Floral hosts of leaf-cutter bees (Megachilidae) in a biodiversity hotspot revealed by pollen DNA metabarcoding of historic specimens.

South Africa is a megadiverse country with three globally recognised biodiversity hotspots within its borders. Bees in particular show high diversity and endemism in the western part of the country. Not much is currently known about the floral host preferences of indigenous bees in South Africa, with data only available from observational studies. Pollen metabarcoding provides provenance information by utilising DNA analyses instead of floral visitation and traditional microscopic identification to identify pollinator food plants, which can be time consuming and imprecise. In this study, we sampled pollen from leaf-cutter bees (Megachilidae) specimens maintained in a historic insect collection (National Collection of Insects, South Africa) that were originally collected from two florally important areas in South Africa (Succulent Karoo and Savanna) and used metabarcoding to determine pollen provenance. We also sampled pollen from leafcutter bee species with wider distributions, that extend across many different biomes, to determine if these 'generalist' species show relaxed floral host specificity in some biomes. Metabarcoding involved sequencing of the nuclear internal transcribed spacer 2 (ITS2) region. Amplicons were compared to a sequence reference database to assign taxonomic classifications to family level. Sequence reads were also clustered to OTUs based on 97% sequence similarity to estimate numbers of plant species visited. We found no significant difference in the mean number of plant taxa visited in the Succulent Karoo and Savanna regions, but the widespread group visited significantly more floral hosts. Bees from the widespread group were also characterised by a significantly different composition in pollen assemblage. The time since specimens were collected did not have an effect on the mean number of taxa visited by any of the bee species studied. This study highlights national history collections as valuable sources of temporal and spatial biodiversity data.

Plant-pollinator interactions over time: Pollen metabarcoding from bees in a historic collection.

Pollination is a key component in agricultural food production and ecosystem maintenance, with plant-pollinator interactions an important research theme in ecological and evolutionary studies. Natural history collections provide unique access to samples collected at different spatial and temporal scales. Identification of the plant origins of pollen trapped on the bodies of pollinators in these collections provides insight into historic plant communities and pollinators' preferred floral taxa. In this study, pollen was sampled from Megachile venusta Smith bees from the National Collection of Insects, South Africa, spanning 93 years. Three barcode regions, the internal transcribed spacer 1 and 2 (ITS1 and ITS2) and ribulose-1,5-biphosphate carboxylase (rbcL), were sequenced from mixed pollen samples using a next-generation sequencing approach (MiSeq, Illumina). Sequenced reads were compared to sequence reference databases that were generated by extracting sequence and taxonomic data from GenBank. ITS1 and ITS2 were amplified successfully across all (or most) samples, while rbcL performed inconsistently. Age of sample had no impact on sequencing success. Plant classification was more informative using ITS2 than ITS1 barcode data. This study also highlights the need for comprehensive reference databases as limited local plant sequence representation in reference databases resulted in higher-level taxon classifications being more confidently interpreted. The results showed that small, insect-carried pollen samples from historic bee specimens collected from as early as 1914 can be used to obtain pollen metabarcodes. DNA metabarcoding of mixed origin pollen samples provided a faster, more accurate method of determining pollen provenance, without the need for expert palynologists. The use of historic collections to sample pollen directly from pollinators provided additional value to these collections. Sampling pollen from historic collections can potentially provide the spatial and temporal scales for investigations into changes in plant community structure or pollinator floral choice in the face of global climate change.

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.

High within-host genetic variation of the nematode Spirocerca lupi in a high-density urban dog population.

The nematode worm Spirocerca lupi has a cosmopolitan distribution and can cause the death of its final canid host, typically dogs. While its life cycle, which involves a coprophagous beetle intermediate host, a number of non-obligatory vertebrate paratenic hosts and a canid final host, is well understood, surprisingly little is known about its transmission dynamics and population genetic structure. Here we sequenced cox1 to quantify genetic variation and the factors that limit gene flow in a 300 km(2) area in South Africa. Three quarters of the genetic variation, was explained by differences between worms from the same host, whereas a quarter of the variation was explained by differences between worms from different hosts. With the help of a newly derived model we conclude that while the offspring from different infrapopulations mixes fairly frequently in new hosts, the level of admixture is not enough to homogenize the parasite populations among dogs. Small infrapopulation sizes along with clumped transmission may also result in members of infrapopulations being closely related.