False ring formation in eastern hemlock branches: impacts of hemlock woolly adelgid and elongate hemlock scale

Herbivores can alter plant physiology through the induction of abnormal wood formation. Feeding by some insects induces the formation of false rings, a band of thick-walled latewood cells within the earlywood portion of the tree ring that reduces water transport. Hemlock woolly adelgid (Adelges tsugae Annand) and elongate hemlock scale (Fiorinia externa Ferris) are invasive insects that both feed on eastern hemlock [Tsuga canadensis (L.) Carrière]. Adelges tsugae has a greater effect on tree health than F. externa, but the mechanism underlying their differential effect is unknown. We explored the effects of these herbivores by assessing growth ring formation in branches of trees that had been experimentally infested for 4 yr with A. tsugae, F. externa, or neither insect. We measured false ring density, ring growth, and earlywood: latewood ratios in the two most recently deposited growth rings. Branches from A. tsugae-infested trees had 30% more false rings than branches from F. externa-infested trees and 50% more than branches from uninfested trees. In contrast, branches from F. externa-infested trees and control trees did not differ in false ring formation. Radial growth and earlywood: latewood ratios did not differ among treatments. Our results show that two invasive herbivores with piercing-sucking mouth parts have differing effects on false ring formation in eastern hemlock. These false rings may be the product of a systemic plant hypersensitive response to feeding by A. tsugae on hemlock stems. If false rings are responsible for or symptomatic of hemlock water stress, this may provide a potential explanation for the relatively large effect of A. tsugae infestations on tree health.     

Distribution and abundance of Adelges tsugae (Hemiptera: Adelgidae) within hemlock trees

We studied the distribution of hemlock woolly adelgid, Adelges tsugae Annand (Hemiptera: Adelgidae), within hemlock trees for three summer (progrediens) and two winter (sistens) generations in northern Georgia. Eastern hemlock, Tsuga canadensis (L.) Carrière, trees were treated with 0, 10, or 25% of 1.5 g of imidacloprid per 2.5 cm of tree diameter at breast height and fertilized or not in a factorial design. Adelgid ovisacs per centimeter of branch were more abundant from June 2007 to June 2008 in the upper tree crown of insecticide untreated trees and when all trees were combined and that was the general trend for most comparisons. However, ovisacs were more abundant in the lower crown of insecticide treated trees in June 2008. More sistens nymphs settled on the upper crown branches than on the lower branches in summers 2007 and 2008. Higher eggs per ovisac were observed in the upper crown in February 2008 and in both the winter and summer 2009. In contrast, adelgids were more fecund in the lower crown in June 2008. On fertilized trees, eggs laid per adult were higher in the upper crown in February 2008. In summer 2008, eggs per ovisac were higher in the lower crown, but this reversed again to the upper crown by summer 2009. New growth of branches also varied among sample dates. These data demonstrate the variable distribution of adelgid and hemlock growth within trees over time and suggest that sampling only one crown area will not provide accurate estimates of adelgid densities.     

The tree crown distribution of hemlock woolly adelgid, Adelges tsugae (Hem., Adelgidae) from randomized branch sampling

Abstract:  An exotic insect, the hemlock woolly adelgid, Adelges tsugae Annand (Hem., Adelgidae), is spreading through eastern North America, killing hemlock trees [ Tsuga canadensis (L.) Carrière], and thereby impoverishing ecosystems. Adelges tsugae , like many alien invasive insects, is difficult to monitor or sample in the forest. Monitoring of A. tsugae has been hampered by lack of information about its distribution within tree crowns. In order to assist future monitoring and biocontrol of A. tsugae , this study investigates the crown distribution of A. tsugae by sampling from the entire height of mature hemlock trees in a forest with an established infestation. In addition to A. tsugae , sampling includes scale insects, which are another group of important pests on hemlock trees. This study demonstrates the utility of a randomized branch sampling (RBS) plan for monitoring both invasive insects as well as native insects that are difficult to sample. Results from the RBS show that in trees with high populations of A. tsugae , branches from the lower crown have slightly higher densities of A. tsugae than upper crown branches. In trees with low A. tsugae populations, the upper crown may have higher A. tsugae densities than the lower crown. North pointing branches also have higher densities of A. tsugae than branches pointing in other cardinal directions. Future sampling efforts for A. tsugae can take advantage of higher densities in certain portions of the crown to increase accuracy.     

