Taxonomic fingerprints of Plantago atrata Hoppe – a Review

Plantago atrata Hoppe (dark plantain; Plantaginaceae) is a lesser-studied narrow-leaved Plantain species and in the present review, we summarize the knowledge about the plant. The distribution of this alpine species and reported subspecies is described. Emphasis is made on P. atrata taxonomic features including botanical characteristics and phylogenetic discrimination via DNA barcoding and metabolic composition. The ecological significance of this plant species is discussed from floristic and nutrient value points of view. The collected information would serve as a basis for further in-depth research on physiological and phytochemical aspects of P. atrata, which would enable the understanding of this plant’s high-altitude adaptation and its possible contribution to beneficial applications for other organisms.


General distribution of plantain and Plantago atrata's habitat
Plantago L. species belongs to the family Plantaginaceae.The representatives of the genus Plantago are perennial herbaceous plants or dwarf shrubs that are usually wind-pollinated [1], [2].There are over 200 species of plantain distributed mainly in temperate areas [3], [4].The plantain grows in meadows, dry grassy and sandy places, along roads, and in yards, up to 2000 m above sea level.The plantain was distributed mainly in Europe, and it was later transferred to other continents.The North American Indians called the plant "the white man's footstep" because it appeared wherever Europeans passed.Hence the name of the genus -"planta" from Latin means "sole" [5].An alternative interpretation is that the name is a reference to the broad leaves touching the ground that are found in basal rosettes in some species [6].
The species is characteristic of regions enriched in Nardus stricta communities at elevations ranging from 1500 to 2500 m a.s.l. with high and prolonged snow cover [24].This habitat is typical of the highest mountains in Europe, especially the subalpine and alpine areas.The soil is acidic, on various rock bases, including limestone, but in the latter case, the topsoil is decalcified due to high annual rainfall [24].

Botanical characteristics
Plantago atrata Hoppe (P.montana sensu Lam.) is a perennial herb, acaulous, erect, 3-30 cm tall.The leaves are 5-21 x 2-7 cm, linear to linear-lanceolate to lanceolate, green, glabrous, or sparsely pubescent, rarely villous at the base, and are positioned in basal rosettes (Figure 2a, b).The peduncles are 4.5-30 cm, as long as or longer than leaves, erect or ascending, sparsely hairy.The spike is 2-5 cm, oblong-ovoid.The bracts are entire, long-ciliate, with glabrous or sparsely pubescent keel and brown margins.The sepals are long-ciliate, with a brownish margin, and nearly equal.The corolla lobes are ovate-lanceolate.The anthers are yellowish or violet.The conical capsule contains seeds that are 3-5 mm ovoid and rugose [1], [2].In comparison with the well-studied P. lanceolata and P. major, the seeds of P. atrata are significantly bigger in size (Figure 2c-e).Pollen was used as a taxonomic feature for P. atrata subsp.carpatica [25].The pollen grains are verrucae with marked granulation and absent annulus; the operculum is not well defined; the aperture membrane is not reticulate or minutely verrucate.According to the pollen, P. atrata showed the closest resemblance to P. media and P. arenaria.The harsh environmental conditions have caused the plant to adapt anatomically and morphologically.A detailed anatomical analysis of P. atrata was performed by Ianovici [26].The leaves were amphistomatic with a high density of non-glandular and capitate trichomes.The decrease in stomatal density at higher altitudes was pointed to facilitate the plant's adjustment.The occurrence of lignified cells was suggested to contribute to more rigid leaves against strong wind.The rhizome served as a reservoir of nutritional reserves and had significant lignification at the cell walls of large cell groups.

Phylogenetics
Phylogenetic studies supported by morphological and molecular data had assigned P. atrata to subgenus Albicans Rahn section Montana Barnéoud [3], [35], [36].The species that were in a cluster with P. atrata Hoppe included P. nivalis Boiss., as well as P. cafra Decne.and P. monosperma Pourr..Among the molecular markers, the nuclear ribosomal ITS2 and the plastid trnL-F intergenic spacer regions appeared as the most corresponding to the morphological characteristics of subg.Albicans [35], [36].
The Barcode of Life Data System (BOLD) database contains DNA barcoding regions for Plantago sp.[37].Figure 3 shows part of the phylogenetic tree based on the ITS marker and positioning P. atrata together with P. nivalis and P. cafra, as well as to the more phylogenetically distant P. lagopus and P. lanceolata (subg.Albicans, sect.Lanceifolia Barnéoud).

