Map showing the sites used for the dietary metabarcoding analysis (Syferkuil and Welverdiend), and collection of plant material (Timbavati, Nkuhlu and Welverdiend) were used for the extraction of POSA species lists.

The map was generated in ArcMap 10.8.1 by W. Muller using open-source shapefiles (SANBI (2012) [56], Municipal Demarcation Board [57] and the National Geographic Society [58]).</p

The composition of the final DNA sequence reference database in terms of the number of genera (histogram) and species (line plot) per family.

The composition of the final DNA sequence reference database in terms of the number of genera (histogram) and species (line plot) per family.</p

Discriminatory power of the <i>rbc</i>L and <i>trn</i>L reference datasets predicted by the application of three distance-based measures: Nearest neighbour, Meier’s best close match, as well as BOLD identification criterion, for both default and optimized thresholds for the inclusion and exclusion of singletons for the respective datasets.

All three analyses were performed to identify queries within the respective identification thresholds to genus level.</p

S1 File -

Success of a metabarcoding study is determined by the extent of taxonomic coverage and the quality of records available in the DNA barcode reference database used. This study aimed to create an rbcL and a trnL (UAA) DNA barcode sequence reference database of plant species that are potential herbivore foraging targets and commonly found in semi-arid savannas of eastern South Africa. An area-specific species list of 765 species was compiled according to plant collection records available and areas comparable to an eastern semi-arid South African savanna. Thereafter, rbcL and trnL sequences of species from this list were mined from GenBank and BOLD sequence databases according to specific quality criteria to ensure accurate taxonomic coverage and resolution. These were supplemented with sequences of 24 species sequenced for this study. A phylogenetic approach, employing Neighbor-Joining, was used to verify the topology of the reference libraries to known angiosperm phylogeny. The taxonomic reliability of these reference libraries was evaluated by testing for the presence of a barcode gap, identifying a data-appropriate identification threshold, and determining the identification accuracy of reference sequences via primary distance-based criteria. The final rbcL reference dataset consisted of 1238 sequences representing 318 genera and 562 species. The final trnL dataset consisted of 921 sequences representing 270 genera and 461 species. Barcode gaps were found for 76% of the taxa in the rbcL barcode reference dataset and 68% of the taxa in the trnL barcode reference dataset. The identification success rate, calculated with the k-nn criterion was 85.86% for the rbcL dataset and 73.72% for the trnL dataset. The datasets for rbcL and trnL combined during this study are not presented as complete DNA reference libraries, but rather as two datasets that should be used in unison to identify plants present in the semi-arid eastern savannas of South Africa.</div

Fig 3 -

Scatterplots of the Barcode gap s for species present (A) in the rbcL dataset and (B) trnL dataset. Scatterplots show the relationships between minimum interspecific distance (y-axis) and maximum intraspecific distance (x-axis) where the barcode gap is indicated by points falling above the 1:1 slope. High densities of plots are indicated by the darker colours.</p

List of species, for which <i>rbc</i>L and/or <i>trn</i>L barcode sequences were not available in public databases, that were sequenced at the NWU with the <i>rbc</i>L and <i>trn</i>L barcode, submitted to GenBank, and included in the <i>rbc</i>L and <i>trn</i>L reference datasets respectively.

List of species, for which rbcL and/or trnL barcode sequences were not available in public databases, that were sequenced at the NWU with the rbcL and trnL barcode, submitted to GenBank, and included in the rbcL and trnL reference datasets respectively.</p

List of species, for which <i>rbc</i>L and/or <i>trn</i>L barcode sequences were available in public databases, included in the <i>rbc</i>L and <i>trn</i>L reference datasets with their selected accession numbers (GenBank) and/or process IDs (BOLD).

List of species, for which rbcL and/or trnL barcode sequences were available in public databases, included in the rbcL and trnL reference datasets with their selected accession numbers (GenBank) and/or process IDs (BOLD).</p

List of species compiled in this study for the Central Bushveld and Lowveld bioregions in the Limpopo and Mpumalanga provinces in South Africa using the POSA database, related studies and field surveys of the two bioregions.

