Physicochemical properties of waterfall stream. Table 1 shows the results of the environmental parameters of the Huay Pah Lahd stream. Rainfall strongly affects physicochemical properties of the water because velocity, depth, and level increased, and nutrient run-off (and/or pollution run-off) changed during the sampling period in the rainy season. The water at the sampling site is shallow (0.4-1 m). Temperatures in water samples range from 20°C to 33°C, while water temperature along the bank of Pharadorn waterfall, located downstream of Romklao waterfall in Phu Hin Rong Kla National Park, is 25°C6. Low water temperature and low light intensity are important for bryophyte development and primary productivity; however, nutrient enrichment in plant media (e.g., soil, sediment) is the most important component for bryophyte growth7.
The quantities of nutrients such as ammonia-nitrogen (NH3-N), nitrate-nitrogen (NO3-N), total Kjeldahl nitrogen (TKN) and phosphate (PO4) were below detectable levels, i.e., 1.01 mg L-1, < 4.0 mg L-1, and 0.02 mg L-1, respectively. Increased concentrations of phosphorus and nitrogen compounds, which are limiting factors and essential nutrients for aquatic life, can cause eutrophication. However, dilution effects during the rainy season in lotic ecosystems is a key factor in causing low nutrient concentrations in water bodies8. Heavy metal concentrations (Cu, Cd, Zn, Fe, Cr, Pb and Mn) in the water sample were relatively low to undetectable except for Ni, which was detected at 0.056 mg L-1. Furthermore, F¯ and Cl¯contents were < 0.15 and 6.2 mg L-1, respectively, which are considered low according to the permissible limits set by the World Health Organization (WHO) at 0.6-1.5 mg L-1 and 250 mg L-1, respectively9. Total organic carbon (TOC) content of the water sample is below the detection limit (< 0.05 mg L-1), which is due to the low content of contaminants in the water sources10.
Total solids is a direct measurement of the total mass of organic and inorganic particles suspended in water, as well as total dissolved ions in the water11. A high total solid content in waters is the most likely cause of increased total hardness. Other possible sources of increased hard water content include Ca, Mg and other heavy metals widely distributed in rocks and sediments12. Pah Lahd stream is naturally soft in this study (31.7 mg L-1) because it has very low amounts of total solids and minerals. Soft water is defined as having a low amount of calcium carbonate in the water sample13.
In this study, the pH value of the stream was near neutral (6.75). Slightly acidic water (~pH 5-6) occurs naturally, enabling some heavy metals that are adsorbed to mineral surfaces to be dissolved in aquatic ecosystems, as well as enhancing mineral dissolution in sediment14. However, in this investigation, slightly acidic water (pH 6.5-6.9) was not shown to be detrimental to aquatic biota or the ecosystem. The DO level of the study site (5.03 mg L-1) was nearly equivalent to the DO water quality standard in a Thai waterfall stream (6 mg L-1); nevertheless, a reduction in DO levels might be linked to human activities, i.e., the presence of relatively large numbers of tourists and improper waste disposal15. High flow velocity and turbulence of a waterfall increases DO content16. Biological oxygen demand (BOD) is another important indicator of water quality, as it measures the quantity of oxygen required for microbial respiration and biological degradation of organic matter in water. Reduced BOD levels imply that the quantity of organic substances is promoting the growth of microbial populations, thus enhancing the available DO content for aquatic life. The current study revealed that the water sample had a low BOD level (<1 mg L-1), indicating that the waterfall ecosystem had good water quality17.
