Is There a Universal Endurance Microbiota?

Billions of microbes sculpt the gut ecosystem, affecting physiology. Since endurance athletes' performance is often physiology-limited, understanding the composition and interactions within athletes' gut microbiota could improve performance. Individual studies describe differences in the relative abundance of bacterial taxa in endurance athletes, suggesting the existence of an "endurance microbiota", yet the taxa identified are mostly non-overlapping. To narrow down the source of this variation, we created a bioinformatics workflow and reanalyzed fecal microbiota from four 16S rRNA gene sequence datasets associated with endurance athletes and controls, examining diversity, relative abundance, correlations, and association networks. There were no significant differences in alpha diversity among all datasets and only one out of four datasets showed a significant overall difference in bacterial community abundance. When bacteria were examined individually, there were no genera with significantly different relative abundance in all four datasets. Two genera were significantly different in two datasets (Veillonella and Romboutsia). No changes in correlated abundances were consistent across datasets. A power analysis using the variance in relative abundance detected in each dataset indicated that much larger sample sizes will be necessary to detect a modest difference in relative abundance especially given the multitude of covariates. Our analysis confirms several challenges when comparing microbiota in general, and indicates that microbes consistently or universally associated with human endurance remain elusive.

Bird nests as botanical time capsules: DNA barcoding identifies the contents of contemporary and historical nests.

Bird nests in natural history collections are an abundant yet vastly underutilized source of genetic information. We sequenced the nuclear ribosomal internal transcribed spacer to identify plant species used as nest material in two contemporary (2003 and 2018) and two historical (both 1915) nest specimens constructed by Song Sparrows (Melospiza melodia) and Savannah Sparrows (Passerculus sandwichensis). A total of 13 (22%) samples yielded single, strong bands that could be identified using GenBank resources: six plants (Angiospermae), six green algae (Chlorophyta), and one ciliate (Ciliophora). Two native plant species identified in the nests included Festuca microstachys, which was introduced to the nest collection site by restoration practitioners, and Rosa californica, identified in a nest collected from a lost habitat that existed about 100 years ago. Successful sequencing was correlated with higher sample mass and DNA quality, suggesting future studies should select larger pieces of contiguous material from nests and materials that appear to have been fresh when incorporated into the nest. This molecular approach was used to distinguish plant species that were not visually identifiable, and did not require disassembling the nest specimens as is a traditional practice with nest material studies. The many thousands of nest specimens in natural history collections hold great promise as sources of genetic information to address myriad ecological questions.

Proteomic analysis of microtubule inner proteins (MIPs) in Rib72 null Tetrahymena cells reveals functional MIPs.

The core structure of motile cilia and flagella, the axoneme, is built from a stable population of doublet microtubules. This unique stability is brought about, at least in part, by a network of microtubule inner proteins (MIPs) that are bound to the luminal side of the microtubule walls. Rib72A and Rib72B were identified as MIPs in the motile cilia of the protist Tetrahymena thermophila. Loss of these proteins leads to ciliary defects and loss of additional MIPs. We performed mass spectrometry coupled with proteomic analysis and bioinformatics to identify the MIPs lost in RIB72A/B knockout Tetrahymena axonemes. We identified a number of candidate MIPs and pursued one, Fap115, for functional characterization. We find that loss of Fap115 results in disrupted cell swimming and aberrant ciliary beating. Cryo-electron tomography reveals that Fap115 localizes to MIP6a in the A-tubule of the doublet microtubules. Overall, our results highlight the complex relationship between MIPs, ciliary structure, and ciliary function.

Interpreting nature's finest insect silks (Order Embioptera): hydropathy, interrupted repetitive motifs, and fiber-to-film transformation for two neotropical species.

