Anthracene-Induced Alterations in Liverwort Architecture In Vitro: Potential for Bioindication of Environmental Pollution.

Polycyclic aromatic hydrocarbons (PAHs) are widespread globally, primarily due to long-term anthropogenic pollution sources. Since PAHs tend to accumulate in soil sediments, liverwort plants, such as Lunularia cruciata, are susceptible to their adverse effects, making them good models for bioindicators. The aim of this study was to probe the impact of anthracene, a three-ring linear PAH, on the growth parameters of L. cruciata and the relationship established with the internalization of the pollutant throughout the phenology of the plant. Intrinsic plant responses, isolated from external factors, were assessed in vitro. L. cruciata absorbed anthracene from the culture medium, and its bioaccumulation was monitored throughout the entire process, from the gemma germination stage to the development of the adult plant, over a total period of 60 days. Consequently, plants exposed to concentrations higher than 50 µM anthracene, decreased the growth area of the thallus, the biomass and number of tips. Moreover, anthracene also impinged on plant symmetry. This concentration represented the maximum limit of bioaccumulation in the tissues. This study provides the first evidence that architectural variables in liverwort plants are suitable parameters for their use as bioindicators of PAHs.

Cell death in bryophytes: emerging models to study core regulatory modules and conserved pathways.

This review summarizes recent progress in our current understanding of the mechanisms underlying the cell death pathways in bryophytes, focusing on conserved pathways and particularities in comparison to angiosperms. Regulated cell death (RCD) plays key roles during essential processes along the plant life cycle. It is part of specific developmental programmes and maintains homeostasis of the organism in response to unfavourable environments. Bryophytes could provide valuable models to study developmental RCD processes as well as those triggered by biotic and abiotic stresses. Some pathways analogous to those present in angiosperms occur in the gametophytic haploid generation of bryophytes, allowing direct genetic studies. In this review, we focus on such RCD programmes, identifying core conserved mechanisms and raising new key questions to analyse RCD from an evolutionary perspective.

Orthotrichumcamanchacanum, a remarkable new moss species from Chile (Bryopsida, Orthotrichaceae).

Orthotrichumcamanchacanum is presented as a newly described species from Chile. The species is primarily distinguished by its emergent capsule with cryptoporous stomata, a double peristome, linear-lanceolate stem leaves with a long hyaline aristae in apex, conspicuously differentiated perichaetial leaves, and a densely hairy vaginula. The species was discovered in the mountain massif of the Andes in the Coquimbo region, notable for its unique climatic conditions. Molecular data and a brief discussion comparing the newly described species with the most closely related taxa are also provided.

Understanding desiccation tolerance and sex-specific responses in Bryum argenteum: insights from phenological phases and physiological analyses.

BACKGROUND AND AIMS: Desiccation tolerance (DT) is crucial for survival in arid environments, where organisms develop strategies in reproduction, maintenance and defence to cope with water scarcity. Therefore, investigating the relationship between reproduction and DT is essential to understand the ecology and adaptive strategies of species. This study explores the connection between the development of male and female gametangia in the moss Bryum argenteum and the decrease in DT during the progression of phenological phases in gametangia and protonema. METHODS: Samples collected from a dry tropical forest in Brazil were cultivated, cloned and subjected to desiccation. Subsequently, the physiological parameters of shoots and protonemata were analysed. Shoot and protonema regeneration were monitored for 28 d after the physiological analyses. Both phases were subjected to control and desiccation treatments. KEY RESULTS: Significant effects of desiccation and sex on the physiological parameters and regeneration capacity of shoots and protonemata were found. Male shoots generally exhibited lower values of Fv/Fm (quantum efficiency of photosystem II) and ϕPSII (effective quantum yield of photosystem II), while females demonstrated higher values and better recovery after desiccation. Protonemata also showed variation in Fv/Fm over time and with sex, with no significant differences in ϕPSII between them. Desiccated male shoots had higher mortality rates and produced fewer new shoots. For females, the regeneration patterns varied between the desiccation-exposed groups and the control, with decreased shoot production, and some protonemata growing into filaments without forming shoots. CONCLUSION: These findings improve our understanding of the ecological responses of bryophytes to desiccation stress and provide insights into their adaptive strategies in challenging environments, such as the possible rarity of males in dioicous moss populations.

