Epiphytic Cryptogams as Bioindicators of Air Quality in a Tropical Andean City

: Air pollution is one of the main environmental problems in developed and developing countries. Epiphytic cryptogams (bryophytes and lichens) are proposed as a reliable indicator to detect environmental changes, given to their sensitivity to pollutants. In this study we evaluated air quality in the city of Ambato using bryophytes and epiphytic lichens on three land uses (urban, peri-urban and control). In each zone we selected ten trees (a total of 90 trees) for each station (a total of nine stations), where we recorded the frequency and cover of epiphytic cryptogams in a quadrat of 10 × 50 cm that was divided into 5 × 5 cm squares. Differences in richness, index of atmospheric purity (IAP) and diversity were analyzed using a generalized linear model (GLM) and changes in species composition using multivariate analysis. We recorded 39 species of cryptogams (25 lichens and 14 bryophytes). Richness, diversity and index of atmospheric purity were higher in the control zone compared to the urbanized zones. Community composition changed between the different zones, with increasing differences between the control and urban zones. The urban areas of the city of Ambato were identiﬁed with high levels of air pollution due to their lower diversity related to higher vehicular trafﬁc and industrial activities (e.g., footwear and textile factories, tanneries). Thus, epiphytic cryptogams are a fast and low-cost method for air quality assessment in tropical areas.


Introduction
Air pollution is currently one of the main environmental problems in urban and rural areas due to rapidly developing industrialization and urbanization worldwide as well as in Latin American cities [1][2][3][4]. Vehicular traffic and industrial activities in emerging economies deteriorate the air quality in large areas where there is no air pollution monitoring network [5], and thus chronic and acute exposures of inhaled pollutants can produce toxic effects in biological systems [6].
Ambato is a city located in the central Andean valley of Ecuador and one the most important cities in terms of productivity (e.g., industrial, commercial and manufacturing engine) and economy at the regional level [7]. Thus, industrial activities (e.g., footwear and textile factories, tanneries) lead to a constant growth in the flow of different types of motorized transport that generate high levels of air pollution [8]. The expansion of the urban areas towards rural areas implies the elimination of green areas, generating environmental impacts in the city, and therefore air pollution becomes one of the main environmental problems [8]. Thus, in the city has carried out evaluations of certain pollutants as sulfur dioxide (SO 2 ) , nitrogen dioxide (NO 2 ) ozone (O 2 ), benzene (C 6 H 6 ) [8], carbon dioxide (CO 2 ) and sulfur oxides (SO x ) related with air pollution. However, there is only one air pollution monitoring station and few studies in this city, which has not made possible the accurate determination of the current state of the air quality in the different zones of city of Ambato [9]. Epiphytic cryptogams, due to their anatomical, morphological, and physiological characteristics (e.g., they obtain nutrients directly from the atmosphere) [10], are very effective as bioindicators of air pollution in many cities around the world [11][12][13][14][15][16][17][18][19][20], and also for programs and protocols of air pollution in several countries. These organisms allow early detection of signs of environmental change given that they are fully exposed to airborne contaminants [11,16,21]. Thus, the application of epiphytic cryptogams are a fast and low-cost method for this purpose.
Studies in Ecuador using bioindicators to detect air pollution are scarce [3,32,37]. Thus, for the first time, epiphytic cryptogams (lichens and bryophytes) were used as biomonitors to assess the air pollution in the city of Ambato. Specifically, we addressed the following questions: (1) Are the richness, IAP and composition of cryptogams (lichens and bryophytes) influenced by land use changes? and (2) Can cryptogams (lichens and bryophytes) be used as indicators of air pollution related to vehicular traffic?

Study Area
The present study was carried out at nine stations of the city of Ambato (Figure 1), in the western mountain cordillera of Ecuador, at 2678 m a.s.l. with an approximate area of 1016.454 km 2 and temperature that fluctuates between 12 and 27 • C. Nine sampling stations located in three land uses (three urban stations, three peri-urban stations and three control stations) were selected. The control zone ( Figure 1) was located in the high areas of the rustic parishes of Ambato, represented by a dense canopy layer of evergreen montane tropical vegetation (e.g., Alnus acuminata) and very little rural traffic (3 vehicles/h). The peri-urban zone ( Figure 1) was located on the periphery of the city with an accelerated advancement of the agricultural frontier, where the disturbed forests are mixed with pastures dominated by planted trees of Eucalyptus globulus; and is subject to relative high levels of traffic (1000 vehicles/h) due to the transit between this area and the city. The urban zone (Figure 1) was located within the central area. This part of the city has a very uniform structure and is dominated by a section of grassland with planted trees of introduced species and palms. This area has higher levels of vehicular traffic (2080 vehicles/h) and industrial and commercial activities than the others. The research was conducted during the months of March to September 2020.

Design and Data Collection
Three stations were selected for each land use (a total of nine stations), 10 trees with similar bark structure and diameter at breast height (DBH) > 10 cm were chosen for each station (90 trees). In each tree, the frequency and cover of bryophytes and epiphytic lichens were recorded with a 10 × 50 cm quadrat that was divided into 20 squares of 5 × 5 cm [30,32], which was placed vertically at 1.20 m.
We obtained vehicular traffic with punctual one-day sampling data related with the number of vehicles for each zone. We considered three different categories: (LV) = light-vehicles: cars and small vans; (HV) = heavy-vehicles: trucks and buses; and (MT) = motorbikes [38].

