Community-wide patterns in pollen and ovule production, their ratio (P/O), and other floral traits along an elevation gradient in southwestern China.

BACKGROUND: As the male and female gametophytes of flowering plants, pollen and ovules largely determine the upper and lower boundaries of plant reproductive success. It is commonly predicted that pollen and ovule number per flower should increase, and pollen-ovule ratio (P/O) per flower should decrease with increasing elevation in response to a more stochastic pollination environment. Here, we aimed to determine the response of pollen number, ovule number, and P/O to other floral traits and elevation gradients for 84 insect-pollinated herbaceous flowering plant species in five sub-alpine and alpine communities (2709 to 3896 m a.s.l.) on Yulong Snow Mountain, southwestern China. RESULTS: Six floral traits, including P/O, floral display area, flower number, tube depth, flower shape, and pollen presentation, were highly correlated with pollen and ovule number per flower. With increasing elevation, pollen number and P/O per flower increased marginally and significantly, respectively; ovule number per individual, flower number per individual, stigma stamen separation, and inflorescence height decreased significantly. However, ovule number per flower and other floral traits (i.e., floral display area, tube depth, stigma height, stamen height, and pollen and P/O per individual) did not change with elevation. We detected significant phylogenetic signals for pollen number, ovule number, and P/O, suggesting that these traits may be highly conserved and with limited response to changing environmental conditions. CONCLUSIONS: Results revealed patterns of plant reproductive character evolution along elevation gradients and the potential factors governing their spatial variation in high-elevation environments. Plant species at high elevations are more likely adapted to cross-pollination, indicated by increased P/O per flower at high elevations on Yulong Mountain. Combined effects of phylogenetic history and plant-pollinator interactions should determine plant trait evolution.

Plants, pollinators and their interactions under global ecological change: The role of pollen DNA metabarcoding.

Anthropogenic activities are triggering global changes in the environment, causing entire communities of plants, pollinators and their interactions to restructure, and ultimately leading to species declines. To understand the mechanisms behind community shifts and declines, as well as monitoring and managing impacts, a global effort must be made to characterize plant-pollinator communities in detail, across different habitat types, latitudes, elevations, and levels and types of disturbances. Generating data of this scale will only be feasible with rapid, high-throughput methods. Pollen DNA metabarcoding provides advantages in throughput, efficiency and taxonomic resolution over traditional methods, such as microscopic pollen identification and visual observation of plant-pollinator interactions. This makes it ideal for understanding complex ecological networks and their responses to change. Pollen DNA metabarcoding is currently being applied to assess plant-pollinator interactions, survey ecosystem change and model the spatiotemporal distribution of allergenic pollen. Where samples are available from past collections, pollen DNA metabarcoding has been used to compare contemporary and past ecosystems. New avenues of research are possible with the expansion of pollen DNA metabarcoding to intraspecific identification, analysis of DNA in ancient pollen samples, and increased use of museum and herbarium specimens. Ongoing developments in sequencing technologies can accelerate progress towards these goals. Global ecological change is happening rapidly, and we anticipate that high-throughput methods such as pollen DNA metabarcoding are critical for understanding the evolutionary and ecological processes that support biodiversity, and predicting and responding to the impacts of change.

Quantitative and qualitative assessment of pollen DNA metabarcoding using constructed species mixtures.

Pollen DNA metabarcoding-marker-based genetic identification of potentially mixed-species pollen samples-has applications across a variety of fields. While basic species-level pollen identification using standard DNA barcode markers is established, the extent to which metabarcoding (a) correctly assigns species identities to mixes (qualitative matching) and (b) generates sequence reads proportionally to their relative abundance in a sample (quantitative matching) is unclear, as these have not been assessed relative to known standards. We tested the quantitative and qualitative robustness of metabarcoding in constructed pollen mixtures varying in species richness (1-9 species), taxonomic relatedness (within genera to across class) and rarity (5%-100% of grains), using Illumina MiSeq with the markers rbcL and ITS2. Qualitatively, species composition determinations were largely correct, but false positives and negatives occurred. False negatives were typically driven by lack of a barcode gap or rarity in a sample. Species richness and taxonomic relatedness, however, did not strongly impact correct determinations. False positives were likely driven by contamination, chimeric sequences and/or misidentification by the bioinformatics pipeline. Quantitatively, the proportion of reads for each species was only weakly correlated with its relative abundance, in contrast to suggestions from some other studies. Quantitative mismatches are not correctable by consistent scaling factors, but instead are context-dependent on the other species present in a sample. Together, our results show that metabarcoding is largely robust for determining pollen presence/absence but that sequence reads should not be used to infer relative abundance of pollen grains.

Pollen DNA barcoding: current applications and future prospects.

Identification of the species origin of pollen has many applications, including assessment of plant-pollinator networks, reconstruction of ancient plant communities, product authentication, allergen monitoring, and forensics. Such applications, however, have previously been limited by microscopy-based identification of pollen, which is slow, has low taxonomic resolution, and has few expert practitioners. One alternative is pollen DNA barcoding, which could overcome these issues. Recent studies demonstrate that both chloroplast and nuclear barcoding markers can be amplified from pollen. These recent validations of pollen metabarcoding indicate that now is the time for researchers in various fields to consider applying these methods to their research programs. In this paper, we review the nascent field of pollen DNA barcoding and discuss potential new applications of this technology, highlighting existing limitations and future research developments that will improve its utility in a wide range of applications.

Review and future prospects for DNA barcoding methods in forensic palynology.

Pollen can be a critical forensic marker in cases where determining geographic origin is important, including investigative leads, missing persons cases, and intelligence applications. However, its use has previously been limited by the need for a high level of specialization by expert palynologists, slow speeds of identification, and relatively poor taxonomic resolution (typically to the plant family or genus level). By contrast, identification of pollen through DNA barcoding has the potential to overcome all three of these limitations, and it may seem surprising that the method has not been widely implemented. Despite what might seem a straightforward application of DNA barcoding to pollen, there are technical issues that have delayed progress. However, recent developments of standard methods for DNA barcoding of pollen, along with improvements in high-throughput sequencing technology, have overcome most of these technical issues. Based on these recent methodological developments in pollen DNA barcoding, we believe that now is the time to start applying these techniques in forensic palynology. In this article, we discuss the potential for these methods, and outline directions for future research to further improve on the technology and increase its applicability to a broader range of situations.

Interspecific seed discounting and the fertility cost of hybridization in an endangered species.

Hybrid fertilizations can have negative demographic effects on taxa by usurping ovules that would otherwise give rise to nonhybrid offspring. The consequent reduction in conspecific matings may be exaggerated in rare taxa and constitutes a fertility cost that has rarely been quantified. Here, the effect of interspecific mating was estimated on the fecundity of locally rare red mulberry (Morus rubra), which hybridizes with introduced white mulberry (Morus alba) and red yen white hybrids. First, the asymmetry in pollen production among red, white and hybrid mulberry in two sympatric populations was quantified. The fertility cost of hybridization was then assessed experimentally by estimating seed production and rates of interspecific mating in red mulberry trees from plots where white and hybrid mulberry trees were selectively removed. On average, the percentage of mulberry pollen per plot produced by red mulberry (8%) was significantly lower than the mean for white and hybrid mulberry combined (92%). Experimentally removing white and hybrid mulberry increased the siring fertility of red mulberry by 14% but produced no change in seed set. Results indicate that seeds of red mulberry, ordinarily sired by conspecific pollen, are being discounted through fertilization of ovules by heterospecific pollen, which may contribute to local decline of red mulberry.