3.1. Morphology and Colour of T. hispida var. coronata Lorica

Loricae in the control sample were usually elliptical in shape, although differently shaped envelopes were also observed (Figure 1). Some observed shapes included longitudinally oval (Figure 1 C, K, and P), almost round (Figure 1 F and T), lemon-shaped (Figure 1 U), or conically tapered toward the posterior end (Figure 1 V). The loricae possessed apical pores that were smooth (Figure 1 D and I) or surrounded by spirally arranged folds (Figure 1 Q and R) and only occasionally by small spines (Figure 1 P and S). There were also cells that formed loricae with short collars or, in contrast, relatively long collars (Figure 1 F–H and J and T and K and N–O, respectively). All these variations also applied, to some extent, to loricae from the experimental sets, both with the addition of Fe or Mn. A common feature of all the observed loricae from the control sample was the lack of spines on the surface, which are the main diagnostic features of the taxon (and the entire species) and are typical of specimens found in the field. There were only small warts/papillae regularly distributed, but they rarely spread on the entire lorica and were visible in the SEM microphotographs (Figure 1 P–W). In the control sample, there were many monads that did not form loricae at all, and there were also monads with a very thin and hyaline lorica hardly visible at first sight (Figure 1 A and B).

In the samples to which we provided Fe at three different concentrations, we observed changes on the surface of the lorica, which mainly consisted of spine formation (Figures 2 and 3). First, the lorica surfaces were densely covered with spines. The spines were visible and prominent under SEM. The spines developed the most with the lowest added amount of FeNa-EDTA (+1.4 mL) (Figure 3 A–H) and the least in the medium amount of FeNa-EDTA (+1.9 mL) (Figure 3 Q–T). Regardless of the concentration, we observed that the lorica wall was very porous, and the pores were large, which gave the impression that the wall had a highly reticulate structure (Figure 3). The development of the collar also differed from that of the control sample. The lorica apical pore was surrounded by numerous and sharp spines forming a crown (which is typical of the variety coronata); in some cases, there were also short collars with spines at the rim (Figure 3 B and J). It is worth mentioning that in samples with Fe supplementation, the loricae were of a little different shapes, from elliptical to round, and the number of oval-round loricae was significant. The colouration differed such that in the sample with the least amount of added Fe, the coverings produced by the monads were the brightest, ranging from hyaline to slightly greenish-brownish (Figure 2 A–E). In the two subsequent higher concentrations, envelopes were darker with more red colour, and this was particularly evident in the empty envelopes, which may demonstrate that Fe deposition in the lorica walls increases with cell aging and/or the duration of exposure (Figure 2 F–O).

In the samples with Mn supplementation, the envelopes were elliptical and, in most cases, more elongated than the structures formed under Fe supplementation conditions. The walls of the loricae were also porous, but the pores were smaller, and the structure was more densely arranged; therefore, the loricae appeared solid and thick, especially at higher concentrations of Mn in the sample (Figure 4 A–Ee). Spines on the surface of the envelopes were developed but were sparsely distributed on the walls and were often reduced to small warts (Figure 4 P–S, U, V, and Cc). This difference was most visible in the samples with the highest concentration of MnCl₂·4H₂O in the medium (+2.2 mL). Another element of the lorica is a usually well-developed collar. We found different stages of collar development. In the vast majority, the collar was very low but distinctly formed and uneven at the rim. This feature was observed in all samples, regardless of the Mn concentration (Figure 4). One observation that should be highlighted is that the loricae from the samples supplemented with Mn were longer and more elongated than those from the Fe-supplemented samples. A comparative analysis of the length of the envelopes showed that there were statistically significant differences in the dimension tested in specimens growing in a manganese-enriched medium (Wilcoxon test, Z = 4.52, p ≤ 0.001). However, no difference was observed in lorica size from the Fe-enriched medium (Wilcoxon test, Z = 0.88, p > 0.05). The box-whisker plot analysis showed the length distribution of the lorica T. hispida var. coronata indicating a clear increase in the parameter under study in individuals cultivated in the medium supplemented by Mn ions (Figure 5). The colouration of the envelopes was darker than that in the samples with Fe supply. They were brown or even deep brown (Figure 4 C–O), although there were also specimens, likely young ones, with brighter loricae (Figure 4 A and B). The percentage of the brightest envelopes was the highest in the sample with +1.4 mL MnCl₂·4H₂O; in the remaining two experimental conditions with higher concentrations, the envelopes were darker in most of the specimens.