Dating the evolution of the complex thalloid liverworts (Marchantiopsida): total-evidence dating analysis supports a Late Silurian-Early Devonian origin and post-Mesozoic morphological stasis.

Divergence times based on molecular clock analyses often differ from those derived from total-evidence dating (TED) approaches. For bryophytes, fossils have been excluded from previous assessments of divergence times, and thus, their utility in dating analyses remains unexplored. Here, we conduct the first TED analyses of the complex thalloid liverworts (Marchantiopsida) that include fossils and evaluate macroevolutionary trends in morphological 'diversity' (disparity) and rates. Phylogenetic analyses were performed on a combined dataset of 130 discrete characters and 11 molecular markers (sampled from nuclear, plastid and mitochondrial genomes). Taxon sampling spanned 56 extant species - representing all the orders within Marchantiophyta and extant genera within Marchantiales - and eight fossil taxa. Total-evidence dating analyses support the radiation of Marchantiopsida during Late Silurian-Early Devonian (or Middle Ordovician when the outgroup is excluded) and that of Ricciaceae in the Middle Jurassic. Morphological change rate was high early in the history of the group, but it barely increased after Late Cretaceous. Disparity-through-time analyses support a fast increase in diversity until the Middle Triassic (c. 250 Ma), after which phenotypic evolution slows down considerably. Incorporating fossils in analyses challenges previous assumptions on the affinities of extinct taxa and indicates that complex thalloid liverworts radiated c. 125 Ma earlier than previously inferred.

Biological Crust Diversity Related to Elevation and Soil Properties at Local Scale in a Montane Scrub of Ecuador.

The montane shrublands of southern Ecuador represent one of the least studied ecosystems, which in the last decade have been seriously threatened by increasing wildfires, deforestation, overgrazing, and conversion to forest plantations. Our main objective was to determine, at the local scale, the diversity of species composing the biological soil crust (BSC) at three elevations (2100, 2300, and 2500 m.a.s.l.) and their possible relationships with soil physical and chemical properties in montane shrublands. For this purpose, three monitoring plots of 100 m2 were established at each elevation, and within each plot, 20 subplots were established (180 subplots sampled in total). In addition, composite soil samples were collected at a depth of 0 to 10 cm, and some physical and biochemical parameters (e.g., bulk density, texture, pH, organic matter, soil organic carbon, total nitrogen, available phosphorus, and potassium) of the soil were analyzed. The results show 35 species (23 lichens, 10 bryophytes and 2 cyanobacteria) at three elevations with a bell-shaped or hump-shaped distribution pattern. This allowed us to point out that the species richness was higher at the intermediate elevations and that the composition showed significant differences in the three elevations related to soil factors. Elevation and soil drivers may help to better chose the more suitable biological soil crust (lichen-dominated and bryophyte-dominated BSC) for the management and conservation of the montane scrub of Ecuador, which is strongly threatened by human activities.

Rehabilitation of eroded trails and gullies on quartzite rock outcrops with native species in a high-altitude grassland.

The quartzite rock outcrops and the native vegetation of grasslands located at the Serra da Calçada Mountain in Minas Gerais State (Brazil) have been severely degraded by extreme sports activities such as motocross and off-road vehicles, greatly damaging the abundant headwaters. The main consequences thereof were hilly and gully erosion processes with soil loss and the deviation of the water from its original paths. However, currently, there is no report of successful restoration efforts in severely eroded outcrops in Brazilian high-altitude grasslands (campo rupestre). Through the Universal Soil Loss Equation (USLE), we found a high general erosion rate in the study site (669.91 t·ha-1·year-1), and the specific soil loss provoked by off-road vehicles on trails was significantly greater (49 m3 per 100 m2) than that caused by mountain bikes and trekking (5.8 m3 per 100 m2). We performed the physical reconstruction of eroded outcrops and surface water flow paths by allocating locally available quartzite rocks. These rocks were inoculated with different species of bryophytes and planted with native species under two treatments: un-inoculated and inoculated with arbuscular mycorrhizal fungi (AMF) spores of the Rhizophagus irregularis species. After 2 years, the bryophyte communities showed a similar pattern to the preserved site, and the AMF inoculation favoured plant establishment of most species, especially of the Asteraceae, Cyperaceae, Fabaceae, Malpighiaceae, Orchidaceae and Poaceae families. The AMF also improved the soil fertility, highlighting soil P, SOM, CEC, NH4+-N as well as soil water content and water retention capacity. Poaceae family species showed an outstanding occupation, which was considered a functional indicator of rehabilitation success, functioning as a "hydraulic carpet" for water exportation, conduction and drainage across the outcrops. This study provides an eco-technology to restore severely eroded outcrops over headwaters using native species in the Brazilian high-altitude grasslands.

