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Insects that feed on plants are widely used for studies of host-plant-associated speciation due to their diversity and tractability. The value of these studies necessarily depends upon an accurate understanding of the diet of the focal insects. In North America, the 12 Phytomyza Fallén (Diptera: Agromyzidae) leafmining fly species feeding on hollies (Ilex spp. (Aquifoliaceae: Ilex)) initially appeared to be primarily a host-plant-associated radiation of largely monophagous species. However, our increased collecting efforts found that a majority of the morphospecies are oligophagous, feeding on multiple Ilex species. Patterns of host-associations suggest that colonization of new hosts followed by host-associated speciation may be an important feature of diversification. Analyses of mitochondrial COI data in three oligophagous species found evidence of host-associated genetic structure consistent with processes of host-associated divergence. However, in one of these species, evidence of geographic divergence as well as host-associated divergence was detected, illustrating complexity in factors relating to speciation and host-use evolution in this radiation of leafmining flies.

Evolutionary radiations have been well documented in plants and insects, and natural selection may often underly these radiations. If radiations are adaptive, the diversity of species could be due to ecological speciation in these lineages. Agromyzid flies exhibit patterns of repeated host-associated radiations. We investigated whether host-associated population divergence and evidence of divergent selection exist in the leaf miner Phytomyza glabricola on its sympatric host plants, the holly species, Ilex coriacea and I. glabra. Using AFLPs and nuclear sequence data, we found substantial genetic divergence between host-associated populations of these flies throughout their geographic range. Genome scans using the AFLP data identified 13 loci under divergent selection, consistent with processes of ecological speciation. EF-1α data suggest that I. glabra is the original host of P. glabricola and that I. coriacea is the novel host, but the AFLP data are ambiguous with regard to directionality of the host shift.

Boxwood (Buxus spp.) are commercially important evergreen ornamental plants with an annual market value of over $103 million in the United States. The recent U.S. incursion of boxwood blight disease caused by the fungus Calonectria pseudonaviculata (syn. Cylindrocladium pseudonaviculatum, Cy. buxicola) threatens the health and productivity of boxwood in both landscape plantings and nurseries. The first confirmed U.S. reports of the disease were made from Connecticut and North Carolina in November 2011 (2,4), followed by diagnoses in 10 additional states during 2012 and 2013. By August 2013, symptoms consistent with boxwood blight had been observed from B. sempervirens in Delaware, Maryland, New Jersey, and southeastern New York. Affected plants showed rapid onset of disease symptoms: dark brown to black spots or diffuse dark areas on leaves, followed by defoliation. Narrow, elongate black cankers also formed on current season shoots. Symptomatic stems and leaves were placed in petri dishes with moistened filter paper at 22°C for 3 days under continuous light. Conidiophores were excised, then placed on potato dextrose agar amended with streptomycin and neomycin (0.3 g/l). Resultant colonies showed dark brown pigmentation at the colony center surrounded by tan to reddish brown rings with white mycelia at the advancing edge. Conidia (n = 30 per isolate) were hyaline, cylindrical, rounded at both ends, with a single septum (45 to 76 × 4 to 6 μm; avg. 63 × 5 μm). Conidiophores (n = 20 per isolate) comprised a stipe, a hyaline septate stipe extension (length 119 to 192 μm; avg. 150 μm) and a terminal ellipsoidal vesicle (diameter 4 to 10 μm; avg. 7 μm). Based on morphological characteristics, the causal agent was identified as C. pseudonaviculata (1,4). Voucher specimens were deposited in the U.S. National Fungus Collections (BPI 892698 to 701). To verify morphological diagnosis, genomic DNA was extracted from fungal biomass grown in liquid cultures of yeast extract peptone dextrose media. A portion of the β-tubulin gene (TUB2) was PCR amplified and sequenced bi-directionally using primers Bta/Bt2b (3). BLASTn searches of NCBI GenBank databases using the TUB2 sequences (Accession Nos. KF785808 to 11) demonstrated 96 to 100% sequence identity with other C. pseudonaviculata isolates. To confirm pathogenicity, 5-month-old B. sempervirens and B. microphylla seedlings were spray-inoculated with a spore suspension of 1 × 104 conidia/ml. One isolate from each state was independently tested with four replicates each. Non-inoculated water-sprayed plants served as negative controls. Plants were maintained in growth chambers at 22°C under constant light. Blight symptoms developed 4 to 5 days post inoculation. C. pseudonaviculata was re-isolated from inoculated plants; no symptoms or signs were observed from control plants. To our knowledge, this is the first report of C. pseudonaviculata in the states of Delaware, Maryland, New Jersey, and New York. This report demonstrates that C. pseudonaviculata is now widespread across the United States eastern seaboard, and represents a substantial threat to boxwood plants in North American landscapes and nurseries. References: (1) P. Crous et al. Sydowia 54:23, 2002. (2) D. F. Farr and A. Y. Rossman. Fungal Databases, USDA-ARS. Retrieved from http://nt.ars-grin.gov/fungaldatabases , 30 August 2013. (3) N. L. Glass and G. C. Donaldson. Appl. Environ. Microbiol. 61:1323, 1995. (4) K. L. Ivors et al. Plant Dis. 96:1070, 2012.