Factors affecting settlement rate of the hemlock woolly adelgid,Adelges tsugae,on eastern hemlock,Tsuga canadensis

1 We assessed the importance of several factors potentially affecting the settlement rate of the invasive hemlock woolly adelgid Adelges tsugae (Hemiptera: Adelgidae) on uninfested foliage of the eastern hemlock, Tsuga canadensis. We conducted our experiments in Massachusetts (U.S.A.) with overwintering sistens adelgids, and applied standard densities of infested foliage to uninfested branches in a planned multiple‐comparison design. 2 Settlement rates of progrediens crawlers produced by the overwintering sistens were highest when adelgid‐infested foliage was loosely attached to uninfested foliage and both branches were then enclosed in a mesh sleeve. 3 Early‐emerging crawlers settled at a higher rate than did late‐emerging crawlers. 4 Increasing the density of infested branches did not affect settlement rates. 5 We also tested whether less severe winter conditions improved settlement, and found that overwintering infested foliage in a refrigerator decreased settlement rate relative to foliage overwintered outdoors. 6 Our results suggest a protocol for adelgid inoculations that could substantially increase the success rate of experimental manipulations and encourage additional research on the population dynamics of this pest.     

Prey suitability and phenology of Leucopis spp. (Diptera: Chamaemyiidae) associated with hemlock woolly adelgid (Hemiptera: Adelgidae) in the Pacific Northwest

Leucopis spp. (Diptera: Chamaemyiidae) from the Pacific Northwest previously were identified as potential biological control agents for the hemlock woolly adelgid, Adelges tsugae Annand (Hemiptera: Adelgidae), in the eastern United States. We collected Leucopis spp. larvae from A. tsugae infested western hemlocks in Oregon and Washington and reared them on an unidentified Pineus spp., Pineus strobi (Hartig), Adelges cooleyi (Gillette), Adelges piceae (Ratzeburg), and A. tsugae in three no-choice tests. Leucopis spp. survival on A. tsugae was significantly higher than on A. piceae during the 2010 progrediens generation test and significantly higher than on P. strobi and A. cooleyi during the 2010 sistens generation test. However, across all three tests, some larvae completed development to adult on all four of the alternative adelgid species. Larvae that survived to the adult stage were identified as Leucopis argenticollis Zetterstedt and Leucopis piniperda Malloch. These results suggest that populations of L. argenticollis and L. piniperda in the Pacific Northwest may not be specific to A. tsugae. We also studied the phenology of Leucopis spp. on fourteen A. tsugae infested western hemlock trees in Oregon and Washington over a period of 14 mo. Leucopis spp. larvae were collected year-round, but highest densities coincided with the presence of progrediens and sistens eggs and adults of A. tsugae. There was a positive correlation between Leucopis spp. and A. tsugae abundance.     

Isolation and characterization of microsatellite markers for Carolina hemlock (Tsuga caroliniana)

We describe the isolation and characterization of 31 polymorphic di- and trinucleotide microsatellite marker loci for Carolina hemlock (Tsuga caroliniana Englem.). In addition, primer pairs for 16 loci amplified scoreable alleles in six other Tsuga species. In eastern North America, both Carolina hemlock and eastern hemlock (Tsuga canadensis [L.] Carr.) populations are declining due to infestation by hemlock woolly adelgid, Adelges tsugae. The markers described here should enhance population genetic studies of hemlocks, providing valuable information for conserving and restoring these important forest tree species.     