Chemotaxonomy
Phytochemical investigation of P. atrata has revealed the presence of iridoid glycosides, phenylpropanoid glycosides, and flavonoids (Table 2).These groups of compounds are useful taxonomic markers for genus Plantago [43], [40], [44].The iridoid glucosides aucubin and catalpol as well as phenylpropanoid glycoside verbascoside (=acteoside) are the most characteristic compounds in Plantago species [40].Among flavonoids, the main constituents are luteolin-and apigenin-7-Oglucoside [43].The iridoid profiles have been shown to exhibit a significant correlation with morphological and other chemical specifications of the representatives of the genus Plantago [39], [40].The analyses relied mostly on isolated compounds identified by nuclear magnetic resonance (NMR) spectroscopy (Table 2).Aucubin was pointed as typical for the whole genus, while bartsioside and catalpol as well as 5substituted iridoids were each characteristic of a subgenus in the family [39].Aucubin content was shown to depend on various aspects of the genotype, soil fertility, etc. [45], [46].On the other hand, analysis of plantain species showed qualitatively constant iridoid patterns that were not influenced by environmental conditions and phenophase according to Janković et al. [40].Comparison between the aucubin content of different plantain species was precisely performed by HPLC, HPTLC, and LC-ESI-MS chromatographic methods, and indicated the highest accumulation in P. atrata [41].Aucubin and catalpol are active compounds possessing biological activities, including anti-cancer, anti-aging, antiinflammatory, antioxidant, hepatoprotective, osteoprotective, and neuroprotective properties [47].
The caffeoyl phenylethanoid glycosides (CPGs) are widely distributed in the genus and have similarly been suggested to be taxonomic markers in the Plantaginaceae [48] -the verbascoside being the main constituent in P. atrata according to [39].Nevertheless, taxonomically useful diversity based on CPGs seemed to be limited within the genus [48].Verbascoside is usually present, sometimes together with plantamajoside.

Environmental interactions
Plantago atrata belongs to the high-altitude mountainous habitats in the sub-alpine and alpine zones, where the flora is very specific [24].The alpine species had been monitored to evaluate the effect of climate and biotic factors on the ecosystems (Table 3).Improved knowledge about the processes shaping species coexistence in high mountain ecosystems should help researchers understand and predict vegetation vulnerability to environmental changes.The habitat of P. atrata consists of medium fertile soils with good moisture content and it was shown to increase in a long-term period in the central Apennines due to climate change and the respective ongoing 'thermophilization' process [53].It was assumed that the expansion of species as P. atrata, typical of medium fertile soils, contributed to the increment of the nutrient and moisture ecological values and subsequent increase in nutrient-and moisture-demanding species.Accordingly, there is a tendency for an increase in thermophilic plant frequency and a decline in several cryophilic species [53].
The climate changes affect plant distribution, growth, and nutritional quality, and on the other hand, an enhanced competition for resources for herbivores would reduce pasture quality and food availability [56].Correspondingly, P. atrata is among the cold-adapted vegetation utilized for feeding by the Apennine chamois.The impact of grazing animals (including red deer, sheep, and dairy cows) in the alpine region is important for floristic maintenance, and recent studies are also focused on the nutritional value of the pastures [55].Several reports support that plantain compounds may influence the rumen microflora of grazing ruminants and ultimately their nutrient utilization [55], [60], [61], [62].Jayanegara et al. [61] investigated the impact of alpine plant species on elevating beneficial fatty acids in the rumen of dairy cows.According to the researchers' hypothesis, the content of plant tannins exhibits toxicity to microbial species that are involved in the biohydrogenation of polyunsaturated fatty acids.The performed in vitro studies could not provide evidence for a ruminal effect of alpine forages including P. atrata that seemed not enriched in tannins.Further in vitro experiments with acteoside suggested that it reduces free ammonia and rather facilitates nitrogen integration into amino acids and respectively proteins and growth [62].The effect of aucubin was less pronounced.Plantago atrata subsp.carpatica (Soó) Soó is considered a Pan-Carpathian endemic that is listed on the Polish, Slovakian, and IUCN Red List of Threatened Plants [59].Presently, this species is maintained and propagated ex situ.In support of conservation programs for the cultivation and propagation of endangered plants, Zubek et al. [59] demonstrated P. atrata to be dependent on arbuscular mycorrhizal fungi (AMF) for their growth as the mycorrhizal dependency was 91%.In Table 3 are listed AMFs and bacteria that positively affected P. atrata growth, as the native AMF isolates were equally active as the used laboratory strains.Accordingly, it was concluded that an effective microbial consortium could significantly assist ex situ propagation and plant productivity.

Conclusion
The dark plantain Plantago atrata has drawn our attention since it is not a well-studied species, while evolutionary, it is a close relative of narrow-leaved plantains used in traditional medicine for the treatment of inflammation, skin conditions, respiratory issues, and wound healing.The harsh highaltitude conditions at which P. atrata grows suggest a specific phytochemical composition and related bioactive potential that could be beneficial for herbivores, including humans.This alpine species is part of an ecosystem that is sensitive to climate change, and it could serve as an indicator for monitoring environmental influences and biotic interactions in the high mountains that are its habitat.The controlled cultivation of the plant would assist in the identification of individual factors affecting its state, which can be further applied for nature monitoring, and for biotechnological studies for agricultural and pharmacological purposes.

Figure 3 .
Figure 3. Plantago TaxonID Tree based on ITS regions in the BOLD database.Phylogenetically close to P. atrata species are shown.

Table 3 .
Groups of coexisting organisms with P. atrata noticed in the literature