Pollen DNA Research Articles

Assessment of Surface Sterilisation Approaches for the Removal of Pollen DNA from Philaenus spumarius.

Dietary analysis of herbivorous insects relies on successfully eliminating surface contamination. If this cannot be performed reliably, then it will not be possible to differentiate between plants that the insect is feeding on and plants the insect has been in contact with, either directly or via pollen. Methods in the literature often use bleach and alcohol washes to remove contamination. We perform a controlled metabarcoding baseline study on a herbivorous, xylem-feeding insect, the Meadow Spittlebug (Philaenus spumarius), using Oxford Nanopore Technologies (ONT) sequencing, and identify possible contamination that persists after washes. Despite the reported success of methods in the literature, we find that contamination is still present, leading to possible false-positive results. We hypothesise that pollen is the main source of contamination, its robust nature making it difficult to remove, and conduct a further three experiments with the goal of removing pollen from the surface of Philaenus spumarius. This study investigates the effectiveness of robust bleach/Tween/alcohol washes, sterile gut excision (including combined with Distel application), and ultraviolet light as alternative sterilisation approaches. Overall, our findings indicate that we are unable to remove surface contamination and still detect signals that may originate in the gut. In no experiment did we unequivocally detect plant DNA that originated in the P. spumarius gut.

Using pollen DNA metabarcoding to trace the geographical and botanical origin of honey from Karangasem, Indonesia

The unique floral fingerprint embedded within honey holds valuable clues to its geographical and botanical origin, playing a crucial role in ensuring authenticity and detecting adulteration. Honey from native Apis cerana and Heterotrigona itama bees in Karangasem, Indonesia, was examined utilizing pollen DNA metabarcoding for honey source identification. In this study, we used ITS2 amplicon sequencing to identify floral DNA in honey samples. The finding reveals distinct pollen signatures for each bee species. Results analysis showed A. cerana honey generated 179,267 sequence reads, assembled into Amplicon Sequence Variants (ASVs) with a total size of 485,932 bp and an average GC content of 59 %. H. itama honey generated 177,864 sequence reads, assembled into ASVs with a total size of 350,604 bp and an average GC content of 57 %. A. cerana honey exhibited a rich tapestry of pollen from eleven diverse genera, with Schleichera genus dominating at an impressive relative read abundance of 72.8 %. In contrast, H. itama honey displayed a remarkable mono-dominance of the Syzygium genus, accounting for a staggering 99.95 % of its pollen composition or relative read abundance, highlighting their distinct foraging preferences and floral resource utilization. Notably, all identified pollen taxa were indigenous to Karangasem, solidifying the geographical link between honey and its origin. This study demonstrates pollen DNA metabarcoding may identify honey floral sources. By using pollen profiles from different bee species and their foraging patterns, we may protect consumers against honey adulteration and promote sustainable beekeeping in Karangasem district. Future research could explore expanding the database of reference pollen sequences and investigating the influence of environmental factors on pollen composition in honey. Investigating this technology's economic and social effects on beekeepers and consumers may help promote fair trade and sustainable beekeeping worldwide.

Mediator kinase subunit CDK8 phosphorylates transcription factor TCP15 during tomato pollen development.

Pollen development in flowering plants has strong implications for reproductive success. Pollen DNA can be targeted to improve plant traits for yield and stress tolerance. In this study, we demonstrated that the Mediator subunit CYCLIN-DEPENDENT KINASE 8 (CDK8) is a key modulator of pollen development in tomato (Solanum lycopersicum). SlCDK8 knockout led to significant decreases in pollen viability, fruit yield, and fruit seed number. We also found that SlCDK8 directly interacts with transcription factor TEOSINTE BRANCHED1-CYCLOIDEA-PCF15 (SlTCP15) using yeast two-hybrid screens. We subsequently showed that SlCDK8 phosphorylates Ser 187 of SlTCP15 to promote SlTCP15 stability. Phosphorylated TCP15 directly bound to the TGGGCY sequence in the promoters of DYSFUNCTIONAL TAPETUM 1 (SlDYT1) and MYB DOMAIN PROTEIN 103 (SlMYB103), which are responsible for pollen development. Consistently, disruption of SlTCP15 resembled slcdk8 tomato mutants. In sum, our work identified a new substrate of Mediator CDK8 and revealed an important regulatory role of SlCDK8 in pollen development via cooperation with SlTCP15.

