Shawn A. Steffan

Using next-generation sequencing approaches to isolate simple sequence repeat (SSR) loci in the plant sciences

The application of next-generation sequencing (NGS) technologies for the development of simple sequence repeat (SSR) or microsatellite loci for genetic research in the botanical sciences is described. Microsatellite markers are one of the most informative and versatile DNA-based markers used in plant genetic research, but their development has traditionally been a difficult and costly process. NGS technologies allow the efficient identification of large numbers of microsatellites at a fraction of the cost and effort of traditional approaches. The major advantage of NGS methods is their ability to produce large amounts of sequence data from which to isolate and develop numerous genome-wide and gene-based microsatellite loci. The two major NGS technologies with emergent application in SSR isolation are 454 and Illumina. A review is provided of several recent studies demonstrating the efficient use of 454 and Illumina technologies for the discovery of microsatellites in plants. Additionally, important aspects during NGS isolation and development of microsatellites are discussed, including the use of computational tools and high-throughput genotyping methods. A data set of microsatellite loci in the plastome and mitochondriome of cranberry (Vaccinium macrocarpon Ait.) is provided to illustrate a successful application of 454 sequencing for SSR discovery. In the future, NGS technologies will massively increase the number of SSRs and other genetic markers available to conduct genetic research in understudied but economically important crops such as cranberry.

Trophic hierarchies illuminated via amino acid isotopic analysis.

Food web ecologists have long sought to characterize the trophic niches of animals using stable isotopic analysis. However, distilling trophic position from isotopic composition has been difficult, largely because of the variability associated with trophic discrimination factors (inter-trophic isotopic fractionation and routing). We circumvented much of this variability using compound-specific isotopic analysis (CSIA). We examined the 15N signatures of amino acids extracted from organisms reared in pure culture at four discrete trophic levels, across two model communities. We calculated the degree of enrichment at each trophic level and found there was a consistent trophic discrimination factor (~7.6‰). The constancy of the CSIA-derived discrimination factor permitted unprecedented accuracy in the measurement of animal trophic position. Conversely, trophic position estimates generated via bulk-15N analysis significantly underestimated trophic position, particularly among higher-order consumers. We then examined the trophic hierarchy of a free-roaming arthropod community, revealing the highest trophic position (5.07) and longest food chain ever reported using CSIA. High accuracy in trophic position estimation brings trophic function into sharper focus, providing greater resolution to the analysis of food webs.

Cascading diversity effects transmitted exclusively by behavioral interactions

Consumer diversity generally increases resource consumption. Consumers can also impact other species by altering their behavior, but it is unclear how such nonconsumptive effects scale with diversity. We independently manipulated predator species richness and the consumptive and nonconsumptive effects of predator communities to measure the role of each factor in protecting Brassica oleracea plants from caterpillar herbivory. Plant biomass was greatest when diverse predator assemblages induced antipredator behaviors in herbivores, an effect not further strengthened when predators could also kill caterpillars. Predators within diverse communities were more likely to forage on plants and to disrupt herbivore feeding, reflecting greater aversion to foraging among conspecific than heterospecific competitors. Predator diversity, therefore, initiated behavioral changes at the predator and then herbivore trophic levels, both to the benefit of plants. Our results indicate that strong, emergent species-richness effects can be transmitted entirely through behavioral interactions, independent of resource consumption.

A new pest species of the mealybug genus Ferrisia fullaway (Hemiptera: Pseudococcidae) from the United States.

Abstract A new pest species of Ferrisia (Hemiptera: Pseudococcidae), Ferrisia gilli Gullan, is described and illustrated based on the adult female, third-instar female, and first- and second-instar nymphs. Observation of pharate third-instar males and females failed to reveal sexual dimorphism of the second instar. Diagnosis of this new species was facilitated by the collection of nucleotide sequence data from fragments of a mitochondrial gene (COI) and two nuclear genes (EF-1α and 28S). The first phylogenetic study of Ferrisia is presented; combined and separate analyses of the three gene regions support monophyly of F. gilli and suggest that Ferrisia virgata (Cockerell) is a species complex. The known distribution of F. gilli from California and the southeastern United States is reported. It may be native to the southeastern states. This mealybug seems to be polyphagous because it feeds on a variety of species of woody plants, both evergreen and deciduous, as well as on monocots. It has at least three generations annually in central California, where it is newly recognized as a pest in pistachio and almond orchards, but has been present in northern California since at least 1968. The main problems caused by this mealybug in pistachio orchards are contamination of foliage and fruit with honeydew and the concomitant promotion of two major fungal pathogens.

