Philippa Kaur

Differentiation within A. pinguis cryptic species A
Genetic studies using combined DNA sequences from five chloroplasts (rbcL-a, matK, rpoC1, trnL-F, trnH-pabA) and 1 (ITS) nuclear genomes (4598 bp) showed some differentiation within the A. pinguis cryptic species A into three distinct groups (lineages) A1, A2 and A3 (Fig. 3). Most of investigated plants belonging to the lineage A1 originated from the Pieniny Mts. (PNN), however two samples A1 were collected at the Beskidy Mts. (BS) and one at the Tatry Mts. (T). All plants identified to A2 and A3 came from the Beskidy Mts. and the Tatry Mts., respectively. Maximum parsimony analyses of combined plastid loci and the nuclear ITS locus produced trees showing that the lineage A3 is genetically the most distinct, while A1 and A2 reveal more similarity. In the K2P mode, the percentage of variation in the sequences between lineages A1 and A2 equals to 0.20%, while for A1 and A3 it raised five times, i.e. 1.0%. The same occurred for the lineages A2 and A3, 1.0%.
Figure 3

Phylogram resulting from maximum likelihood (ML) analysis based on combined data of all sequences and showing genetic similarity and differentiation between lineages A1, A2 and A3 of A. pinguis cryptic species A. Bootstrap values are given at branches. A. maxima was used as an outgroup for tree rooting.

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Total and water soluble (active) alkaline elements
The total content of alkaline elements (Table 2) shows that the content of calcium (Ca) prevails over magnesium (Mg), potassium (K) and sodium (Na) at any investigated site, i.e. Pieniny Mts. (PNN), Beskidy Mts. (BS) and Tatry Mts. (T). Soil samples collected under the lineage A1 covered the whole three geographical distributions, where the site BS exhibited the highest Ca concentrations (31 459.6 mg kg−1) followed by PNN (16 178.8 mg kg−1) and finally T with 7 398.7 mg kg−1. Interestingly, the lineage A2 occurred only at the site BS characterised by high Ca content (27 303.4 mg kg−1), whereas A3 at the Tatry Mts. (T) where the level 22 227.6 mg kg−1 was recorded. These data imply that the lineage A1 may have developed site-specific adaptation mechanisms to various concentrations of calcium. In the case of A2 and A3, the observed Ca concentrations amounted to 27,303.4 and 22,227.6 mg kg−1, respectively and should be described as high.
Table 2 Total content of alkaline elements (Ca, Mg, K, Na) in the growth media of A. pinguis cryptic species A genetic lineage A1, A2, A3 at Pieniny, Beskidy and Tatry Mts.
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Variations in magnesium (Mg) concentrations for the lineage A1 followed another pattern differing from that observed in the case of calcium. Its contents varied accordingly: T  > BS  > PNN, with the highest levels recorded for A3 and A1 at the Tatry MTs. (T), respectively. It should be mentioned that both Ca and Mg are in most cases responsible (Ca much more) for geochemical reactions controlling the pH of the growth media. The role of potassium (K) as well as sodium (Na) is generally less pronounced in these reactions, but also their contents, which were very low appeared as the proof.
The evaluation of site-specific occurrence of the lineages A1, A2 and A3 should not be performed on the basis of total content solely of alkaline elements, since this fraction is mostly informative on the current status of Ca, Mg, K and Na. Therefore, we have tested the soil samples for recovering the concentrations expressed as active fractions (Table 3) potentially involved in the growth process of these lineages. The levels (percentage share into the total content) of active Ca are significantly low and varied as follows: PNN (3.27%)  > T (0.89%)  > BS (0.73%) for A1, but raised to 1.34% (BS) in the case of A2. The lineage A3 has recorded a concentration of 0.71%, slightly comparable to A1, but at the same site (T).
Table 3 Content of active forms of alkaline elements (Ca, Mg, K, Na) in the growth media of A. pinguis cryptic species A genetic lineage A1, A2, A3 at Pieniny, Beskidy and Tatry Mts.
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Should these Ca concentrations reflect any trend in site-specific behavior of Aneura pinguis cryptic species A. three lineages? Preliminary observations may be indicative of the calciphilous character of A1, specifically for the PNN site, followed by A2 in the case of BS. Lineages identified at the relatively lower share of active Ca, that is below 1.00% may fall into the acidophilous range. The percentage share of active Mg into its total concentrations followed similar distribution patterns like active Ca, with A1 recording 2.53% at the PNN site. Magnesium and calcium are divalent elements, which significantly control the alkalinity of soil environment.
In the case of the current study, the occurrence of this lineage (i.e. A1) at this site is not a random process. By applying the same criteria like for active Ca, it appeared that A1, A2 and A3 at the Beskidy as well as Tatry Mts. met the rule of active Mg  T (0.87).
Lineage A2 index: BS = 4.34.
Lineage A3 index: T = 1.31.
The respective pH values changed quite accordingly to the indices as shown below:
Lineage A1 site pH: BS (8.05)  > PNN (7.50)  > T (6.45).
Lineage A2 site pH: BS = 7.85.
Lineage A3 site pH: T = 7.08.
These ranges imply that genetic lineages A1 and A2 are by essence both calciphilous biotypes and may occur on sites rich in Ca, mostly alkaline as confirmed by the PNN and BS sites. On the other hand, some biotypes of the lineage A1 may be easily adapting also to low Ca concentrations, indicative of acidophilous features, as in the case of A3. Both (A1 and A3) occur at the Tatry MTs.
A detailed distribution of indices as well as respective pH is illustrated by the Figs. 4, 5 and 6, specifically for the genetic lineages A1, A2 and A3, respectively. The mean index values for the PNN site is 3.24 which discriminates the data into two groups: 60%  3.24. In the case of BS, the mean value amounted to 2.70, but for only two sampling sites. Therefore, the mean values of the singular site specific index shows a clear pattern, which strengthens the preferential adaptation of A1 in prevalence to alkalinity as follows: PPN (3.24)  > BS (2.70)  > T (0.87).
Figure 4