Predators associated with the hemlock woolly adelgid (Hemiptera: Adelgidae) in the Pacific Northwest

The hemlock woolly adelgid, Adelges tsugae Annand (Hemiptera: Adelgidae), is causing widespread mortality of eastern hemlock, Tsuga canadensis L. Carrière, in the eastern United States. In western North America, feeding by A. tsugae results in negligible damage to western hemlock, Tsuga heterophylla (Raf.) Sargent. Host tolerance and presence of endemic predators may be contributing to the relatively low levels of injury to T. heterophylla caused by A. tsugae. Field surveys of the predator community associated with A. tsugae infestations on 116 T. heterophylla at 16 sites in Oregon and Washington were conducted every 4-6 wk from March 2005 through November 2006. Fourteen uninfested T. heterophylla were also surveyed across 5 of the 16 sites. Each sample tree was assigned an A. tsugae population score ranging from 0 to 3. Predators collected from A. tsugae-infested T. heterophylla represent 55 species in 14 families, listed in order of abundance: Derodontidae, Chamaemyiidae, Hemerobiidae, Coccinellidae, Cantharidae, Reduviidae, Miridae, Syrphidae, Chrysopidae, Coniopterygidae, Staphylinidae, Anthocoridae, Nabidae, and Raphidiidae. Laricobius nigrinus Fender (Coleoptera: Derodontidae), Leucopis argenticollis Zetterstedt (Diptera: Chamaemyiidae), and Leucopis atrifacies (Aldrich) (Chamaemyiidae) were the most abundant predators; together comprising 59% of predator specimens recovered. Relationships among predators and A. tsugae were determined through community structure analysis. The abundances of Laricobius spp. larvae, L. nigrinus adults, Leucopis spp. larvae, and L. argenticollis adults were found to be positively correlated to A. tsugae population score. Predators were most abundant when the two generations of A. tsugae eggs were present. L. argenticollis and L. atrifacies were reared on A. tsugae in the laboratory, and host records show them to feed exclusively on Adelgidae.     

Effects of fertilizer and low rates of imidacloprid on Adelges tsugae (Hemiptera: Adelgidae)

Healthy hemlock trees, Tsuga canadensis (L.) Carrière, and hemlock woolly adelgid, Adelges tsugae Annand (Hemiptera: Adelgidae), populations should favor retention and population growth of adelgid predators such as Laricobius nigrinus Fender (Coleoptera: Derodontidae) and Sasajiscymnus tsugae (Sasaji & McClure) (Coleoptera: Coccinellidae). Eastern hemlock trees between 15 and 38 cm diameter at breast height (dbh) were treated with 0, 10, or 25% of 1.5 g imidacloprid (Merit 75 WP) per 2.5 cm dbh and were either fertilized or not, in a 3 by 2 factorial design. After 2 yr, imidacloprid reduced the numbers of ovisacs and eggs found on trees in a dosage-dependent manner, while enhancing tree growth parameters such as new shoots or needles and the length of new shoots. Fertilized trees had greater adelgid fecundity, which was positively correlated with total foliar N in both winter generations. In February 2009 (27 mo after imidacloprid treatment), higher imidacloprid dosages to unfertilized trees resulted in reduced adelgid fecundity. Concentrations of N, P, and K were higher in the foliage of trees treated with insecticide, whereas foliar aluminum concentrations were consistently lower in trees with higher insecticide dosages. Trees treated with low rates of imidacloprid were healthier than untreated trees, but only trees treated with the 0.1 x dosage had sufficient adelgids to possibly sustain predators over extended periods.     