Silks produced by webspinners (Order Embioptera) interact with water by transforming from fiber to film, which then becomes slippery and capable of shedding water. We chose to explore this mechanism by analyzing and comparing the silk protein transcripts of two species with overlapping distributions in Trinidad but from different taxonomic families. The transcript of one, Antipaluria urichi (Clothodidae), was partially characterized in 2009 providing a control for our methods to characterize a second species: Pararhagadochir trinitatis (Scelembiidae), a family that adds to the taxon sampling for this little known order of insects. Previous reports showed that embiopteran silk protein (dubbed Efibroin) consists of a protein core of repetitive motifs largely composed of glycine (Gly), serine (Ser), and alanine (Ala) and a highly conserved C-terminal region. Based on mRNA extracted from silk glands, Next Generation sequencing, and de novo assembly, P. trinitatis silk can be characterized by repetitive motifs of Gly-Ser followed periodically by Gly-Asparagine (Asn-an unusual amino acid for Efibroins) and by a lack of Ala which is otherwise common in Efibroins. The putative N-terminal domain, composed mostly of polar, charged and bulky amino acids, is ten amino acids long with cysteine in the 10th position-a feature likely related to stabilization of the silk fibers. The 29 amino acids of the C-terminus for P. trinitatis silk closely resemble that of other Efibroin sequences, which show 74% shared identity on average. Examination of hydropathicity of Efibroins of both P. trinitatis and An. urichi revealed that these proteins are largely hydrophilic despite having a thin lipid coating on each nano-fiber. We deduced that the hydrophilic quality differs for the two species: due to Ser and Asn for P. trinitatis silk and to previously undetected spacers in An. urichi silk. Spacers are known from some spider and silkworm silks but this is the first report of such for Embioptera. Analysis of hydropathicity revealed the largely hydrophilic quality of these silks and this feature likely explains why water causes the transformation from fiber to film. We compared spun silk to the transcript and detected not insignificant differences between the two measurements implying that as yet undetermined post-translational modifications of their silk may occur. In addition, we found evidence for codon bias in the nucleotides of the putative silk transcript for P. trinitatis, a feature also known for other embiopteran silk genes.

Changes at a Critical Branchpoint in the Anthocyanin Biosynthetic Pathway Underlie the Blue to Orange Flower Color Transition in Lysimachia arvensis.

Anthocyanins are the primary pigments contributing to the variety of flower colors among angiosperms and are considered essential for survival and reproduction. Anthocyanins are members of the flavonoids, a broader class of secondary metabolites, of which there are numerous structural genes and regulators thereof. In western European populations of Lysimachia arvensis, there are blue- and orange-petaled individuals. The proportion of blue-flowered plants increases with temperature and daylength yet decreases with precipitation. Here, we performed a transcriptome analysis to characterize the coding sequences of a large group of flavonoid biosynthetic genes, examine their expression and compare our results to flavonoid biochemical analysis for blue and orange petals. Among a set of 140 structural and regulatory genes broadly representing the flavonoid biosynthetic pathway, we found 39 genes with significant differential expression including some that have previously been reported to be involved in similar flower color transitions. In particular, F3'5'H and DFR, two genes at a critical branchpoint in the ABP for determining flower color, showed differential expression. The expression results were complemented by careful examination of the SNPs that differentiate the two color types for these two critical genes. The decreased expression of F3'5'H in orange petals and differential expression of two distinct copies of DFR, which also exhibit amino acid changes in the color-determining substrate specificity region, strongly correlate with the blue to orange transition. Our biochemical analysis was consistent with the transcriptome data indicating that the shift from blue to orange petals is caused by a change from primarily malvidin to largely pelargonidin forms of anthocyanins. Overall, we have identified several flavonoid biosynthetic pathway loci likely involved in the shift in flower color in L. arvensis and even more loci that may represent the complex network of genetic and physiological consequences of this flower color polymorphism.

Two cryptic species of California mustard within Caulanthus lasiophyllus.

PREMISE: Cryptic species are evolutionarily distinct lineages lacking distinguishing morphological traits. Hidden diversity may be lurking in widespread species whose distributions cross phylogeographic barriers. This study investigates molecular and morphological variation in the widely distributed Caulanthus lasiophyllus (Brassicaceae) in comparison to its closest relatives. METHODS: Fifty-two individuals of C. lasiophyllus from across the species' range were sequenced for the nuclear ribosomal internal transcribed spacer region (ITS) and the chloroplast trnL-F region. A subset of these samples were examined for the chloroplast ndhF gene. All 52 individuals were scored for 13 morphological traits, as well as monthly and annual climate conditions at the collection locality. Morphological and molecular results are compared with the closest relatives-C. anceps and C. flavescens-in the "Guillenia Clade." To test for polyploidy, genome size estimates were made for four populations. RESULTS: Caulanthus lasiophyllus consists of two distinct lineages separated by eight ITS differences-eight times more variation than what distinguishes C. anceps and C. flavescens. Fewer variable sites were detected in trnL-F and ndhF regions, yet these data are consistent with the ITS results. The two lineages of C. lasiophyllus are geographically and climatically distinct; yet morphologically overlapping. Their genome sizes are not consistently different. CONCLUSIONS: Two cryptic species within C. lasiophyllus are distinguished at the molecular, geographic, and climatic scales. They have similar genome sizes and are morphologically broadly overlapping, but an ephemeral basal leaf character may help distinguish the species.

