Fighting and aggressive sound determines larger male to win male-male competition in a bark beetle.

Intrasexual selection occurs in male-male competition over access to females and usually results in the larger male winning. While much research has documented that size matters, little is known about how the larger male wins. Dendroctonus valens is an aggregating monogamous bark beetle in which males have large variation in body size and display intense competition over females. Behavioral observation showed two males fight each other within the gallery by pushing/shoving and stridulated more when two males encountered each other. Experiments using two different-sized males synchronously competing showed that larger males won 95% of contests. Reciprocal displacement experiments using muted and intact males of different or equal size were used to simulate male-male competition. Larger males displaced the smaller resident male in 90% of contests, while smaller males prevailed over larger residents in 6.7% of contests. With both males silenced, larger males displaced smaller males in 80% of contests, while smaller males prevailed in 8% of contests. Further experiments using equal-sized males showed aggressive sound-emitting males displaced muted males in 67% of contests, yet intact males displaced other intact males in only 37.5% of contests. Sound analysis showed sound pressure level is an honest signal of body size and males chose soft sounds over loud aggressive sounds in assays. Therefore, D. valens males have evolved dual behaviors, fighting and aggressive sounds associated with body size, to assess rivals to compete for a partner, gaining insights in male-male competition for this species and for other animals.

New Approaches in Urban Forestry to Minimize Invasive Species Impacts: The Case of Xiongan New Area in China.

China is implementing an extensive urban forestry plan in Xiongan New Area (XNA), a new city in Hebei province. The city has been designated to serve Beijing's noncapital functions and promote the integration of the broader Beijing-Tianjin-Hebei city-region. As part of a green initiative to minimize environmental impacts and its carbon footprint, a massive urban forestry system has been planned on an unprecedented scale, expected to cover over 600 km2 by 2030. Using science to inform policy, one major goal is to simultaneously minimize impacts of invasive species, while making urban forests more resilient to potential invasive species threats. In this review, we introduce these urban forestry plans such as basic concepts and principles for afforestation, tree species to be planted, delineation of existing pests already established, and expected forest invasive species of concern threatening the new area. Finally, we introduce a framework for invasive pest management strategies in XNA based on a "big data" approach and decision system to minimize impacts of invasive species. This new approach to urban forestry has the potential to become an exemplary global model for urban forestry planning, one that integrates research activities focused on forest health surveys and monitoring with sustainable forestry management. Finally, we provide an overview of the forest health policy required for the design of an unprecedentedly large new urban forest from initial planning to full implementation of an integrated forest management program.

Physical contact, volatiles, and acoustic signals contribute to monogamy in an invasive aggregating bark beetle.

The behavioral strategies and mechanisms by which some insects maintain monogamous mating systems are not well understood. We investigated the mating system of the bark beetle Dendroctonus valens, and identified several contributing mechanisms. Field and laboratory observations suggest the adults commonly form permanent bonds during host colonization. Moreover, it showed mated females that remained paired with males produced more offspring than mated females that were alone in galleries. In bioassays, a second female commonly entered a gallery constructed by a prior female. Videos show she commonly reached the location of the first female, but they did not engage in actual fighting. Rather, the second female typically departs to form her own gallery. Acoustic signaling likewise does not appear to influence female-female encounters, based on controlled muting experiments. Instead, the intruder appears to perceive the resident's presence by physical contact. Both acoustic signals and volatiles released by females during gallery constructing were shown to attract males. After a male joined a female in a gallery, the male-produced aggressive sounds, which were shown by playback to deter other males from entering the gallery. Unlike female-female interactions, resident males use their head and rear to push intruders out of galleries. Additionally, volatiles released by males during feeding repelled intruding males, discouraging them from entering the gallery. Males also construct plugs that block the entrance, which may prevent subsequent males and predators from entering the gallery. Thus, D. valens has evolved multifaceted mechanisms contributing to single pairings that confer benefits to both sexes.

Immune tolerance of vector beetle to its partner plant parasitic nematode modulated by its insect parasitic nematode.