Bryophyte taxa in the study site. Based on their microhabitats and life modes, a total of eight bryophytes were collected from two major taxonomic groupings i.e., (1) moss: epilithic bryophytes or rupicolous (three acrocarpous mosses: Hyophila involuta (Hook.) A. Jaeger (Pottiaceae), S. cataractae and Bryum sp. (Bryaceae); and three aquatic mosses (one acrocarpous moss, Fissidens crispulus Brid. var. crispulus (Fissidentaceae) and two pleurocarpous mosses: Claopodium prionophyllum (Müll. Hal.) Broth. (Leskeaceae), and Ectropothecium zollingeri (Müll. Hal.) A. Jaeger (Hypnaceae); and (2) liverworts: one thalloid liverwort (Marchantia emarginata Reinw. Blume & Nees var. emarginata (Marchantiaceae), and one aquatic leafy liverwort (Porella acutifolia (Lehm. & Lindenb.) Trevis. var. birmanica S. Hatt. (Porellaceae).
Scopelophila cataractae is a rare taxon found in Thailand that is listed as endangered in the IUCN Red List’s threatened category18. Few specimens of this moss taxa have been discovered in the forests of Northern Thailand19. Scopelophila cataractae is found in various parts of the world including China, Korea, Japan, Papua New Guinea North and South America20,21. A protonemal colony of S. cataractae was observed at the same period of time (June, during the rainy season) and at the same study site as in the previous study19. Narrow, light-green patches of the protonemata occur along the stone base (c. 50 cm height) in streams, together with colonies of other bryophytes such as H. involuta. The colony consisted of numerous filamentous protonema which produced shoots of S. cataractae with numerous gemma-like cells on the axils of younger leaves.
Porella acutifolia var. birmanica was firstly discovered in Burma and is mostly distributed in the Indochina regions including Burma, Vietnam, Laos, and Thailand22,23. This taxon has been reported in Doi (mountain) Suthep and Ru See (Hermit) cave in Doi Suthep-Pui National Park, Chiang Mai Province, at elevations of about 1,100–1,200 m22. In open and urban environments, Hyophila involuta, Bryum sp., and M. emarginata may be termed pioneer taxa. Hyophila involuta can be located in a variety of habitats including deserts, soil in humid regions, soil, wet rocks, and waterfall stream banks, as well as on concrete buildings in urban settings24,25. Unfortunately, Bryum sp. specimen lacked sporophyte materials, thus it was not possible to identify it to the species level. Members of the Bryaceae family, on the other hand, are abundant in urban and disturbed regions across the world, and can be seen growing with potted plants26. Marchantia emarginata, a cosmopolitan taxon of thalloid liverwort27, is abundant on soils and rocks near stream banks and other locations in Chiang Mai Province, and is not restricted to National Parks. There were three lithophytic mosses, which are aquatic, semi-aquatic, or found on soil and rock near the Huay Pah Lahd falls seasonally dry streams. Ectropothecium zollingeri, F. crispulus var. crispulus, and C. prionophyllum, for example, may thrive on muddy, debris-covered rocks that are inundated during the rainy season and dry during the hot-dry season. Many bryologists have found mosses such as E. zollingeri28; F. crispulus var. crispulus20; and C. prionophyllum29 in various moist or semi-wet locations including aquariums.
Heavy metal concentrations in bryophyte tissues and substrates. Bryophytes do not have true roots, stems, and leaves; rather, they possess multicellular rhizoids at the lowest part of the structure, which are responsible for water and nutrient absorption30. Bryophyte rhizoids facilitate uptake of available minerals and water to the stems through capillary action. Because bryophytes lack roots, they can readily absorb heavy metals throughout their entire surface of rhizoids31. Furthermore, phyllids (leaf-like structure) and thalli of bryophytes have highly absorbent surfaces and an absence of waxy cuticle over the laminal surfaces. As a consequence, cell walls easily absorb moisture and a wide range of minerals and metal ions from the water that flows over the plant32. Heavy metal accumulation in bryophyte tissues in this study appear in Table 2. Copper levels in tissue ranged from 8.5 mg kg-1 (P. acutifolia var. birmanica gametophyte) to 530.1 mg kg-1 (S. cataractae protonema); Cd from 4.8 mg kg-1 (E. zollingeri gametophyte) to 16.9 mg kg-1 (S. cataractae protonema); Zn from 129.4 mg kg-1 (H. involuta gametophyte) to 1,187.2 mg kg-1 (S. cataractae protonema); Fe from 3,962.5 mg kg-1 (H. involuta gametophyte) to 8,026.7 mg kg-1 (S. cataractae protonema); and Mn from 143.3 mg kg-1 (S. cataractae gametophyte) to 504.6 mg kg-1 (C. prionophyllum gametophyte).