The ITS region provides a reliable DNA barcode for identifying reishi/lingzhi (Ganoderma) from herbal supplements.

The dietary supplement industry is rapidly growing yet, a recent study revealed that up to 60% of supplements may have substituted ingredients, some of which can be harmful contaminants or additives. When ingredients cannot be verified morphologically or biochemically, DNA barcoding complemented with a molecular phylogenetic analysis can be a powerful method for species authentication. We employed a molecular phylogenetic analysis for species authentication of the commonly used fungal supplement, reishi (Ganoderma lingzhi), by amplifying and sequencing the nuclear ribosomal internal transcribed spacer regions (ITS) with genus-specific primers. PCR of six powdered samples and one dried sample all sold as G. lucidum representing independent suppliers produced single, strong amplification products in the expected size-range for Ganoderma. Both best-hit BLAST and molecular phylogenetic analyses clearly identified the presence of G. lingzhi DNA in all seven herbal supplements. We detected variation in the ITS sequences among our samples, but all herbal supplement samples fall within a large clade of G. lingzhi ITS sequences. ITS-based phylogenetic analysis is a successful and cost-effective method for DNA-based species authentication that could be used in the herbal supplement industry for this and other fungal and plant species that are otherwise difficult to identify.

UV radiation increases phenolic compound protection but decreases reproduction in Silene littorea.

Plants respond to changes in ultraviolet (UV) radiation both morphologically and physiologically. Among the variety of plant UV-responses, the synthesis of UV-absorbing flavonoids constitutes an effective non-enzymatic mechanism to mitigate photoinhibitory and photooxidative damage caused by UV stress, either reducing the penetration of incident UV radiation or acting as quenchers of reactive oxygen species (ROS). In this study, we designed a UV-exclusion experiment to investigate the effects of UV radiation in Silene littorea. We spectrophotometrically quantified concentrations of both anthocyanins and UV-absorbing phenolic compounds in petals, calyces, leaves and stems. Furthermore, we analyzed the UV effect on the photosynthetic activity in hours of maximum solar radiation and we tested the impact of UV radiation on male and female reproductive performance. We found that anthocyanin concentrations showed a significant decrease of about 20% with UV-exclusion in petals and stems, and a 30% decrease in calyces. The concentrations of UV-absorbing compounds under UV-exclusion decreased by approximately 25% in calyces and stems, and 12% in leaves. Photochemical efficiency of plants grown under UV decreased at maximum light stress, reaching an inhibition of 58% of photosynthetic activity, but their ability to recover after light-stress was not affected. In addition, exposure to UV radiation did not affect ovule production or seed set per flower, but decreased pollen production and total seed production per plant by 31% and 69%, respectively. Our results demonstrate that UV exposure produced opposing effects on the accumulation of plant phenolic compounds and reproduction. UV radiation increased the concentration of phenolic compounds, suggesting a photoprotective role of plant phenolics against UV light, yet overall reproduction was compromised.

Genome skimming and microsatellite analysis reveal contrasting patterns of genetic diversity in a rare sandhill endemic (Erysimum teretifolium, Brassicaceae).

Barriers between islands often inhibit gene flow creating patterns of isolation by distance. In island species, the majority of genetic diversity should be distributed among isolated populations. However, a self-incompatible mating system leads to higher genetic variation within populations and very little between-population subdivision. We examine these two contrasting predictions in Erysimum teretifolium, a rare self-incompatible plant endemic to island-like sandhill habitats in Santa Cruz County, California. We used genome skimming and nuclear microsatellites to assess the distribution of genetic diversity within and among eight of the 13 remaining populations. Phylogenetic analyses of the chloroplast genomes revealed a deep separation of three of the eight populations. The nuclear ribosomal DNA cistron showed no genetic subdivision. Nuclear microsatellites suggest 83% of genetic variation resides within populations. Despite this, 18 of 28 between-population comparisons exhibited significant population structure (mean FST = 0.153). No isolation by distance existed among all populations, however when one outlier population was removed from the analysis due to uncertain provenance, significant isolation by distance emerged (r2 = 0.5611, p = 0.005). Population census size did not correlate with allelic richness as predicted on islands. Bayesian population assignment detected six genetic groupings with substantial admixture. Unique genetic clusters were concentrated at the periphery of the species' range. Since the overall distribution of nuclear genetic diversity reflects E. tereifolium's self-incompatible mating system, the vast majority of genetic variation could be sampled within any individual population. Yet, the chloroplast genome results suggest a deep split and some of the nuclear microsatellite analyses indicate some island-like patterns of genetic diversity. Restoration efforts intending to maximize genetic variation should include representatives from both lineages of the chloroplast genome and, for maximum nuclear genetic diversity, should include representatives of the smaller, peripheral populations.

