A method for studying the influence of Mn oxyhydroxides on the trace element content of aquatic bryophytes.

The main objective of this study was to develop and test a method of separating externally deposited Mn oxyhydroxides and co-precipitated elements from samples of aquatic moss (the moss Fontinalis antipyretica). The method, which uses 0.1 M hydroxylamine to dissolve the oxyhydroxides, was tested with samples collected in rivers with slightly acidic, well­oxygenated waters, where high rates of Mn precipitation occur. The method was effective (it extracted up to 84 % of the Mn) and selective (Fe oxyhydroxides were not extracted). The elements Ba, Cd, Zn and Ni were associated with the Mn oxyhydroxides, while Al, As, Cr, Cu, Fe, Hg and Pb were not. Deposition of Mn therefore increased the concentration of some elements in the moss samples. However, as Mn precipitation depends on Eh and pH, which are independent of the concentrations of the elements in water, the relationship between water and moss element concentrations is not clear (i.e. the data are noisy). This is a problem in biomonitoring studies, which assume a close relationship between element concentrations in moss and water. The value of the proposed extraction method is that it can be used to correct the effect of Mn deposition. We present an example of this correction applied to the Cd concentrations in the test data. We found that the noise introduced by the Mn, including age-related effects (observed by comparing concentrations in 0-2.5 and 2.2-5.0 cm sections from the shoot apex), can be reduced. Additionally, the correction revealed recent increases in Cd concentrations in one site that were not observed in the uncorrected data. Another finding of interest was the low content of total Mn and different extractability (of most elements) observed in moss samples collected in alkaline waters. Finally, we discuss how future studies designed for different environmental scenarios can benefit from application of the proposed method.

A review of 210Pb and 210Po in moss.

Among environment contaminants, 210Pb and 210Po have gained significant research attention due to their radioactive toxicity. Moss, with its exceptional adsorption capability for these radionuclides, serves as an indicator for environmental 210Pb and 210Po pollution. The paper reviews a total of 138 articles, summarizing the common methods and analytical results of 210Pb and 210Po research in moss. It elucidates the accumulation characteristics of 210Pb and 210Po in moss, discusses current research challenges, potential solutions, and future prospects in this field. Existing literature indicates limitations in common measurement techniques for 210Pb and 210Po in moss, characterized by high detection limits or lengthy sample processing. The concentration of 210Pb and 210Po within moss display substantial variations across different regions worldwide, ranging from

Moss as a passive biomonitoring tool for the atmospheric deposition and spatial distribution pattern of toxic metals in an industrial city.

Anthropogenic pollution impacts human and environmental health, climate change, and air quality. Karabük, an industrial area from the Black Sea Region in northern Türkiye, is vulnerable to environmental pollution, particularly soil and air. In this research on methodological aspects, we analyzed the concentrations of six potential toxic metals in the atmospheric deposition of the city using the passive method of moss biomonitoring. The ground-growing terrestrial moss, Hypnum cupressiforme Hedw., was collected during the dry season of August 2023 at 20 urban points. The concentrations of Cr, Cu, Cd, Ni, Pb, and Co were determined in mosses by the ICP-MS method. Descriptive statistical analysis was employed to evaluate the status and variance in the spatial distribution of the studied metals, and multivariate analysis, Pearson correlation, and cluster analysis were used to investigate the associations of elements and discuss the most probable sources of these elements in the study area. Cd and Co showed positive and significant inter-element correlations (r > 0.938), representing an anthropogenic association mostly present in the air particles emitted from several metal plants. The results showed substantial impacts from local industry, manufactured activity, and soil dust emissions. Steel and iron smelter plants and cement factories are the biggest emitters of trace metals in the Karabük area and the primary sources of Cr, Cd, Ni, and Co deposition.

Understanding mercury accumulation in mosses of two subalpine forests in China.