Volatile emissions of eastern hemlock,Tsuga canadensis,and the influence of hemlock woolly adelgid

The volatile emissions of eastern hemlock, Tsuga canadensis Carriere, were identified and quantified using standard and chiral gas chromatography and mass spectrometry. All of the identified compounds were monoterpenes, and included alpha-pinene, myrcene, tricyclene, camphene, alpha-phellandrene, beta-pinene, limonene, beta-phellandrene, terpinolene, and bornyl acetate. alpha-Pinene, myrcene, and camphene comprised greater than 75% by mass of the total release. Infestation by the exotic insect, hemlock woolly adelgid (HWA, Adelges tsugae Annand), resulted in an increased release rate of monoterpenes from branch tips. Release rate was negatively correlated to the amount of the branch tip sample that was new growth, suggesting that release rate is greater from previous-year foliage. Additionally the percent composition of the volatile profile is slightly altered by infestation, with alpha-pinene comprising 57% of volatiles from infested foliage and 66% from uninfested foliage.     

Comparison of numerical response and predation effects of two coccinellid species on hemlock woolly adelgid (Homoptera: Adelgidae)

The hemlock woolly adelgid, Adelges tsugae Annand, is an introduced pest in North America that is native to Asia, and is causing extensive damage to eastern hemlock (Tsuga canadensis Carriere) and Carolina hemlock (Tsuga caroliniana Englemann) in the eastern United States. We compared two coccinellids imported for biological control of the adelgid: Scymnus ningshanensis Yu et Yao from China and Pseudoscymnus tsugae Sasaji and McClure from Japan. In a laboratory study, we measured the numerical response of each beetle species to a range of prey densities, and in field studies we examined the reproductive success and ability of the coccinellids to reduce populations of the hemlock woolly adelgid. In the laboratory, S. ningshanensis showed a positive numerical response as hemlock woolly adelgid density increased, and P. tsugae showed a density-independent response. In field cages, the presence of S. ningshanensis resulted in negative hemlock woolly adelgid population growth, in contrast to positive growth in both control cages and cages containing P. tsugae. Both our laboratory and field experiments suggest that S. ningshanensis has good potential as a biological control agent of hemlock woolly adelgid.     

Effects of fertilization of four hemlock species on Adelges tsugae (Hemiptera: Adelgidae) growth and feeding preference of predators

Understanding how fertilization affects host resistance to hemlock woolly adelgid, Adelges tsugae Annand (Hemiptera: Adelgidae), is important because fertilizers are often used to grow resistant selections to a suitable size for testing. We evaluated four hemlock species (Tsuga) under three different fertilizer regimes to assess whether fertility affected resistance to the adelgid and to determine whether it affected feeding preferences of the adelgid predators Laricobius nigrinus Fender and Sasajiscymnus tsugae (Sasaji & McClure). Treatments were long-term fertilization (from June 2008 to June 2009), short-term fertilization (from March to June 2009), and no fertilizer. Fertilizer was applied biweekly with 240 ppm N by using water-soluble fertilizer (N-P-K, 20:20:20). Plants (>1 yr old) were artificially infested with adelgids on 31 March 2009. Among unfertilized hemlocks (n=10 per species), foliar N was highest in Tsuga mertensiana (Bong.) CarriBre and lowest in T. chinensis (Franch.) E. Pritz. Significantly more progredien ovisacs or sisten eggs were present on T. mertensiana than on the other hemlock species with none on unfertilized T. chinensis. A. tsugae adults on T. heterophylla (Raf.) Sarg. were unaffected by fertility, but densities of developing A. tsugae nymphs were higher on unfertilized T. heterophylla plants than on fertilized T. heterophylla plants regardless of fertilizer treatment. Both L. nigrinus and S. tsugae consumed more adelgid eggs that developed on fertilized T. canadensis than from unfertilized plants. The predators did not exhibit this preference for adelgid eggs from females that developed on T. heterophylla or T. mertensiana.     