Microbes are trophic analogs of animals

Significance We report evidence that microbes are trophically equivalent to animals. When bacteria or fungi are fed the same diets as animals, the microbes register the same trophic position as animals. This discovery reframes how microbes can be viewed within food chains and facilitates the inclusion of the microbiome in functional diversity studies. To demonstrate the broad applicability of our approach, we investigated the ancient symbioses represented by leaf-cutter ant fungus gardens, revealing four discrete trophic levels within this community and providing evidence that fungi, not ants, are the dominant herbivores of the Neotropics. Altogether, we show that microbes can be integrated with plants and animals in a food chain, thereby unifying the macro- and microbiome in studies of trophic ecology. In most ecosystems, microbes are the dominant consumers, commandeering much of the heterotrophic biomass circulating through food webs. Characterizing functional diversity within the microbiome, therefore, is critical to understanding ecosystem functioning, particularly in an era of global biodiversity loss. Using isotopic fingerprinting, we investigated the trophic positions of a broad diversity of heterotrophic organisms. Specifically, we examined the naturally occurring stable isotopes of nitrogen (15N:14N) within amino acids extracted from proteobacteria, actinomycetes, ascomycetes, and basidiomycetes, as well as from vertebrate and invertebrate macrofauna (crustaceans, fish, insects, and mammals). Here, we report that patterns of intertrophic 15N-discrimination were remarkably similar among bacteria, fungi, and animals, which permitted unambiguous measurement of consumer trophic position, independent of phylogeny or ecosystem type. The observed similarities among bacterial, fungal, and animal consumers suggest that within a trophic hierarchy, microbiota are equivalent to, and can be interdigitated with, macrobiota. To further test the universality of this finding, we examined Neotropical fungus gardens, communities in which bacteria, fungi, and animals are entwined in an ancient, quadripartite symbiosis. We reveal that this symbiosis is a discrete four-level food chain, wherein bacteria function as the apex carnivores, animals and fungi are meso-consumers, and the sole herbivores are fungi. Together, our findings demonstrate that bacteria, fungi, and animals can be integrated within a food chain, effectively uniting the macro- and microbiome in food web ecology and facilitating greater inclusion of the microbiome in studies of functional diversity.

Diet quality influences isotopic discrimination among amino acids in an aquatic vertebrate.

Stable nitrogen isotopic composition of amino acids (δ15NAA) has recently been employed as a powerful tool in ecological food web studies, particularly for estimating the trophic position (TP) of animal species in food webs. However, the validity of these estimates depends on the consistency of the trophic discrimination factor (TDF; - Δδ15NAA at each shift of trophic level) among a suite of amino acids within the tissues of consumer species. In this study, we determined the TDF values of amino acids in tadpoles (the Japanese toad, Bufo japonicus) reared exclusively on one of three diets that differed in nutritional quality. The diets were commercial fish-food pellets (plant and animal biomass), bloodworms (animal biomass), and boiled white rice (plant carbohydrate), representing a balanced, protein-rich, and protein-poor diet, respectively. The TDF values of two “source amino acids” (Src-AAs), methionine and phenylalanine, were close to zero (0.3–0.5‰) among the three diets, typifying the values reported in the literature (∼0.5‰ and ∼0.4‰, respectively). However, TDF values of “trophic amino acids” (Tr-AAs) including alanine, valine, leucine, isoleucine, and glutamic acid varied by diet: for example, the glutamic acid TDF was similar to the standard value (∼8.0‰) when tadpoles were fed either the commercial pellets (8.0‰) or bloodworms (7.9‰), but when they were fed boiled rice, the TDF was significantly reduced (0.6‰). These results suggest that a profound lack of dietary protein may alter the TDF values of glutamic acid (and other Tr-AAs and glycine) within consumer species, but not the two Src-AAs (i.e., methionine and phenylalanine). Knowledge of how a nutritionally poor diet can influence the TDF of Tr- and Src-AAs will allow amino acid isotopic analyses to better estimate TP among free-roaming animals.

The American cranberry mitochondrial genome reveals the presence of selenocysteine (tRNA-Sec and SECIS) insertion machinery in land plants.

