Nickel excess affects phenology and reproductive attributes of Asterella wallichiana and Plagiochasma appendiculatum growing in natural habitats

Bryophytes are potent metal absorbers, thriving well on heavy metal (HM)-polluted soils. Mechanisms controlling uptake, compartmentalization and impacts of HMs on bryophytes life cycle are largely unknown. The current study is an effort to decipher mechanisms of nickel (Ni) excess-induced effects on the phenological events of two bryophytes, Asterella wallichiana and Plagiochasma apendiculatum growing in natural habitats. Observations revealed Ni-excess induced negative impacts on abundance, frequency of occurrence of reproductive organs, population viability and morphological traits, spore viability and physiological attributes of both the liverworts. Results led us conclude that P. appendiculatum survived better with the lowest impact on its life cycle events than A. wallichiana under Ni excess in natural habitats. Our findings collectively provide insights into the previously unknown mechanisms of Ni-induced responses in liverworts with respect to phenological attributes, as well as demonstrate the potential of P. appendiculatum to survive better in Ni excess habitats.

. Soil analyses of sites inhabited by Asterella wallichiana and Plagiochasma appendiculatum. Concentrations (mg kg -1 ) of macronutrients (organic carbon, phosphorus, nitrogen and organic matter), micronutrients (copper, zinc, iron and manganese) and trace elements (nickel, Ni) present in soil inhabited by A. wallichiana and P. appendiculatum under field conditions. Samples from five collection sites were individually taken. Sites with lower Ni concentrations (10.25 ± 0.79 mg kg -1 ) were referred as 'control site' (CS, n = 5) and sites with higher Ni concentrations (125 ± 5.16 mg kg -1 ) were referred as 'Ni-excess site' (NS, n = 5). Data presented are means ± standard errors. Different letters (a, b) within a row indicate significant differences from each other (Tukey's test, P ≤ 0.05). www.nature.com/scientificreports/ Ni uptake potential of liverworts. Asterella wallichiana and Plagiochasma appendiculatum grown in their natural habitats were examined for their Ni uptake potential at the gametophytic stage. Results showed that Ni uptake potential of A. wallichiana and P. appendiculatum varied significantly in NS compared with CS. About 12.6-and 7.7-fold increases in Ni uptake potential (Niup) were noted in the gametophytic thalli of A. wallichiana and P. appendiculatum, respectively, in the NS relative to that of the CS under natural conditions (Table 3). These observations provided us a stimulus to investigate the morphological and physiological changes in the gametophytic thalli of A. wallichiana and P. apendiculatum during their life cycle events in response to Ni excess under field conditions. Table 2. Single pollution index (PI), contamination factor (Cf), sum of contamination (PIsum) and Nemerow pollution index (PI Nemerow ) of nickel-excess (NS) and control sites (CS). For the PI and Cf, concentrations of Ni, Cu and Zn measured in the CS and NS (n = 5) were compared with their respective world mean value 37 . PIsum was calculated using the geometric mean of PI of each metal in the CS and NS. Different letters (a and b) within a column indicate significant differences from each other in all combinations (Tukey's test, P ≤ 0.05).  Table 3. Physiological indices of young gametophytic thalli of Asterella wallichiana and Plagiochasma appendiculatum. DB (mg kg -1 , dry weight), dry biomass ; Niup, nickel (Ni) uptake (mg kg -1 , dry weight); Ti, Ni tolerance index (Ti); RSA, rhizoid surface area; malondialdehye (MDA) and H 2 O 2 contents (µmol g -1 fresh weight); relative electrical conductance (REC %), chlorophyll a (Chl a), chlorophyll b (Chl b), carotenoid (CAR), ascorbic acid (ASA), glutathione (GSH) and proline (PL) contents (mg g -1 fresh weight) in young gametophytic thalli of A. wallichiana and P. appendiculatum grown in Ni-excess sites (NS, n = 5) and control sites (CS, n = 5) were measured. Different letters (a and b) within a row indicate significant differences from each other in all combinations (Tukey's test, P ≤ 0.05). Bold letters A and P represent A. wallichiana and P. appendiculatum, respectively.  (Fig. 1a,b). In general, A. wallichiana showed more abundance than P. appendiculatum throughout the year in CS. However, in NS, A. wallichiana exhibited a higher reduction in abundance as compared to P. appendiculatum. The maximum reduction in abundance was observed in December, January, and February month in A. wallichiana by the difference in the average number mature thalli by 40, 40 and 40, respectively than in P. appendiculatum by 20, 20 and 30, respectively (Fig. 1a,b). Furthermore, principal component analysis (PCA) also indicated that the variation in the abundances of mature gametophytic thalli was more evident in P. appendiculatum than in A. wallichiana grown in Ni excess sites in NS (Fig. 1c). Population viability analysis (PVA) carried out on the young gametophytic thalli of A. wallichiana and P. appendiculatum grown on the CS and NS showed visible differences in the distribution of reproductively active archegonia and antheridia. Transition of the young gametophytic thalli into archegonia and antheridia production was counted during a period of four weeks. During this time, NS showed significant impact on lowering the number (N) of reproductively active archegonia and antheridia in both the liverworts compared to CS (Fig. 2a,b). However, A. wallichiana exhibited a higher reduction in the number of reproductively active archegonia and antheridia compared to P. appendiculatum (Fig. 2a,b). Furthermore, the quasi-extinction (QE) (i.e., the probability calculated versus threshold level) also showed a negative effect of Ni excess on the distribution patterns www.nature.com/scientificreports/ of the reproductively active archegonia and antheridia in both the liverworts (Fig. 2c,d). Importantly, on the basis of the PVA and QE data, effect of Ni excess on the number of reproductively archegonia and antheridia was found to be more evident in A. wallichiana than in P. appendiculatum ( Fig. 2a-d).
Effect of Ni excess on physiological indices. Ni excess showed negative impact on the physiological indices of both the liverworts. Such as, effect of Ni excess was evident on the gametophytic thallus dry biomass (DB), with 24% and 10% reduction in A. wallichiana and P. appendiculatum, respectively, recorded in NS compared to CS (Table 3). Ni tolerance index (Ti) in NS was 0.79 (A. wallichiana) and 0.9 (P. appendiculatum) as compared with their respective value in CS (Table 3). Rhizoids surface area (RSA) of the A. wallichiana and P. appendiculatum gametophytic thalli grown in NS showed reduction by 23.7% and 7%, respectively, compared with their corresponding CS (Table 3). Abiotic stresses exert secondary oxidative damages to plants besides the ionic toxicity, which results in peroxidation of the cell membrane lipids 29 . About 2.3-and 2-fold increase in malondialdehyde (MDA) content was recorded in young gametophytic thalli of A. wallichiana and P. appendiculatum, respectively, grown in NS compared with CS (Table 3). Additionally, the relative electrolyte conductance (REC), showed maximum increase of 40% and 5% in A. wallichiana and P. appendiculatum respectively in NS relative to that of CS (Table 3). H 2 O 2 content in young gametophytic thalli of both A. wallichiana and P. appendiculatum were more in NS compared to CS (Table 3). Among the examined photosynthesis pigments, the levels of chloro-   (Table 3). A dramatic decrease in Chl b content was observed in A. wallichiana (32%) and P. appendiculatum (20%) in NS, when compared with CS ( Table 3). The carotenoid (CAR) content was decreased in A. wallichiana by 32% and increased in P. appendiculatum by 38% in NS compared with CS (Table 3). However, in CS, P. appendiculatum showed higher CAR content than A. wallichiana. We have also studied the level of non-enzymatic antioxidants such as ascorbic acid (ASA) and glutathione (GSH). About 41% and 12% increase in ascorbic acid (ASA) content was recorded in A. wallichiana and P. appendiculatum, respectively, in NS Compared to CS (Table 3). Glutathione (GSH) content was enhanced by 42% and 73% in A. wallichiana and P. appendiculatum, respectively, in NS as compared to CS. Proline content was increased by 46% and 57% in A. wallichiana and P. appendiculatum in NS as compared to CS ( Table 3).

Effect of Ni excess on the development of gametophytic stages of liverworts.