Singular site specific index of active forms of alkaline elements (Ca, Mg, K, Na) and pH in the growth media of A. pinguis cryptic species A genetic lineage A1 at Pieniny, Beskidy and Tatry Mts.

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Figure 5

Singular site specific index of active forms of alkaline elements (Ca, Mg, K, Na) and pH in the growth media of A. pinguis cryptic species A genetic lineage A2 at Beskidy Mts.

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Figure 6

Singular site specific index of active forms of alkaline elements (Ca, Mg, K, Na) and pH in the growth media of A. pinguis cryptic species A genetic lineage A3 at Tatry Mts.

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The genetic lineage A2 outlines a great variability in terms of the site specific index, which was slightly high (4.62) only for the sampling site BS 3–28. It should be mentioned that the mean value at this site raised up to 4.34, hence being 56% lower than the highest and next 49% higher than the lowest index. Curiously, the respective pH values did not vary significantly (7.83–7.89), which implies that A2 is decidedly calciphilous.
Indices reported in the Fig. 6 fluctuated widely from 0.66 to 2.38 with a mean of 1.31. Only two values were higher but the remaining, i.e. about 67% placed below. Such high share reveals that the genetic lineage A3 is basically acidophilus. This is decidedly outlined by significantly low values of indices as a consequence of low concentrations of active Ca. More