Cuticle structure and chemical composition of waxes in Phaeoceros laevis (L.) Prosk (Notothyladaceae, Anthocerotophyta).

The development of a hydrophobic cuticle covering the epidermis was a crucial evolutionary novelty ensuring the establishment of land plants. However, there is little information about its structure and chemical composition, as well as its functional implications in avascular lineages such as Anthocerotophyta. The main goal of the present study was to compare the gametophyte and sporophyte cuticles of Phaeoceros laevis. Semithin sections were analyzed through light microscopy (LM), cuticle structure was evaluated by transmission electron microscopy (TEM) and epicuticular wax morphology was analyzed by scanning electron microscopy (SEM). Total waxes were analyzed by CG/MS, and the components were identified based on the mass spectra. A thin lipophilic layer was detected on the sporophyte surface, structured as a stratified cuticular layer, similar to the well-known structure described for vascular plants. On the other hand, the gametophyte cuticle was observed only with TEM as a thin osmiophilic layer. SEM analyses showed a film-type wax on the surface of both life phases. The wax layer was eight-fold thicker on the sporophyte (0.8 µg cm-2) than on gametophyte (0.1 µg cm-2). Possible mechanical and/or drought protection are discussed. Fatty acids, primary alcohols, and steroids were identified in both life phases, while the kauren-16-ene diterpene (3%) was detected only on the sporophyte. Although no alkanes were detected in P. laevis, our findings unveil great similarity of the sporophyte cuticle of this hornwort species with the general data described for vascular plants, reinforcing the conservative condition of this character and supporting the previous idea that the biosynthetic machinery involved in the synthesis of wax compounds is conserved since the ancestor of land plants.

Volatile chemical composition of Octoblepharum albidum Hedw. (Bryophyta) from the Brazilian Amazon.

Bryophytes have a variety of bioactive compounds that can be used in biotechnological processes. The objective of this study was to know the volatile chemical composition of Octoblepharum albidum Hedw. from the Amazon and investigate its association with possible bioactive effects on insects. The volatile concentrate of O. albidum was obtained by micro-scale simultaneous distillation-extraction and analyzed by gas chromatography coupled to mass spectrometry and the identification of the compounds was based on system libraries and specialized literature. Twelve organic compounds (92.44% of the total) were identified. Hexadecanoic acid, oleic acid, E-isoeugenol, 1-octen-3-ol, and stearic acid were the major compounds. Most of the compounds have already been reported from bryophytes, while others have an unprecedented occurrence in the group. All identified compounds have biological activities reported in the literature and may participate in plant defense mechanisms against insects, causing mortality or developmental inhibition. In this study, we describe for the first time the volatile chemical composition of O. albidum from Brazil and provide evidence that this species is a source of bioactive compounds. The identified compounds have been reported in the literature to cause mortality or affect the biological parameters of insects, what suggests the possibility of their usage in the formulation of bioinsecticides.

Biological soil crusts and how they might colonize other worlds: insights from these Brazilian ecosystem engineers.

When bryophytes, lichens, eukaryotic algae, cyanobacteria, bacteria, and fungi live interacting intimately with the most superficial particles of the soil, they form a complex community of organisms called the biological soil crust (BSC or biocrust). These biocrusts occur predominantly in drylands, where they provide important ecological services such as soil aggregation, moisture retention, and nitrogen fixation. Unfortunately, many BSC communities remain poorly explored, especially in the tropics. This review summarizes studies about BSCs in Brazil, a tropical megadiverse country, and shows the importance of ecological, physiological, and taxonomic knowledge of biocrusts. We also compare Brazilian BSC communities with others around the world, describe why BSCs can be considered ecosystem engineers, and propose their use in the colonization of other worlds.

Metabolome of Ceratodon purpureus (Hedw.) Brid., a cosmopolitan moss: the influence of seasonality.