List of species compiled in this study for the Central Bushveld and Lowveld bioregions in the Limpopo and Mpumalanga provinces in South Africa using the POSA database, related studies and field surveys of the two bioregions.</p

Data_Sheet_1_Assessing the Likelihood of Gene Flow From Sugarcane (Saccharum Hybrids) to Wild Relatives in South Africa.ZIP

<p>Pre-commercialization studies on environmental biosafety of genetically modified (GM) crops are necessary to evaluate the potential for sexual hybridization with related plant species that occur in the release area. The aim of the study was a preliminary assessment of factors that may contribute to gene flow from sugarcane (Saccharum hybrids) to indigenous relatives in the sugarcane production regions of Mpumalanga and KwaZulu-Natal provinces, South Africa. In the first instance, an assessment of Saccharum wild relatives was conducted based on existing phylogenies and literature surveys. The prevalence, spatial overlap, proximity, distribution potential, and flowering times of wild relatives in sugarcane production regions based on the above, and on herbaria records and field surveys were conducted for Imperata, Sorghum, Cleistachne, and Miscanthidium species. Eleven species were selected for spatial analyses based on their presence within the sugarcane cultivation region: four species in the Saccharinae and seven in the Sorghinae. Secondly, fragments of the nuclear internal transcribed spacer (ITS) regions of the 5.8s ribosomal gene and two chloroplast genes, ribulose-bisphosphate carboxylase (rbcL), and maturase K (matK) were sequenced or assembled from short read data to confirm relatedness between Saccharum hybrids and its wild relatives. Phylogenetic analyses of the ITS cassette showed that the closest wild relative species to commercial sugarcane were Miscanthidium capense, Miscanthidium junceum, and Narenga porphyrocoma. Sorghum was found to be more distantly related to Saccharum than previously described. Based on the phylogeny described in our study, the only species to highlight in terms of evolutionary divergence times from Saccharum are those within the genus Miscanthidium, most especially M. capense, and M. junceum which are only 3 million years divergent from Saccharum. Field assessment of pollen viability of 13 commercial sugarcane cultivars using two stains, iodine potassium iodide (IKI) and triphenyl tetrazolium chloride, showed decreasing pollen viability (from 85 to 0%) from the north to the south eastern regions of the study area. Future work will include other aspects influencing gene flow such as cytological compatibility and introgression between sugarcane and Miscanthidium species.</p

Image_1_Assessing the Likelihood of Gene Flow From Sugarcane (Saccharum Hybrids) to Wild Relatives in South Africa.pdf

<p>Pre-commercialization studies on environmental biosafety of genetically modified (GM) crops are necessary to evaluate the potential for sexual hybridization with related plant species that occur in the release area. The aim of the study was a preliminary assessment of factors that may contribute to gene flow from sugarcane (Saccharum hybrids) to indigenous relatives in the sugarcane production regions of Mpumalanga and KwaZulu-Natal provinces, South Africa. In the first instance, an assessment of Saccharum wild relatives was conducted based on existing phylogenies and literature surveys. The prevalence, spatial overlap, proximity, distribution potential, and flowering times of wild relatives in sugarcane production regions based on the above, and on herbaria records and field surveys were conducted for Imperata, Sorghum, Cleistachne, and Miscanthidium species. Eleven species were selected for spatial analyses based on their presence within the sugarcane cultivation region: four species in the Saccharinae and seven in the Sorghinae. Secondly, fragments of the nuclear internal transcribed spacer (ITS) regions of the 5.8s ribosomal gene and two chloroplast genes, ribulose-bisphosphate carboxylase (rbcL), and maturase K (matK) were sequenced or assembled from short read data to confirm relatedness between Saccharum hybrids and its wild relatives. Phylogenetic analyses of the ITS cassette showed that the closest wild relative species to commercial sugarcane were Miscanthidium capense, Miscanthidium junceum, and Narenga porphyrocoma. Sorghum was found to be more distantly related to Saccharum than previously described. Based on the phylogeny described in our study, the only species to highlight in terms of evolutionary divergence times from Saccharum are those within the genus Miscanthidium, most especially M. capense, and M. junceum which are only 3 million years divergent from Saccharum. Field assessment of pollen viability of 13 commercial sugarcane cultivars using two stains, iodine potassium iodide (IKI) and triphenyl tetrazolium chloride, showed decreasing pollen viability (from 85 to 0%) from the north to the south eastern regions of the study area. Future work will include other aspects influencing gene flow such as cytological compatibility and introgression between sugarcane and Miscanthidium species.</p