EnTAP: Bringing faster and smarter functional annotation to non-model eukaryotic transcriptomes.

EnTAP (Eukaryotic Non-Model Transcriptome Annotation Pipeline) was designed to improve the accuracy, speed, and flexibility of functional gene annotation for de novo assembled transcriptomes in non-model eukaryotes. This software package addresses the fragmentation and related assembly issues that result in inflated transcript estimates and poor annotation rates of protein-coding transcripts. Following filters applied through assessment of true expression and frame selection, open-source tools are leveraged to functionally annotate the reduced set of translated proteins. Downstream features include fast similarity search across five repositories, protein domain assignment, orthologous gene family assessment, and Gene Ontology (GO) term assignment. The final annotation integrates across multiple databases and selects an optimal assignment from a combination of weighted metrics describing similarity search score, taxonomic relationship, and informativeness. Researchers have the option to include additional filters to identify and remove contaminants, identify associated pathways, and prepare the transcripts for enrichment analysis. This fully featured pipeline is easy to install, configure, and runs significantly faster than comparable annotation packages. EnTAP is optimized to generate extensive functional information for the gene space of organisms with limited or poorly characterized genomic resources.

Comparative Transcriptomics Among Four White Pine Species.

Conifers are the dominant plant species throughout the high latitude boreal forests as well as some lower latitude temperate forests of North America, Europe, and Asia. As such, they play an integral economic and ecological role across much of the world. This study focused on the characterization of needle transcriptomes from four ecologically important and understudied North American white pines within the Pinus subgenus Strobus The populations of many Strobus species are challenged by native and introduced pathogens, native insects, and abiotic factors. RNA from the needles of western white pine (Pinus monticola), limber pine (Pinus flexilis), whitebark pine (Pinus albicaulis), and sugar pine (Pinus lambertiana) was sampled, Illumina short read sequenced, and de novo assembled. The assembled transcripts and their subsequent structural and functional annotations were processed through custom pipelines to contend with the challenges of non-model organism transcriptome validation. Orthologous gene family analysis of over 58,000 translated transcripts, implemented through Tribe-MCL, estimated the shared and unique gene space among the four species. This revealed 2025 conserved gene families, of which 408 were aligned to estimate levels of divergence and reveal patterns of selection. Specific candidate genes previously associated with drought tolerance and white pine blister rust resistance in conifers were investigated.

Warming and provenance limit tree recruitment across and beyond the elevation range of subalpine forest.

Climate niche models project that subalpine forest ranges will extend upslope with climate warming. These projections assume that the climate suitable for adult trees will be adequate for forest regeneration, ignoring climate requirements for seedling recruitment, a potential demographic bottleneck. Moreover, local genetic adaptation is expected to facilitate range expansion, with tree populations at the upper forest edge providing the seed best adapted to the alpine. Here, we test these expectations using a novel combination of common gardens, seeded with two widely distributed subalpine conifers, and climate manipulations replicated at three elevations. Infrared heaters raised temperatures in heated plots, but raised temperatures more in the forest than at or above treeline because strong winds at high elevation reduced heating efficiency. Watering increased season-average soil moisture similarly across sites. Contrary to expectations, warming reduced Engelmann spruce recruitment at and above treeline, as well as in the forest. Warming reduced limber pine first-year recruitment in the forest, but had no net effect on fourth-year recruitment at any site. Watering during the snow-free season alleviated some negative effects of warming, indicating that warming exacerbated water limitations. Contrary to expectations of local adaptation, low-elevation seeds of both species initially recruited more strongly than high-elevation seeds across the elevation gradient, although the low-provenance advantage diminished by the fourth year for Engelmann spruce, likely due to small sample sizes. High- and low-elevation provenances responded similarly to warming across sites for Engelmann spruce, but differently for limber pine. In the context of increasing tree mortality, lower recruitment at all elevations with warming, combined with lower quality, high-provenance seed being most available for colonizing the alpine, portends range contraction for Engelmann spruce. The lower sensitivity of limber pine to warming indicates a potential for this species to become more important in subalpine forest communities in the coming centuries.

Linking carbon and water relations to drought-induced mortality in Pinus flexilis seedlings.