MAIN CONCLUSION: Ceratodon purpureus showed changes in disaccharides, flavonoids, and carotenoids throughout annual seasons. These changes indicate harsher environmental conditions during the dry period, directing metabolic precursors to enhance the antioxidant system. Bryophytes are a group of land plants comprising mosses (Bryophyta), liverworts (Marchantyophyta), and hornworts (Antocerotophyta). This study uses the molecular networking approach to investigate the influence of seasonality (dry and rainy seasons) on the metabolome and redox status of the moss Ceratodon purpureus (Hedw.) Brid., from Campos do Jordão, Brazil. Samples of C. purpureus were submitted to three extraction methods: 80% methanol producing the soluble fraction (intracellular compounds), followed by debris hydrolysis using sodium hydroxide producing the insoluble fraction (cell wall conjugated compounds), both analyzed by HPLC-MS; and extraction using pre-cooled methanol, separated into polar and non-polar fractions, being both analyzed by GC-MS. All fractions were processed using the Global Natural Product Social Molecular Network (GNPS). The redox status was assessed by the analysis of four enzyme activities combined with the analysis of the contents of ascorbate, glutathione, carotenoids, reactive oxygen species (ROS), and malondialdehyde acid (MDA). During the dry period, there was an increase of most biflavonoids, as well as phospholipids, disaccharides, long-chain fatty acids, carotenoids, antioxidant enzymes, ROS, and MDA. Results indicate that C. purpureus is under harsher environmental conditions during the dry period, mainly due to low temperature and less water availability (low rainfall).

Moss transcription factors regulating development and defense responses to stress.

Transcription factors control gene expression, leading to regulation of biological processes that determine plant development and adaptation to the environment. Land colonization by plants occurred 450-470 million years ago and was accompanied by an increase in the complexity of transcriptional regulation associated to transcription factor gene expansions. AP2/ERF, bHLH, MYB, NAC, GRAS, and WRKY transcription factor families increased in land plants compared with algae. In angiosperms, they play crucial roles in regulating plant growth and responses to environmental stressors. However, less information is available in bryophytes and only in a few cases is the functional role of moss transcription factors in stress mechanisms known. In this review, we discuss current knowledge of the transcription factor families involved in development and defense responses to stress in mosses and other bryophytes. By exploring and analysing the Physcomitrium patens public database and published transcriptional profiles, we show that a high number of AP2/ERF, bHLH, MYB, NAC, GRAS, and WRKY genes are differentially expressed in response to abiotic stresses and during biotic interactions. Expression profiles together with a comprehensive analysis provide insights into relevant transcription factors involved in moss defenses, and hint at distinct and conserved biological roles between bryophytes and angiosperms.

Autophagy modulates growth and development in the moss Physcomitrium patens.

Physcomitrium patens apical growing protonemal cells have the singularity that they continue to undergo cell divisions as the plant develops. This feature provides a valuable tool to study autophagy in the context of a multicellular apical growing tissue coupled to development. Herein, we showed that the core autophagy machinery is present in the moss P. patens, and characterized the 2D and 3D growth and development of atg5 and atg7 loss-of-function mutants under optimal and nutrient-deprived conditions. Our results showed that 2D growth of the different morphological and functional protonemata apical growing cells, chloronema and caulonema, is differentially modulated by this process. These differences depend on the protonema cell type and position along the protonemal filament, and growth condition. As a global plant response, the absence of autophagy favors the spread of the colony through protonemata growth at the expense of a reduction of the 3D growth, such as the buds and gametophore development, and thus the adult gametophytic and reproductive phases. Altogether this study provides valuable information suggesting that autophagy has roles during apical growth with differential responses within the cell types of the same tissue and contributes to life cycle progression and thus the growth and development of the 2D and 3D tissues of P. patens.

The regulatory activities of microRNAs in non-vascular plants: a mini review.