Crop Evolution of Foxtail Millet.

Studies on the domestication, genetic differentiation, and crop evolution of foxtail millet are reviewed in this paper. Several genetic studies were carried out to elucidate the genetic relationships among foxtail millet accessions originating mainly from Eurasia based on intraspecific hybrid pollen semi-sterility, isozymes, DNA markers, and single-nucleotide polymorphisms. Most studies suggest that China is the center of diversity of foxtail millet, and landraces were categorized into geographical groups. These results indicate that this millet was domesticated in China and spread over Eurasia, but independent origin in other regions cannot be ruled out. Furthermore, the evolution of genes was reviewed (i.e., the Waxy gene conferring amylose content in the endosperm, the Si7PPO gene controlling polyphenol oxidase, the HD1 and SiPRR37 genes controlling heading time, the Sh1 and SvLes1 genes involved in grain shattering, and the C gene controlling leaf sheath pigmentation), and the variation and distribution of these genes suggested complex patterns of evolution under human and/or natural selection.

Stepping towards pollen DNA metabarcoding: A breakthrough in forensic sciences.

This review is engaged in determining the capability of plant pollen as a significant source of evidence for the linkage between suspects and crime location in forensic sciences. Research and review articles were collected from Google Scholar, the Web of Science, and PubMed. Articles were searched using specific keywords such as "Forensic Palynology," "Pollen metabarcoding," "Plant forensics," and "Pollen" AND "criminal investigation." Boolean logic was also utilized to narrow the articles to be included in this review article. Through the literature and exploratory research, it has been observed in the current study that with advancements in technology, forensic palynology has found its application in creating an association between the crime scene and suspected individuals to have a link to it, as pollen DNA is a long-lasting investigative tool that can effectively help forensic investigations. Moreover, the literature shows that the DNA of pollen and spores has helped forensic scientists link suspects to crime scenes, and the introduction of pollen DNA metabarcoding tools has eased the efforts of palynologists to analyze pollen DNA. The introduction of DNA metabarcoding techniques to analyze pollen from plants has helped identify the geological locations of the plants and ultimately identify the culprit.

Complementarity of lacustrine pollen and sedimentary DNA in representing vegetation on the central-eastern Tibetan Plateau

Complementarity of lacustrine pollen and sedimentary DNA in representing vegetation on the central-eastern Tibetan Plateau

Transcriptome analysis provides insight into the regulatory mechanisms underlying pollen germination recovery at normal high ambient temperature in wild banana (Musa itinerans).

Cultivated banana are polyploid, with low pollen fertility, and most cultivars are male sterile, which leads to difficulties in banana breeding research. The selection of male parent with excellent resistance and pollen fertility is therefore essential for banana breeding. Wild banana (Musa itinerans) have developed many good characteristics during natural selection and constitute an excellent gene pool for breeding. Therefore, research on wild banana breeding is very important for banana breeding. In the current analysis, we examined the changes in viability of wild banana pollens at different temperatures by in vitro germination, and found that the germination ability of wild banana pollens cultured at 28°C for 2 days was higher than that of pollens cultured at 23°C (pollens that could not germinate normally under low temperature stress), 24°C (cultured at a constant temperature for 2 days) and 32°C (cultured at a constant temperature for 2 days). To elucidate the molecular mechanisms underlying the germination restoration process in wild banana pollens, we selected the wild banana pollens that had lost its germination ability under low temperature stress (23°C) as the control group (CK) and the wild banana pollens that had recovered its germination ability under constant temperature incubation of 28°C for 2 days as the treatment group (T) for transcriptome sequencing. A total of 921 differentially expressed genes (DEGs) were detected in CK vs T, of which 265 were up-regulated and 656 were down-regulated. The combined analysis of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed that the activation, metabolism of various substances (lipids, sugars, amino acids) play a major role in restoring pollen germination capacity. TCA cycle and the sesquiterpenoid and triterpenoid biosynthetic pathways were also significantly enriched in the KEGG pathway. And we found that some DEGs may be associated with pollen wall formation, DNA methylation and DNA repair. The cysteine content, free fatty acid (FFA) content, H2O2 content, fructose content, and sucrose content of pollen were increased at treatment of 28°C, while D-Golactose content was decreased. Finally, the GO pathway was enriched for a total of 24 DEGs related to pollen germination, of which 16 DEGs received targeted regulation by 14 MYBs. Our study suggests that the balance between various metabolic processes, pollen wall remodelling, DNA methylation, DNA repairs and regulation of MYBs are essential for germination of wild banana pollens.