Data Analysis
In order to determine the sampling effort in the three zones, a species accumulation curve based on samples and the Chao 2 nonparametric richness estimator was used. Species richness and diversity (Shannon-Weaver and Simpson indices) were determined. The Index of Atmospheric Purity (IAP) was used to determine air quality in the city of Ambato. The IAP was calculated based on the summary of the frequencies of all the species present in each sampled tree [30].
IAP tree = Summary of the frequency of each species. Values from 0 to 20. IAP area = Average IAP values of trees for each area. Box plots were used to visualize changes in richness, diversity, and index of atmospheric purity in the different zones. Changes in richness, diversity, and the index of atmospheric purity of epiphytic cryptogams related with land use and station were analyzed with generalized linear models (GLM), using a Poisson error distribution and a logarithmic link function [46]. We applied non-parametric correlations (Spearman, p < 0.05) between richness, IAP, diversity and vehicular traffic.
Non-metric multidimensional scaling (NMDS) was performed to detect patterns of species composition in relation to land uses. The NMDS was run using the Bray-Curtis distance and 999 Monte Carlo permutations. To test whether the three land uses had significantly different compositions of epiphytic cryptogams and to detect the effects of station and vehicular traffic, we performed a permutational multivariate analysis of variance (PERMANOVA). All analyses were performed with R statistical software version 3.6.3 [47] and the statistical package "vegan" [48].  Species richness at tree level showed a high value for the control zone (14) compared with the peri-urban (9) and urban (5)   Richness, species diversity and index of atmospheric purity were influenced by zone, where the control zone has a positive effect and the peri-urban and urban zones have a negative effect (Table 1). A similar pattern was observed at the station level, with a positive effect at the stations in the control zone (Table 1). Table 1. Results of the Generalized Linear Model between richness, index of atmospheric purity and diversity of epiphytic cryptogams as a function of the different land uses and station. S1 = Celiano Monge, S2 = San Francisco, S3 = Atocha-Ficoa, S4 = SB Pinllo, S5 = Ambatillo, S6 = Quisapincha, S7 = Pasa, S8 = San Fernando and S9 = Pilahuin.

Beta Diversity
The NMDS ordination showed that the community composition of epiphytic cryptogams (lichen and bryophyte) is different in the in urban areas compared to the control areas ( Figure 4). The multivariate statistical analyses showed that epiphytic composition of cryptogams was structured according to land use changes, with a large component of variation (i.e., 30%) associated with station, followed by zone and vehicular traffic, with 15% and 12 %, respectively (Table 2).

Discussion
The results indicated changes in the diversity (alpha and beta) and the index of atmospheric purity of epiphytic cryptogam communities as moving from rural (control) to urban areas. Similarly, previous studies found that diversity and IAP decreases in urban areas as a result of land use, industrial activity and increase in the flow of different types of motorized transport that generate a greater amount of polluting emissions [15,26,32,49].
In this context, the index of atmospheric purity, richness, and diversity (Shannon-Weaver and Simpson diversity indices) showed a significant increase in the control zone, indicating better air quality, as reported in previous studies [14,26,28,50,51]. In addition, in the control zone there was a higher richness of species sensitive to contamination (e.g., Neckeropsis undulata) and lichen species (e.g., Leptogium marginellum). An opposite pattern was observed in the urban area, with low diversity and IAP values related to higher pollution, mainly due to the vehicle fleet and industrial activities in urban zones [26,27,29,30] and the dominance of species resistant to atmospheric pollution, such as Candelaria concolor, Physcia sorediosa and Physcia aipolia.
Following the same pattern, the composition of the cryptogam communities revealed significant changes between control and urban zones. Control zones were dominated by bryophyte species (e.g., Neckeropsis undulata and Syntrichia fragilis) and some sensitive lichen species (e.g., Teloschistes chrysophthalmus) that were classified as sensitive to contamination in previous studies [16,26]. Conversely, in urban areas there is a greater coverage of tolerant species, such as Candelaria concolor and Physcia aipolia that are closely related to increased vehicular traffic and industrial activities. Supporting this idea, several studies have documented that these species are characteristic of areas with a high degree of pollution [16,26,32].
However, the Atocha-Ficoa urban station showed IAP values and community composition very similar to the control station, which may be related to vegetation cover (nine hectares) with a high diversity of tree species (Cedrela odorata L. and Jubaea chilensis Baill). Similarly to our results, previous studies have shown that urban areas with large vegetation coverage and native wooded species imply more habitat available for the establishment of epiphytic cryptogams [32]. Thus, it is important to consider that changes in diversity, IAP, and community composition in the epiphytic cryptogam communities in the city of Ambato also occurred at the station level.
Although our study demonstrates the efficacy of cryptogam diversity in biomonitoring of land use changes and air quality in the city of Ambato, we suggest that future work should evaluate the effects of microclimatic changes and host tree traits in epiphytic cryptogams as part of biomonitoring, given that previous studies have shown that urbanization related with microclimatic changes (e.g., canopy cover), host tree traits including bark texture, pH, and tree diameter show significative effects on cryptogam diversity in tropical urban environments [52][53][54].

Conclusions
In conclusion, the index of atmospheric purity, alpha and beta diversity of epiphytic cryptogams were drastically affected by changes in land use (e.g., urbanization), related to increased vehicular traffic and industrial activities (e.g., footwear, tanneries, textile). The urban area of the city of Ambato was identified as having high levels of air pollution due to its high vehicular traffic, low diversity and IAP values. This work showed that epiphytic cryptogams are suitable for monitoring air pollution in Ecuador, and that the combined use of alpha, beta diversity and index of atmospheric purity with epiphytic cryptogams proved to be a fast and low-cost effective method for air quality assessment in tropical areas. Acknowledgments: We thank Ministerio del Ambiente y Agua del Ecuador by providing access to the study areas and anonymous reviewers for constructive comments on the manuscript.