MAIN CONCLUSION: MicroRNA-mediated gene regulation in non-vascular plants is potentially involved in several unique biological functions, including biosynthesis of several highly valuable exclusive bioactive compounds, and those small RNAs could be manipulated for the overproduction of essential bioactive compounds in the future. MicroRNAs (miRNAs) are a class of endogenous, small (20-24 nucleotides), non-coding RNA molecules that regulate gene expression through the miRNA-mediated mechanisms of either translational inhibition or messenger RNA (mRNA) cleavage. In the past years, studies have mainly focused on elucidating the roles of miRNAs in vascular plants as compared to non-vascular plants. However, non-vascular plant miRNAs have been predicted to be involved in a wide variety of specific biological mechanisms; nevertheless, some of them have been demonstrated explicitly, thus showing that the research field of this plant group owns a noteworthy potential to develop novel investigations oriented towards the functional characterization of these miRNAs. Furthermore, the insights into the roles of miRNAs in non-vascular plants might be of great importance for designing the miRNA-based genetically modified plants for valuable secondary metabolites, active compounds, and biofuels in the future. Therefore, in this current review, we provide an overview of the potential roles of miRNAs in different groups of non-vascular plants such as algae and bryophytes.

Chemical Composition and Cytotoxic Evaluation of the Essential Oil of Phyllogonium viride (Phyllogoniaceae, Bryophyta).

The present study aimed to determine the chemical composition and biological activity of the essential oil obtained from Phyllogonium viride Brid. (Phyllogoniaceae, Bryophyta), whose samples were collected in southern Brazil. For the first time, the cytotoxic activity of the essential oil of P. viride in breast and colorectal tumor cells (MCF-7 and HCT-116) was evaluated, as well as the cytotoxic potential of this oil in non-tumoral cells of human immortalized keratinocytes (HaCaT) via MTT assay. The compounds majorly found in P. viride essential oil were ß-bazzanene (20.30 %), ß-caryophyllene (17.06 %), ß-chamigrene (14.02), and germacrene B (11.72 %). Treatment with P. viride essential oil in the different tested cell lines did not induce any toxicity in most of the tested concentrations. These data contribute to generating new scientific information about this understudied plant species. Furthermore, the chemical characterization of the compounds present in the essential oil of P. viride can lead to greater elucidation of its biotechnological potential.

Molecular mechanisms involved in functional macroevolution of plant transcription factors.

Transcription factors (TFs) are key components of the transcriptional regulation machinery. In plants, they accompanied the evolution from unicellular aquatic algae to complex flowering plants that dominate the land environment. The adaptations of the body plan and physiological responses required changes in the biological functions of TFs. Some ancestral gene regulatory networks are highly conserved, while others evolved more recently and only exist in particular lineages. The recent emergence of novel model organisms provided the opportunity for comparative studies, producing new insights to infer these evolutionary trajectories. In this review, we comprehensively revisit the recent literature on TFs of nonseed plants and algae, focusing on the molecular mechanisms driving their functional evolution. We discuss the particular contribution of changes in DNA-binding specificity, protein-protein interactions and cis-regulatory elements to gene regulatory networks. Current advances have shown that these evolutionary processes were shaped by changes in TF expression pattern, not through great innovation in TF protein sequences. We propose that the role of TFs associated with environmental and developmental regulation was unevenly conserved during land plant evolution.

Platyhypnidium aquaticum as Bioindicator of Metal and Metalloid Contamination of River Water in a Neotropical Mountain City.

Water contamination is a major environmental problem in many cities of the world. Most water contamination results from industry and human activities that generate toxic substances (e.g., metals). Rheophilic and aquatic mosses are found in lotic ecosystems, and their morphological and physiological traits are responsive to ecological and pollution gradients. Here we hypothesized that the native rheophilic moss Platyhypnidium aquaticum (A. Jaeger) M. Fleisch exposed to polluted waters can bioaccumulate greater amounts of metals, and a metalloid, than P. aquaticum exposed to pollution-free water. To this aim, we tested the bioindicator capacity of the aquatic P. aquaticum for 15 metals (Cd, Pb, Zn, Fe, K, Ca, Na, Mn, V, Co, Ba, Cr, Al, Sr, and Mg) and one metalloid (As), in twelve river samples coming from three urban and one control zone along the Zamora river in the city of Loja. When compared to the control, our results showed that P. aquaticum in the Southern, Central, and Northern zones of the city bioaccumulated higher concentrations of Ba, Cd, Co, Fe, Mg, Mn, Na, Sr, Zn, and the metalloid As. On the other hand, concentrations of Al, Ca, Cr, Pb, and V in P. aquaticum tended to be lower in the control zone, but these differences were not significant. We suggest that the presence of these contaminants may be related to water pollution (e.g., residual discharges and a lack of treatment systems) along urban zones of the river. We report for the first time the utility of P. aquaticum as a model species for development of long-term biomonitoring programs of water contamination in South America. Passive biomonitoring with P. aquaticum can be a simple and low-cost method to obtain reliable data of the current state of water contamination with metals and metalloids in tropical regions.