Immune response of insect vectors to transmitted pathogens or insect hosts against parasites are well studied, whereas the mechanism of tripartite interactions remains elusive. In this study, we investigated the immune interactions of the vector beetle Monochamus alternatus ( Ma) to the devastating plant parasitic nematode Bursaphelenchus xylophilus ( Bx) and the insect parasitic nematode Howardula phyllotretae ( Hp). We report the unique immune mechanism by which the vector beetle tolerates many devastating Bx in its trachea, yet that immune tolerance is compromised by the parasitic nematode Hp. Contact with either nematode species triggers epithelial reactive oxygen species (ROS) production in Ma. Only the entry of Bx, not Hp, infection, induces increased expression of antioxidative genes, through which the ROS levels are balanced in the trachea of beetles. Furthermore, we found that up-regulation of antioxidative genes was induced by the interaction of Toll receptors. In contrast, beetles infected by Hp retain high levels of oxidative stress and melanization in trachea, and as a result, decrease Bx loading. This study highlights the role of Toll receptors in mediating the activation of antioxidative genes in immune tolerance to plant parasitic nematodes, and suggests the use of insect parasites as a biologic control.-Zhou, J., Zhao, L.-L., Yu, H.-Y., Wang, Y.-H., Zhang, W., Hu, S.-N., Zou, Z., Sun, J.-H. Immune tolerance of vector beetle to its partner plant parasitic nematode modulated by its insect parasitic nematode.

Comparative analysis of the Monochamus alternatus immune system.

The pine sawyer beetle, Monochamus alternatus, is regarded as a notorious forest pest in Asia, vectoring an invasive pathogenic nematode, Bursaphelenchus xylophilus, which is known to cause pine wilt disease. However, little sequence information is available for this vector beetle. This hampered the research on its immune system. Based on the transcriptome of M. alternatus, we have identified and characterized 194 immunity-related genes in M. alternatus, and compared them with homologues molecules from other species known to exhibit immune responses against invading microbes. The lower number of putative immunity-related genes in M. alternatus were attributed to fewer C-type lectin, serine protease (SP) and anti-microbial peptide (AMP) genes. Phylogenetic analysis revealed that M. alternatus had a unique recognition gene, galectin3, orthologues of which were not identified in Tribolium castaneum, Drosophila melanogastor, Anopheles gambiae and Apis mellifera. This suggested a lineage-specific gene evolution for coleopteran insects. Our study provides the comprehensive sequence resources of the immunity-related genes of M. alternatus, presenting valuable information for better understanding of the molecular mechanism of innate immunity processes in M. alternatus against B. xylophilus.

Invasive bark beetle-associated microbes degrade a host defensive monoterpene.

Conifers respond to herbivore attack with defensive chemicals, which are toxic to both insects and their associated microorganisms. Microorganisms associated with insects have been widely reported to metabolize toxic chemicals, which may help both microorganisms and host insects overcome host conifer defense. Dendroctonus valens LeConte, an introduced exotic pest from North America to China, has killed millions of healthy pines. Alpha-pinene is the most abundant defensive monoterpene in Chinese Pinus tabuliformis. Although microorganisms associated with D. valens have already been investigated, little is known about their bioactivities when encountering host defensive monoterpenes. In this study, we evaluated the influences of different concentrations of α-pinene to D. valens and the three most frequently isolated yeasts and bacteria of D. valens, and further assayed microorganisms' capabilities to degrade α-pinene. Results showed that the gallery lengths and body weight changes of bark beetles were significantly affected by 6 mg/mL and 12 mg/mL of α-pinene applied in media compared to controls. The tolerance of experimental microorganisms to α-pinene varied depending on the microbial species. Two out of three yeast strains and all three bacterial strains degraded 20%-50% of α-pinene compared to controls in 24 h in vitro. The microorganisms capable of α-pinene degradation in vitro and their tolerance to high levels of α-pinene suggested that D. valens-associated microorganisms may help both microorganisms and the bark beetle overcome host α-pinene defense.

