Chemical Fingerprinting of Cryptic Species and Genetic Lineages of Aneura pinguis (L.) Dumort. (Marchantiophyta, Metzgeriidae)

Abstract

Aneura pinguis (L.) Dumort. is a representative of the simple thalloid liverworts, one of the three main types of liverwort gametophytes. According to classical taxonomy, A. pinguis represents one morphologically variable species; however, genetic data reveal that this species is a complex consisting of 10 cryptic species (named by letters from A to J), of which four are further subdivided into two or three evolutionary lineages. The objective of this work was to develop an efficient method for the characterisation of plant material using marker compounds. The volatile chemical constituents of cryptic species within the liverwort A. pinguis were analysed by GC-MS. The compounds were isolated from plant material using the HS-SPME technique. Of the 66 compounds examined, 40 were identified. Of these 40 compounds, nine were selected for use as marker compounds of individual cryptic species of A. pinguis. A guide was then developed that clarified how these markers could be used for the rapid identification of the genetic lineages of A. pinguis. Multivariate statistical analyses (principal component and cluster analysis) revealed that the chemical compounds in A. pinguis made it possible to distinguish individual cryptic species (including genetic lineages), with the exception of cryptic species G and H. The classification of samples based on the volatile compounds by cluster analysis reflected phylogenetic relationships between cryptic species and genetic lineages of A. pinguis revealed based on molecular data.

Seasonal Variability of Volatile Components in Calypogeia integristipula

“…The samples collected in spring and autumn differ in the content of compounds 4 (positive loading), 13, and 76 (negative loading), which contribute greatly to PC3. The plants collected in spring had a higher content of 1-hexanol (4), while the concentration of benzeneethanol (13) and germacra-4(15),5,10( 14)-trien-1-α-ol (76) was lower than in those collected in autumn (Table 7). The loading plots showing the contribution of individual variables to the first three principal components are presented in Figures S4-S6 and S11-S13.…”

“…However, liverworts do not always show variability in the composition of metabolites depending on environmental factors. An example would be liverworts belonging to the species Aneura pinguis, which do not show such variability [13]. The factor that differentiates them is their structure.…”

“…The analysis of volatile compounds was performed using a previously described gas chromatography-mass spectrometry (GC-MS) method [13,21]. GC-MS analyses using a silphenylene phase were performed on a Trace 1310 (Thermo Scientific, Waltham, MA, USA) equipped with a Quadrex 007-5MS column (30 m, 0.25 mm, 0.25 µm).…”

“…Chemotaxonomic studies of liverworts have shown the presence of specific compounds at the family, genus, and even species level [9][10][11]. Furthermore, chemical studies of complex species, e.g., Conocephalum conicum and Anaura pinguis, revealed marker compounds enabling the identification of cryptic species [12,13]. Studies conducted on various plant species have shown that the accumulation of specialised metabolites depends on various environmental factors, including temperature, light, and soil moisture [14].…”

“…However, in recent times, species identification has often been performed based on molecular tools, including the fast-growing DNA barcoding proposed by [28]. Another method to identify taxonomically difficult species of liverworts is an analysis of the chemical composition of the compounds contained in plants [8,12,13]. Chemical composition analysis is a method for classifying and identifying plants based on confirmed differences and similarities in their biochemical composition.…”