from apple in the Pacific northwest.

Susceptibilities to the neonicotinyl insecticide imidacloprid were determined for clones of apple aphid, Aphis pomi De Geer, and spirea aphid, Aphis spiraecola Patch, collected from conventional and organic apple orchards and from crab apple and wild apple in Washington state and British Columbia over a period of 6 yr. For aphids collected during 1996--1998, adults were dipped in test solutions by using the Food and Agriculture Organization protocol, and third instars and adults were reared on treated apple leaf disks. During the final 3 yr of study, bioassays involved only third instars on treated leaf material. Tests showed that A. spiraecola was significantly more tolerant to imidacloprid compared with A. pomi. Depending on the bioassay method and aphid developmental stage, average LC50 values for A. spiraecola were 4.4 -5.7 times higher than those for A. pomi established under the same test conditions. Clones of both species from Washington were marginally more tolerant to imidacloprid than clones from British Columbia, but the differences were generally not significant. Average measures of susceptibility for clones from organic orchards or unsprayed trees also did not differ from those for clones from conventional orchards, and there was no evidence for increasing LC50 values over the 6 yr of study. Differences in susceptibility to insecticides between these two anatomically similar species should be considered during the testing of new products for use on apple.