The role of forest ecosystems in the global mercury (Hg) biogeochemical cycle is widely recognized; however, using litterfall as a surrogate to assess the Hg sink function of forests encounters limitations. We investigated the accumulation characteristics and influencing factors of Hg in mosses from two remote subalpine forests in southwestern China. The results indicated that there was high Hg accumulation in subalpine forest mosses, with average concentrations of 82 ± 49 ng g-1 for total mercury (THg) and 1.3 ± 0.8 ng g-1 for methylmercury (MeHg). We demonstrated that the accumulation capacity of Hg in mosses was significantly dependent on species and substrates (micro-habitats), the mosses on tree trunks exhibited significantly elevated Hg accumulation levels (THg 132 ± 56 ng g-1, MeHg 1.6 ± 0.2 ng g-1) compared to mosses in other substrates. The surface morphologies and biochemical components of leaf (phyllidia), such as cation exchange capacity (CEC), pectin, uronic acid, and metallothionein, play a crucial role in the accumulation of Hg by mosses. These findings provide valuable insights into Hg accumulation in forest mosses. Suggesting that the contribution of mosses Hg accumulation should be considered when assessing atmospheric Hg sinks of forests.

Assessment of Air Pollution in Ulaanbaatar Using the Moss Bag Technique.

Active moss biomonitoring, the so-called moss bag technique, widely applied in many countries, for the first time, was applied to assess the air quality in Ulaanbaatar (Mongolia). Moss bags with Sphagnum girgensohnii Russow were exposed in triplicate in three different periods: December-February, March-May, and December-May at 13 governmental air quality monitoring stations located in the vicinity of thermal power plants and residential areas. The plant tissue content of Al, Ba, Co, Cd, Cr, Cu, Fe, Mn, P, Pb, Sr, S, V, As, and Zn was determined using inductively coupled plasma-optical emission spectrometry, and a direct mercury analyzer was used to determine the Hg content. The samples in residential areas and near thermal power plants that were exposed for 3 months in winter and for 6 months (winter to spring) were characterized by the highest accumulation of the elements. In the moss bags exposed during spring, maximum accumulation of the determined elements was noted in residential areas and near main roads. Regardless of the exposure time and duration, the highest accumulation of Al, Fe, and V was determined at Dambadarjaa air quality station located near a highway and of Hg near the Amgalan power plant. Significant differences in element accumulation between seasons were observed, thus, the accumulation of Al, Ba, As, Co, Cr, Fe, Pb, V, and Zn was higher in spring, while P and S had higher content in the moss samples exposed during winter. The accumulation of elements over the 6-month exposure period was 1.1-6.7 times higher than that of the 3-month periods. Thus, the 6-month exposure can be considered a reliable deployment period as it ensures an adequate signal in terms of enrichment of pollutants. Factor analysis was applied to highlight the association of elements and to link them with possible sources of emission. Three factors were determined, the first one included Al, As, Ba, Co, Cr, Fe, Mn, Pb, Sr, and V and was identified as a geogenic-anthropogenic, the second (Cu, P, and S) and third (Cd and Zn) factors suggested anthropogenic origin. The Relative accumulation factor and enrichment factor were calculated to evaluate the level of air pollution and possible element sources. Considerable contributors to air pollution were Zn, Fe, As, V, Cr, and Al, which may originate from airborne soil particles of crustal matter or transport, as well as coal combustion for heating and cooking.

Combined effect of fungicide, herbicide and plant elicitor used in apple orchards on non-target epiphytic moss Hypnum cupressiforme.

Apple production is a dynamic agricultural system in which pesticides are applied recurrently to control pests and diseases in the orchards. Understanding the impact of such agents on non-target organisms is crucial to minimise unintended consequences while maintaining their use in crop protection. The aim was to test how fungicide, herbicide, elicitor, and their combinations affect the physiology of the epiphytic moss Hypnum cupressiforme that naturally occurs in orchards. Our results showed that both dodine and diflufenican applied separately had a strong negative effect on moss physiology reflected in significantly decreased photosynthetic pigment contents, maximum quantum yield of PSII photochemistry, cell membrane integrity and dehydrogenase activity, and increased membrane lipid peroxidation, which indicates a high physiological stress. Furthermore, the combined use of herbicide and fungicide resulted in further deterioration of the physiological condition compared to the effects of both agents used separately. In many cases, the application of chitosan together with a diflufenican or dodine resulted in a reduction of the negative effects triggered by these agents. The compensatory effect was particularly pronounced in maintaining a low level of cell membrane permeability. Consequently, it can be concluded that chitosan could have a protective function against cell membrane damage in non-target mosses.

