Karen L. Bell

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.

New Genetic and Linguistic Analyses Show Ancient Human Influence on Baobab Evolution and Distribution in Australia

This study investigates the role of human agency in the gene flow and geographical distribution of the Australian baobab, Adansonia gregorii. The genus Adansonia is a charismatic tree endemic to Africa, Madagascar, and northwest Australia that has long been valued by humans for its multiple uses. The distribution of genetic variation in baobabs in Africa has been partially attributed to human-mediated dispersal over millennia, but this relationship has never been investigated for the Australian species. We combined genetic and linguistic data to analyse geographic patterns of gene flow and movement of word-forms for A. gregorii in the Aboriginal languages of northwest Australia. Comprehensive assessment of genetic diversity showed weak geographic structure and high gene flow. Of potential dispersal vectors, humans were identified as most likely to have enabled gene flow across biogeographic barriers in northwest Australia. Genetic-linguistic analysis demonstrated congruence of gene flow patterns and directional movement of Aboriginal loanwords for A. gregorii. These findings, along with previous archaeobotanical evidence from the Late Pleistocene and Holocene, suggest that ancient humans significantly influenced the geographic distribution of Adansonia in northwest Australia.

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.

Applying Pollen DNA Metabarcoding to the Study of Plant-Pollinator Interactions

Premise of the study: To study pollination networks in a changing environment, we need accurate, high-throughput methods. Previous studies have shown that more highly resolved networks can be constructed by studying pollen loads taken from bees, relative to field observations. DNA metabarcoding potentially allows for faster and finer-scale taxonomic resolution of pollen compared to traditional approaches (e.g., light microscopy), but has not been applied to pollination networks. Methods: We sampled pollen from 38 bee species collected in Florida from sites differing in forest management. We isolated DNA from pollen mixtures and sequenced rbcL and ITS2 gene regions from all mixtures in a single run on the Illumina MiSeq platform. We identified species from sequence data using comprehensive rbcL and ITS2 databases. Results: We successfully built a proof-of-concept quantitative pollination network using pollen metabarcoding. Discussion: Our work underscores that pollen metabarcoding is not quantitative but that quantitative networks can be constructed based on the number of interacting individuals. Due to the frequency of contamination and false positive reads, isolation and PCR negative controls should be used in every reaction. DNA metabarcoding has advantages in efficiency and resolution over microscopic identification of pollen, and we expect that it will have broad utility for future studies of plant–pollinator interactions.

An rbcL Reference Library to Aid in the Identification of Plant Species Mixtures by DNA Metabarcoding

Premise of the study: DNA metabarcoding has broad-ranging applications in ecology, aerobiology, biosecurity, and forensics. A bioinformatics pipeline has recently been published for identification using a comprehensive database of ITS2, one of the common plant DNA barcoding markers. There is, however, no corresponding database for rbcL, the other primary marker used in plants. Methods: Using publicly available data, we compiled a reference library of rbcL sequences and trained databases for use with UTAX and RDP classifier algorithms. We used this reference library, along with the existing bioinformatics pipeline and ITS2 reference library, to identify species in an artificial mixture of nine species of pollen. We have made this database publicly available in multiple formats, to allow use with multiple bioinformatics pipelines, now and in the future. Results: Using the rbcL database, in addition to the ITS2 database, we succeeded in making species-level identifications for eight species and a family-level identification of the ninth species. This is an improvement on ITS2 sequence alone. Discussion: The reference library described here will assist with identification of plant species using rbcL. By making another gene region available for standard barcoding, this will increase the resolution and accuracy of identifications.