A negative correlation was found between Ni uptake and reproductive potential of the gametophytic stages of the two liverworts. Developments of male and female reproductive organs were affected by Ni excess (Table 4). Though no visible changes in the development of antheridia and archegonia were recorded in A. wallichiana and P. appendiculatum in both NS and CS, approximately 32% and 21% decrease in frequency of occurrence (FOC) of antheridia (total of antheridia/per 10 × 10 cm 2 patch) was noted for A. wallichiana and P. appendiculatum, respectively (Table 4). While FOC of normal antheridia encounter (NAntE) of A. wallichiana and P. appendiculatum declined by 20.7% and 14.4%, respectively, in NS compared to CS. Development of archegonia was also negatively affected in NS (Table 4), about 25% and ~ 13% reduction in FOC of archegonia (total of archegonia/per 10 × 10 cm 2 patch) was noted for A. wallichiana and P. appendiculatum, respectively, relative to that of CS. While, FOC of normal archegonia encounter (NArcE) of A. wallichiana and P. appendiculatum showed reduction by 25% and 13%, respectively, in NS compared to CS (Table 4). Spore viability was also reduced in both the liverworts in NS compared to CS (Table 4).

Effect of Ni excess on intracellular localization of ROS in young gametophytic thalli. Results
of current study showed higher accumulation of reactive oxygen species (ROS) in both A. wallichiana and P. appendiculatum in NS compared to their respective CS (Fig. 4a-d). Grey values measured for the region of interest (ROI) in A. wallichiana showed 76% increase, while mere 14% increase in grey value was noted in P. appendiculatum thalli in NS compared to their respective CS (Fig. 4e). On the basis of grey values, reactive oxygen species intensity (ROSi) calculated showed 50% and 33% increase in A. wallichiana and P. appendiculatum, respectively, in NS compared to their respective CS (Fig. 4f).
Ni excess changed urease activity in young gametophytic thalli of A. wallichiana and P. appendiculatum. Urease (EC 3.5.1.5) is a key enzyme regulating the hydrolysis of urea into ammonia and bicarbonate. Understanding the role of urease in facilitating Ni uptake and assimilation into urease could be predicted via determining its activity [30][31][32] . Results of current study revealed differential responses in terms of urease activity in young gametophytic thalli of A. wallichiana and P. appendiculatum grown in NS and CS (Fig. 5). The urease enzyme activity was increased in both the liverworts in NS compared to CS. About 275% increase in urease Table 4. Nickel impacts frequency of occurrence of antheridia and archegonia and spore viability in Astrella wallichiana and Plagiochasma appendiculatum. Visible effects of Ni excess (NS, n = 5) observed on frequency of occurrence (FOC) of normal antheridia and archegonia of A. wallichiana and P. appendiculatum compared to control sites (CS, n = 5). Data presented are FOC taking control as 100%. Abbreviations: FOC, frequency of occurrence; NAntE, normal antheridia encounter; NArcE, normal archegonia encounter; T, total spore count; V, viable spore count; NV, non viable spore count. Spore count values represent 100 spore count made per slide, with three independent biological replicates (n = 3).  www.nature.com/scientificreports/ activity was noted in P. appendiculatum compared to 96% in A. wallichiana in NS compared to their respective CS (Fig. 5).

Discussion
Nickel (Ni) phytotoxicity has been proven to be more disastrous than its deficiency in plants 6   www.nature.com/scientificreports/ The HM-uptake potential of a plant depends on several morphological and physiological attributes, such as surface area exposed to HMs, physiological status and thickness of the epidermis 10 . Present study, in natural habitats (Control sites, CS and Nickel excess sites, NS) has shown that A. wallichiana is endowed with higher Ni-uptake potential than P. appendiculatum. In general, soil pH has been associated with mobility of metal ions in soil solution and in the rhizosphere zone of a plant 18 . Bryophytes, being hyper-accumulator of metal ions like Cu, Zn, Ni and Pb, uses several mechanisms to reduce metal uptake and/or adsorb metal ions on the surface alone 18 . In the current study, the higher accumulation and availability of more Ni 2+ ions in NS, A. wallichiana and P. appendiculatum could be linked to decrease in pH value from 8.3 in CS to 7.7 in Ni-excess NS ( Table 1).
