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%.
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.
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.
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).
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 < 1.00%.
Potassium (K) and sodium (Na) have shared the most in their total content which was several times lower as compared mostly to total Ca. Despite this fact, active K represented from 10.6 to 24.8% whereas for Na, the values were significantly higher, i.e., 13.9 to 43.4%, even. It should be pointed out that these two monovalent elements are mostly responsible for regulating diffusion processes, hence their geochemical solubility raises; but this is not to alter the effects controlled by both active Ca and Mg.
Interactions for lineages versus alkaline elements versus pH
Multiple comparisons of mean ranks in the ANOVA using the post hoc Scheffe tests showed statistically significant (p < 0.05) differences between growth sites of lineages A1, A2, A3 with respect to active forms of Ca, Mg, K, Na and pH of soil materials. The greatest similarity was observed for the site identified with lineages A. pinguis A1 and A2, but differed significantly for two factors, i.e. pH and Ca.
A. pinguis lineage A3 grew at the most distinct site. It differed statistically and significantly from the lineage A1 in terms of the total content of Ca, Mg and Na as well as active forms of Mg, K and Na. In the case of A2, the same statistical pattern was observed for total content of Ca, Mg and active forms of Mg, Na, respectively. The similarities and differences which emerged from statistical evaluation corroborated strictly those observed from genetic data (Fig. 3). This appears as an unquestionable proof of chemical characteristics of sites in the internal speciation of A. pinguis cryptic species A.
Genetic lineage adaptability index versus site alkalinity and acidity (pH)
Data reported earlier have shown some trends in the site preference of the particular A. pinguis lineages growing at Pieniny (PNN), Beskidy (BS) and Tatry (T) Mts. Next, the concentrations of the alkaline elements (Ca, Mg, K, Na) in terms of their total as well as active forms should be supported by a comparative index applicable for any site. We have suggested the active forms of Ca/(Mg + K + Na), (Table 4) which should reflect the dynamic character of site reaction (pH) resulting from an equilibrium among these elements. Then, the growth response of genetic lineages A1, A2 and A3 has been considered to be strongly or weakly integrated to high or low values of this index. Its mean values along with pH are listed below:
Lineage A1 index: PNN (3.24) > BS (2.70) > 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 and 40% > 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).
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.
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.
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.
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.
Source: Ecology - nature.com