Orientation behavior of the predator Laricobius nigrinus (Coleoptera: Derodontidae) to hemlock woolly adelgid and host tree odors in a multi-chambered olfactometer

We studied the adult ambulatory response of the predator, Laricobius nigrinus Fender (Coleoptera: Derodontidae), to odors from its prey, Adelges tsugae Annand, the hemlock woolly adelgid, and foliage of hemlock woolly adelgid, host hemlocks (Tsuga spp.), and other conifers. Both the predator and hemlock woolly adelgid are apparently native to western North America, but the predator is being released in the eastern United States, which has different hemlock species, for biological control of a lineage of hemlock woolly adelgid inadvertently introduced from Japan. L. nigrinus responded to odors from hemlock woolly adelgid host trees, but not to odors from hemlock woolly adelgid. L. nigrinus collected from hemlock woolly adelgid-infested western hemlock were more strongly attracted to odors from western hemlock [Tsuga heterophylla (Rafinesque) Sargent] than eastern hemlock [Tsuga canadensis (L.) Carrière] in most trials. Odors from western white pine (Pinus monticola Douglas ex D. Don) and white spruce [Picea glauca (Moench) Voss] were as attractive as western hemlock odors whereas odors from Douglas-fir [Pseudotsuga menziesii variety menziesii (Mirbel)] and ponderosa pine (Pinus ponderosa Douglas ex Lawson) were avoided. L. nigrinus reared on hemlock woolly adelgid-infested eastern hemlock in the laboratory were lethargic and were not attracted to either eastern or western hemlock odors. Predators collected in the field and tested monthly from December to March responded similarly each month, except February, when they flew rather than walked in the olfactometer, suggesting a period of dispersal or mate finding at that time of year. The implications of these results for programs to release L. nigrinus in the eastern United States for control of hemlock woolly adelgid are discussed.     

Arboreal spiders in eastern hemlock

Eastern hemlock [Tsuga canadensis (L.) Carrière] is a foundation species in forests of eastern North America that plays a key role in ecosystem function. It is highly susceptible to the exotic invasive hemlock woolly adelgid (Adelges tsugae Annand), which is causing widespread hemlock mortality. We surveyed the spider communities of eastern hemlock and deciduous canopies over 2 yr, collecting over 4,000 spiders from 21 families. We found that eastern hemlock canopies harbored a more abundant, rich, and diverse spider community than did deciduous canopies. Five spider families were present in our hemlock collections that were absent from the deciduous collections, including Mysmenidae, Theridiosomatidae, Mimetidae, Lycosidae, and Agelenidae. In hemlock canopies there were 4× the number of web builders, consisting primarily of the Tetragnathidae and Araneidae, than active hunters, consisting primarily of the Anyphaenidae and the Salticidae. Ours is the first in depth study of the spider community in eastern hemlock. Spider abundance in hemlock canopies suggest that they may play a role regulating herbivore populations, and could possibly affect the invasive hemlock woolly adelgid, either through direct consumption of the adelgids themselves or through interactions with classical biological control agents.     

Feeding preference of three lady beetle predators of the hemlock woolly adelgid (Homoptera: Adelgidae)

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.     

When one is not necessarily a lonely number: initial colonization dynamics of Adelges tsugae on eastern hemlock, Tsuga canadensis

The ability to establish successfully in a new area can vary considerably among species. In addition to the well-recognized importance of propagule pressure in driving the rates of establishment of biological invaders, the life history strategy of a species can also affect establishment success, such as in the extent to which Allee effects (positive density-dependence), and environmental and demographic stochasticity manifest themselves. We quantified the establishment success of Adelges tsugae, a non-native insect currently invading North American hemlock. We inoculated eastern hemlock host trees with varying densities of this parthenogenetic insect, from 1 to >500 progrediens ovisacs. The number of settled sistens (the subsequent generation) was positively related to the initial density. More interesting, however, was that we recorded successful establishment from released progrediens ovisacs, and the subsequent initiation of the next generation (sistens), in ≈39 % of host trees inoculated with 1 ovisac. The observation that successful establishment can be accomplished by a single ovisac produced by a single individual has important implications in the invasion dynamics and management of A. tsugae.     