Abundance of higher plants is negatively affected by HMs 35 . Among the lower plants, impact of Ni-Cu complex on boreal forest vegetation has been evaluated along the Russian-Norwegian-Finnish border 36 . Air pollution loaded with Ni has been shown to negatively affect the species richness and abundance of bryophytes. In addition, high pH and high total phosphorous (P) concentrations and low C/N values in the humus have also been advocated as factors causing decline in abundance and species richness of bryophytes 36 . In current study, NS (n = 5) showed reduced abundance of A. wallichiana and P. appendiculatum compared with CS (n = 5) (Fig. 1a-c). This finding could be supported by an explanation that the NS having higher Ni excess showed positive correlation with total Cu content and pH value, thus leading to reduced abundance of A. wallichiana and P. appendiculatum compared to CS (Table 1).
Soil pollution indices, including PI, Cf, PIsum and PI Nemerow , are widely used to evaluate the threat level of a particular element in soil 37 . These indices revealed strong contamination of Ni metal in the NS soils compared with CS soils among the three heavy metals (Cu, Zn and Ni), as only Ni scored higher values of PI, Cf and PI Nemerow in the NS soils than in CS soils ( Table 2). Contribution of Ni towards increasing the PIsum value was much higher in the NS than in CS, further supporting that Ni is the only metal causing contamination of the NS soil (Table 2). Cf indices have been used to classify the soils into different categories based on the levels of excess HMs (Cd, Pb, Co, Cr, Ni, V, Cu, Zn, Mo, As, Th, and U) 38 . Application of Cf has also used for the background determination of pollution assessment of HMs in sediments and soils 39 . Similarly, PI Nemerow is used for the assessment of HM contamination in surface layers of Roztocze National Park forest soils (South East Poland) 40 . Additionally, our population viability analysis (PVA) of the data obtained under field conditions also indicated negative impact of Ni excess on the number of reproductively active archegonia and antheridia of both the liverworts, with higher negative Ni effect being observed on A. wallichiana than P. appendiculatum (Fig. 2a,b). In support of the PVA result, the Quasi-extinction (QE) analysis also showed negative effect of Ni excess on the numbers of reproductively active archegonia and antheridia of the two liverworts under field conditions, of which A. wallichiana suffered higher Ni effect than P. appendiculatum (Fig. 2c,d).
The HM pollution has been shown to induce changes in the surface properties of mosses. A study conducted in Pleurozium schreberi (a moss) proved that exposure of moss to Ni could reduce the canopy size 41 . Reductions in rhizoids surface area (RSA) of A. wallichiana and P. appendiculatum thalli in NS could be a morphological adaptation of these liverworts to reduce the areas exposed to Ni excess (Table 3). Besides affecting RSA, Ni excess was found to reduce DB and FOC of antheridia and archegonia of both the liverworts, with A. wallichiana being most affected in the later (Tables 3,4). Successful survival of HM-hyper-accumulator plants grown in HM-excess soils has been linked to their higher tolerance index Ti 42 . For example, higher Ti potential of Raphanus sativus L. compared with Brassica napus L. grown on multimetal-excess soils advocates its uses in phyto-remediation and better survival in HM excess soils 42 . Higher rhizoids surface area (RSA) has been shown to improve Ti potential by adsorption of metal ions on the surface of rhizoids 18 . Such that, reduced values of Ti for A. wallichiana in NS could be attributed to more reduction in RSA in comparison with P. appendiculatum compared to CS ( Table 3). The FOC of antheridia and archegonia in both A. wallichiana and P. appendiculatum were lower in NS (Table 4), which might be linked to the negative impact of Ni excess on the growth and development of male and female gametopyhtes; and this was more clearly observed in A. wallichiana than P. appendiculatum. www.nature.com/scientificreports/ Accumulation of HMs and their localization to floral organs have serious implications on reproductive potential of a plant 12 . In Cucurbita pepo, HMs (e.g., Zn, Cu, Ni and Pb) were shown to translocate from soil into floral organs, such as pistil, anther and nectary 12 . This HM translocation was found to negatively impact pollen viability, pollen removal and deposition, thereby affecting the overall plant fitness in C. pepo 12 . Similarly, HM translocation and accumulation have been shown to impact pollen germination and pollen tube length in tobacco plants 43 . Parallel to these observations, current study also pointed negative impact of Ni excess on the reproductive behavior of A. wallichiana and P. appendiculatum. Accumulation of Ni ions in the sporophytes and gametophytes of both the liverworts were observed (Fig. 3 Panel A and B). Prominent changes observed included reduction in the number of FOC of normal antheridia and archegonia in NS, particularly in A. wallichiana compared to CS (Table 4).