Unexpected predicted length variation for the coding sequence of the sleep related gene, BHLHE41 in gorilla amidst strong purifying selection across mammals.

There is a molecular basis for many sleep patterns and disorders involving circadian clock genes. In humans, "short-sleeper" behavior has been linked to specific amino acid substitutions in BHLHE41 (DEC2), yet little is known about variation at these sites and across this gene in mammals. We compare BHLHE41 coding sequences for 27 mammals. Approximately half of the coding sequence was invariable at the nucleotide level and close to three-quarters of the amino acid alignment was identical. No other mammals had the same "short-sleeper" amino acid substitutions previously described from humans. Phylogenetic analyses based on the nucleotides of the coding sequence alignment are consistent with established mammalian relationships confirming orthology among the sampled sequences. Significant purifying selection was detected in about two-thirds of the variable codons and no codons exhibited significant signs of positive selection. Unexpectedly, the gorilla BHLHE41 sequence has a 318 bp insertion at the 5' end of the coding sequence and a deletion of 195 bp near the 3' end of the coding sequence (including the two short sleeper variable sites). Given the strong signal of purifying selection across this gene, phylogenetic congruence with expected relationships and generally conserved function among mammals investigated thus far, we suggest the indels predicted in the gorilla BHLHE41 may represent an annotation error and warrant experimental validation.

Whole Plastome Sequencing Within Silene Section Psammophilae Reveals Mainland Hybridization and Divergence With the Balearic Island Populations.

Reconstructing the phylogenetic relationships within Caryophyllaceae tribe Sileneae has been obscured by hybridization and incomplete lineage sorting. Silene is the largest genus in the Caryophyllaceae, and unraveling its evolutionary history has been particularly challenging. In order to infer the phylogenetic relationships among the five species in Silene section Psammophilae, we have performed a genome skimming approach to acquire the complete plastid genome (cpDNA), nuclear ribosomal cistron (nrDNA), and partial mitochondrial genome (mtDNA). We have included 26 populations, representing the range of each species' distribution. This section includes five morphologically similar species endemic to the Iberian Peninsula and Balearic Islands (Ibiza and Formentera), yet some of them occupy distinct edaphic habitats (e.g. maritime sands, calcareous sandstones). In addition to phylogeographic analyses, genetic structuring using the chloroplast data set was inferred with Discriminant Analysis of Principal Components (DAPC), analyses of molecular variance (AMOVA), and a partial Mantel test. Reference-guided assembly of 50 bp single-end and 250 bp paired-end Illumina reads produced the nearly complete cpDNA genome (154 kbp), partial mtDNA genome (from 81 to 114 kbp), and the nrDNA cistron (6.4 kbp). Selected variable regions of the cpDNA and mtDNA assemblies were confirmed by Sanger sequencing. Phylogenetic analyses of the mainland populations reveal incongruence among the three genomes. None of the three data sets produced relationships consistent with taxonomy or geography. In contrast, Silene cambessedesii, present in the Balearic Islands, is the only species that forms a strongly supported monophyletic clade in the cpDNA genome and is strongly differentiated with respect to the remaining taxa of the Iberian Peninsula. These results contrast with those obtained for mainland populations. Across the entire analysis, only one well-supported mainland clade of Silene littorea and Silene stockenii emerges from the southern region of the Iberian Peninsula. DAPC and AMOVA results suggest the absence of genetic structure among mainland populations of Silene section Psammophilae, whereas partial Mantel test discarded spatial correlation of genetic differentiation. The widespread incongruence between morphology-based taxonomic boundaries and phylogeography suggests a history of interspecific hybridization, in which only a substantial geographic barrier, like isolation by the Mediterranean Sea, was sufficient to create and maintain species boundaries in Silene section Psammophilae.