Investigating the use of pollen DNA metabarcoding to quantify bee foraging and effects of threshold selection.

DNA metabarcoding of pollen is a useful tool for studying bee foraging ecology. However, several questions about this method remain unresolved, including the extent to which sequence read data is quantitative, which type of sequence count removal threshold to use and how that choice affects our ability to detect rare flower visits, and how sequence artefacts may confound conclusions about bee foraging behavior. To address these questions, we isolated pollen from five plant species and created treatments comprised of pollen from each species alone and combinations of pollen from multiple species that varied in richness and evenness. We used ITS2 and rbcL metabarcoding to identify plant species in the samples, compared the proportion of pollen by mass to the proportion of sequencing reads for each plant species in each treatment, and analyzed the sequencing data using both liberal and conservative thresholds. We collected pollen from foraging bees, analyzed metabarcoding data from those samples using each threshold, and compared the differences in the pollinator networks constructed from the data. Regardless of the threshold used, the relationship between the proportion of pollen by mass and sequencing reads was inconsistent, suggesting that the number of sequence reads is a poor proxy for pollen abundance in mixed-species samples. Using a liberal threshold resulted in greater detection of original plant species in mixtures but also detected additional species in mixtures and single-species samples. The conservative threshold reduced the number of additional plant species detected, but several species in mixtures were not detected above the threshold, resulting in false negatives. Pollinator networks produced using the two thresholds differed and illustrated tradeoffs between detection of rare species and estimation of network complexity. Threshold selection can have a major effect on conclusions drawn from studies using metabarcoding of bee pollen to study plant-pollinator interactions.

A primer on pollen assignment by nanopore-based DNA sequencing

The possibility to identify plants based on the taxonomic information coming from their pollen grains offers many applications within various biological disciplines. In the past and depending on the application or research in question, pollen origin was analyzed by microscopy, usually preceded by chemical treatment methods. This procedure for identification of pollen grains is both time-consuming and requires expert knowledge of morphological features. Additionally, these microscopically recognizable features usually have a low resolution at species-level. Since a few decades, DNA has been used for the identification of pollen taxa, as sequencing technologies evolved both in their handling and affordability. We discuss advantages and challenges of pollen DNA analyses compared to traditional methods. With readers with little experience in this field in mind, we present a hands-on primer for genetic pollen analysis by nanopore sequencing. As our lab mainly works with pollen collected within agroecological research projects, we focus on pollen collected by pollinating insects. We briefly consider sample collection, storage and processing in the laboratory as well as bioinformatic aspects. Currently, pollen metabarcoding is mostly conducted with next-generation sequencing methods that generate short sequence reads (<1 kb). Increasingly, however, pollen DNA analysis is carried out using the long-read generating (several kb), low-budget and mobile MinION nanopore sequencing platform by Oxford Nanopore Technologies. Therefore, we are focusing on aspects for palynology with the MinION DNA sequencing device.

Climate and human impacts inferred from a 1500-year multi-proxy record of an alpine peatland in the South-Eastern Alps

In the last decades, climate and human activities significantly affected ecosystems, particularly in mountain areas. Whereas the Alps have been extensively studied for palaeoclimatic reconstructions, little information is available about ecological changes, especially in the Southeastern Alps. This study presents a palaeoclimatic and palaeoecological reconstruction from a 1500-years-old mountain peatland record: Wölflmoor, Bozen (Italy).Through Physico-chemical proxies, testate amoeba, pollen, plant macro-fossils, and environmental DNA (eDNA) analyses, we pursued a twofold objective: 1) reconstructing the palaeoenvironmental variations of the peatland and the surrounding environment to identify the main dry/wet periods, and their correlations with climate and human activities; 2) testing the use of eDNA from a peatland in palaeoecological reconstructions. The dating highlighted a distinct change of age at a depth of 55.5–50.5 cm (ca. 800–930 calibrated years), which suggests a depositional gap (hiatus), at least in this section, occurred during the Medieval Warm Period, due to the increase in air temperature. However, the possible exploitation of the peatland by humans cannot be excluded. Indeed, the increase of Poaceae, Cerealia type, and Secale cereale, and the reduction of Fagus sylvatica and Quercus presented a high human pressure starting around 720 cal. CE. Environmental DNA metabarcoding results showed similar trends. However, it gave limitations such as the preferential amplification of the most abundant species, leading to a relatively small number of detected taxa. This is one of the first studies of eDNA metabarcoding from peatland; thus, we argue that ample room for improvement is expected in a short time, making eDNA metabarcoding a valuable complementary approach, primarily when both flora and fauna taxa are targeted.