HMs induce production of ROS, which causes extensive damage to lipids, reducing membrane fluidity, and elevates membrane leakiness, as evidenced by increased MDA contents in stressed plants 44 . In current study, a significant increase in MDA content was recorded in young gametophytic thalli of A. wallichiana and P. appendiculatum in NS compared to CS (Table 3). Our findings are in agreement with those of Choudhury and Panda 45,46 , which also observed a gradual increase in MDA content in Taxithelium nepalense, a moss subjected to Pb or Cr stress. Besides MDA content, both the liverworts in NS also showed increased membrane leakiness as revealed by higher values of relative electric conductance (REC) in A. wallichiana than in P. appendiculatum compared to CS ( Table 3). Observations of fluorescence microscopy revealed that Ni excess in NS could significantly induce ROS production, as evidenced by increases in ROS intensity (ROSi) when compared with CS ( Fig. 4a-f).
Photosynthetic pigments are one of the main sites of HM-induced injury in plants 47 . HMs have been shown to reduce photosynthetic pigment (Chl a and Chl b) contents 44,48 . Ni-induced negative impacts on the photosynthetic pigments were more evident in A. wallichiana as compared to P. appendiculatum in NS (Table 3). Higher urease enzyme activity in P. appendiculatum than A. wallichiana in NS compared to CS also advocates efficient Ni management by the former than the later (Fig. 5).
The non-enzymatic antioxidant molecules such as glutathione (GSH) and ascorbic acid (ASA), play vital role in tolerance to HMs 49,50 . GSH and ASA both are potential ROS scavenger molecules in plants 49 . In the present study, Ni excess enhanced the GSH and ASA content in both A. wallichiana and P. appendiculatum. GSH also acts as a precursor of phytochelatins and helps in the chelation of HMs which are then often sequestered in the vacuoles 50 . P. appendiculatum showed higher increase in GSH content compared to A. wallichiana which indicates that P. appendiculatum has better HMs sequestering and detoxification capacities. The relative abundance of proline is important biochemical indicators for abiotic stress tolerance 51 . Both A. wallichiana and P. appendiculatum accumulated higher proline in NS than CS. Proline regulates the accumulation of usable nitrogen, which might contribute to membrane stability and mitigates the disruptive effect of HMs stress.
This comprehensive morphological, physiological and reproductive investigations in A. wallichiana and P. appendiculatum has helped to understand mechanisms operative in liverworts for the management of Ni-induced oxidative stress under field conditions. Findings are of immense significance in establishing the mechanistic pathway of Ni-induced damage on the life cycles of the liverworts (Fig. 6). The mechanistic model developed on current observations clearly shows that Ni excess can induce morphological, physiological and reproductive changes in A. wallichiana and P. appendiculatum. These changes have the potential to negatively impact the phenological events of examined liverworts. Excessive production of ROS under Ni excess in NS, which induced membrane damage, and brought significant changes in A. wallichiana and P. appendiculatum and overall plant fitness. Translocations of Ni 2+ ions and their accumulation in gametophytes impact the maturation of gametophyte to sporophyte.