Stability of petal color polymorphism: the significance of anthocyanin accumulation in photosynthetic tissues.

BACKGROUND: Anthocyanins are the primary source of colour in flowers and also accumulate in vegetative tissues, where they have multiple protective roles traditionally attributed to early compounds of the metabolic pathway (flavonols, flavones, etc.). Petal-specific loss of anthocyanins in petals allows plants to escape from the negative pleiotropic effects of flavonoid and anthocyanins loss in vegetative organs, where they perform a plethora of essential functions. Herein, we investigate the degree of pleiotropy at the biochemical scale in a pink-white flower colour polymorphism in the shore campion, Silene littorea. We report the frequencies of pink and white individuals across 21 populations and underlying biochemical profiles of three flower colour variants: anthocyanins present in all tissues (pink petals), petal-specific loss of anthocyanins (white petals), and loss of anthocyanins in all tissues (white petals). RESULTS: Individuals lacking anthocyanins only in petals represent a stable polymorphism in two populations at the northern edge of the species range (mean frequency 8-21%). Whereas, individuals lacking anthocyanins in the whole plant were found across the species range, yet always at very low frequencies (< 1%). Biochemically, the flavonoids detected were anthocyanins and flavones; in pigmented individuals, concentrations of flavones were 14-56× higher than anthocyanins across tissues with differences of > 100× detected in leaves. Loss of anthocyanin pigmentation, either in petals or in the whole plant, does not influence the ability of these phenotypes to synthesize flavones, and this pattern was congruent among all sampled populations. CONCLUSIONS: We found that all colour variants showed similar flavone profiles, either in petals or in the whole plant, and only the flower colour variant with anthocyanins in photosynthetic tissues persists as a stable flower colour polymorphism. These findings suggest that anthocyanins in photosynthetic tissues, not flavonoid intermediates, are the targets of non-pollinator mediated selection.

Resolving two Haploembia (Embioptera: Oligotomidae) cryptic species: molecular data confirms parthenogenetic females can be distinguished by their antisocial behavior.

The names of two cryptic species of Haploembia found in California are resolved and methods for identification are summarized. Molecular data of the Histone III subunit was used to evaluate color and behavior as species identifiers, confirming that antisocial behavior is a good identifier for the parthenogenetic species (Haploembia tarsalis), whereas the more variable coloration patterns were helpful, but less so. A genome size ratio of 1.44 between the parthenogenetic H. tarsalis and the sexually reproducing H. solieri was observed, along with higher genetic variation within the asexual lineage. This, and the identification of what appears to be a putative hybrid, contributes to current work examining mutation rates and selective pressures on genome size in parthenogenetic populations.

Digital photography provides a fast, reliable, and noninvasive method to estimate anthocyanin pigment concentration in reproductive and vegetative plant tissues.

Anthocyanin pigments have become a model trait for evolutionary ecology as they often provide adaptive benefits for plants. Anthocyanins have been traditionally quantified biochemically or more recently using spectral reflectance. However, both methods require destructive sampling and can be labor intensive and challenging with small samples. Recent advances in digital photography and image processing make it the method of choice for measuring color in the wild. Here, we use digital images as a quick, noninvasive method to estimate relative anthocyanin concentrations in species exhibiting color variation. Using a consumer-level digital camera and a free image processing toolbox, we extracted RGB values from digital images to generate color indices. We tested petals, stems, pedicels, and calyces of six species, which contain different types of anthocyanin pigments and exhibit different pigmentation patterns. Color indices were assessed by their correlation to biochemically determined anthocyanin concentrations. For comparison, we also calculated color indices from spectral reflectance and tested the correlation with anthocyanin concentration. Indices perform differently depending on the nature of the color variation. For both digital images and spectral reflectance, the most accurate estimates of anthocyanin concentration emerge from anthocyanin content-chroma ratio, anthocyanin content-chroma basic, and strength of green indices. Color indices derived from both digital images and spectral reflectance strongly correlate with biochemically determined anthocyanin concentration; however, the estimates from digital images performed better than spectral reflectance in terms of r2 and normalized root-mean-square error. This was particularly noticeable in a species with striped petals, but in the case of striped calyces, both methods showed a comparable relationship with anthocyanin concentration. Using digital images brings new opportunities to accurately quantify the anthocyanin concentrations in both floral and vegetative tissues. This method is efficient, completely noninvasive, applicable to both uniform and patterned color, and works with samples of any size.