Survival of tree seedlings at high elevations has been shown to be limited by thermal constraints on carbon balance, but it is unknown if carbon relations also limit seedling survival at lower elevations, where water relations may be more important. We measured and modeled carbon fluxes and water relations in first-year Pinus flexilis seedlings in garden plots just beyond the warm edge of their natural range, and compared these with dry-mass gain and survival across two summers. We hypothesized that mortality in these seedlings would be associated with declines in water relations, more so than with carbon-balance limitations. Rather than gradual declines in survivorship across growing seasons, we observed sharp, large-scale mortality episodes that occurred once volumetric soil-moisture content dropped below 10%. By this point, seedling water potentials had decreased below -5 MPa, seedling hydraulic conductivity had decreased by 90% and seedling hydraulic resistance had increased by >900%. Additionally, non-structural carbohydrates accumulated in aboveground tissues at the end of both summers, suggesting impairments in phloem-transport from needles to roots. This resulted in low carbohydrate concentrations in roots, which likely impaired root growth and water uptake at the time of critically low soil moisture. While photosynthesis and respiration on a leaf area basis remained high until critical hydraulic thresholds were exceeded, modeled seedling gross primary productivity declined steadily throughout the summers. At the time of mortality, modeled productivity was insufficient to support seedling biomass-gain rates, metabolism and secondary costs. Thus the large-scale mortality events that we observed near the end of each summer were most directly linked with acute, episodic declines in plant hydraulic function that were linked with important changes in whole-seedling carbon relations.

Resin duct characteristics associated with tree resistance to bark beetles across lodgepole and limber pines.

Bark beetles have recently killed billions of trees, yet conifer defenses are formidable and some trees resist attack. A primary anti-insect defense of pines is oleoresin from a system of resin ducts throughout the tree. Resin defense traits are heritable, and evidence suggests that resin duct characteristics are associated with resistance to insects. However, comparisons of resin ducts in trees killed by bark beetles to trees that resisted attack are unavailable. We compared vertical resin duct characteristics (number, density, and size) and growth rates from trees that were "resistant" (survived mass attack) versus "susceptible" (killed by attack) to bark beetles in lodgepole (Pinus contorta) and limber (Pinus flexilis) pines. Resistant trees of both species had significantly more resin ducts in recent growth than susceptible trees. Discriminant analysis (DA) correctly categorized 84% of lodgepole and 92% of limber pines as susceptible/resistant based on combinations of resin duct and growth characteristics from recent 5- through 20-year growth intervals. DA models using measures from only the most recent 5 years of growth correctly categorized 72 and 81% of lodgepole and limber pines, respectively. Comparing resistant to susceptible trees independent of species identity led to the correct categorization of 82% of trees based on factors from 5- to 20-year intervals, and 73% of trees using only resin duct counts from the most recent 5 years. We conclude that resin duct characteristics can be used to assess tree resistance to bark beetles across pine species, and offer a metric for management to enhance pest resistance.

Mountain pine beetle develops an unprecedented summer generation in response to climate warming.

The mountain pine beetle (MPB; Dendroctonus ponderosae) is native to western North America, attacks most trees of the genus Pinus, and periodically erupts in epidemics. The current epidemic of the MPB is an order of magnitude larger than any previously recorded, reaching trees at higher elevation and latitude than ever before. Here we show that after 2 decades of air-temperature increases in the Colorado Front Range, the MPB flight season begins more than 1 month earlier than and is approximately twice as long as the historically reported season. We also report, for the first time, that the life cycle in some broods has increased from one to two generations per year. Because MPBs do not diapause and their development is controlled by temperature, they are responding to climate change through faster development. The expansion of the MPB into previously inhospitable environments, combined with the measured ability to increase reproductive output in such locations, indicates that the MPB is tracking climate change, exacerbating the current epidemic.

Subsurface microbial diversity in deep-granitic-fracture water in Colorado.