In addition to nutrient and light availability, sedimentary biogeochemical processes can play an essential role in seagrass productivity. Previous investigations of the interactions between seagrasses and their underlying sediments have failed to clearly identify the spatio-temporal variability of the major geochemical parameters involved. Dissolved and solid phase chemical parameters in eelgrass vegetated and nearby non-vegetated sediments were investigated in this study to determine their vertical, lateral, and temporal distributions. Solid-state microelectrodes were used to investigate dissolved O2, ΣH2S, Fe2+, and Mn2+ on mm space scales. In this study, spatial heterogeneity was assessed and diurnal “ventilation” by seagrass productivity (i.e., the translocation of photosynthetically produced oxygen to the anoxic sedimentary environment) was not observed probably because benthic infaunal activity (bioturabation and bioirrigation) and microzones established by microbial processes led to highly heterogeneous sediment geochemistry where temporal variability was obscured by small-scale spatial variability. Non-vegetated sediments were less geochemically variable laterally than vegetated sediments, however, in some cases, they had similar vertical variability, possibly because they had been vegetated at an earlier time. This study demonstrates that in vegetated sediments where there is also substantial benthic macrofaunal activity it is difficult to separate the impacts of the two types of biota on sediment geochemistry and their spatial patterns, and it also raises the question of the applicability of traditional one-dimensional diagenetic models for such spatially–temporally complex sediments.

Interactions between seagrasses and associated sediment pore water chemistry in response to light–dark conditions were investigated in this study. Vertical profiling using a solid-state, gold–mercury amalgam made possible the observation of fine-scale (

Recently diverged taxa often show discordance in genetic divergence among genomic loci, where some loci show strong divergence and others show none at all. Genetic studies alone cannot distinguish among the possible mechanisms but experimental studies on other aspects of divergence may provide guidance in the inference of causes of observed discordances. In this study, we used no-choice mating trials to test for the presence of reproductive isolation between host races of the leaf-mining fly, Phytomyza glabricola on its two holly host species, Ilex coriacea and I. glabra. These trials inform our effort to determine the cause of significant differences in the degree of divergence of nuclear and mitochondrial loci of flies collected from the two host plants. We present evidence of reproductive isolation between host races in a controlled greenhouse setting: significantly more mate pairs consisting of flies from the same host plant species produced offspring than inter-host mate pairs, which produced no offspring. We also tested whether the presence of the natal or non-natal host plant affects reproductive success. Flies collected from I. coriacea were more likely to produce offspring when in the presence of the natal host, whereas the presence or absence of either the natal or non-natal host had no effect on flies collected from I. glabra. The results indicate discordant patterns of nuclear and mitochondrial divergence among host races of P. glabricola are likely due to incomplete lineage sorting, and the host races may be well on their way to becoming biological species.

To probe the effects of N-glycosylation on the fibrin-dependent plasminogenolytic activity of tissue-type plasminogen activator (t-PA), we have expressed a human recombinant t-PA (rt-PA) gene in Chinese hamster ovary (CHO) cells and in a murine C127 cell line. The resulting rt-PA glycoproteins were isolated and their associated N-linked oligosaccharide structures determined by using a combination of high-resolution Bio-Gel P-4 gel filtration chromatography, sequential exoglycosidase digestion, and methylation analysis. The results show that CHO rt-PA is N-glycosylated differently from murine C127 derived rt-PA. Further, both rt-PA's are N-glycosylated differently from t-PA derived from a human colon fibroblast and the Bowes melanoma cell line (Parekh et al., 1989), confirming that N-glycosylation of the human t-PA polypeptide is cell-type-specific. Both CHO and murine rt-PA were fractionated on lysine-Sepharose chromatography. The N-glycosylation of the major forms was analyzed and their fibrin-dependent plasminogenolytic activity determined by using an indirect amidolytic assay with Glu-plasminogen and a chromogenic plasmin substrate. The results suggest that the various forms of rt-PA differ from one another with respect to the kinetics of their fibrin-dependent activation of plasminogen. Together, these data support the notion (Wittwer et al., 1989) that N-glycosylation influences the fibrin-dependent catalytic activity of t-PA and that t-PA when expressed in different cell lines may consist of kinetically and structurally distinct glycoforms.

The locations of three cleavages that can occur in mouse 45S pre-rRNA were determined by Northern blot hybridization and S1 nuclease mapping techniques. These experiments indicate that an initial cleavage of 45S pre-rRNA can directly generate the mature 5' terminus of 18S rRNA. Initial cleavage of 45S pre-rRNA can also generate the mature 5' terminus of 5.8S rRNA, but in this case cleavage can occur at two different locations, one at the known 5' terminus of 5.8S rRNA and another 6 or 7 nucleotides upstream. This pattern of cleavage results in the formation of cytoplasmic 5.8S rRNA with heterogeneous 5' termini. Further, our results indicate that one pathway for the formation of the mature 5' terminus of 28S rRNA involves initial cleavages within spacer sequences followed by cleavages which generate the mature 5' terminus of 28S rRNA. Comparison of these different patterns of cleavage for mouse pre-rRNA with that for Escherichia coli pre-rRNA implies that there are fundamental differences in the two processing mechanisms. Further, several possible cleavage signals have been identified by comparing the cleavage sites with the primary and secondary structure of mouse rRNA (see W. E. Goldman, G. Goldberg, L. H. Bowman, D. Steinmetz, and D. Schlessinger, Mol. Cell. Biol. 3:1488-1500, 1983).