Reproductive success through high pollinator visitation rates despite self incompatibility in an endangered wallflower.

PREMISE OF THE STUDY: Self incompatibility (SI) in rare plants presents a unique challenge-SI protects plants from inbreeding depression, but requires a sufficient number of mates and xenogamous pollination. Does SI persist in an endangered polyploid? Is pollinator visitation sufficient to ensure reproductive success? Is there evidence of inbreeding/outbreeding depression? We characterized the mating system, primary pollinators, pollen limitation, and inbreeding/outbreeding depression in Erysimum teretifolium to guide conservation efforts. METHODS: We compared seed production following self pollination and within- and between-population crosses. Pollen tubes were visualized after self pollinations and between-population pollinations. Pollen limitation was tested in the field. Pollinator observations were quantified using digital video. Inbreeding/outbreeding depression was assessed in progeny from self and outcross pollinations at early and later developmental stages. KEY RESULTS: Self-pollination reduced seed set by 6.5× and quadrupled reproductive failure compared with outcross pollination. Pollen tubes of some self pollinations were arrested at the stigmatic surface. Seed-set data indicated strong SI, and fruit-set data suggested partial SI. Pollinator diversity and visitation rates were high, and there was no evidence of pollen limitation. Inbreeding depression (δ) was weak for early developmental stages and strong for later developmental stages, with no evidence of outbreeding depression. CONCLUSIONS: The rare hexaploid E. teretifolium is largely self incompatible and suffers from late-acting inbreeding depression. Reproductive success in natural populations was accomplished through high pollinator visitation rates consistent with a lack of pollen limitation. Future reproductive health for this species will require large population sizes with sufficient mates and a robust pollinator community.

Transcriptome and Biochemical Analysis of a Flower Color Polymorphism in Silene littorea (Caryophyllaceae).

Flower color polymorphisms are widely used as model traits from genetics to ecology, yet determining the biochemical and molecular basis can be challenging. Anthocyanin-based flower color variations can be caused by at least 12 structural and three regulatory genes in the anthocyanin biosynthetic pathway (ABP). We use mRNA-Seq to simultaneously sequence and estimate expression of these candidate genes in nine samples of Silene littorea representing three color morphs (dark pink, light pink and white) across three developmental stages in hopes of identifying the cause of flower color variation. We identified 29 putative paralogs for the 15 candidate genes in the ABP. We assembled complete coding sequences for 16 structural loci and nine of ten regulatory loci. Among these 29 putative paralogs, we identified 622 SNPs, yet only nine synonymous SNPs in Ans had allele frequencies that differentiated pigmented petals (dark pink and light pink) from white petals. These Ans allele frequency differences were further investigated with an expanded sequencing survey of 38 individuals, yet no SNPs consistently differentiated the color morphs. We also found one locus, F3h1, with strong differential expression between pigmented and white samples (>42x). This may be caused by decreased expression of Myb1a in white petal buds. Myb1a in S. littorea is a regulatory locus closely related to Subgroup 7 Mybs known to regulate F3h and other loci in the first half of the ABP in model species. We then compare the mRNA-Seq results with petal biochemistry which revealed cyanidin as the primary anthocyanin and five flavonoid intermediates. Concentrations of three of the flavonoid intermediates were significantly lower in white petals than in pigmented petals (rutin, quercetin and isovitexin). The biochemistry results for rutin, quercetin, luteolin and apigenin are consistent with the transcriptome results suggesting a blockage at F3h, possibly caused by downregulation of Myb1a.

On flavonoid accumulation in different plant parts: variation patterns among individuals and populations in the shore campion (Silene littorea).