Significance of moss pretreatments in active biomonitoring surveys.

The present study examines the impact of pretreatment procedures on the metal concentrations in bags that are to be exposed. We examine Mn, Fe, Cu, Zn, Cd, and Pb amounts in Sphagnum fallax and Dicranum polysetum mosses using atomic absorption spectrometry. The concentration of Hg was also determined using a mercury analyzer. Two sample preparation ways were tested (with and without rinsing) and their influence was evaluated by determining the coefficient of variation (CV). Chlorophyll content was also determined in mosses collected from three habitats (deep woodland, forest road, and wood lot). The results indicate, that the concentration of elements deposited in mosses depends on the species and the habitat where they were collected (ANOVA, p < 0.001). Rinsing of mosses reduces the CV for Mn, Fe, Cu, and Zn and uniform the material prior to exposure (CV for the majority of metals <10%). Selected correlations were found for element concentrations with chlorophyll content. Photosynthetic activity of mosses decreased by about 80% during their one-month storage in the laboratory. Due to the varying concentration of metals in the collected samples, proper, and standardized preparation of mosses before exposure, they can be effectively used in active biomonitoring.

Compared to other biomonitoring work the novel approach is the simultaneous study of two moss species, the analysis of three different habitats and the tie-in of accumulated trace elements by mosses and their vitality by measuring chlorophyll content and photosynthetic activity.

Influence of phytocenosis on the medical potential of moss extracts: the Pleurozium schreberi (Willd. ex Brid.) Mitt. case.

The question was asked "whether plant phytocenosis has an impact on the medical potential of the extracts from Pleurozium schreberi". Moss samples were collected from four different phytocoenoses: mixed forest (oak-pine forest), a forest tract in pine forest, 5-15-year-old pine forest and 50-year-old pine forest. Chemical composition of the extracts, antioxidative capacity (FRAP and ABTS·+ assays), as well as biological activities including cytotoxicity for the mouse fibroblasts L929 line (MTT reduction assay), biostatic/biocidal effect against selected bacteria and fungi (broth microdilution method followed by culture on solid media), and regenerative properties on human fibroblasts HFF-1 line (scratch assay) were tested. The conducted research clearly proves that phytocenosis determines the quality of moss extracts. The analyses showed that in every examined aspect the IV-7 extract (obtained from a specimen collected in a Pinus sylvestris L. forest, monoculture up to 15 years old) exhibited the highest values and the strongest activity. Other extracts of the same species but growing in other phytocenoses-in a mixed forest (IV-5), a forest tract in a Pinus sylvestris monoculture forest (IV-6) and in a P. sylvestris forest of pine monoculture about 50 years old (IV-8) showed much weaker activity and lower values of the above-mentioned parameters. At the same time, none of the tested extracts exerted a pro-regenerative effect. The P. schreberi extracts were characterized by a varied total content of phenolic compounds in the range from 0.63 ± 0.02 to 14.01 ± 0.25 mg/g of plant material. UPLC/MS analysis showed a varied phenolic profile of the extracts, with caffeoylquinic acid and quercetin triglucoside predominating in all of them.

Moss-bag technique as an approach to monitor elemental concentration indoors.