Web orientation and prey resources for web-building spiders in eastern hemlock

We examined the arthropod community on eastern hemlock, Tsuga canadensis (L.) Carr, in the context of its role in providing potential prey items for hemlock-associated web-weaving spiders. Using sticky traps simulating spider webs, we evaluated what prey items are available to web-weaving spiders in eastern hemlock based on web orientation (horizontal versus vertical) and cardinal direction. We found that the overwhelming majority (>70%) of prey items available to spiders in hemlock canopies were Diptera. Psocoptera, Hymenoptera, and Hemiptera comprised most of the remaining potential prey. A significant direction × orientation interaction, and greater trap capture in some direction-orientation combinations, suggests that spiders might locate their webs in eastern hemlock canopies for thermoregulatory purposes, ultimately optimizing prey capture. We also evaluated these findings in the context of hemlock infestation by the invasive hemlock woolly adelgid, Adelges tsugae Annand. The adelgid is a sedentary insect with a mobile crawler stage that provides a readily available, easily obtained food source for predators in hemlock canopies. However, an abundance of alternative prey will affect within canopy spider distribution and the potential intensity with which spiders consume these prey. Understanding the response of spiders to potential prey availability is essential to understanding the trophic interactions involving these predators and their potential for influencing herbivore populations.     

Forest dynamics following eastern hemlock mortality in the southern Appalachians

Understanding changes in community composition caused by invasive species is critical for predicting effects on ecosystem function, particularly when the invasive threatens a foundation species. Here we focus on dynamics of forest structure, composition and microclimate, and how these interact in southern Appalachian riparian forests following invasion by hemlock woolly adelgid, HWA, Adelges tsugae. We measured and quantified changes in microclimate; canopy mortality; canopy and shrub growth; understory species composition; and the cover and diversity in riparian forests dominated by eastern hemlock Tsuga canadensis over a period of seven years. Treatments manipulated hemlock mortality either through invasion (HWA infested stands) or girdling (GDL) hemlock trees. Mortality was rapid, with 50% hemlock tree mortality occurring after six years of invasion, in contrast to more than 50% mortality in two years following girdling. Although 50% of hemlock trees were still alive five years after infestation, leaf area lost was similar to that of girdled trees. As such, overall responses over time (changes in light transmittance, growth, soil moisture) were identical to girdled stands with 100% mortality. Our results showed different growth responses of the canopy species, shrubs and ground layer, with the latter being substantially influenced by presence of the evergreen shrub, rhododendron Rhododendron maximum. Although ground layer richness in the infested and girdled stands increased by threefold, they did not approach levels recorded in hardwood forests without rhododendron. Increased growth of co‐occurring canopy trees occurred in the first few years following hemlock decline, with similar responses in both treatments. In contrast, growth of rhododendron continued to increase over time. By the end of the study it had a 2.6‐fold higher growth rate than expected, likely taking advantage of increased light available during leaf‐off periods of the deciduous species. Increased growth and dominance of rhododendron may be a major determinant of future responses in southern Appalachian ecosystems; however, our results suggest hemlock will be replaced by a mix of Acer, Betula, Fagus and Quercus canopy genera where establishment is not limited by rhododendron.     

Somatic embryogenesis and cryostorage of eastern hemlock and Carolina hemlock for conservation and restoration

Key message

Embryogenic cultures of eastern and Carolina hemlocks could be initiated, and somatic embryos and plantlets produced using standard conifer protocols and media. Embryogenic hemlock cultures were cryostored and recovered.