Materials and methods
Experimental sites. The area selected for the present study was district Reasi of the Union territory of Jammu and Kashmir, India which is geographically situated between 33° 4′ 58.1016′′ N latitude and 74° 49′ 59.9268′′ E longitude with an area spanning over 151,701 hectares.  A. wallichiana and P. appendiculatum. Soils inhabited by A. wallichiana and P. appendiculatum were collected and analyzed for the presence of various macro-and micronutrients. Estimation of macronutrients in soil samples was performed by the methods of Subbiah and Asija 52 for nitrogen (N 2 ) and Olsen et al. 53 for phosphorus (P). Determination of micronutrients (Cu, Zn, Fe, Mn and Ni) was estimated by atomic absorption spectroscopy method using standard procedures provided with the instrument (Perkin Elmer 3110, Germany). Soil pollution indices, including single pollution index (PI), contamination factor (Cf), sum of contamination (PIsum) and Nemerow pollution index (PI Nemerow ), were calculated for Cu, Zn and Ni using the standard procedures described in Qingjie et al. 54 and Kowalska et al. 37 . These indices were used to classify soils into different classes depending upon the degree of the contamination of a specific HM. For instance, soils with PI < 1, < 2, < 3, < 5 and > 5 indicated absent, low, moderate, strong and very strong soil pollution. For the Cf, soils were classified as follows: Cf < 1 = low contamination, Cf between 1 and 3 = moderate contamination, Cf between 3 and 6 = considerable contamination, Cf > 6 = very high contamination. For PIsum, geometric mean of PI of each HM present in the soil was taken. For PI Nemerow , the following criterion was applied for the soil classification: Class I (PI Nemerow values ≤ 0.7), II (PI Nemerow values between 0.7 and 1), III (PI Nemerow values between 1 and 2), IV (PI Nemerow values between 2 and 3) and V (PI Nemerow values > 3). Class I, II, III, IV and V refers to clean, warning limit, slight pollution, moderate pollution and heavy pollution, respectively. www.nature.com/scientificreports/ Nickel uptake potential of A. wallichiana and P. appendiculatum under field conditions. The young gametophytic stages of both the liverworts collected from the natural sites designated as CS (control sites, S1-S5, thereafter called as CS, n = 5, having minimum Ni concentration) and NS (NS1-NS5, thereafter called as NS, Ni excess sites, n = 5, having high concentration of Ni) sites were washed with tap water followed by distilled water to remove soil particles and other vegetation. To determine the Ni uptake potential of liverworts collected from the field, about 300 mg of oven dried samples of A. wallichiana and P. appendiculatum collected from CS and NS were placed in the muffle furnace (300-400 °C for 5 h) to ash, which was then digested using wet-digestion procedure in a mixture of HNO 3 and HClO 4 (4:1, v/v) as described elsewhere. The concentrations of Ni (in mg kg −1 tissue) were determined using Atomic Absorption Spectrometry (Shimazdu, AA7000, Japan) following manufacturer instructions.

Soil analysis and soil pollution indices of sites inhabited by
Collection of young gametophytic thalli of A. wallichiana and P. appendiculatum. Fresh samples of A. wallichiana and P. appendiculatum at young gametophytic stage (marked with absence of mature antheridia and archegonia) were collected from the CS (n = 5) an NS (n = 5) in district Reasi and brought to the laboratory in polyethylene bags. At this stage, samples were divided into two parts for short-term physiological and biochemical, and long-term morphological analyses: appendiculatum. Subsequent effects of Ni accumulation also caused disturbance in nitrogen metabolism (urease enzyme activity), while reduction in photosynthetic pigments in both the liverworts, with higher effects on A. wallichiana may be linked to reduced photosynthesis. Ni-induced physiological changes led to reduction in dry biomass, rhizoid surface area, abundance, frequency of occurrence of mature gametophytes (male and female) and frequency distribution and number of reproductively active archegonia and antheridia in both two liverworts, with higher effects on A. www.nature.com/scientificreports/ (i) Short-term analyses: For short-term physiological analyses, gametophytic stages of A. wallichiana and P. appendiculatum collected from NS and CS were used for physiological and biochemical parameters such as malondialdehye content (MDA, µmol g -1 fresh weight), H 2 O 2 content (µmol g -1 fresh weight) and relative electric conductance (REC%) 29,55 .
(ii) Long-term analyses were carried on the young gametophytic thalli of A. wallichiana and P. appendiculatum growing on NS and CS. These sites were visited every week for a period of 1-2 months to observe the development of young gametophyte into mature gametophytes bearing antheridia and archegonia, and development of sporophyte.
Population viability analysis of A. wallichiana and P. appendiculatum under field conditions. For measuring the impact of Ni excess on the survival and normal functioning of the reproductive structures (antheridia and archegonia) of A. wallichiana and P. appendiculatum, a comprehensive population viability analysis (PVA) was carried using the Vortex ver. 10.3.1 56 . The impact of Ni excess on the frequency distribution and number of the reproductively active archegonia and antheridia of the A. wallichiana and P. appendiculatum was determined for the young gametophytic thalli of the two liverworts growing in CS and NS. Vortex was also used to determine the quasi-extinction risk imposed by Ni using several input parameters, such as initial population size (number of mature gametophytes of A. wallichiana and P. appendiculatum in CS and NS), mortality (number of gametophytes perished under Ni excess in NS), catastrophe (Ni excess considered as a catastrophe reducing number of reproductively active antheridia and archegonia in NS compared to CS) and reproductive potential of the antheridia and archegonia of both the liverworts.
