Investigating floral choice in bees (megachilidae) using pollen metabarcoding.

Doctor of Philosophy in Genetics. University of KwaZulu-Natal, Durban 2017Interactions between plants and their pollinators are often poorly understood, specifically in a species-diverse country such as South Africa. Traditional methods of studying plant-pollinator interactions are time-consuming and imprecise. This study aimed to develop a technique that uses genetic analyses to identify pollen provenance directly from bees (Megachilidae) housed in a historic collection and to apply this technique to investigate floral choice differences in species of megachilid bees from three regionally important areas in South Africa: the Succulent Karoo, Savanna, and a widespread group, with bees occurring throughout the country. To develop the technique to accurately identify provenance, pollen was sampled from Megachile venusta specimens in the collection. Three DNA barcode regions were amplified and sequenced on an Illumina MiSeq instrument: the internal transcribed spacer 1 (ITS1) and internal transcribed spacer 2 (ITS2) regions, and the ribulose-1,5-biphosphate carboxylase (rbcL) gene. Sequenced reads were compared to sequence reference databases that were generated by extracting sequence and taxonomic data from GenBank. ITS2 reads were also compared to an established ITS2 database for Viridiplantae. More diverse plant classifications were obtained with ITS2 compared to ITS1. Amplification and sequencing of rbcL was inconsistent on pollen sampled from historic specimens. To study how floral choice differed in three South African regions, ITS2 was sequenced on Illumina HiSeq and MiSeq from pollen sampled from two different bee species from each region. Sequence reads were compared to the previously published ITS2 sequence reference database. Generalised linear models (GLM) indicated that the mean number of both plant families and species varied significantly between bee species. No significant effect of the time since bee collection was found. Taxon identifications were only confidently interpreted on family-level due to very limited local plant representation in sequence reference databases. 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 also 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 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.publishersversionPeer reviewe

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 km2 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.AG was funded by the National Research Foundation (NRF) of South Africa grant GUN 2072974 to JMG.Running costs were covered by JMG and a Research Development grant from the University of Pretoria to PdW.www.elsevier.com/locate/vetparhb201

Quantification of the genetic variation in Spirocerca lupi from the Pretoria area

Dissertation (MSc)--University of Pretoria, 2010.Geneticsunrestricte

Pollination: Impact, role-players, interactions and study – A South African perspective

Plant–pollinator interactions are essential for maintaining both pollinator and plant communities in native and agricultural environments. Animal-instigated pollination can be complex. Plants are usually visited by a number of different animal species, which in turn may visit flowers of several plant species. Therefore, the identification of the pollen carried by flower visitors is an essential first step in pollination biology. The skill and time required to identify pollen based on structure and morphology has been a major stumbling block in this field. Advances in the genetic analysis of DNA, using DNA barcoding, extracted directly from pollen offers an innovative alternative to traditional methods of pollen identification. This technique, which is reviewed in detail, can be used on pollen loads sampled from bees in the field and from specimens in historic collections. Here the importance of pollination, the role-players involved, their management and the evolution of their interactions, behaviour and morphology are reviewed – with a special focus on South African bees. Significance:  Pollen metabarcoding will enable the identification of pollen for a multitude of uses, including agriculture, conservation and forensics. Plant–pollinator interaction documentation through pollen identification gives a more certain record of a visitor being a pollinator rather than a flower visitor that could be a nectar gatherer

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