The moss-bag technique has been used for many decades to monitor outdoor pollution. More recently, however, the method has been used to monitor indoor air pollution (IAP), as humans spend the majority of their time indoors. The purpose of the research conducted was to evaluate indoor air pollution using active moss biomonitoring. Pleurozium schreberi moss bags were exposed for two seasons (summer and winter), hanging over tile stoves and coal stoves. The selected elements: Al, Cu, Cd, Co, Pb, Zn, V, Ba, Cr, Fe, Mn, Sr, P, Ni, and S were determined by Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES) and, for Hg, by a direct mercury analyzer. The study found the exposure season affected the concentrations of selected elements in 62.5% of cases, and their source was identified. The average concentrations of Co, Ba, Cr, and Sr were higher, and statistically significant, in winter, after a 12-week exposure period of the mosses, regardless of the type of heating or cooking stove owned. The higher phosphorus concentrations obtained in summer indicate physiological stress caused by unfavorable winter exposure conditions. In the future, the number of species used to assess indoor air pollution should be increased and the range of pollutants expanded, along with the identification of their sources, taking residents' lifestyles into account.

Mapping spatial and temporal trends of atmospheric deposition of nitrogen at the landscape level in Germany 2005, 2015 and 2020 and their comparison with emission data.

Mosses are particularly suitable for recording the accumulation of atmospheric substance inputs in large areas at relatively many locations. In Europe, this has been done every five years since 1990 as part of the European Moss Survey. In this framework, mosses were collected at up to 7312 sites in up to 34 countries and chemically analyzed for metals (since 1990), nitrogen (since 2005), persistent organic pollutants (since 2010) and microplastic (since 2015). The present investigation aimed at determining the nitrogen accumulated in three-year-old shoots from mosses collected in Germany in 2020 by quality-controlled sampling and chemical analysis according to the European Moss Survey Protocol (ICP Vegetation 2020). The spatial structure of the measurement values was analyzed by means of Variogram Analysis, and the respective function was used for Kriging-Interpolation. In addition to mapping the nitrogen values according to the international classification, maps based on 10 percentile classes were calculated. Maps for the Moss Survey 2020 data were compared with respective maps produced from the 2005 and 2015 Moss Survey data. Trends in Germany-wide nitrogen medians over the past three campaigns (2005, 2015 and 2020) show that nitrogen medians decreased by -2 % between 2005 and 2015 and increased by +8 % between 2015 and 2020. These differences are not significant and do not match the emission trends. Therefore, emission register data needs to be controlled by monitoring nitrogen deposition with technical and biological samplers and deposition modelling.

Are Mosses Used in Atmospheric Trace Metal Deposition Surveys Impacted by Their Substrate Soils? A National Study in Albania.

This research used moss biomonitoring to assess the atmospheric deposition of selected trace metals across the whole territory of Albania, a country of diverse lithology, and topography. Here, we assess three elements (Cr, Ni, and Co) that were identified in high concentrations compared to values reported by European moss surveys of 2010 and 2015. The possibility of element uptake by moss from substrate soils was assessed by analyzing moss and topsoil samples from the same areas. For this purpose, moss (Hypnum cupressiforme (Hedw.)) and topsoil samples were collected throughout Albania. Higher concentrations of elements in moss were found in areas of very high element content in soil characterized by no/or thin humus layer and sparse vegetation that stimulates soil dust generation. To compensate for the natural variation of the elements and to show their anthropogenic variation, geochemical normalization was conducted as the ratio of Co, Cr, and Ni concentration data to be concentration. Associations between elements in moss and soil samples, investigated by Spearman-Rho correlation analysis, indicated strong and significant correlations (r > 0.81, p = 0.000) between elements' data in moss or soil, and weak or no correlations (r < 0.4, p > 0.05) between the same data of moss and soil. Factor analysis revealed two main factors that selectively affect the elements in moss and top soil samples. Findings from this research suggested negligible interactions between moss and substrate soils, with the exception of soils with high concentrations of elements.

The occurrence of 40K and 137Cs radioactivity in mosses during 2015-2019 in the Djerdap National Park (E. Serbia).