Pollen metabarcoding reveals the floral resources and niche overlap of honeybees in forest areas

AbstractWe investigated the floral resources of the honeybee (Apis mellifera) by pollen DNA metabarcoding as the first step in honeybee conservation. We attempted to identify as many floral resources and as wide resource availability as possible in areas and compared the richness of floral resources between forests and national parks. We found that both forest and national parks can sufficiently support honeybee floral resources; however, national parks were more stable habitats with more complex vegetation structure than those of forests. Also, honeybee's preferences for floral resources were determined using niche breadth, niche overlap, and network analysis among the floral resources in forest areas. Quercus and Actinidia were the most frequently detected taxa of pollen resources in both forests and national parks by pollen metabarcoding. Even though we anticipated that more various pollen resource taxa would be found in national parks than forests, no differences in the number pollen resource taxa were detected between them. According to the niche overlap analysis result, the honeybee preferred the pollen resources in both forests and national parks, and the co‐occurrence pattern analysis of pollen resource taxa in both forests and national parks showed a simple network. Altogether, our findings suggested that DNA metabarcoding of pollen collected by the honeybee can provide useful information on the flowering plants in a given area. As the use of floral pollen resources varies spatially and temporally, periodic monitoring of pollen resources can identify which pollen is required to maintain the health of honeybee colonies.

Temporal change in floral availability leads to periods of resource limitation and affects diet specificity in a generalist pollinator.

Generalist species are core components of ecological networks and crucial for the maintenance of biodiversity. Generalist species and networks are expected to be more resilient, and therefore understanding the dynamics of specialization and generalization in ecological networks is a key focus in a time of rapid global change. Whilst diet generalization is frequently studied, our understanding of how it changes over time is limited. Here we explore temporal variation in diet specificity in the honeybee (Apis mellifera), using pollen DNA metabarcoding of honey samples, through the foraging season, over two years. We find that, overall, honeybees are generalists that visit a wide range of plants, but there is temporal variation in the degree of specialization. Temporal specialization of honeybee colonies corresponds to periods of resource limitation, identified as a lack of honey stores. Honeybees experience a lack of preferred resources in June when switching from flowering trees in spring to shrubs and herbs in summer. Investigating temporal patterns in specialization can identify periods of resource limitation that may lead to species and network vulnerability. Diet specificity must therefore be explored at different temporal scales in order to fully understand species and network stability in the face of ecological change.

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.

Pollen DNA metabarcoding reveals cryptic diversity and high spatial turnover in alpine plant-pollinator networks.

Alpine plant-pollinator communities play an important role in the functioning of alpine ecosystems, which are highly threatened by climate change. However, we still have a poor understanding of how environmental factors and spatiotemporal variability shape these communities. Here, we investigate what drives structure and beta diversity in a plant-pollinator metacommunity from the Australian alpine region using two approaches: pollen DNA metabarcoding (MB) and observations. Individual pollinators often carry pollen from multiple plant species, and therefore we expected MB to reveal a more diverse and complex network structure. We used two gene regions (ITS2 and trnL) to identify plant species present in the pollen loads of 154 insect pollinator specimens from three alpine habitats and construct MB networks, and compared them to networks based on observations alone. We compared species and interaction turnover across space for both types of networks, and evaluated their differences for plant phylogenetic diversity and beta diversity. We found significant structural differences between the two types of networks; notably, MB networks were much less specialized but more diverse than observation networks, with MB detecting many cryptic plant species. Both approaches revealed that alpine pollination networks are very generalized, but we estimated a high spatial turnover of plant species (0.79) and interaction rewiring (0.6) as well as high plant phylogenetic diversity (0.68) driven by habitat differences based on the larger diversity of plant species and species interactions detected with MB. Overall, our findings show that habitat and microclimatic heterogeneity drives diversity and fine-scale spatial turnover of alpine plant-pollinator networks.