In this investigation, gametophytes of S. cataractae had considerably greater Cu accumulation (p < 0.05) or approximately 3.7-59 × than did other bryophytes, although protonema of S. cataractae had a slightly higher Cu concentration than gametophytes of S. cataractae (p > 0.05). Because it accumulated Cu primarily in gametophyte tissue, S. cataractae, often known as “rare Cu moss,” is categorized as a hyperaccumulator33. Copper is an essential nutrient that is required for plant development and growth. This element plays a significant role in regulating physiological functions such as the photosynthetic and respiratory electron transport chains, nitrogen fixation, protein metabolism, antioxidant production, the ROS defense system, cell wall metabolism, and hormone perception, and acts as an essential cofactor for numerous metalloproteins34. At the cellular level, however, excessive Cu concentrations are harmful to plants because binding to different enzymes results in inactivation and disruption of enzyme activity or protein functions34. Gametophytes of S. cataractae accumulated substantial amounts of Cd, Zn and Fe, with concentrations of 9.2 mg kg-1, 846.1 mg kg-1 and 5,434.3 mg kg-1, respectively. S. cataractae has been shown to accumulate substantial amounts of different heavy metals such as Cd, Cu and Zn in contaminated soils (e.g., Cu tailings)4. Remarkably low Cu concentrations were detected in C. prionophyllum and H. involuta (10.3 kg-1 and 9.6 mg kg-1, respectively).
The highest Cu concentrations were found in sediment substrate of shoot colonies and protonemal colonies of S. cataractae (251.6 mg kg-1 and 239.4 mg kg-1, respectively) (p < 0.05), whereas substantial Fe concentrations were found in sediment substrate of gametophyte colonies of H. involuta (3,127.1 mg kg-1) (p < 0.05) and sediment substrate of protonemal colonies, shoot colonies and decayed moss of S. cataractae (2,345.3, 2,289.4 and 1,963.7 mg kg-1, respectively) (Table 3). Copper concentrations in substrates of S. cataractae and water were generally in the following order: sediment substrate > rock > water. According to recent research, growth substrate is a key source of heavy metals in stream environments. This may have led to increased absorption and accumulation of Al, Cu and Zn in S. cataractae gametophytes19. Cadmium and Zn concentrations in rock substrates of S. cataractae and P. acutifolia var. birmanica were low(0.5 and 0.3 mg kg-1 for Cd, and 34.9 and 31.2 mg kg-1 for Zn, respectively). Heavy metals (Cu, Fe and Mn) in rock substrates of P. acutifolia var. birmanica were found to be similar to in the rock substrates of S. cataractae. This comparable distribution of heavy metals in rock substrates may be attributed to the fact that they are located in a similar environment and so receive heavy metals from similar sources and mechanisms. This trend is consistent with the findings of the previous study19.