The radiation status of the national park (NP) Djerdap (Eastern Serbia) is characterized using bioindicators (mosses). Mosses (16 species, 156 samples) were collected randomly within the area of NP Djerdap during the month of June, periodically from 2015 to 2019. Samples were collected in the regions of Dobra, Donji Milanovac, and Tekija. Within the mosses, the presence of 40 K and 137Cs is regarded as a good indicator of radionuclides present in the environment due to their easy interchangeability with 39 K highly present in living moss organisms. The activity concentrations of gamma ray-emmiting radionuclides in samples were determined using high-resolution gamma ray spectrometry, an HPGe-ORTEC/Ametek detector. The determining activity concentrations of 40 K and 137Cs in collected samples were as follows: for 40 K from 31.4 to 721 Bq kg-1and for 137Cs varied from 2.6 to 908 Bq kg-1. The average activity concentrations of 40 K and 137Cs (Bq kg-1) in mosses in the period 2015-2019 were the lowest in moss samples collected in the Dobra region, and the highest in the area of Donji Milanovac. The most prevalent collected moss species within the Djerdap National Park was Hypnum cupressiforme. The determined activity concentrations in H. cupressiforme of 137Cs for the whole region of NP Djerdap for all 5 years was 78.1 ± 70.3 Bq kg-1 and of 40 K was 181 ± 86.1 Bq kg-1.

Vanadium Contamination in Soil and Atmospheric Deposition in Albania.

By examining and evaluating the vanadium content in topsoil and moss samples, this study sought to better understand vanadium contamination in soil and atmospheric deposition. In the research area, Hypnium cupressiforme sps. moss is used. According to different distribution patterns and the lack of a link between vanadium in moss and soil samples studied by correlation analysis, no interactions between substrate soil and moss samples were investigated. Maximum vanadium concentrations (13.2 mg/kg and 250 mg/kg, respectively) were found in both moss and soil samples near the Cu mineral-rich Gjegjan area. Using lithium-normalized data on vanadium, the effect of anthropogenic activity on the vanadium in moss and soil samples is examined. There were no relationships between concentration and normalized data in moss and soil samples, showing the simultaneous effects of natural and anthropogenic sources of vanadium in the research area. Country-specific trends revealed no change for vanadium since 2010 in Albania.

Mosses in the Kopacki Rit Nature Park, Croatia, as bioindicators of a potential radioactive contamination of the middle Danube River basin.

We studied activity concentrations of radionuclides in the Kopacki Rit Nature Park using mosses as bioindicators. This area of intact nature is at the tripoint of Croatia, Hungary, and Serbia, being located basically at the centre of the middle Danube River basin. Therefore, it can be easily affected by airborne pollution from various locations in the Middle Europe and beyond. The goal of our research was to assess whether the Park could serve as a location where any new radioactive contamination could be sensitively detected, which implied a necessity for low activity concentrations at the present time. Our gamma-ray spectrometry revealed the presence of only one anthropogenic gamma emitter, that is, 137Cs. Its activity concentration in the mosses ranged from 0.7 to 13.1 Bq kg-1, being low indeed. Another radionuclide in our focus was 210Pb. Generally, its elevated concentrations may signify ecologically undesirable human activities that involve naturally occurring radioactive matter. The activity concentration of 210Pb in the mosses was in the range from 183 to 690 Bq kg-1. This did not depart from the results of other similar studies and was again low enough for a detection of possible excess amounts of this radionuclide in the future.

Moss biomonitoring of air pollution with potentially toxic elements in the Kumanovo Region, North Macedonia.