Assessing the lysis of diverse pollen from bulk environmental samples for DNA metabarcoding

Pollen is ubiquitous year-round in bulk environmental samples and can provide useful information on previous and current plant communities. Characterization of pollen has traditionally been completed based on morphology, requiring significant time and expertise. DNA metabarcoding is a promising approach for characterizing pollen from bulk environmental samples, but accuracy hinges on successful lysis of pollen grains to free template DNA. In this study, we assessed the lysis of morphologically and taxonomically diverse pollen from one of the most common bulk environmental sample types for DNA metabarcoding, surface soil. To achieve this, a four species artificial pollen mixture was spiked into surface soils collected from Colorado, North Carolina, and Pennsylvania, and subsequently subjected to DNA extraction using both the PowerSoil and PowerSoil Pro Kits (Qiagen) with a heated incubation (either 65 °C or 90 °C). Amplification and Illumina sequencing of the internal transcribed spacer subunit 2 (ITS2) was completed in duplicate for each sample (total n, 76), and the resulting sequencing reads taxonomically identified using GenBank. The PowerSoil Pro Kit statistically outperformed the PowerSoil Kit for total DNA yield. When using either kit, incubation temperature (65 °C or 90 °C) used had no impact on the recovery of DNA, plant amplicon sequence variants (ASVs), or total plant ITS2 reads. This study highlighted that lysis of pollen in bulk environmental samples is feasible using commercially available kits, and downstream DNA metabarcoding can be used to accurately characterize pollen DNA from such sample types.

Forest‐permafrost feedbacks and glacial refugia help explain the unequal distribution of larch across continents

AbstractAimThe continental‐scale distribution of plant functional types, such as evergreen and summergreen needle‐leaf forest, is assumed to be determined by contemporary climate. However, the distribution of summergreen needle‐leaf forest of larch (Larix Mill.) differs markedly between the continents, despite relatively similar climatic conditions. The reasons for these differences are little understood. Our aim is to identify potential triggers and drivers of the current distribution patterns by comparing species' bioclimatic niches, glacial refugia and postglacial recolonization patterns.LocationNorthern hemisphere.TaxonSpecies of the genus Larix (Mill.).MethodsWe compare species distribution and dominance using species ranges and sites of dominance, as well as their occurrence on modelled permafrost extent, and active layer thickness (ALT). We compare the bioclimatic niches and calculate the niche overlap between species, using the same data in addition to modern climate data. We synthesize pollen, macrofossil and ancient DNA palaeo‐evidence of past Larix occurrences of the last 60,000 years and track differences in distribution patterns through time.ResultsBioclimatic niches show large overlaps between Asian larch species and American Larix laricina. The distribution across various degrees of permafrost extent is distinctly different for Asian L. gmelinii and L. cajanderi compared to the other species, whereas the distribution on different depths of ALT is more similar among Asian and American species. Northern glacial refugia for Larix are only present in eastern Asia and Alaska.Main ConclusionThe dominance of summergreen larches in Asia, where evergreen conifers dominate most of the rest of the boreal forests, is dependent on the interaction of several factors which allows Asian L. gmelinii and L. cajanderi to dominate where these factors coincide. These factors include the early postglacial spread out of northern glacial refugia in the absence of competitors as well as a positive feedback mechanism between frozen ground and forest.

Individual-level specialisation and interspecific resource partitioning in bees revealed by pollen DNA metabarcoding.

It is increasingly recognised that intraspecific variation in traits, such as morphology, behaviour, or diet is both ubiquitous and ecologically important. While many species of predators and herbivores are known to display high levels of between-individual diet variation, there is a lack of studies on pollinators. It is important to fill in this gap because individual-level specialisation of flower-visiting insects is expected to affect their efficiency as pollinators with consequences for plant reproduction. Accordingly, the aim of our study was to quantify the level of individual-level specialisation and foraging preferences, as well as interspecific resource partitioning, in three co-occurring species of bees of the genus Ceratina (Hymenoptera: Apidae: Xylocopinae), C. chalybea, C. nigrolabiata, and C. cucurbitina. We conducted a field experiment where we provided artificial nesting opportunities for the bees and combined a short-term mark-recapture study with the dissection of the bees’ nests to obtain repeated samples from individual foraging females and complete pollen provisions from their nests. We used DNA metabarcoding based on the ITS2 locus to identify the composition of the pollen samples. We found that the composition of pollen carried on the bodies of female bees and stored in the brood provisions in their nests significantly differed among the three co-occurring species. At the intraspecific level, individual females consistently differed in their level of specialisation and in the composition of pollen carried on their bodies and stored in their nests. We also demonstrate that higher generalisation at the species level stemmed from larger among-individual variation in diets, as observed in other types of consumers, such as predators. Our study thus reveals how specialisation and foraging preferences of bees change from the scale of individual foraging bouts to complete pollen provisions accumulated in their nests over many days. Such a multi-scale view of foraging behaviour is necessary to improve our understanding of the functioning of plant-flower visitor communities.