Reproductive Isolation of Ips nitidus and I. shangrila in Mountain Forests of Western China: Responses to Chiral and Achiral Candidate Pheromone Components.

Eastern Palearctic conifers are subject to frequent bark beetle outbreaks. However, neither the species responsible nor the semiochemicals guiding these attacks are well understood. Two high-mountain Ips species on Qinghai spruce, Picea crassifolia, I. shangrila and I. nitidus, are typical in this regard. Six synthetic candidate pheromone components that we earlier identified from hindguts of unmated males of these two Ips species were tested for field activity in Qinghai province, P. R. China. For I. nitidus, racemic ipsdienol ((±)-Id) could replace the naturally-produced blend of enantiomers containing 74% (-)-(S)-Id (74:26 S:R), in attractive ternary or binary blends. In contrast, sympatric I. shangrila were attracted mainly to blends including Id of opposite chirality, 97%-(+)-(R)-Id. Of the verbenols, (-)-trans-verbenol was inactive for I. nitidus or inhibitory for I. shangrila, but (-)-cis-verbenol (cV) was a key component of the pheromone in both species. Two fully factorial experiments demonstrated that (±)-Id, cV, and 2-methyl-3-buten-2-ol (MB) are components of the aggregation pheromone of I. nitidus, whereas only (+)-Id and cV are essential components of the aggregation pheromone of I. shangrila. While MB is not necessary for attraction of I. shangrila, it is an active antagonist and likely functions in species isolation. A review of the pheromone production and responses in Palearctic Ips and Pseudoips showed that cV is more common than methylbutenols, and both elicit qualitatively variable responses. Ipsdienol is the most common component with variable chirality, and is a necessary, but often not sufficient, factor for determining pheromone specificity.

Planting sentinel European trees in eastern Asia as a novel method to identify potential insect pest invaders.

Quarantine measures to prevent insect invasions tend to focus on well-known pests but a large proportion of the recent invaders were not known to cause significant damage in their native range, or were not even known to science before their introduction. A novel method is proposed to detect new potential pests of woody plants in their region of origin before they are introduced to a new continent. Since Asia is currently considered to be the main supplier of insect invaders to Europe, sentinel trees were planted in China during 2007-2011 as an early warning tool to identify the potential for additional Asian insect species to colonize European trees. Seedlings (1-1.5 m tall) of five broadleaved (Quercus petraea, Q. suber, Q. ilex, Fagus sylvatica, and Carpinus betulus) and two conifer species (Abies alba and Cupressus sempervirens) were planted in blocks of 100 seedlings at two widely separated sites (one in a nursery near Beijing and the other in a forest environment near Fuyang in eastern China), and then regularly surveyed for colonization by insects. A total of 104 insect species, mostly defoliators, were observed on these new hosts, and at least six species were capable of larval development. Although a number of the insects observed were probably incidental feeders, 38 species had more than five colonization events, mostly infesting Q. petraea, and could be considered as being capable of switching to European trees if introduced to Europe. Three years was shown to be an appropriate duration for the experiment, since the rate of colonization then tended to plateau. A majority of the identified species appeared to have switched from agricultural crops and fruit trees rather than from forest trees. Although these results are promising, the method is not appropriate for xylophagous pests and other groups developing on larger trees. Apart from the logistical problems, the identification to species level of the specimens collected was a major difficulty. This situation could be improved by the development of molecular databases.

Sentinel trees as a tool to forecast invasions of alien plant pathogens.

Recent disease outbreaks caused by alien invasive pathogens into European forests posed a serious threat to forest sustainability with relevant environmental and economic effects. Many of the alien tree pathogens recently introduced into Europe were not previously included on any quarantine lists, thus they were not subject to phytosanitary inspections. The identification and description of alien fungi potentially pathogenic to native European flora before their introduction in Europe, is a paramount need in order to limit the risk of invasion and the impact to forest ecosystems. To determine the potential invasive fungi, a sentinel trees plot was established in Fuyang, China, using healthy seedlings of European tree species including Quercus petreae, Q. suber, and Q. ilex. The fungal assemblage associated with symptomatic specimens was studied using the tag-encoded 454 pyrosequencing of the nuclear ribosomal internal transcribed spacer-1 (ITS 1). Taxa with probable Asiatic origin were identified and included plant pathogenic genera. These results indicate that sentinel plants may be a strategic tool to improve the prevention of bioinvasions.

