Nathan P. Havill

Mitochondrial DNA from Hemlock Woolly Adelgid (Hemiptera: Adelgidae) Suggests Cryptic Speciation and Pinpoints the Source of the Introduction to Eastern North America

Abstract The hemlock woolly adelgid, Adelges tsugae Annand (Hemiptera: Adelgidae), is an introduced pest of unknown origin that is causing severe mortality to hemlocks (Tsuga spp.) in eastern North America. Adelgids also occur on other Tsuga species in western North America and East Asia, but these trees are not significantly damaged. The purpose of this study is to use molecular methods to clarify the relationship among hemlock adelgids worldwide and thereby determine the geographic origin of the introduction to eastern North America. Adelgids were collected from multiple locations in eastern and western North America, mainland China, Taiwan, and Japan, and 1521 bp of mitochondrial DNA was sequenced for each sample. Phylogenetic analyses suggest that the source of A. tsugae in eastern North America was likely a population of adelgids in southern Japan. A single haplotype was shared among all samples collected in eastern North America and samples collected in the natural range of T. sieboldii in southern Honshu, Japan. A separate adelgid mitochondrial lineage was found at higher elevations in the natural range of T. diversifolia. Adelgids from mainland China and Taiwan represent a lineage that is clearly diverged from insects in North America and Japan. In contrast to eastern North America, there is no conclusive evidence for a recent introduction of A. tsugae into western North America, where multiple haplotypes are found. Implications for hemlock woolly adelgid control, taxonomy, and plant–insect coevolution are discussed.

Phylogeny and Biogeography of Tsuga (Pinaceae) Inferred from Nuclear Ribosomal ITS and Chloroplast DNA Sequence Data

Abstract Hemlock, Tsuga (Pinaceae), has a disjunct distribution in North America and Asia. To examine the biogeographic history of Tsuga, phylogenetic relationships among multiple accessions of all nine species were inferred using chloroplast DNA sequences and multiple cloned sequences of the nuclear ribosomal ITS region. Analysis of chloroplast and ITS sequences resolve a clade that includes the two western North American species, T. heterophylla and T. mertensiana, and a clade of Asian species within which one of the eastern North American species, T. caroliniana, is nested. The other eastern North American species, T. canadensis, is sister to the Asian clade. Tsuga chinensis from Taiwan did not group with T. chinensis from mainland China, and T. sieboldii from Ullung Island did not group with T. sieboldii from Japan suggesting that the taxonomic status of these distinct populations should be reevaluated. The Himalayan species, T. dumosa, was in conflicting positions in the chloroplast and ITS trees, suggesting that it may be of hybrid origin. Likelihood-based biogeographic inference with divergence time estimates infers an Eocene basal crown group diversification and an initial widespread circumpolar distribution with subsequent vicariance and extinction events leading to the current disjunct distribution.

DNA barcodes to identify species and explore diversity in the Adelgidae (Insecta: Hemiptera: Aphidoidea)

The Adelgidae are relatively small, cryptic insects, exhibiting complex life cycles with parthenogenetic reproduction. Due to these characteristics, the taxonomy of the group is problematic. Here, we test the effectiveness of the standard 658‐bp barcode fragment from the 5′‐end of the mitochondrial cytochrome c oxidase 1 gene (COI) in differentiating among 17 species of Adelgidae, in associating life‐cycle stages, and in assessing patterns of geographical variation in selected species. Species of Adelgidae are well‐differentiated by DNA barcodes, enabling the identification of different morphological forms, immature stages and individuals on different hosts and at different periods of the life cycle. DNA barcodes have uncovered cryptic diversity within taxa and, in other cases, a lack of sequence divergence in species pairs previously separated by life‐cycle characteristics, indicating a need for further taxonomic analysis.

Lonesome George is not alone among Galápagos tortoises

Document S1. Supplemental Experimental Procedures and Results, One Table, One Figure and Supplemental ReferencesxDownload (.21 MB ) Document S1. Supplemental Experimental Procedures and Results, One Table, One Figure and Supplemental References

A New Species of Laricobius (Coleoptera: Derodontidae) From Japan With Phylogeny and a Key for Native and Introduced Congeners in North America

ABSTRACT Laricobius osakensis Montgomery and Shiyake sp. nov., collected from Adelges tsugae Annand on hemlock [Tsuga sieboldii Carr. and Tsuga diversifolia (Maxim.) Mast.] in Japan, is described and illustrated. The new species was collected from several localities on Honshu, Shikokou, and Kyushu Islands. The genus has not been reported previously from Japan. Morphological features, a molecular phylogeny, and diagnostic DNA sites are provided to distinguish this new species from previously described species of the genus. Because of plans to release L. osakensis for the biological control of A. tsugae in eastern North America, a key and discussion are provided to differentiate it from the native North American species, Laricobius nigrinus Fender, Laricobius laticollis Fall, and Laricobius rubidus LeConte, and from two previously imported species—Laricobius erichsonii Rosenhauer, which is endemic in Europe, and Laricobius kangdingensis Zilahi-Balogh & Jelinek, which is endemic in China.