The presence of anthocyanins in flowers and fruits is frequently attributed to attracting pollinators and dispersers. In vegetative organs, anthocyanins and other non-pigmented flavonoids such as flavones and flavonols may serve protective functions against UV radiation, cold, heat, drought, salinity, pathogens, and herbivores; thus, these compounds are usually produced as a plastic response to such stressors. Although, the independent accumulation of anthocyanins in reproductive and vegetative tissues is commonly postulated due to differential regulation, the accumulation of flavonoids within and among populations has never been thoroughly compared. Here, we investigated the shore campion (Silene littorea, Caryophyllaceae) which exhibits variation in anthocyanin accumulation in its floral and vegetative tissues. We examined the in-situ accumulation of flavonoids in floral (petals and calyxes) and vegetative organs (leaves) from 18 populations representing the species' geographic distribution. Each organ exhibited considerable variability in the content of anthocyanins and other flavonoids both within and among populations. In all organs, anthocyanin and other flavonoids were correlated. At the plant level, the flavonoid content in petals, calyxes, and leaves was not correlated in most of the populations. However, at the population level, the mean amount of anthocyanins in all organs was positively correlated, which suggests that the variable environmental conditions of populations may play a role in anthocyanin accumulation. These results are unexpected because the anthocyanins are usually constitutive in petals, yet contingent to environmental conditions in calyxes and leaves. Anthocyanin variation in petals may influence pollinator attraction and subsequent plant reproduction, yet the amount of anthocyanins may be a direct response to environmental factors. In populations on the west coast, a general pattern of increasing accumulation of flavonoids toward southern latitudes was observed in calyxes and leaves. This pattern corresponds to a gradual increase of UV-B radiation and temperature, and a decrease of rainfall toward the south. However, populations along the southern coast exposed to similar climatic stressors showed highly variable flavonoid contents, implying that other factors may play a role in flavonoid accumulation.

Transcriptome analysis of a petal anthocyanin polymorphism in the arctic mustard, Parrya nudicaulis.

Angiosperms are renown for their diversity of flower colors. Often considered adaptations to pollinators, the most common underlying pigments, anthocyanins, are also involved in plants' stress response. Although the anthocyanin biosynthetic pathway is well characterized across many angiosperms and is composed of a few candidate genes, the consequences of blocking this pathway and producing white flowers has not been investigated at the transcriptome scale. We take a transcriptome-wide approach to compare expression differences between purple and white petal buds in the arctic mustard, Parrya nudicaulis, to determine which genes' expression are consistently correlated with flower color. Using mRNA-Seq and de novo transcriptome assembly, we assembled an average of 722 bp per gene (49.81% coding sequence based on the A. thaliana homolog) for 12,795 genes from the petal buds of a pair of purple and white samples. Our results correlate strongly with qRT-PCR analysis of nine candidate genes in the anthocyanin biosynthetic pathway where chalcone synthase has the greatest difference in expression between color morphs (P/W = ∼7×). Among the most consistently differentially expressed genes between purple and white samples, we found 3× more genes with higher expression in white petals than in purple petals. These include four unknown genes, two drought-response genes (CDSP32, ERD5), a cold-response gene (GR-RBP2), and a pathogen defense gene (DND1). Gene ontology analysis of the top 2% of genes with greater expression in white relative to purple petals revealed enrichment in genes associated with stress responses including cold, drought and pathogen defense. Unlike the uniform downregulation of chalcone synthase that may be directly involved in the loss of petal anthocyanins, the variable expression of several genes with greater expression in white petals suggest that the physiological and ecological consequences of having white petals may be microenvironment-dependent.

Reproductive ecology and severe pollen limitation in the polychromic tundra plant, Parrya nudicaulis (Brassicaceae).

Pollen limitation is predicted to be particularly severe in tundra habitats. Numerous reproductive patterns associated with alpine and arctic species, particularly mechanisms associated with reproductive assurance, are suggested to be driven by high levels of pollen limitation. We studied the reproductive ecology of Parrya nudicaulis, a species with relatively large sexual reproductive investment and a wide range of floral pigmentation, in tundra habitats in interior montane Alaska to estimate the degree of pollen limitation. The plants are self-compatible and strongly protandrous, setting almost no seed in the absence of pollinators. Supplemental hand pollinations within pollinator exclusion cages indicated no cage effect on seed production. Floral visitation rates were low in both years of study and particularly infrequent in 2010. A diversity of insects visited P. nudicaulis, though syrphid and muscid flies composed the majority of all visits. Pollen-ovule ratios and levels of heterozygosity are consistent with a mixed mating system. Pollen limitation was severe; hand pollinations increased seed production per plant five-fold. Seed-to-ovule ratios remained low following hand pollinations, indicating resource limitation is likely to also be responsible for curtailing seed set. We suggest that pollen limitation in P. nudicaulis may be the result of selection favoring an overproduction of ovules as a bet-hedging strategy in this environmental context of highly variable pollen receipt.