This is the first de novo assembly and annotation of a complete mitochondrial genome in the Ericales order from the American cranberry (Vaccinium macrocarpon Ait.). Moreover, only four complete Asterid mitochondrial genomes have been made publicly available. The cranberry mitochondrial genome was assembled and reconstructed from whole genome 454 Roche GS-FLX and Illumina shotgun sequences. Compared with other Asterids, the reconstruction of the genome revealed an average size mitochondrion (459,678 nt) with relatively little repetitive sequences and DNA of plastid origin. The complete mitochondrial genome of cranberry was annotated obtaining a total of 34 genes classified based on their putative function, plus three ribosomal RNAs, and 17 transfer RNAs. Maternal organellar cranberry inheritance was inferred by analyzing gene variation in the cranberry mitochondria and plastid genomes. The annotation of cranberry mitochondrial genome revealed the presence of two copies of tRNA-Sec and a selenocysteine insertion sequence (SECIS) element which were lost in plants during evolution. This is the first report of a land plant possessing selenocysteine insertion machinery at the sequence level.

15N-enrichment of plant tissue to mark phytophagous insects, associated parasitoids, and flower-visiting entomophaga

New techniques are presented on the use of 15N to mark insects. 15N, a stable isotope of nitrogen, was enriched above natural abundance in plant and insect tissues. Two laboratory studies demonstrated that enriched 15N‐concentrations could be tracked from plant to insect using mass spectrometry. In the first study, adult Cotesia plutellae (Kurdjimov) (Hymenoptera: Braconidae) and Hippodamia convergens Guérin‐Méneville (Coleoptera: Coccinellidae) were allowed to feed at the flowers of rapid‐cycling Chinese cabbage plants that had been fertilized with 15N‐enriched potassium nitrate (KNO3‐15NO3). Both insect groups were found to have significantly elevated 15N levels after visiting the flowers of the 15N‐enriched plants for 48 hours. In the second study, 15N‐enriched bean plant (Phaseolus vulgaris L.) tissue was incorporated into an insect diet and fed to navel orangeworms, Amyelois transitella (Walker) (Lepidoptera: Pyralidae). When the navel orangeworm larvae were 4th instars, they were removed from the diet and exposed to the parasitoid, Goniozus legneri Gordh (Hymenoptera: Bethylidae). Results indicated that the enriched 15N‐concentration of the bean plants was transferred to the navel orangeworms and, subsequently, to the parasitoids. This work may provide useful techniques to help establish whether agriculturally important entomophaga visiting 15N‐enriched flowers or parasitizing enriched sentinel larvae in the field can be effectively marked with 15N.

A syndrome of mutualism reinforces the lifestyle of a sloth.

Arboreal herbivory is rare among mammals. The few species with this lifestyle possess unique adaptions to overcome size-related constraints on nutritional energetics. Sloths are folivores that spend most of their time resting or eating in the forest canopy. A three-toed sloth will, however, descend its tree weekly to defecate, which is risky, energetically costly and, until now, inexplicable. We hypothesized that this behaviour sustains an ecosystem in the fur of sloths, which confers cryptic nutritional benefits to sloths. We found that the more specialized three-toed sloths harboured more phoretic moths, greater concentrations of inorganic nitrogen and higher algal biomass than the generalist two-toed sloths. Moth density was positively related to inorganic nitrogen concentration and algal biomass in the fur. We discovered that sloths consumed algae from their fur, which was highly digestible and lipid-rich. By descending a tree to defecate, sloths transport moths to their oviposition sites in sloth dung, which facilitates moth colonization of sloth fur. Moths are portals for nutrients, increasing nitrogen levels in sloth fur, which fuels algal growth. Sloths consume these algae-gardens, presumably to augment their limited diet. These linked mutualisms between moths, sloths and algae appear to aid the sloth in overcoming a highly constrained lifestyle.

Discrimination of American Cranberry Cultivars and Assessment of Clonal Heterogeneity Using Microsatellite Markers

Cranberries (Vaccinium macrocarpon Ait.) are an economically important fruit crop derived from a North American native species. We report the application of 12 simple sequence repeats (SSR) or microsatellite markers to assess the genetic diversity of cranberry cultivars. We studied 164 samples of 21 different cranberry cultivars, 11 experimental hybrids, and 6 representative accessions of wild species. Genetic cluster analysis, based on 117 SSR alleles, differentiated the major cranberry cultivars. However, some cranberry cultivar subclone variants and mislabeled samples were observed. Consensus genetic profiles identified the most likely clonal representatives of several important cranberry cultivars (e.g., “Ben Lear,” “Howes,” and “Stevens”). The markers were further used to confirm putative parents of several hybrid progenies. The long-term goal of our studies is to identify, preserve, and utilize unique genetic materials to breed improved cranberries. Attaining this goal will help growers maintain sustainability under changing economic and environmental conditions.