Trachelomonas bituricensis var. lotharingia M.L. Poucques 1952, a morphologically interesting, rare euglenoid new to the algal flora of the Czech Republic

Abstract This report describes the discovery of the rare euglenoid taxon Trachelomonas bituricensis var. lotharingia in a small mesotrophic pond in the Czech Republic. Only limited data are available on the distribution of this taxon as same as for typical variety of Trachelomonas bituricensis, even though this taxon is morphologically very well defined. I provide a brief discussion of the taxonomic validity of this taxon based only on morphological features, which are characteristic for the taxonomy of the genus Trachelomonas. This finding is completely new for the algal flora of the Czech Republic. This report provides new information about the worldwide distribution of this taxon and its ecology.


Introduction
Floristics studies have had a long tradition in the Czech Republic beginning in the second half of the 19th century, when several works about alga fl ora in this country were published. Since the publication of pioneering works by Kirchner (1878) and Hansgirg (1892Hansgirg ( , 1899, Lhotský and Rosa (1955) published a list of algal and cyanobacterial species from the Moravia region. Th e current knowledge about algal diversity in this region was summarised by Poulíčková et al. (2004). All of these reports describe fl oristic studies examining the prevalence of all groups of algae in our state. Th ese studies, combined with recent reports about the distribution of various groups of algae (e.g., Caisová and Gąbka 2009, Kaštovský et al. 2010, Kučera et al. 2008and Šťastný 2010, provide comprehensive information about algal fl ora. Unfortunately, little is known about several of these groups and their distribution, e.g., diatoms, dinophytes and euglenoids. I performed a nearly 5 year survey during my bachelors and masters studies investigating the diversity of photosynthetic euglenophytes (Juráň 2010(Juráň , 2012) based on extensive literature reviews and my own fl oristic studies across the country. As a result, I constructed a preliminary checklist of euglenoid taxa of the Czech Republic including several species that were newly reported in our state. Th is checklist will be published soon. One of these new species is Trachelomonas bituricensis var. lotharingia from Ďáblík pond in South Bohemia.
Th e genus Trachelomonas includes euglenoids with their cells enclosed in envelopes (loricas) comprising polysaccharides with a content of iron and several inorganic compounds (Pereira et al. 2003). Th ese loricas are highly variable in shape and surface morphology. Listed features are traditional markers used in Trachelomonas taxonomy, as proposed by Defl andre (1926). Since the original description of Trachelomonas by Ehrenberg (1834), more than 1700 taxa have been identifi ed due to this high morphological variability in the genus (Guiry and Guiry 2015).
Trachelomonas bituricensis was originally described by Wurtz (1947), who identifi ed this species in plankton from the pond of the Hardouine in La Brenne, France. In the typical species, the lorica is covered with long, sharp, curved spines. One variety of this species, Trachelomonas bituricensis var. lotharingia, was fi rst described by Poucques (1952). Th e description of new taxon was based on material from a wetland in the Woëvre region in France. Th is variety has shorter collars (see Fig. 3) than the typical forma described by Wurtz (1947). In addition, the edge of the collar is more undulated.

Methods
Th e aim of this study was to survey the algal fl ora of the small mesotrophic pond Ďáblík (South Bohemia region, Czech Republic, 48°49'41.0"N, 14°35'49.6"E, see Figs 1 and 2). Th is habitat is a small pond that functions as a primary retention pond, without fi shery management. Th is pond contains huge communities of Carex spp. and Sphagnum spp. and a stable population of the charophycean alga Nitella fl exilis. Th e pond is progressively becoming overgrown with vegetation. Th e locality is favourable for the growth of threatened vascular plants, e.g., Nymphaea candida, Calla palustris and Menyanthes trifoliata. Wetland plants Utricularia australis and Potamogeton obtusifolius are common in this locality (Albrecht 2003).  In 2012 and 2013, a pilot survey was taken, and in 2014, a more detailed survey with monthly sampling at fi ve chosen locations was performed. Th e aim of this survey was to record algal diversity, especially for loricate euglenoid genera Trachelomonas and Strombomonas. Samples were taken using a 20 μm plankton net, and the material was fi xed in formaldehyde solution and Lugol's iodine solution. Samples were examined microscopically under an Olympus BX51 microscope and photographed with an Olympus DP-71 camera with DP Controller 3.1.267 software. Samples fi xed in iodine solution were used for scanning electron microscopy. Materials for scanning electron microscopy were prepared as described by Conforti (2009). Th e material was fi ltered through polycarbonate membrane fi lters (1 μm pore size) and air-dried. Filter pieces were attached to stubs and subsequently coated with gold. Th e specimens were then saved for future scanning electron microscopy analysis or examined under a JEOL JSM-7401F scanning electron microscope (Institute of Parasitology, Biology centre of AS CR, České Budějovice).