Phenological attributes of A. wallichiana and P. appendiculatum in field conditions. For field conditions, ecological attributes of the habitats of two liverworts, including mean temperature and mean relative humidity (Supplementary Table 1) and pH were recorded. Developmental stages of the liverworts: vegetative (young gametophyte) and reproductive stages (mature gametophyte bearing antheridia and archegonia) and abundance, frequency of occurrence (FOC, number of reproductive structures encountered per 10 × 10 cm 2 patch of area under study) were recorded. Plants were photographed in the field using a digital camera (Cybershot DSC-H10, Sony, USA). Each sample was divided into two parts; one part was kept for the preparation of herbarium and another for the identification of thalli using gametophytic and sporophytic characters.
Dimethyl glyoxime test for Ni absorption in young and mature gametophytic thalli and sporophytes. To visualize Ni 2+ ions absorption and its accumulation in A. wallichiana and P. appendiculatum in thalli, sporophyte and rhizoids, the dimethyl glyoxime (DMG) staining procedure was used 57,58 . In brief, A. wallichiana and P. appendiculatum gametophyte and sporpophytic stages collected from CS and NS were thoroughly washed with double distilled water. Later, tissues were air dried and then placed in petri plates containing dimethyl glyoxime (DMG) solution for 10 min, followed by washing with distilled water to remove any surface retention of DMG. Accumulation of Ni 2+ ions upon DMG staining was observed as pink color.
Morphometeric and reproductive parameters in mature gametophytic thalli and sporophytes. The male and female mature gametophyte developmental attributes like archegonia and antheridia numbers were counted and dry biomass was measured. Sporophyte viability was determined by staining the spores with 2,3,5-triphenyltetrazolium chloride (TTC). For anatomical studies, ventral section of thallus stained with DMG for CS and NS of both the A. wallichiana and P. appendiculatum were cut manually. Sections were mounted in glycerin before photomicrography using a NIKON ECLIPSE E400 (Nikon Corporation, Tochigi, Japan) camera.

Stress indicators.
Reactive oxygen species measurement in young gametophytic thalli. Reactive oxygen species (ROS) detection in young gametophytic thalli of A. wallichiana and P. appendiculatum was done using 2′,7′-dichlorodihydrofluorescein diacetate (H 2 DCFDA) based fluorescence microscopy 59 . Briefly, the thalli were placed on glass Petri plate containing Ni solution (0.1 mM, mocking NS habitat, NS) for 10 min and distilled water (mocking control habitat, CS). They were then allowed to float on a 60 µM H 2 DCFDA solution prepared in buffer (1 mM KCl, 1 mM MgCl 2 and 5 mM MES, pH 6.1) for 10 min in dark. After a brief wash with buffer, the thalli were observed using Leica DM1000 fluorescence microscope under GFP filter having 470/40 nm bandpass excitation and emission of 525 nm fluorescence microscopy. The whole thalli were imaged, and the grey values were calculated by drawing ROI (region of interest) bearing similar area for all the samples. The average intensities were used to calculate the ROS concentration.
Non-enzymatic antioxidant profiles of A. wallichiana and P. appendiculatum. Estimations of ascorbic acid (ASA), glutathione (GSH) and proline (PL) were done as described in Choudhary et al. 29 .
Estimation of Ni-specific metalloenzyme urease (EC 3.5.1.5) activity. Among the Ni-specific metalloenzymes, urease enzyme activity was estimated in A. wallichiana and P. appendiculatum collected from the NS and CS following the method of Kandeler and Gerber 32 .
Statistical analysis. Otherwise stated, for each experiment, five biological repetitions were designed, and the resulting data were expressed as mean values ± standard errors. In entire experiments, each biological repetition had three technical repeats. One-way analysis of variance (ANOVA) was carried out, and data were