The history of introduction of the African baobab (Adansonia digitata, Malvaceae: Bombacoideae) in the Indian subcontinent

To investigate the pathways of introduction of the African baobab, Adansonia digitata, to the Indian subcontinent, we examined 10 microsatellite loci in individuals from Africa, India, the Mascarenes and Malaysia, and matched this with historical evidence of human interactions between source and destination regions. Genetic analysis showed broad congruence of African clusters with biogeographic regions except along the Zambezi (Mozambique) and Kilwa (Tanzania), where populations included a mixture of individuals assigned to at least two different clusters. Individuals from West Africa, the Mascarenes, southeast India and Malaysia shared a cluster. Baobabs from western and central India clustered separately from Africa. Genetic diversity was lower in populations from the Indian subcontinent than in African populations, but the former contained private alleles. Phylogenetic analysis showed Indian populations were closest to those from the Mombasa-Dar es Salaam coast. The genetic results provide evidence of multiple introductions of African baobabs to the Indian subcontinent over a longer time period than previously assumed. Individuals belonging to different genetic clusters in Zambezi and Kilwa may reflect the history of trafficking captives from inland areas to supply the slave trade between the fifteenth and nineteenth centuries. Baobabs in the Mascarenes, southeast India and Malaysia indicate introduction from West Africa through eighteenth and nineteenth century European colonial networks.

Elusive Traces: Baobabs and the African Diaspora in South Asia

The history of botanical exchanges between Africa and the Indian subcontinent reaches back in time over 5000 years. Recent advances in archaeobotany have revealed these connections through evidence of food crops of African origin found at various archaeological sites in the subcontinent. However, little is known about the people that brought the crops to these places and other parts of the Indian Ocean world. This is also the case with other plants from Africa such as the charismatic baobab tree (Adansonia digitata L.) that appears to have had a longstanding presence in South Asia. Most scholarly accounts assume that ‘Arab traders’ were responsible for introducing baobabs to this region but do not offer any reasons for their doing so. Few scholars, if any, have sought to relate the dispersal of baobabs with the history of African migrations to the region. This paper reveals the elusive traces of their entwined environmental histories by linking baobab genetics with historical accounts and cultural evidence of the presence of African diasporic communities in South Asia.

Genetic diversity and biogeography of the boab Adansonia gregorii (Malvaceae: Bombacoideae)

The Kimberley region of Western Australia is recognised for its high biodiversity and many endemic species, includingthecharismaticboabtree,AdansoniagregoriiF.Muell.(Malvaceae:Bombacoideae).Inordertoassesstheeffects ofbiogeographicbarriersonA.gregorii,weexaminedthegeneticdiversityandpopulationstructureofthetreespeciesacross itsrangeintheKimberleyandadjacentareastotheeast.Geneticvariationatsixmicrosatellitelociin220individualsfromthe entire species range was examined. Five weakly divergent populations, separated by west-east and coast-inland divides, were distinguished using spatial principal components analysis. However, the predominant pattern was low geographic structure and high gene flow. Coalescent analysis detected a population bottleneck and significant gene flow across these inferred biogeographic divides. Climate cycles and coastline changes following the last glacial maximum are implicated in decreasesinancientA.gregoriipopulationsize.Ofallthepotentialgene flowvectors,variousmacropodspeciesandhumans are the most likely.

Isolation, via 454 Sequencing, and Characterization of Microsatellites for Vachellia farnesiana (Fabaceae: Mimosoideae)

Premise of the study: We isolated 15 polymorphic microsatellite markers from Vachellia farnesiana for use in population genetic studies to determine the native range of the species. Methods and Results: Initially, 454 shotgun sequencing was used to identify and design primers for 68 microsatellite loci. Of these, we trialed 47 loci in the target species, and 42 (89%) amplified a product of expected size. Fifteen of the 47 loci were screened for variation in 21 individuals from the native range of V. farnesiana in southern Mexico and 20 from northwestern Australia. Fourteen loci were polymorphic, with observed heterozygosity ranging from 0.026 to 1.00 (mean = 0.515) and two to 12 alleles per locus (average = 5.2). Cross-amplification was successful in four to 11 loci in three other Vachellia species. Conclusions: The new microsatellite loci will be useful in understanding genetic variation and investigating the role of human-mediated dispersal in the current distribution of V. farnesiana.

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.