Results
Th e algal fl ora of the Ďáblík pond is dominated by dinophytes (Peridinium spp.), together with desmids and metaphytic communities of Spirogyra spp. steril., as well as diatoms (especially Gomphonema species). Taxon-rich fl ora of the loricate euglenoids will be studied in detail using optical and scanning electron microscopy in conjunction with comparisons of the chemical and physical parameters of pond water. Based on the preliminary results, some of the taxa appear to represent new algal fl ora of the Czech Republic, but these taxa require further study. Th e most interesting fi nding is the discovery of Trachelomonas bituricensis var. lotharingia, a newly recorded variety of algal fl ora in the Czech Republic. Th is variety is quite common in Ďáblík pond, which contains a stable population of this alga. Th is fi nding represents the fi rst record of this taxon among algal fl ora of the Czech Republic based on a detailed literature review and the fl oristic survey across the Czech Republic that I performed during my bachelors and masters studies (Juráň 2010(Juráň , 2012. Th e loricas in this species are (41 -)45-46 μm in length and (30 -)33-35 μm wide. Th e cell contains numerous disc-like chloroplasts, probably without pyrenoids. Th e surface of the lorica in this taxon from Ďáblík pond is punctate and covered with well-developed hollow, sharp spines (see Fig. 3A-D), most of which are slightly curved and cover the body of the lorica. Th e caudal region contains very long, curved spines, and only a few narrow, conical spines surround the apical pore, which has a collar (see Fig. 3B, marked with arrows). Th e collar of the lorica is short, with an undulated edge (see Fig. 3D, marked with arrow). Th is taxon has always been found during the spring at shallow sites with populations of Nitella fl exilis near the banks of the pond. Th ese fi ndings suggest that Trachelomonas bituricensis var. lotharingia is probably a benthic or metaphytic taxon.
A comparison of a typical variety of Trachelomonas bituricensis, the variety described by Poucques (1952) and the material found in Ďáblík pond is presented in Table 1. Figure 3. Visualisation of the morphology of Trachelomonas bituricensis var. lotharingia by optical and electron scanning microscopy. A Overall appearance of lorica under an optical microscope, with a short collar with undulated edge (marked with arrow) B Detailed image of the lorica surface with three types of spines: long, curved spines at the antapical part of the lorica; shorter spines on the lorica's body; and several straight spines near the apical pore (marked with arrows). Note the numerous disc-like chloroplasts in the cell C, D Loricas viewed by scanning electron microscopy, with clearly visible punctuation at the surface, and short collar with undulated edge (marked with arrow). Scale bars = 10 μm.