Yeast diversity associated with invasive Dendroctonus valens killing Pinus tabuliformis in China using culturing and molecular methods.

Bark beetle-associated yeasts are much less studied than filamentous fungi, yet they are also considered to play important roles in beetle nutrition, detoxification, and chemical communication. The red turpentine beetle, Dendroctonus valens, an invasive bark beetle introduced from North America, became one of the most destructive pests in China, having killed more than 10 million Pinus tabuliformis as well as other pine species. No investigation of yeasts associated with this bark beetle in its invaded ranges has been conducted so far. The aim of this study was to assess the diversity of yeast communities in different microhabitats and during different developmental stages of Den. valens in China using culturing and denaturing gradient gel electrophoresis (DGGE) approaches and to compare the yeast flora between China and the USA. The yeast identity was confirmed by sequencing the D1/D2 domain of LSU ribosomal DNA (rDNA). In total, 21 species (13 ascomycetes and eight basidiomycetes) were detected by culturing method, and 12 species (11 ascomycetes and one basidiomycetes) were detected by molecular methods from China. The most frequent five species in China were Candida piceae (Ogataea clade), Cyberlindnera americana, Candida oregonensis (Metschnikowia clade), Candida nitratophila (Ogataea clade) and an undescribed Saccharomycopsis sp., detected by both methods. Seven species were exclusively detected by DGGE. Ca. oregonensis (Metschnikowia clade) was the most frequently detected species by DGGE method. Eight species (all were ascomycetes) from the USA were isolated; seven of those were also found in China. We found significant differences in yeast total abundance as well as community composition between different developmental stages and significant differences between the surface and the gut. The frass yeast community was more similar to that of Den. valens surface or larvae than to the community of the gut or adults. Possible functions of the yeast associates are discussed.

Large shift in symbiont assemblage in the invasive red turpentine beetle.

Changes in symbiont assemblages can affect the success and impact of invasive species, and may provide knowledge regarding the invasion histories of their vectors. Bark beetle symbioses are ideal systems to study changes in symbiont assemblages resulting from invasions. The red turpentine beetle (Dendroctonus valens) is a bark beetle species that recently invaded China from its native range in North America. It is associated with ophiostomatalean fungi in both locations, although the fungi have previously been well-surveyed only in China. We surveyed the ophiostomatalean fungi associated with D. valens in eastern and western North America, and identified the fungal species using multi-gene phylogenies. From the 307 collected isolates (147 in eastern North America and 160 in western North America), we identified 20 species: 11 in eastern North America and 13 in western North America. Four species were shared between eastern North America and western North America, one species (Ophiostoma floccosum) was shared between western North America and China, and three species (Grosmannia koreana, Leptographium procerum, and Ophiostoma abietinum) were shared between eastern North America and China. Ophiostoma floccosum and O. abietinum have worldwide distributions, and were rarely isolated from D. valens. However, G. koreana and L. procerum are primarily limited to Asia and North America respectively. Leptographium procerum, which is thought to be native to North America, represented >45% of the symbionts of D. valens in eastern North America and China, suggesting D. valens may have been introduced to China from eastern North America. These results are surprising, as previous population genetics studies on D. valens based on the cytochrome oxidase I gene have suggested that the insect was introduced into China from western North America.

Do novel genotypes drive the success of an invasive bark beetle-fungus complex? Implications for potential reinvasion.