A microbial community analysis using 16S rRNA gene sequencing was performed on borehole water and a granite rock core from Henderson Mine, a >1,000-meter-deep molybdenum mine near Empire, CO. Chemical analysis of borehole water at two separate depths (1,044 m and 1,004 m below the mine entrance) suggests that a sharp chemical gradient exists, likely from the mixing of two distinct subsurface fluids, one metal rich and one relatively dilute; this has created unique niches for microorganisms. The microbial community analyzed from filtered, oxic borehole water indicated an abundance of sequences from iron-oxidizing bacteria (Gallionella spp.) and was compared to the community from the same borehole after 2 weeks of being plugged with an expandable packer. Statistical analyses with UniFrac revealed a significant shift in community structure following the addition of the packer. Phospholipid fatty acid (PLFA) analysis suggested that Nitrosomonadales dominated the oxic borehole, while PLFAs indicative of anaerobic bacteria were most abundant in the samples from the plugged borehole. Microbial sequences were represented primarily by Firmicutes, Proteobacteria, and a lineage of sequences which did not group with any identified bacterial division; phylogenetic analyses confirmed the presence of a novel candidate division. This "Henderson candidate division" dominated the clone libraries from the dilute anoxic fluids. Sequences obtained from the granitic rock core (1,740 m below the surface) were represented by the divisions Proteobacteria (primarily the family Ralstoniaceae) and Firmicutes. Sequences grouping within Ralstoniaceae were also found in the clone libraries from metal-rich fluids yet were absent in more dilute fluids. Lineage-specific comparisons, combined with phylogenetic statistical analyses, show that geochemical variance has an important effect on microbial community structure in deep, subsurface systems.

Genetic variation in piñon pine, Pinus edulis, associated with summer precipitation.

Three previous reports of microgeographical variation of glycerate dehydrogenase (Gly) frequencies in piñon, Pinus edulis, established the hypothesis that Gly frequencies contribute to adaptation to heterogeneous environments, specifically to variation in soil moisture. In each of these studies, the frequency of the Gly-3 allele or of Gly-33 homozygotes was higher on dry sites than on nearby moist sites. Here we attempt to extend these observations by testing the hypothesis that Gly frequencies respond to soil moisture variation on a range-wide scale. Gly frequencies were surveyed in 11 natural populations, and the frequency of the Gly-3 allele varied from 0.27 to 0.65 among the sample sites. Elevation varied from 1650 to 3100 m, and summer precipitation, defined as precipitation from April to August, varied from 13.7 to 26.4 cm. The soil types at the collection sites were schist, quaternary volcanic or a mixture of shale and sandstone. Logistic regression revealed that Gly frequencies did not respond to either elevation or soil type, but were related to summer precipitation (P < 0.01). The correlation between summer precipitation and the frequency of the Gly-3 allele was r = -0.92 (P < 0.001). Thus, the patterns of differentiation on microgeographical scales are consistent with greater differentiation on a range-wide scale.

HISTORICAL SEPARATION AND PRESENT GENE FLOW THROUGH A ZONE OF SECONDARY CONTACT IN PONDEROSA PINE.

I examined the effects of historical division and secondary contact between eastern and western varieties of ponderosa pine (Pinus ponderosa Laws Pinaceae) on extant patterns of genetic variation. Fossil and biogeographic evidence both indicate that the current point of contact between these two varieties represents secondary contact following historical separation during the Wisconsin glaciation. Current gene flow was assessed by observing the degree of introgression of paternally inherited cpDNA and maternally inherited mtDNA polymorphisms. Both seeds and pollen are wind dispersed in ponderosa pine. Introgression was primarily from west to east, the direction of the prevailing wind, for both organelles, but introgression of cpDNA far exceeded that of mtDNA. Thus pollen is the main agent of contemporary gene flow between the two varieties. Neither seeds nor pollen showed enough introgression since secondary contact to have homogenized the two gene pools. However, allozyme differentiation was minimal. This calls into question assumptions of selective neutrality for at least some of the markers. Theory predicts that nuclear markers will show a high locus-to-locus variance of FST following historical separation. This prediction is confirmed by the allozyme data for ponderosa pine, and may provide a useful means of identifying historical separations from allele frequency data.

GENETIC DIFFERENTIATION AND HETEROZYGOSITY IN PINYON PINE ASSOCIATED WITH RESISTANCE TO HERBIVORY AND ENVIRONMENTAL STRESS.