Discussion
Th ese two taxa (Trachelomonas bituricensis and Trachelomonas bituricensis var. lotharingia) are commonly mentioned in works about euglenophytes (e.g., Huber-Pestalozzi 1955, Popova 1966, Starmach 1983and Vetrova 1986. Th ese taxa have distinct morphology that is easy to distinguish from that of other species, the descriptions of which are commonly used in taxonomic keys. However, data about the occurrence of these taxa are very limited. A typical form of Trachelomonas bituricensis was only recently reported to occur in several places, including benthos in a fi shpond in Poleski National Park, Poland (Kočárková et al. 2002) and two rivers in Kosovo (Kurteshi et al. 2013a(Kurteshi et al. , 2013b. Th e second reported location of lotharingia variety was a bog in the Russian taiga, where this taxon was found in a wetland containing saw grass (Carex sp.) (Safonova 1987). In addition, Vetrova (1986) identifi ed this variety among algal fl ora of Ukraine. Perhaps the most recently reported occurrence of this taxon was in the Southeastern US, where it was rarely observed in ditches, lakes and ponds (Wolowski and Walne 2007).
Several species of Trachelomonas have the similar morphology as Trachelomonas bituricensis. Th e most resembling taxa are Trachelomonas horrida, which was fi rst described by Palmer (1905), as well as T. megalacantha (DaCunha, 1914) and T. spectabilis (Defl andre, 1926). All of these species have loricas covered with well-developed long, sharp spines, but their spines are straight, unlike the curved spikes on the loricas of Trachelomonas bituricensis. Huber-Pestalozzi (1955) argued that Trachelomonas bituricensis var. lotharingia is highly similar to Trachelomonas spinosa, which is found in ponds containing aquatic plants (Stokes 1890), and that it probably represents a larger form of these taxa. Stokes' (1890) description of the surface and shape of the Trachelomonas spinosa lorica is similar to that of Trachelomonas bituricensis, whereas its collar is described as a "… short, smooth, truncate extension", with no mention of the undulated edge or additional cylindrical spines. In addition, Tell and Conforti (1986) described Trachelomonas spinosa from Argentina as having a similar morphology, with an apical pore with a short, thick neck surrounded by spines; again, an undulated edge is not mentioned. Vetrova (1986) noted the similarity between Trachelomonas bituricensis and lotharingia-variety with T. horrida and T. spinosa, although they did not mention the taxonomy of Trachelomonas bituricensis taxa in a subsequent report and the validity of this variety could be discussed. Finally, Wolowski and Walne (2007) reported that Trachelomonas bituricensis var. lotharingia from the US has a dentate collar.
Th e high morphological variability of the genus Trachelomonas makes classifi cation based only on morphology (e.g., shape, surface, absence or presence of various forms of collar and sometimes colour of the lorica) quite diffi cult and has led to the description of morphologically similar forms as separate taxa. As a result of morphological variability, the traditional taxonomy of Trachelomonas based only on a description of lorica characters is problematic for several reasons. First, there are several examples of the morphology of euglenoids changing depending on environmental conditions. Th is variability in shape was observed in several euglenoid genera in culture (Conforti 1998) and those cultivated in situ under natural conditions (Bauer et al. 2012). Th e cells of these taxa exhibited morphological changes depending on the level of organic carbon in the medium or the environment. Pringsheim (1953) and Singh (1956a, b) found that in some Trachelomonas species, the loricas of cells in clonal culture (i.e., descendants of a single cell) have morphological diversity in shape and surface, which probably depends on their specifi c ontogeny. Th is diversity represents the main problem with classifi cation based solely on morphology. Indeed, in discussing Euglena taxonomy, Pringsheim (1956) noted that, "Certain authors could not resist the temptation to give a name to every minor deviation from the 'type' previously described or prevailing at the same or at similar places". Th is comment accurately describes the state of taxonomy of the genus Trachelomonas. Th erefore, the large number of currently described taxa within the genus Trachelomonas likely refl ects the morphological variability created by environmental conditions. Many of these taxa may instead be recognised as "eco-morphs" of the typical form. Furthermore, Pringsheim (1953Pringsheim ( , 1956 claimed that unless individual species are cultured separately, the eff ects of external factors aff ecting heritable morphological traits cannot be distinguished. In light of these fi ndings, perhaps Trachelomonas bituricensis var. lotharingia is only an eco-morph of the typical forma, or perhaps it represents a bituricensis eco-morph of Trachelomonas spinosa. Alternatively, this alga may represent a complex of species. More detailed morphological studies are needed to solve this taxonomic puzzle, including basic morphometric analysis and especially molecular analysis. Many reports about euglenoid taxonomy based on morphological and molecular data (e.g., Karnkowska-Ishikawa et al. 2010, 2012, 2013and Kosmala et al. 2005, 2007a, 2007b, 2009 show that several species or other intraspecifi c taxa may instead only represent eco-morphs or ontogenetic stages of individual species, as discussed in a report about Monomorphina genus taxonomy (Kosmala et al. 2007b). Unfortunately, similar studies about the taxonomy of the genus Trachelomonas remain to be performed. Several recent works (e.g., Nejmová et al. 2011, Škaloud et al. 2015and Veselá et al. 2012) about species concept in algae refl ect a combination of molecular and morphological data. Th erefore, a combination of ecological, morphological and molecular data can be used to defi ne the species concept of a genus with worldwide distribution such as Trachelomonas. However, this issue requires further study.

Conclusion
Th is report describes what is likely the third record of Trachelomonas bituricensis var. lotharingia in Europe and the fi rst record of this taxon in the Czech Republic. Th e description of this taxon is morphologically and ecologically similar to the original description. Th e taxonomic validity of this taxon remains to be validated, as it may represent part of a complex of morphologically similar species together with T. spinosa and T. horrida. Nonetheless, the discovery of this taxon sheds light on the distribution of algae of the Czech Republic and European algal fl ora in general.