Computational protocol: Evaluation of six candidate DNA barcode loci for identification of five important invasive grasses in eastern Australia

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Protocol publication

[…] A total of 66 specimens representing five invasive grass species in Eastern Australia (Chloris gayana, Eragrostis curvula, Hyparrhenia hirta, Nassella neesiana, Nassella trichotoma) and seven native grass species (Austrostipa densiflora, Anthosachne scabra, Microlaena stipoides, Poa sieberiana, Rytidosperma caespitosum, Rytidosperma pallidum and Themeda triandra) were collected from the Australian Capital Territory, New South Wales, Queensland and Victoria in eastern Australia under the permit issued by Department of Primary Industry (Permit No. INT14/8307) (see for collection information and GenBank accession numbers). The seven native grasses were selected for examination here, as each is morphologically similar to one or more of the invasive grasses and potentially affected by their presence in areas of Eastern Australia where they overlap. These samples included field sampled specimens (N = 62) and vouchered herbarium specimens from the Australian National Herbarium (N = 2) and the National Herbarium of Victoria (N = 2). Leaf samples (appropriately 0.3 cm2 in size) were preserved in > 70% EtOh and stored at the Wagga Wagga Agricultural Institute (NSW Department of Primary Industries) and allocated unique specimen identifiers (e.g. ww00001) for sample tracking. All specimen records and associated gene sequences have been submitted into the Barcode of Life Data systems (BOLD) []. [...] Forward and reverse sequence chromatograms at each locus were assembled and checked for signal quality using SeqMan (DNA STAR package, DNAStar Inc., Madison, WI, USA). At each locus, specimen consensus sequences were exported into BioEdit [] for alignment using ClustalW [] with default parameters. The aligned sequences were also manually edited in BioEdit to remove primer reads.DNA barcode gap analysis was determined at each locus by plotting maximum intraspecific distance (Dintra) against minimum nearest neighbour distance (DNN). Intra and inter-specific pairwise genetic distances used in barcode gap analyses were generated at BOLD and adjusted by the Kimura 2-parameter (K2P) model of nucleotide evolution.At each locus, the nearest neighbour minimum interspecific p–distances (DNN) was plotted against maximum intraspecific p–distances (Dintra) to determine presence or absence of a DNA barcode gap among species ().Species monophyly was tested at each locus using both Neighbor-joining (NJ) and Maximum Likelihood (ML) tree construction methods. K2P pairwise distances used in NJ tree constructions were computed in MEGA 6.0 []. ML trees were constructed using PhyML 3.1 [], incorporating a GTR nucleotide substitution model (plus Gamma distribution). Bootstrap replication (N = 1000) was used to assess confidence of NJ clusters and ML clades.The same phylogenetic analyses were also performed on multi locus combinations (di, tri, tetra and penta) of the six loci to determine if any locus combination outperformed single loci for resolving species monophyly. […]

Pipeline specifications

Software tools BOLD, BioEdit, Clustal W, MEGA, PhyML
Applications Phylogenetics, WGS analysis