3.2. Element Composition

The SEM-EDX spectroscopy analysis revealed differences in the elemental composition of T. hispida var. coronata loricae. In the envelopes from the control sample, there was a higher percentage share of Mn than of Fe compounds, 2.36% and 1.01%, respectively (Figure 6a). In our study, we observed that both ions were incorporated into the walls of loricae, regardless of their concentration. The increasing supplementation of any ion and its incorporation manifested as a high percentage in lorica composition, resulting in the other ion also being incorporated in a high amount. However, with the increasing content of Mn in the medium and in the wall of the envelopes, the Fe content was small, usually constituting no more than 15% of the Mn content (Figure 6e, 6f, and 6g). In the case of Fe supplementation, Mn still constituted more than 50% of the total amount of Fe in the studied loricae (Figure 6b, 6c, and 6d). It should be noted that with increasing concentrations of a given ion, its higher content was also manifested in the chemical composition of the lorica walls. Only at the lowest experimental concentration of the ferric ion was the concentration of manganese in the lorica wall higher than that of iron (Figure 6b). The remaining constituent elements of the studied envelopes were Ca, Mg, and K, as well as small amounts of P, S, and Cl (Figure 6). All the results obtained during the study are summarized in Table 1.

4.1. Lorica Morphology

According to available sources for taxon identification, the typical T. hispida var. coronata forms a crown around the apical pore. This crown consists of sharp, conical spines, usually longer (according to Sena et al. [32] up to even 3.5 μm long) than on the rest of the lorica, or sometimes a short collar equipped with distinct spines [1, 4, 6, 15, 33, 34]. This structure is the feature that distinguishes the variety from the nominative variety hispida, which has spines around the apical pore that are as long as those on the lorica surface and from other varieties such as the most commonly recorded, crenulatocollis, (which means collar around apical pore with some sort of crenulated [toothed] rim) or spinulosa (with a distinct dentate collar) [9, 10, 35]. Our specimens formed either crowns of spines, especially in the samples enriched with Fe or short collars, without spines, mainly in samples with Mn, or they did not form any structured element around the apical pore. Thus, these characteristics are highly variable and dependent, at least to some extent, on the chemical composition of the medium. Our observations concurred with those of Rosowski et al. [36], who, studying the same taxon under laboratory conditions, found differently developed collars from inconspicuous to distinct. This is not different from other diagnostic features. The presence of spines on the surface is an important factor. Using the available key guides, one can see that the lorica surface of specimens belonging to the species T. hispida is covered with spines or can only be punctuated without spines (nominative variety, var. crenulatocollis, var. volicensis). For the remaining varieties, the available information mentions that they have spines or warts, or information on the ornamentation is absent; thus, implicitly, these specimens have spines similar to those of the nominative variety [9–11, 37]. Nevertheless, it is very difficult to find any record of T. hispida from natural ecosystems without spines (alternatively warts) on the envelope surface, and they would have only been punctuated (e.g., [1, 4, 6, 8, 13, 26, 32, 38, 39]). For comparison, we presented microphotographs (LM and SEM) of T. hispida cells from environmental samples (Figure 7). In our study, we observed loricae, which were always punctuated to a greater or lesser extent and were completely spineless, with small warts, delicate papillae, or spines on the lorica surface. It must be highlighted, however, that the spines did not look like those on loricae observed in natural collections found in freshwater. However, there are only a few records showing cells of spiny Trachelomonas species in culture conditions that might not form spines. The first evidence, in the form of LM and SEM pictures, was provided by West and Walne [13], who reported spineless loricae of T. hispida var. coronata in the majority of the samples under culture conditions. Kim, Shin, and Boo [15] transferred T. hispida cells from Korean ponds and lakes into the laboratory and observed both spiny and spineless loricae during the research conducted. Similarly, Poniewozik, Sajnaga, and Zięba [40] used T. hispida in an experimental study that investigated the development of loricated monads in water from various natural water bodies, filtered through a GF/C and a 0.22-μm Millipore syringe filter. After the experiment, they did not observe loricae that formed conical sharp spines covering the envelopes. Rosowski et al. [36] provided surprising insights into Trachelomonas oblonga var. punctata. This taxon is known from the taxonomic description as having punctuated loricae, being placed in Pringsheim’s T. volvocina group of “never spiny” lorica, and not having a collar around the apical pore. Under the culture conditions, the cells formed heavily ornamented envelopes, equipped with short spines and/or extremely polymorphic papillae [36]. Our research shows that the availability of basic ions with which the lorica of trachelomonads saturates is essential for the formation of a robust structure, such as a collar, and for the development of spines on the wall surface. This was particularly evident in cultures grown in the presence of increasing amounts of Fe chelate and less so in cultures grown with an additional pool of Mn. In the latter, the lorica formed shorter spines, had a smaller number of pores, and appeared to have a denser and more rigid internal architecture. This suggests that the presence of increased amounts of these two elements in the water or medium affects lorica formation differently in T. hispida var. coronata. The structures surrounding the apical pore were also differently developed in specimens grown in Fe- or Mn-supplemented media (long crown-forming spines vs. short collars with fine serrations at the edge). We must acknowledge, however, that although individuals from Fe-enriched cultures, regardless of the ion concentration, had numerous and long spines on the surface of the lorica, these spines were not as strong and thick as those reported and documented from environmental samples [6, 8, 25, 34, 41, 42]. To investigate the influence of different medium compositions on the lorica formation process, we also attempted to test another species, assuming that supplementation with the basic components of lorica, which Fe and Mn are considered to be, would allow the development of additional structures in individuals that, in the natural environment, produce smooth lorica without any ornamentation on the surface. For this purpose, we used the strain T. volvocina (Ehrenb.) Perty (CCAP 1283/4B) and placed samples under the same conditions as T. hispida var. coronata. However, during the course of observation, we noticed that the lorica formation process was almost completely stopped. Most cells functioned as naked monads, devoid of lorica. If this structure was formed, it was only by a few cells, and the lorica was very thin, delicate, transparent, and completely colourless, as observed under LM (Figure 8). The lorica was such a delicate structure that despite several attempts, we failed in observing it under SEM by preparing the material using the method commonly used for this type of structure. Therefore, we believe that the studies showing spineless “spiny” trachelomonads from culture conditions and the present study on T. hispida var. coronata provide evidence regarding triggers of the process of lorica formation as a solid structure equipped with strong spines. Additional drivers besides chemical composition are needed or, more likely, the entire environmental complexity must occur to cause an effect of distinct spines on the surface of the lorica. The absence of a lorica, or a different model for the formation of spines that are consequently usually thin and fragile under laboratory conditions, may suggest a role for a well-developed, rigid structure, such as lorica, in protection against predators in the natural environment, as has been suggested by Poniewozik, Sajnaga, and Zięba [40].