Identification of Seasonal Honey Based on Quantitative Detection of Typical Pollen DNA

Monofloral honey is produced from the nectar of a single predominant botanical species in a particular season and has certain unique properties. Valuable monofloral honey produced in a particular season with unique properties is often targeted for adulteration. Herein, a method for the identification of monofloral honey and determination of its production season was developed. Major nectar plants, including Prunus sp., Robinia pseudoacacia, Castanea sp., and Kalopanax sp., were selected to evaluate the honey produced between April and July in South Korea. Results showed that the highest amount of DNA from each plant was detected in the corresponding flowering season. The pollens tended to accumulate in the honeycomb after the flowering season. The accumulations result in an increase in the diversity of pollen detected in honey. Additionally, DNA quantity of each plant decreased in the samples as the number of plant DNA types increased from May to July. Moreover, the authenticity of the commercial monofloral honey samples showed only cherry blossom honey was found authentic, which exhibited the expected high amount of Prunus sp. DNA. This molecular tool is expected to be useful in verifying the origin of monofloral honey and its production season.

Temporal Patterns of Honeybee Foraging in a Diverse Floral Landscape Revealed Using Pollen DNA Metabarcoding of Honey.

SynopsisUnderstanding the plants pollinators use through the year is vital to support pollinator populations and mitigate for declines in floral resources due to habitat loss. DNA metabarcoding allows the temporal picture of nectar and pollen foraging to be examined in detail. Here, we use DNA metabarcoding to examine the forage use of honeybees (Apis mellifera L.) within a florally diverse landscape within the UK, documenting the key forage plants used and seasonal progression over two years. The total number of plant taxa detected in the honey was 120, but only 16 of these were found with a high relative read abundance of DNA, across the main foraging months (April–September). Only a small proportion of the available flowering genera in the landscape were used by the honeybees. The greatest relative read abundance came from native or near-native plants, including Rubus spp., Trifolium repens, the Maleae tribe including Crataegus, Malus, and Cotoneaster, and Hedera helix. Tree species were important forage in the spring months, followed by increased use of herbs and shrubs later in the foraging season. Garden habitat increased the taxon richness of native, near-native and horticultural plants found in the honey. Although horticultural plants were rarely found abundantly within the honey samples, they may be important for increasing nutritional diversity of the pollen forage.

Host plants and pollination regions for the long-distance migratory noctuid moth, Hadula trifolii Hufnagel in China.

Nocturnal moths are important pollinators of plants. The clover cutworm, Hadula trifolii, is a long‐distance migratory nocturnal moth. Although the larvae of H. trifolii are polyphagous pests of many cultivated crops in Asia and Europe, the plant species pollinated by the adult are unclear. Pollen species that were attached to individual migrating moths of H. trifolii were identified based on pollen morphology and DNA to determine their host plants, geographic origin, and pollination areas. The moths were collected on their seasonal migration pathway at a small island, namely Beihuang, in the center of the Bohai Sea of China during 2014 to 2018. Pollen was detected on 28.60% of the female moths and 29.02% of the male, mainly on the proboscis, rarely on compound eyes and antennae. At least 92 species of pollen from 42 plant families, mainly from Asteraceae, Amaranthaceae, and Pinaceae, distributed throughout China were found on the test moths. Migratory H. trifolii moths visited herbaceous plants more than woody plants. Pollen of Macadamina integrifolia or M. tetraphylla was found on moths early in the migratory season. These two species are distributed in Guangdong, Yunnan, and Taiwan provinces in China, indicating that migratory moths probably traveled about 2000 km from southern China to the Beihuang Island in northern China. Here, by identifying plant species using pollen, we gained a better understanding of the interactions between H. trifolii moths and a wide range of host plants in China. This work provides valuable and unique information on the geographical origin and pollination regions for H. trifolii moths.