Many bryophyte taxa have been tested for their tolerance and accumulation capabilities at both laboratory and field scales. For example, B. radiculosum Brid. (Bryaceae) grown in industrial areas of Portoscuso (Sardinia, Italy) has been used as bioindicator for trace elements such as Pb, Cd and Zn, with accumulation rates at 61-2141 mg kg-1, 3-40.6 mg kg-1 and 32-2,360 mg kg-1, respectively35. Bryum radiculosum growing in areas which received heavy metals from Cu-containing pesticides in vineyards, accumulated considerably lower amounts of Cu than Bryum sp. in this study (135.3 mg kg-1), or less than 1.4-13.5 fold35. Furthermore, P. acutifolia var. birmanica, C. prionophyllum and Bryum sp. accumulated substantial Fe (6,877.6 mg kg-1, 6,370 mg kg-1 and 5,869.9 mg kg-1, respectively), as well as substantial Cd (12 mg kg-1, 8.2 mg kg-1 and 6.2 mg kg-1, respectively), and modest amounts of Zn (161.3 mg kg-1, 226.9 mg kg-1 and 225.5 mg kg-1, respectively). Cadmium is a hazardous metal and Cd exposure in moss media at 10 mM inhibited photosynthesis and caused nutrient deficiencies, which can lead to chlorosis in gametophyte tissues of Physcomitrium patens (Hedw.) Mitt. (Funariaceae) and aquatic moss, Fontinalis antipyretica Hedw. (Fontinalaceae)36. Zinc and Fe often occur in high concentrations in the lithosphere. Both are major components of numerous enzymes and proteins in plants and are thus essential for biota. High concentrations of Zn and Fe, on the other hand, can be toxic to moss cells, affecting the entire plant by decreasing moss growth and development37.
In this study, substantial Mn concentrations were detected in gametophytic tissues of C. prionophyllum, Bryum sp., H. involuta and F. crispulus var. crispulus (504.6 mg kg-1, 482.6 mg kg-1, 467.2 mg kg-1 and 448.6 mg kg-1, respectively). Manganese accumulations in the study bryophytes were much higher (144.3-504.6 mg kg-1) when compared to four moss taxa Bryum argenteum Hedw. (Bryaceae), Bryum capillare Hedw. (Bryaceae), Brachythecium sp. (Brachytheciaceae), and Hypnum cupressiforme Hedw. (Hypnaceae) grown in various locations (roadside, populated areas, forests, croplands), with a wide range of Mn accumulation (0.1-8.6 mg kg-1)38. Excessive Mn concentration in plant tissues can induce oxidative stress, alter enzymatic activity, absorption and accumulation of nutrients, and translocation of certain elements including calcium (Ca), magnesium (Mg), Fe and phosphorus (P)39.
The rediscovery of P. acutifolia var. birmanica in Huay Pah Lahd stream after a half-century22 may suggest that the Doi Suthep-Pui National Park still serves as a haven for sensitive bryophytes, or it could indicate that the park is minimally affected by anthropogenic activities and thus can support a suitable habitat for bryophytes. However, recent reports suggest that anthropogenic activities around the sampling location may have introduced heavy metals into sediment and water, resulting in increased heavy metal absorption and accumulation in bryophyte tissue19. Because P. acutifolia var. birmanica also accumulated substantial heavy metals, particularly Cd, the presence of this leafy liverwort may be used as bioindicator in future research for monitoring changes in environmental patterns of stream ecosystems.
Enrichment factors. The EFs of all heavy metals examined (Fig. 1) were very low (< 2). For example, the EFs of Cd and Mn in the study site were lower than 12.4 times and 34.5 times those of mosses grown along a major road in Serbia, suggesting that the sources of these metals are lithologic, i.e., sediment, water, and rock40. Anthropogenic activities are certainly sources of heavy metals, particularly in locations of domestic dwellings, industry, and other human activities41.
Each bryophyte taxon has specific habitat and environmental preferences and a different ecological niche. These factors, in combination with their sensitivity to environmental change, makes bryophyte taxa distribution a useful indicator of vegetation alteration and climate change42. Thus, bryophytes are commonly used to evaluate the health status of a habitat, as they have the propensity to take up and accumulate pollutants from soil and water. Many reports have indicated that certain sensitive bryophyte taxa accumulate trace metals (Cd, Pb, Ni, and Cr) from the atmosphere, soil and water in contaminated areas across many regions worldwide43. Unfortunately, to date, there exist few studies investigating bryophytes as bioindicators of the heavy metals in both terrestrial and aquatic environments in Thailand19.