4.2. Element Composition and Lorica Colour

The trachelomonad lorica mainly comprises Ca, Mg, K, P, S, Cl, Fe, and Mn, of which the last two are the most important depositional elements providing colour and determining the type of internal microarchitecture of the envelopes (granular or microcrystalline) [14, 25, 43–46]. Sometimes, the amount of some elements is higher or lower. Poniewozik, Sajnaga, and Zięba [40] observed a lack of Mn in young loricae and a lack of Fe in old loricae in studies conducted on T. hispida. West and Walne [13] demonstrated special cell selectivity for Mn, which was incorporated faster than Fe was in the envelopes of T. hispida var. coronata. Our results are in accordance with the results [13, 40] as we observed that Mn was incorporated in a significant amount, even in the samples with Fe supplementation, whereas in the samples with Mn supplementation, its amount was considerably high with a concurrent low amount of Fe. Another aspect is the colour of loricae. We observed differently coloured envelopes of T. hispida var. coronata at different stages of lorica development. In the control sample and in both sets of experiments, young, bright, and thin loricae formed by the monads shortly after cell division were observed. We also observed older loricae with a reddish-brown colour (in samples with Fe chelate) and those that were brown or even brownish-black (in samples with Mn in the form of hydrated manganese chloride). Generally, we observed that cells growing in Mn-supplemented medium were darker than those grown in Fe-rich medium, which took on a reddish colour, with the colour becoming increasingly intense for cells growing in increasing ion concentrations in the medium and over time. Therefore, the change in colour was due to both the age of the cell and the duration of exposure to the chemical element. Similar previous observations suggest that the aging of cells is a main factor of envelope darkening [13, 40] or the chemical composition of the medium [17]. In general, in trachelomonads in nature, the formation of very thin and transparent loricae is a rare phenomenon. Of the widely distributed species, only T. oblonga is described as having a thin, bright lorica [9–11], but it still appears different from the specimens we observed in culture. These tended to be yellowish, slightly reddish, and, above all, considerably thicker. There was no difficulty in answering the question of whether a found individual likely has or does not have a lorica, a question we sometimes asked ourselves when conducting our observations. However, during our research, we had the opportunity to identify young cells that were in the process of forming a new lorica. A question that remains unanswered is why cells with hyaline and thin lorica are rarely found in environmental samples. Further research in this area is required to identify the chemical composition and microarchitecture of the envelopes of different taxa. The range of the amounts of different elements present in loricae and how various physical and chemical parameters affect lorica composition should also be investigated.

