Super-Mitobarcoding in Plant Species Identification? It Can Work! The Case of Leafy Liverworts Belonging to the Genus Calypogeia

Abstract

Molecular identification of species is especially important where traditional taxonomic methods fail. The genus Calypogeia belongs to one of the tricky taxons. The simple morphology of these species and a tendency towards environmental plasticity make them complicated in identification. The finding of the universal single-locus DNA barcode in plants seems to be 'the Holy Grail'; therefore, researchers are increasingly looking for multiloci DNA barcodes or super-barcoding. Since the mitochondrial genome has low sequence variation in plants, species delimitation is usually based on the chloroplast genome. Unexpectedly, our research shows that super-mitobarcoding can also work! However, our outcomes showed that a single method of molecular species delimitation should be avoided. Moreover, it is recommended to interpret the results of molecular species delimitation alongside other types of evidence, such as ecology, population genetics or comparative morphology. Here, we also presented genetic data supporting the view that C. suecica is not a homogeneous species.

Keywords: Calypogeia; liverworts; mitogenome; species identification; super-barcoding.

Figure 1

The five most variable regions in plastome and mitogenome of Calypogeia obtained by 500 bp-long sliding window. The chart shows the π values.

Figure 2

The phylogram based on plastid and mitochondrial CDSs derived from a Bayesian analysis. The posterior probability value lower than 1 is given at the node.

Figure 3

Species delimitation by GMYC analysis. Red lines indicate the same species group.

Figure 4

Species delimitation by ASAP analysis for mitochondrial (A) and chloroplast (B) genomes. The colorful fields indicate the groups of species. Every field contains the number of individuals. Above the colorful bars, the coefficient asap-score (the lower value) and number of species (the upper value) recognized for whole dataset are presented. The cladogram supports the species delimitation presented on the bar charts.

Figure 5

Results of several analyses across the mitochondrial sequences of Calypogeia sp. using the sliding-window method. (A) The plot of the mean Kimura 2-parameter distance matrix for each 500 bp-long window. (B) The proportion of zero non-conspecific distances. (C) The proportion of zero cells in the distance matrix. The unbroken horizontal line crossing the y-axis at 0 is the proportion of zero cells in the distance matrix created from the full dataset. (D) The proportion of clades that are identical between the windows and the full dataset. (E) The sum of diagnostic nucleotide positions for all species. (F) The proportion of species that are monophyletic.