Abstract

Eastern hemlock (Tsuga canadenesis) and Carolina hemlock (Tsuga caroliniana) are threatened with extirpation from their native ranges in eastern North America by the introduction of the hemlock woolly adelgid (HWA; Adelges tsugae), an exotic insect pest that has already killed millions of hemlock trees. Efforts to conserve and restore these members of the Pinaceae could be greatly enhanced by the availability of an in vitro propagation system. We conducted experiments to initiate embryogenic cultures from eastern and Carolina hemlock zygotic embryos at different stages of development using three media supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-Benzylaminopurine (BA). Cone collection date, medium and source tree had significant effects on induction of embryogenic tissue from zygotic embryo explants of both species, which ranged as high as 52 % for eastern hemlock and 17 % for Carolina hemlock. Embryogenic hemlock cultures could be cryostored using a protocol employing sorbitol and DMSO, and recovered following several months of frozen storage. Transfer of embryogenic tissue from proliferation media containing 2, 4-D and BA to a Litvay medium with abscisic acid promoted the development of somatic embryos, which were stimulated to mature by slow drying under semi-permeable plastic film. Embryos moved to an imbibition-germination medium without plant growth regulators and incubated in the light elongated and subsequently germinated. A small number of germinated embryos survived transfer to ex vitro conditions and grew into somatic seedlings. The embryogenesis and cryostorage systems developed in the study are already being integrated with hemlock breeding efforts to develop clones with resistance or tolerance to HWA.     

Widespread inbreeding and unexpected geographic patterns of genetic variation in eastern hemlock (Tsuga canadensis), an imperiled North American conifer

Eastern hemlock (Tsuga canadensis [L.] Carr.) is an ecologically important tree species experiencing severe mortality across much of its eastern North American distribution, caused by infestation of the exotic hemlock woolly adelgid (Adelges tsugae Annand). To guide gene conservation strategies for this imperiled conifer, we conducted a range-wide genetic variation study for eastern hemlock, amplifying 13 highly polymorphic nuclear microsatellite loci in 1,180 trees across 60 populations. The results demonstrate that eastern hemlock exhibits moderate inbreeding, possibly a signature of a prehistoric decline associated with a widespread insect outbreak. Contrary to expectations, populations in formerly glaciated regions are not less genetically diverse than in the putative southern refugial region. As expected, peripheral disjunct populations are less genetically diverse than main-range populations, but some are highly genetically differentiated or contain unique alleles. Spatially explicit Bayesian clustering analyses suggest that three or four Pleistocene glacial refuges may have existed in the Southeastern United States, with a main post-glacial movement into the Northeast and the Great Lakes region. Efforts to conserve eastern hemlock genetic material should emphasize the capture of broad adaptability that occurs across the geographic range of the species, as well as genetic variability within regions with the highest allelic richness and heterozygosity, such as the Southern Appalachians and New England, and within disjunct populations that are genetically distinct. Much genetic variation exists in areas both infested and uninfested by the adelgid.     

Insect guild structure associated with eastern hemlock in the southern Appalachians

An assessment of the insect guild structure associated with immature and mature eastern hemlock, Tsuga canadensis (L.) Carrière, at high and low elevations was made before the invasion by the hemlock woolly adelgid, Adelges tsugae (Annand), and elongate hemlock scale, Fiorinia externa Ferris. Guild dynamics were determined among 243 insect species associated with eastern hemlock in the southern Appalachians. Detritivore, hematophage, herbivore, fungivore, parasitoid, predator, scavenger, and transient guild assignments were made. The herbivore and transient guilds were subdivided into chewers, sapsuckers, flower feeders, pollen feeders, pollen/nectar/sap feeders, and seed feeders. A significant association was found between guilds on immature and mature hemlocks at low elevations, but no significant association was found at high elevations. There was also a strong association of phytophagous insects on immature hemlocks and scavengers on mature hemlocks. The observed transient, scavenger, and predator guilds had a significantly lower co-occurrence among species indicating segregation of the species within these guilds. The herbivore guild had a significantly higher co-occurrence among species than the simulated index indicating aggregation of species within this guild across sites. Detritivore, hematophage, and parasitoid guilds did not differ significantly from the simulated community. These results document a diverse and dynamic insect community on eastern hemlock before invasion by hemlock woolly adelgid and elongate hemlock scale.