Molecular delimitation of European leafy liverworts of the genus Calypogeia based on plastid super-barcodes

Abstract

Background: Molecular research revealed that some of the European Calypogeia species described on the basis of morphological criteria are genetically heterogeneous and, in fact, are species complexes. DNA barcoding is already commonly used for correct identification of difficult to determine species, to disclose cryptic species, or detecting new taxa. Among liverworts, some DNA fragments, recommend as universal plant DNA barcodes, cause problems in amplification. Super-barcoding based on genomic data, makes new opportunities in a species identification. Results: On the basis of 22 individuals, representing 10 Calypogeia species, plastid genome was tested as a superbarcode. It is not effective in 100%, nonetheless its success of species discrimination (95.45%) is still conspicuous. It is not excluded that the above outcome may have been upset by cryptic speciation in C. suecica, as our results indicate. Having the sequences of entire plastomes of European Calypogeia species, we also discovered that the ndhB and ndhH genes and the trnT-trnL spacer identify species in 100%. Conclusions: This study shows that even if a super-barcoding is not effective in 100%, this method does not close the door to a traditional single-or multi-locus barcoding. Moreover, it avoids many complication resulting from the need to amplify selected DNA fragments. It seems that a good solution for species discrimination is a development of so-called "specific barcodes" for a given taxonomic group, based on plastome data.

Sequencing of Organellar Genomes of Nowellia curvifolia (Cephaloziaceae Jungermanniales) Revealed the Smallest Plastome with Complete Gene Set and High Intraspecific Variation Suggesting Cryptic Speciation

“…As in the closest known relatives with a plastome sequence mentioned above, the plastome of N. curvifolia consists of 121 unique genes, including 81 protein-coding genes, 6 genes of unknown function (ycf genes), 4 ribosomal RNAs and 30 transfer RNAs (Supplementary Table S2). The gene order and content seem to be stable in the most of leafy and simple thalloid liverworts [4,5,10,18,29,30], with some exceptional gene deletions as in the mentioned above Cololejeunea lanciloba [4] or mycoheterotrophic A. mirabilis [8].…”

“…Top-five most variable genes include rpoB (98 SNPs), ycf 2 (81 SNPs), ycf 1 (51), ndhD (51), ndhF (46) and ndhB (46). Four of these genes (ycf 1, ycf 2, ndhB, ndhD) were among most variable plastid genes of the leafy liverwort genus Calypogeia [5] and ycf 1 and ycf 2 were the most variable genes of simple thalloid Aneura pinguis complex [18]. Both ycf genes are often considered as promising barcodes in many taxa of vascular plants [31][32][33][34].…”

“…The comparative data for genus or species levels are very limited, since most sequenced genera consist of genome sequence of single species [4,10,29,30]. The number of SNPs between closely related Calypogeia species (C. muelleriana and C. integristipula) detected for the whole genome was one third of the amount detected for CDS for Nowellia specimens, and the intraspecific differentiation was limited to few SNPs, however all the samples had European origin [5]. Lower genetic differentiation was also detected at interand intraspecific level of complex thalloid liverwort genera Marchantia and Conocephalum [9], but this lineage is known from low genetic differentiation and slow evolutionary rate of organellar genomes [35].…”

“…Organellar genomes of early land plants are known from their stable structure and almost identical gene order present in all main evolutionary lineages [1][2][3][4][5], which include complex thalloids, simply thalloids, leafy and early divergent leafy liverworts from the order Treubiales and Haplomitriales. Despite the fact that the organellar genomes of model liverwort Marchantia polymorpha were among first sequenced plant plastomes [6] and mitogenomes [7], the number of complete genomes sequences was very limited, till the end of the second decade of XXI century, when several paper were published significantly expanding our knowledge about evolutionary dynamics of these molecules [2][3][4][5]. The current genomic resources comprise 61 mitogenomes and 59 plastomes complete sequences, but many liverwort families do not have their representatives.…”

“…The well-developed morphological features are one of the reason of overlooked cryptic diversity, which often lead to description of new species [15][16][17]. Despite recent publication of several papers dealing with liverwort plastomes [4,5,9,10,18], the GenBank entries didn't include any representative of the Cephaloziaceae family.…”