Arizona's Sunset Crater began erupting in 1064 AD and for the next 200 years buried over 2,000 km2 in ash, cinders, and lava. Soil analyses indicate that pinyon pines (Pinus edulis) currently colonizing the cinder fields are faced with a highly stressful environment. Many of these pinyons suffer chronic, intense insect herbivory that reduces plant growth and eliminates female cone production. In contrast, herbivory among pinyons growing in neighboring sandy-loam soils is minimal. Furthermore, numerous trees within the heavily infested cinder field population suffer relatively low herbivory and maintain normal growth and reproduction. We used four polymorphic enzymes to examine the relationship between herbivore attack, environmental stress and genotypes of the adjacent cinder field, and sandy-loam soil pinyon populations. Our results demonstrate that 1) resistant trees display significant genetic differences and are more heterozygous for two enzymes associated with herbivory than susceptible trees; and 2) the cinder-soil pinyons exhibit significant genetic differences and are more heterozygous for an enzyme associated with environmental stress than the neighboring sandy-loam soil pinyons. We conclude that heterozygosity of specific or closely linked loci may facilitate pinyon resistance to herbivory and environmental stress, and that strong selection across narrow geographic boundaries resulted in rapid genetic differentiation of pinyon populations.

RELATIONSHIPS BETWEEN GENETIC VARIABILITY AND LIFE-HISTORY FEATURES OF BONY FISHES.

Correlations between genetic variation and life-history variables were obtained for 80 species of bony fishes as a means of testing the hypothesis that genetic variation is directly related to 1) opportunity for balancing selection, as indicated by fecundity, and 2) environmental variation, as indicated by capacity for population increase. Genetic data were taken from the literature, and data on longevity, age at maturity, egg size, body size, and lifetime fecundity were taken from the literature where available and were otherwise estimated from other variables. Average heterozygosity does not increase significantly with increasing fecundity. However, heterozygosity is significantly associated with short generation times, quick maturation, small maximum size, and small eggs. Thus, heterozygosity appears to increase on a demographic continuum toward maximum values in species that are most strongly selected for maximizing the intrinsic rate of increase. Such species are associated with less stable environments. Thus, the results indicate a predominate role for environmental variation in controlling genetic variation of bony fishes.

ALLOZYME AND MORPHOLOGICAL DIFFERENTIATION OF MOUNTAIN PINE BEETLES DENDROCTONUS PONDEROSAE HOPKINS (COLEOPTERA: SCOLYTIDAE) ASSOCIATED WITH HOST TREE.

The purpose of this study was to determine whether mountain pine beetles utilizing different host species were differentiated for either morphological or protein variation. Genetic differentiation among host species has been reported for the southern pine beetle, the Douglas-fir beetle, the jeffrey pine beetle, and the mountain pine beetle. However, in these studies, the host trees were sampled at separate sites, and hence geographic variation and variation due to host tree were confounded. The mountain pine beetle occasionally utilizes three host trees (ponderosa pine, lodgepole pine, and limber pine) at single sites in Colorado. Five polymorphic enzyme loci and six morphological characters were used to describe beetles resident in different hosts. Differentiation within a site among host trees was detected at two of five polymorphic proteins, and for both size and morphological shape. The magnitude of genetic differentiation among hosts within a site was approximately equivalent to the magnitude of differentiation among sites. These data suggest that the species of host tree may be an important biotic factor associated with the genetic structure of bark beetle communities. The results are discussed in terms of their potential role in the process of speciation by host race formation.

Life-history patterns and sociality in canids: Body size, reproduction, and behavior.

Empirical associations among co-adapted traits such as body size and patterns of reproduction, development, and behavior are unknown for most animal species, despite numerous theories suggesting otherwise. One way to study these complex relationships is first to consider closely related species and then to generalize findings to other groups. In the present study, relationships among body size, reproductive patterns, development, and sociality were examined in 17 members of the family Canidae (canids). Large canids are more social than smaller species, and offspring of large species achieve independence and tend to breed first at a later age. Large females give birth to absolutely larger young, but relative to their own body weight they allocate fewer resources to bringing a large pup to term. Overall, sexual dimorphism in size is small to moderate, and this is associated with monogamous mating habits and paternal care of young.