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The chemical components analysis of different extracts of A. argyi
In our preliminary study, we accidentally found that A. argyi powder significantly inhibited the germination and reduced the varieties and biomass of weeds in the field, when it was applied as a fertilizer originally. Therefore, we speculated that certain allelochemicals present in A. argyi might inhibit the growth of weeds. To investigated the possible allelochemicals in A. argyi, three solvents (water, 50% ethanol and pure ethanol) were used to extract the metabolites in A. argyi leaves. The three type of extracts were analysed by UPLC-Q-TOF-MS and the components were confirmed by comparison with synthetic standards and MS data in literatures9,10,11. As shown in Table 1 and supplement Fig. 1, we have identified a total of 29 components in A. argyi. Six main compound mass signals were identified in the water extract: caffeic acid, schaftoside, 4-caffeoylquinic acid, 5-caffeoylquinic acid, 3,5-dicaffeoylquinic acid and 3-caffeoylquinic acid. The main compounds of the 50% ethanol extract were 4,5-dicaffeoylquinic acid, 3-caffeoylquinic acid, schaftoside, rutin, kaempferol 3-rutinoside, 3,4-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, 3-caffeoy,1-p-coumaroylquinic acid, 1,3,4-tri-caffeoylquinic acid and eupatilin. The metabolites with higher contents in the pure ethanol extract were eupatilin, jaceosidin and casticin. Among these compounds, caffeic acid is very unique in water extract. Higher contents of schaftoside, 4-caffeoylquinic acid and 3-caffeoylquinic acid were observed in water extract and 50% ethanol extract, but very low concentrations were detected in the pure ethanol extract. 3,4-dicaffeoylquinic acid, jaceosidin, eupatilin and casticin were present at higher concentrations in the 50% ethanol extract and pure ethanol extract, but were detected at very low concentrations or were absent in the water-soluble extract. In a word, we have preliminarily identified the chemical components of different extracts.
Table 1 The chemical composition of different solvent extracts of A. argyi.
Full size table

Comparison of the allelopathic effects of different extracts of A. argyi
To explore the allelopathic effects of three different extracts of A. argyi, seed germination and seedling growth of B. pekinensis, L. sativa and O. sativa were investigated after treatment of A. argyi powder extracts. The results showed that the allelopathic inhibition increased in a concentration dependent manner. When seeds were incubated with extracts in a range of concentrations, the water-soluble extract of A. argyi powder exerted an extremely significant inhibitory effect on the germination index of all the three plants (Fig. 1a,b). While the 50% ethanol extract also showed striking allelopathic inhibitory effects on the germination index of B. pekinensis and L. sativa, but moderately inhibitory effects on O. sativa (Fig. 1c,d). Similarly, the pure ethanol extract only showed powerful inhibitory effects on the germination index of B. pekinensis and L. sativa, but no effects on O. sativa (Fig. 1e,f). Additionally, the water-soluble extract of A. argyi powder displayed extremely inhibition of the biomass of the three plants (Fig. 2a), while the 50% ethanol extract also exerted extremely significant allelopathic inhibitory effects on the biomass of B. pekinensis and L. sativa but inhibited O. sativa moderately (Fig. 2b). However, the pure ethanol extract exerted inhibitory effects on the biomass of these three plants only in high concentrations (Fig. 2c). Based on these results, the allelopathic intensity of the three different extracts of A. argyi was in the order of water-soluble extract  > 50% ethanol extract  > pure ethanol extract.
Figure 1

The different solvent exracts of A. argyi: (a,b) the water-soluble extract, (c,d) the 50% ethanol extract,and (e,f) the pure ethanol extract exert allelopathic effects on germination index of different plants. (n = 3,*P  germination index  > biomass  > germination rate  > root length  > stem length. All allelopathy response indexes reached -1.00 when plants were treated with 150 mg/ml extract. For O. sativa, the six physiological indexes also could be inhibited by a low concentration of extract (50 mg/ml), but the changes were not as obvious as the changes in B. pekinensis and L. sativa. The intensity of inhibition on the six indexes was root length  > stem length  > biomass  > germination index  > germination speed index  > germination rate. When O. sativa seeds treated with 100 mg/ml of extract, the allelopathic response index of root length and stem length were -1.00. The germination rate, germination speed index, germination index and biomass were -0.79, -0.91, -0.91 and -0.84, respectively, under the treatment with 150 mg/ml of extract. In brief, according to the comprehensive allelopathy index of the 6 indicators , the order in which they were sensitive to water-soluble extract of A. argyi were B. pekinensis (Cruciferae)  > L. sativa (Compositae)  > O. sativa (Gramineae).
Figure 3

The water-soluble extract of A. argyi inhibits the germination and growth of Brassica pekinensis, Lactuca sativa and Oryza sativa. Specific performance is in a series of indicators: (a) the germination rate, (b) the germination speed index, (c) the root length, (d) the stem length. (n = 3,*P  More