Novel genotypes often arise during biological invasions, but their role in invasion success has rarely been elucidated. Here we examined the population genetics and behavior of the fungus, Leptographium procerum, vectored by a highly invasive bark beetle, Dendroctonus valens, to determine whether genetic changes in the fungus contributed to the invasive success of the beetle-fungal complex in China. The fungus was introduced by the beetle from the United States to China, where we identified several novel genotypes using microsatellite markers. These novel genotypes were more pathogenic to Chinese host seedlings than were other genotypes and they also induced the release of higher amounts of 3-carene, the primary host attractant for the beetle vector, from inoculated seedlings. This evidence suggests a possible mechanism, based on the evolution of a novel genotype during the two or three decades since its introduction, for the success of the beetle-fungal complex in its introduced region.

Immunocompetence of the red turpentine beetle, Dendroctonus valens LeConte (Coleoptera: Curculionidae, Scolytinae): variation between developmental stages and sexes in populations in China.

Immune defense imposes fitness costs as well as benefits, so organisms should optimize, not maximize, their immune function through their life cycle. We investigated this issue in the red turpentine beetle, Dendroctonus valens LeConte (Coleoptera: Curculionidae, Scolytinae), which is a pine-killing invasive beetle in China, though it is usually considered as a secondary pest in its native range of North America. We hypothesized that pathogen pressure may affect these beetles differently throughout their life history. We measured the insect's immunocompetence throughout life, determining encapsulation ability and phenoloxidase activity in larval stages, pupae and adults. Pupae had the highest encapsulation ability, but encapsulation was not different between final instar larvae and adults. Phenoloxidase (PO) activity was highest in final instar larvae and pupae, followed by the second instar larvae and adults. Total phenoloxidase activity increased significantly from the second instar larval stage to pupae, and then decreased in adults. Although the second instar larvae had the lowest phenoloxidase activity, more than 90% of total PO existed in the hemolymph in the form of the active enzyme, as compared with pupae, in which over 60% of PO occurred as a proenzyme. Both active PO and total PO were much higher in females than in males, though no significant differences were detected between the encapsulation ability of male and female adults. This result suggests the existence of a sexual dimorphism of immunocompetence in D. valens adults. Variations in immunocompetence across developmental stages suggest that D. valens adopts diverse investment strategies in immunocompetence during different stages. Potential reasons for variation in immunocompetence among developmental stages and between the sexes of D. valens are discussed.

Complex interactions among host pines and fungi vectored by an invasive bark beetle.

*Recent studies have investigated the relationships between pairs or groups of exotic species to illustrate invasive mechanisms, but most have focused on interactions at a single trophic level. *Here, we conducted pathogenicity tests, analyses of host volatiles and fungal growth tests to elucidate an intricate network of interactions between the host tree, the invasive red turpentine beetle and its fungal associates. *Seedlings inoculated with two strains of Leptographium procerum isolated from Dendroctonus valens in China had significantly longer lesions and higher mortality rates than seedlings inoculated with other fungal isolates. These two strains of L. procerum were significantly more tolerant of 3-carene than all other fungi isolated there, and the infection of Chinese pine (Pinus tabuliformis) seedlings by these two strains enhanced the production and release of 3-carene, the main attractant for D. valens, by the seedlings. *Our results raise the possibility that interactions among the fungal associates of D. valens and their pine hosts in China may confer advantages to these strains of L. procerum and, by extension, to the beetles themselves. These interactions may therefore enhance invasion by the beetle-fungal complex.

Aggregation pheromone of the Qinghai spruce bark beetle, Ips nitidus eggers.