Feeding Preference of Three Lady Beetle Predators of the Hemlock Woolly Adelgid (Homoptera: Adelgidae)

Abstract In a laboratory study, we tested the feeding preferences of three coccinellid predators of hemlock woolly adelgid, Adelges tsugae Annand, an introduced pest of hemlock in the eastern United States. The species tested were Sasajiscymnus tsugae Sasaji & McClure (formerly Pseudoscymnus tsugae) from Japan, Scymnus ningshanensis Yu & Yao from China, and Harmonia axyridis (Pallas), a generalist species introduced from Asia that is currently widespread in eastern hemlock, Tsuga canadensis Carriere, forests. We measured the feeding preference of each beetle species when given the choice of A. tsugae and either 1) Pineus strobi (Hartig) on Pinus strobus L.; 2) Adelges laricis Vallot on Larix decidua Mill.; 3) Adelges cooleyi (Gillette) on Pseudotsuga menziesii (Mirb.) Franco; or 3) Paraprociphilus tessellatus (Fitch) on Alnus serrulata (Ait.) Willd. We evaluated beetle preference for adults, nymphs, and eggs of each prey species. Generally, when adult or nymphal prey stages were compared, S. tsugae preferred A. tsugae adults to P. strobi, A. cooleyi, A. laricis, and P. tessellatus. S. ningshanensis showed less preference between adelgid species, but it did not prefer P. tessellatus nymphs. When preferences for adelgid eggs were assayed, S. tsugae and S. ningshanensis showed no preference between A. tsugae and A. cooleyi or P. strobi, but S. tsugae did prefer A. tsugae to A. laricis. Larvae of S. tsugae were unable to survive on P. tessellatus nymphs. H. axyridis adults readily consumed both A. tsugae and P. tessellatus, but H. axyridis larvae did not complete their life cycle on A. tsugae. Our host range tests suggest that S. ningshanensis and S. tsugae may feed on several species of Adelgidae and that A. tsugae is often preferred.

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.

Asymmetric hybridization between non-native winter moth, Operophtera brumata (Lepidoptera: Geometridae), and native Bruce spanworm, Operophtera bruceata, in the Northeastern United States, assessed with novel microsatellites and SNPs.

The European winter moth, Operophtera brumata, is a non-native pest in the Northeastern USA causing defoliation of forest trees and crops such as apples and blueberries. This species is known to hybridize with O. bruceata, the Bruce spanworm, a native species across North America, although it is not known if there are hybrid generations beyond F1. To study winter moth population genetics and hybridization with Bruce spanworm, we developed two sets of genetic markers, single nucleotide polymorphisms (SNPs) and microsatellites, using genomic approaches. Both types of markers were validated using samples from the two species and their hybrids. We identified 1216 SNPs and 24 variable microsatellite loci. From them we developed a subset of 95 species-diagnostic SNPs and ten microsatellite loci that could be used for hybrid identification. We further validated the ten microsatellite loci by screening field collected samples of both species and putative hybrids. In addition to confirming the presence of F1 hybrids reported in previous studies, we found evidence for multi-generation asymmetric hybridization, as suggested by the occurrence of hybrid backcrosses with the winter month, but not with the Bruce spanworm. Laboratory crosses between winter moth females and Bruce spanworm males resulted in a higher proportion of viable eggs than the reciprocal cross, supporting this pattern. We discuss the possible roles of population demographics, sex chromosome genetic incompatibility, and bacterial symbionts as causes of this asymmetrical hybridization and the utility of the developed markers for future studies.

Phylogeny of Rhus gall aphids (Hemiptera : Pemphigidae) based on combined molecular analysis of nuclear EF1α and mitochondrial COII genes

Rhus gall aphids (Fordinae : Melaphidini) have a disjunct distribution in East Asia and North America and have specific host plant relationships. Some of them are of economic importance and all species form sealed galls which show great variation in shape, size, structure, and galling‐site. We present a phylogeny incorporating ten species and four subspecies of Rhus gall aphids based on 1694 base pairs of nuclear elongation factor‐1α (EF1α) and mitochondrial cytochrome oxidase subunit II (COII) DNA sequence data. The results suggest that Melaphidini is monophyletic and at the genus level, Schlechtendalia, Nurudea, and Floraphis were each monophyletic. Kaburagia and Meitanaphis were not monophyletic and therefore inconsistent with the current classification. The North American sumac gall aphid, Melaphis rhois, was most closely related to the East Asian Floraphis species, although this was poorly supported. The conservation of gall morphology with respect to aphid phylogeny rather than their host plants suggests that gall morphology is largely determined by the aphids. While there is no evidence of strict co‐speciation between the aphids and their primary host plants, switching between recently diverged host plants may be involved in the speciation process in Melaphidini.

Post release recovery of hemlock woolly adelgid predators in the North Georgia mountains.

Abstract Eastern hemlock, Tsuga canadensis (L.) Carrière, and Carolina hemlock, Tsuga caroliniana Engelmann, provide unique habitat that is threatened by the invasive hemlock woolly adelgid, Adelges tsugae Annand, which arrived in Georgia in 2003. In an attempt to conserve a portion of the mature hemlocks in north Georgia, the USDA Forest Service created over 100 Hemlock Conservation Areas throughout the Chattahoochee National Forest and designated them to receive chemical and/or biological control. Sasajiscymnus tsugae Sasaji and McClure, Laricobius nigrinus Fender, and Scymnus sinunodulus Yu and Yao are predatory beetles reared in the laboratory and released in these areas. To determine establishment of these predators, infested hemlock trees were sampled during spring 2010 - 2012 at some of these release sites. Additionally, nonrelease sites, 0.4 -1.6 km from release areas, were sampled in 2012 to evaluate predator spread from release trees. Five hundred ninety-two S. tsugae, 232 L. nigrinus, 262 native L. rubidus, and 58 Laricobius hybrids were recovered at multiple sites over those years. Sasajiscymnus tsugae was found at 3 sites, 3 years after release and at 2 other sites, 2 years after release. Laricobius nigrinus was found at 1 site, 3 years after release and at 2 sites, 2 years after release. Scymnus sinunodulus was never recovered. Our results demonstrate that S. tsugae and L. nigrinus are established in north Georgia, and that the native L. rubidus is commonly associated with A. tsugae and is hybridizing with L. nigrinus; however, the population sizes, efficacy, and survival rates of all these predators are still unknown.