A study was carried out to investigate air deposition and to explore the natural distribution and contamination with potentially toxic elements (PTEs) in the Kumanovo Region, North Macedonia, by using moss samples as biomonitors for air pollution. The distribution of 51 elements was detected in 42 moss samples collected from this area. Moss samples were analyzed following microwave digestion by inductively coupled plasma-atomic emission spectrometry (ICP-AES) and inductively coupled plasma-mass spectrometry (ICP-MS). It was found that the atmospheric deposition for some PTEs in the moss samples in specific parts of the study area is influenced by anthropogenic and urban activities. R-mode factor analysis was used to identify and characterize element associations, and six associations of elements were determined. Four factors were separated from the group of macroelements determined by ICP-AES: Factor 1 (Al, Cr, Fe, Li, Ni, V, and Zn), F2 (K, Mg, and P), F3 (Ba and Sr), and F4 (Cd, Pb, and Zn); and two associations were separated from the group of trace elements determined by ICP-MS: Factor 1 (As, Co, Cs, Ga, Ge, Sc, Ti, Y, Zr, and rare earth elements-REEs) and Factor 2 (Bi, Br, Hg, I, Sb, Sn, and W).

Pharmacological, Cytotoxic, Immunostimulant, and Auto-Immune Activity of Thalloid Liverworts: An Overview.

Bryophytes have historically been employed as verdant medicine in China, Native America and India. Phenolics, glycosides, fatty acids, other rare aromatic compounds and Terpenoids found in bryophytes may help prevent cancer and other chronic disorders. Liverworts have historically been utilized in traditional medicine and also as immu-nomodulators or immunostimulants. Diterpenoids, Lipophilic mono-, sesqui- and aromatic compounds assisting to the biological activities of liverworts. For their biological functions more than 220 aromatic compounds and 700 terpenoids and other chemicals discovered in liverworts scrutinized for their pharmacological, cytotoxic, immunostimulant and auto-immune efficacies.

The Application of Active Biomonitoring with the Use of Mosses to Identify Polycyclic Aromatic Hydrocarbons in an Atmospheric Aerosol.

The use of biological indicators of environmental quality is an alternative method of monitoring ecosystem pollution. Various groups of contaminants, including organic ones, can be measured in environmental samples. Polycyclic aromatic hydrocarbons (PAHs) have not yet been determined by the moss bag technique. This technique uses several moss species simultaneously in urban areas to select the best biomonitoring of these compounds, which are dangerous to humans and the environment. In this research, a gas chromatography coupled with mass spectrometry was used for the determination of selected PAHs in three species of mosses: Pleurozium schreberi, Sphagnum fallax and Dicranum polysetum (active biomonitoring) and for comparison using an air filter reference method for atmospheric aerosol monitoring. The chlorophyll fluorescence of photosystem II (PSII) was also measured to assess changes in moss viability during the study. As a result of the study, the selective accumulation of selected PAHs by mosses was found, with Pleurozium schreberi being the best bioindicator-9 out of 13 PAHs compounds were determined in this species. The photosynthetic yield of photosystem (II) decreased by 81% during the exposure time. The relationship between PAHs concentrations in mosses and the total suspended particles (TSP) on the filter indicated the possibility of using this bioindicator to trace PAHs in urban areas and to apply the moss bag technique as a method supporting classical instrumental air monitoring.

Cytotoxicity, antimicrobial and antioxidant activities of mosses obtained from open habitats.

Mosses are mainly the object of ecological and taxonomic research. This group of plants are still underestimated by scientists in other aspects of research. Recent research has shown that these plants contain remarkable and unique substances with high biological activity. Five species of mosses from a large urban ecosystem were identified for present study. In order to determine their biological potential, multifaceted studies were carried out, including: total phenolics content, antioxidant activity, antimicrobial and antifungal study, cytotoxicity evaluation, and scratch assay to assess pro-regenerative effect in the context of their possible use as the ingredients of biologically active cosmetics. Additionally, determination of individual phenolic compounds in selected extracts of the tested mosses was made. Research showed that Ceratodon purpureus and Dryptodon pulvinatus extracts had the greatest potential as antioxidants and antimicrobial activity. The cytotoxicity assessment indicated that the extracts from Dryptodon pulvinatus and Rhytidiadelphus squarossus exerted the strongest negative effect on mouse fibroblast line L929 viability at higher concentrations. While, the extract from Tortulla muralis best stimulated human foreskin fibroblast line HFF-1 proliferation and wound healing. The research on individual phenolic compounds content in the extracts tested indicated over 20 peaks on UPLC chromatograms. The conducted study has shown that mosses, especially so far unexplored species of open ecosystems, and e.g. epilytic habitats, may be a valuable source of biologically active substances and thus may constitute important medical and cosmetic possibilities.