Volatiles from hindgut extracts of males of the Qinghai spruce bark beetle, Ips nitidus, from different attack phases (phase 1: unpaired males and phases 2-4: males joined with one to three females) and hindgut extracts of mated females were analyzed by gas chromatography-mass spectrometry (GC-MS)/flame ionization detection (FID) with both polar and enantioselective columns. The GC-MS/FID analyses demonstrated that unpaired males from attack phase 1 (nuptial chamber constructed) produced 2-methyl-3-buten-2-ol, approx. 74%-(-)-ipsdienol, and (-)-cis-verbenol as major hindgut components, and (-)-trans-verbenol, (-)-ipsenol, (-)-verbenone, myrtenol, and 2-phenylethanol as minor or trace components. The quantities of 2-methyl-3-buten-2-ol and especially ipsdienol decreased after mating during phases 2-4, whereas the quantities of (-)-cis- and (-)-trans-verbenol did not change. In contrast, the quantity of (-)-ipsenol seemed to increase as mating activity progressed. After mating with three females (harem size = 3; phase 4), only trace to small amounts of male-specific compounds were detected from I. nitidus male hindguts. Chemical analysis of the hindgut extracts of mated females showed only trace amounts of semiochemicals. A field-trapping bioassay in Qinghai, China showed that the four-component "full blend" containing the three major components, 2-methyl-3-buten-2-ol, (+/-)-ipsdienol, and (-)-cis-verbenol, plus a minor component, (-)-trans-verbenol, caught significantly more I. nitidus (male/female = 1:2.2) than did the unbaited control and two binary blends. The replacement of (+/-)-ipsdienol with nearly enantiomerically pure (-)-ipsdienol in the "full blend" significantly reduced trap catches, which suggests that both enantiomers are needed for attraction. On the other hand, removal of (-)-trans-verbenol from the active "full blend" had no significant effect on trap catches. Our results suggest that the three major components, 2-methyl-3-buten-2-ol, 74%-(-)-ipsdienol, and (-)-cis-verbenol (at 7:2:1), produced by unpaired fed males, are likely the aggregation pheromone components of I. nitidus, thus representing the first characterization of an aggregation pheromone system of a bark beetle native solely to China.

Host specificity of Argopistes tsekooni (Coleoptera: Chrysomelidae), a potential biological control agent of Chinese privet.

Chinese privet, Ligustrum sinense Lour., is a perennial semi-evergreen shrub that is a serious invasive weed in the United States. Classical biological control offers the best hope for controlling it in an economic, effective, and persistent way. Host specificity of one of the most promising biological control agents of Chinese privet, a flea beetle, Argopistes tsekooni Chen (Coleoptera: Chrysomelidae), was evaluated in China by using laboratory no-choice and choice tests on 13 species of Oleaceae and eight species in other families that have important economic value. In adult no-choice survival and oviposition tests, the flea beetle fed and survived for 30 d on Syringa oblata Lindl., Jasminum nudiflorum Lindl., and three species in the genus Ligustrum. Females also oviposited on these species, but only larvae from eggs laid on S. oblata and Ligustrum spp. developed successfully. In addition, the beetles did not feed or oviposit on the species of economic importance. In choice tests, adults preferred L. sinense for feeding and oviposition. These results show that A. tsekooni is relatively host specific and warrants further testing as a biocontrol agent of Chinese privet in the United States.

Cross-attraction between an exotic and a native pine bark beetle: a novel invasion mechanism?

BACKGROUND: Aside from the ecological impacts, invasive species fascinate ecologists because of the unique opportunities that invasives offer in the study of community ecology. Some hypotheses have been proposed to illustrate the mechanisms that allow exotics to become invasive. However, positive interactions between exotic and native insects are rarely utilized to explain invasiveness of pests. METHODOLOGY/PRINCIPAL FINDINGS: Here, we present information on a recently formed association between a native and an exotic bark beetle on their shared host, Pinus tabuliformis, in China. In field examinations, we found that 35-40% of P. tabuliformis attacked by an exotic bark beetle, Dendroctonus valens, were also attacked by a native pine bark beetle, Hylastes parallelus. In the laboratory, we found that the antennal and walking responses of H. parallelus to host- and beetle-produced compounds were similar to those of the exotic D. valens in China. In addition, D. valens was attracted to volatiles produced by the native H. parallelus. CONCLUSIONS/SIGNIFICANCE: We report, for the first time, facilitation between an exotic and a native bark beetle seems to involve overlap in the use of host attractants and pheromones, which is cross-attraction. The concept of this interspecific facilitation could be explored as a novel invasive mechanism which helps explain invasiveness of not only exotic bark beetles but also other introduced pests in principle. The results reported here also have particularly important implications for risk assessments and management strategies for invasive species.