How do sexual expression, reproductive phenology and reproductive success relate to sexual systems in Fissidens Hedw. (Fissidentaceae)? A case study comparing two different sexual systems in mosses.

Reproductive performance is known to differ between co-sexual and non-co-sexual species. Thus, our aim was to determine whether: (i) the distance between sex structures is negatively associated with sex expression; (ii) male gametangia take longer to mature in rhizautoicous species than in gonioautoicous species; and (iii) the gonioautoicous sexual system has greater reproductive success (i.e. percentage of ramets with sporophyte) than the rhizautoicous sexual system. One population each of the mosses Fissidens scariosus and F. submarginatus, rhizautoicous and gonioautoicous, respectively, in a remnant of Atlantic Forest in Brazil were sampled monthly from September 2016 until August 2017. The number and phenophases of antheridia, archegonia and sporophytes were analysed for each species. Sexual expression and reproductive success were calculated, and reproductive phenology compared across environmental variables. As expected, sexual expression was significantly higher for the gonioautoicous species, which produced antheridia throughout the year and archegonia over many months, while gametangia production by the rhizautoicous species occurred only during the rainy season. Mean number of gametangia per perigonium and perichaetium were slightly higher for the rhizautoicous species (6.84 antheridia; 11.38 archegonia) than for the gonioautoicous species (4.39; 7.62). Gametangia and sporophyte production in the rhizautoicous species were markedly seasonal compared to that of the gonioautoicous species, although reproductive success did not differ. Therefore, we infer that the rhizautoicy (i.e. a functional dioicy) promotes lower expression of gametangia compared to gonioautoicy but is more efficient and so obtains the same reproductive success.

Volatile Metabolites in Liverworts of Ecuador.

Volatile metabolites from Frullania brasiliensis Raddi, Herbertus juniperoideus (Sw.) Grolle, Leptoscyphus hexagonus (Nees) Grolle, and Syzygiella anomala (Lindenb. & Gottsche) Steph collected in the south of Ecuador were investigated. Volatile secondary metabolites were extracted by hydrodistillation and analyzed by gas chromatography/flame ionization detector (GC/FID) and Gas chromatography/Mass spectrometry (GC/MS). Sixty-seven volatile compounds were identified in the four species, which represent between 80.12-90.17% of the total chemical composition. The major components were τ-muurolol (32.14%) and Germacrene-D (11.98%) in the essential oil of F. brasiliensis, bicyclogermacrene (18.23%), and Caryophyllene oxide (15.29%) in the oil of H. juniperoideus, Cabreuva oxide D (33.77%) and Elemol (18.55%) in the oil of Leptoscyphus hexagonus, and Silphiperfola-5,7(14)-diene (25.22%) and Caryophyllene oxide (8.98%) in the oil of Syzygiella anomala. This is the first report on volatile compounds for the species Herbertus juniperoideus, Leptoscyphus hexagonus, and Syzygiella anomala.

Assessment of urban environmental quality through the measurement of lead in bryophytes: case study in a medium-sized city.

The use of bryophytes as an indicator of environmental quality has been addressed on numerous occasions and in different places of the world under a variety of conditions. However, in Latin America their use is still limited. In the study described here, the presence and distribution of the bioaccumulation of lead in bryophytes has been evaluated in both contaminated and uncontaminated sites in Villavicencio (Colombia) and its surroundings. Villavicencio is a medium-sized city that is similar in size to many other cities in Latin America. A total of 52 samples of bryophytes were collected, of which 43 belong to points distributed in urban areas of the city (residential, commercial, highway), and the remaining nine are from uncontaminated areas (reference) taken in the surroundings of the city. The samples were treated with acid (nitric and hydrochloric) and subsequently measured by atomic absorption spectrometry. It was found that Pb concentrations in the commercial sector are between 1 and 6 times higher than in the residential and highway sectors. Spatial distribution maps of lead over the sampled territory were created using Arc-GIS 10.1. It is noteworthy that the values obtained are higher than those found in European cities.