Ecological stoichiometric characteristics of soil-moss C, N, and P in restoration stages of karst rocky desertification.

Rocky desertification is the most serious ecological disaster in karst areas. Comprehensive control of rocky desertification plays an important role in promoting the economic development of karst areas. Studying the stoichiometric characteristics of mosses and soil can provide a powerful reference for the ecological restoration and evaluation of ecosystems experiencing rocky desertification. Soil and mosses were collected from sites representing different stages of ecological restoration (bare rock, grassland, shrubland, and secondary forest), and the contents of carbon (C), nitrogen (N), and phosphorus (P) were detected for ecological stoichiometric analysis. The results indicate that in different restoration stages following karst rocky desertification, the contents of soil organic carbon (SOC), total nitrogen (TN), and total phosphorus (TP) and the stoichiometric ratios in the shrub habitat are higher than those in the bare rock, grassland, and secondary forest habitats. However, the TP and available P contents were low at all stages (0.06 g/kg and 0.62 mg/kg, respectively). The N and P contents and stoichiometric ratios in the mosses showed no significant differences among the succession stages. The C contents in the mosses had a significant positive correlation with SOC and TN and TP content, and the P content had a significant positive correlation with the soil available P. However, there was a significant negative correlation between the C: N and C:P ratios of the bryophytes and soil C: N. In summary, during the process of natural restoration of karst rocky desertification areas, SOC and soil TN contents accumulate with each succession stage. Soil nutrients are higher in shrub habitats than in other succession stages. Mosses have a strong effect on improving soil nutrients in rocky desertification areas.

Untargeted In Silico Compound Classification-A Novel Metabolomics Method to Assess the Chemodiversity in Bryophytes.

In plant ecology, biochemical analyses of bryophytes and vascular plants are often conducted on dried herbarium specimen as species typically grow in distant and inaccessible locations. Here, we present an automated in silico compound classification framework to annotate metabolites using an untargeted data independent acquisition (DIA)-LC/MS-QToF-sequential windowed acquisition of all theoretical fragment ion mass spectra (SWATH) ecometabolomics analytical method. We perform a comparative investigation of the chemical diversity at the global level and the composition of metabolite families in ten different species of bryophytes using fresh samples collected on-site and dried specimen stored in a herbarium for half a year. Shannon and Pielou's diversity indices, hierarchical clustering analysis (HCA), sparse partial least squares discriminant analysis (sPLS-DA), distance-based redundancy analysis (dbRDA), ANOVA with post-hoc Tukey honestly significant difference (HSD) test, and the Fisher's exact test were used to determine differences in the richness and composition of metabolite families, with regard to herbarium conditions, ecological characteristics, and species. We functionally annotated metabolite families to biochemical processes related to the structural integrity of membranes and cell walls (proto-lignin, glycerophospholipids, carbohydrates), chemical defense (polyphenols, steroids), reactive oxygen species (ROS) protection (alkaloids, amino acids, flavonoids), nutrition (nitrogen- and phosphate-containing glycerophospholipids), and photosynthesis. Changes in the composition of metabolite families also explained variance related to ecological functioning like physiological adaptations of bryophytes to dry environments (proteins, peptides, flavonoids, terpenes), light availability (flavonoids, terpenes, carbohydrates), temperature (flavonoids), and biotic interactions (steroids, terpenes). The results from this study allow to construct chemical traits that can be attributed to biogeochemistry, habitat conditions, environmental changes and biotic interactions. Our classification framework accelerates the complex annotation process in metabolomics and can be used to simplify biochemical patterns. We show that compound classification is a powerful tool that allows to explore relationships in both molecular biology by "zooming in" and in ecology by "zooming out". The insights revealed by our framework allow to construct new research hypotheses and to enable detailed follow-up studies.