Chemotaxis of the pinewood nematode, Bursaphelenchus xylophilus, to volatiles associated with host pine, Pinus massoniana, and its vector Monochamus alternatus.

The pinewood nematode (PWN), Bursaphelenchus xylophilus, the most important invasive species in pine forests of Asia, is transported to new pine hosts by beetles of the genus Monochamus. Third-stage dispersal juveniles (J(III)) aggregate in pupal chambers around the vector as it matures. We demonstrated that the ratio of three terpenes (alpha-pinene, beta-pinene, and longifolene at 1:2.7:1.1) released by larval Monochamus alternatus strongly attract J(III), whereas the different ratio (1:0.1:0.01) of these three terpenes found in healthy xylem of Pinus massoniana attracts only the propagative stage (J(n)) of the nematode. We suggest that the volatiles produced by the host plants could be the basis of a chemoecological relationship between plant parasitic nematodes and their vector insects. Capture of J(III) with terpene-baited trap tubes deployed for 2 hr in the field was demonstrated. This technique may lead to the development of rapid sampling methodologies for use at either ports-of-entry or in the field.

Biology and preliminary host range assessment of two potential kudzu biological control agents.

Two insect species from China, Gonioctena tredecimmaculata (Jacoby) (Coleoptera: Chrysomelidae) and Ornatalcides (Mesalcidodes) trifidus (Pascoe) (Coleoptera: Curculionidae), were studied in quarantine in the United States as potential biological control agents for kudzu, Pueraria montana variety lobata (Willd.) Maesen and S. Almeida. Adults of G. tredecimmaculata were ovoviviparous and reproduced throughout the summer, producing offspring that had an obligate adult diapause. In no-choice tests, adult and larval G. tredecimmaculata rejected most of the plant species tested, but consumed foliage and completed their life cycle on soybean (Glycine max L. Merr.) and on a native woodland plant, hog-peanut (Amphicarpaea bracteata L. Fernald), which are in the same subtribe as kudzu (Glycininae). Insects showed similar responses to field- and greenhouse-grown soybean and kudzu foliage, despite measurable differences in leaf traits: field-grown foliage of both plants had greater leaf toughness, higher total carbon content, higher trichome density, and lower water content than greenhouse foliage. O. trifidus adults also rejected most of the plants tested but fed on and severely damaged potted soybean and hog-peanut plants in addition to kudzu. Further tests in China are needed to determine whether these species will accept nontarget host plants under open-field conditions.

DNA based cladograms augment the discovery of a new Ips species from China (Coleoptera: Curculionidae: Scolytinae).

The implementation of DNA in taxonomic study is in its infancy because the association of the amount and type of nucleotide change with species boundaries has not been fully examined for most taxa. Mitochondrial cytochrome c oxidase I (COI) nucleotide data is currently the most popular molecular marker for delimiting species boundaries and a standard pair-wise nucleotide divergence between groups of individuals has been suggested for the recognition of new species. It is unlikely that such a standard would be applicable across animal species, but the association of the amount and type of nucleotide change with species boundaries could help with the establishment of a taxon-specific DNA taxonomy. This study utilizes DNA data from nuclear and mitochondrial genes to improve the taxonomy of an important forest beetle pest, Ips. Amount and type of nucleotide difference are associated with monophyletic species based on a cladistic analysis of these data. As a result, a new species from China is described for a clade of beetles whose nucleotide differences exceeded the amount of evolutionary change observed within currently recognized species. The COI data are analyzed independently with an expanded taxon data set, including pair-wise nucleotide differences between recognized sister species. The wide range of average intraspecific pair-wise nucleotide difference (0-10.0%) suggests limitations to the application of a standard percent nucleotide difference as a means to identify species boundaries. At most, average COI nucleotide intraspecific difference provides an informal guide to identify potential clades that may warrant further systematic investigation.