4.3. Taxonomic Ambiguity

In light of the research presented, we consider that trachelomonad loricae, although still a basic taxonomic criterion, are not an entirely good criterion. We are not in a position to claim definitively whether from among specimens that are found in environmental samples, those that do not have spines on the surface of the lorica would not be T. hispida (having the other taxonomic features of the species, i.e., a more or less elliptical/oval lorica, several chloroplasts with double-sheathed pyrenoids, and within the size range for the species). Hypothetically, it could then be labelled as, for example, T. oblonga or T. botanica, which differ from T. hispida primarily in the absence of spines. It is also impossible to transfer the specimens to laboratory conditions and observe how the lorica will form after new monads have been produced under other laboratory conditions. Regarding T. hispida, we suppose that most of the functioning varieties or forms in which this species is unusually rich—there are close to 50 of them—are merely within phenotypic plasticity. The differences in the keys to identification, later reported in scientific studies from different parts of the world, are subtle. An example is the variety caudata [47] and acuminata [19], described several decades ago, of which descriptions are very brief, coinciding with each other and not showing the differences between the taxa. Another example is the varieties crenulatocollis and coronata, both frequently reported globally, where the differences are only in the form of the structure surrounding the apical pore; as shown in the present study, this feature is highly variable, and individuals of one strain (CCAP 1283/2) can produce both types of this structure. Many of these varieties have not been reported for several years, and the differences between them are so subtle that different individuals may represent different varieties in the subjective judgement of researchers. The existence of many taxa, especially those distinguished by Skvortzov [48], who described new varieties of T. hispida: papillata, punctulosa, simplex, charkoviensis, hyalina, or spinopunctulosa, disagreed with the description of Van Oye [49], who considered them to be within the variation of the species. There are many examples of the introduction of multiple varieties based on minor differences in cell morphology. Another example is the work of Dreżepolski [50], who, in a study of euglenoids of water bodies from selected regions of Poland, Lithuania, and Ukraine, in one paper alone described as many as five new varieties of T. hispida: granulo-spina, irregularis, niezabitowskii, setosa, verrucosa, and volicensis. These taxa were, in most cases (an exception being variety volicensis), not subsequently found in other studies by other researchers. The same doubts concerning the overestimated number of species within the genus have been raised and published in papers on two other species within Trachelomonas: the cosmopolitan Trachelomonas caudata, which is morphologically very similar to several other species [51] and the morphologically convergent Trachelomonas volzii and Trachelomonas dubia, for which many varieties and forms have been described from various ecosystems in different parts of the world [16]. This type of research on Trachelomonas loricae, the process of its formation, and the drivers triggering its development is not common, and there is very little data on the subject; hence, there are many areas on this topic that should be developed to propose a different or improved method of identification within Trachelomonas. In the recent 20 years, advanced molecular studies altering taxonomy within the euglenoid genera Euglena, Lepocinclis, Phacus, or Monomorphina have been published [52–58]. These have reordered the taxonomic position of many species, although it must be acknowledged that their value, though undoubtedly great, in environmental studies makes the identification process even more complex. For correct identification of many species within these genera, it becomes indispensable to transfer the cells found to laboratory conditions and carry out molecular studies. Only then can it be said with certainty which species we are dealing with. A similar path probably awaits Trachelomonas (and Strombomonas), especially considering what we believe to be too many taxa introduced into the taxonomy, mainly based on the description of lorica morphology. Pringsheim [17] suggested reducing the number of taxa described based on lorica morphology. We believe that we should proceed in this direction in the taxonomy of Trachelomonas.