The genus Eunotia Ehrenb. (Bacillariophyta) in the Cheremsky Nature Reserve, Ukrainian Polissya, and refined terminology relevant to the raphe system morphology

Abstract Numerous species of Eunotia Ehrenb., widely distributed in the world flora, prefer acidic, dystrophic or oligotrophic freshwater habitats with low conductivity and usually occur in epiphytic or epilithic hydrotopes. In Ukraine, only 32 species and eight varieties of Eunotia were known until this study. For the first time, 9 more species have been recorded mainly from the Cheremsky Nature Reserve, located in Ukrainian Polissya. New findings include 2 species widely distributed in the world flora on most continents and 7 rare species known from several locations, among them E.genuflexa, E.jarensis and E.ruzickae, which are probably European endemics as they have not been reported from other continents. For the present time in the Cheremsky Nature Reserve, the 20 species recorded here, the highest species richness of Eunotia in Ukraine, bring the total number of Eunotia in Ukraine to 41 species, which comprises only 7% of Eunotia species in the world flora. This is indirect evidence of insufficient investigation of the wetlands in Ukraine where Eunotia usually is represented with high species richness. Several definitions are suggested to describe morphological features that are peculiar to the diatom frustule particular to the Eunotia species. The genus Eunotia possesses a mirror-symmetric, mantle-offset, brevisslit raphe system, which may or may not have terminal raphe fissures. Morphological analysis provided in this study revealed the absence of terminal raphe fissures for many species of Eunotia. Instead, the distal ends of the raphe slits finish on the outer valve surface by funnel holes, sometimes pore-like ones, connected with the helictoglossae. However, in the literature those distal ends of the raphe slits were described erroneously as terminal raphe fissures. For the first time different types of raphe system are grounded. Two species Eunotiaimplicata Nörpel-Schempp et al. in Alles et al. and Eunotiaincisa W. Smith ex Gregory were lectotypified.


Introduction
The Cheremsky Nature Reserve, located in Ukrainian Polissya, Volyn region, in the interfluve of Stokhid and Veselukha rivers, occupies about 3 thousand ha. The reserve includes large areas of untouched forests and unique wetlands which take up about 34% of the territory (Figs 1, 2). An eumesotrophic Cheremske bog (total area of about one thousand ha, peat deposition up to 10 m), relates to peripheral-oligotrophic type of development characteristic of bogs formed in lake-like basins. Two lakes within the Cheremsky Nature Reserve Cheremske (7,7 ha, max. depth 7 m) and Redychi (14,0 ha, max. depth 4,5 m) originate from glacial-karst (Konischuk andDidukh 2004, Konishchuk 2005).
As a part of documenting the Eunotia taxa it was necessary to describe the raphe system's particular properties which have taxonomical value on species rank of taxonomy. In recently published terminological glossary the following definition for the raphe was proposed: "Raphe (Lat.) -an elongated slit or pair of slits through the valve wall. When a pair of slits is present each individual slit is a branch of the raphe" (Gogorev et al. 2018: p. 299), which does not include the position of the raphe system in the diatom frustule hierarchic structure and any of its functions. In another glossary about the same definition appears: "The raphe system is composed of one or two slits, or fissures, that penetrate the valve of some diatoms. If two slits are present, each is called a branch of the raphe. Branches may be separated by a silica thickening called the central nodule" and it was indicated that raphe allows diatom cells to move (Diatoms of North America 2019). In the latest one the possible raphe position on the valve is defined as axial (along the apical axis), eccentric (along one margin) or circumferential (around the whole margin of the valve) none of which does not specify raphe position on the valves of the Eunotia species. In both definitions several structural elements of the raphe system are omitted. Moreover, in the second, no distinction is made between the raphe slits and fissures, as both terms look like synonyms but they define different elements of the raphe system. The Eunotia taxa possess unique raphe system the morphology of which has not been studied yet in detail.
This study provides detailed information on the species of Eunotia Ehrenb. found in the Cheremsky Nature Reserve, including rare species recorded in Ukraine for the first time. Revised terminology to highlight morphological features of the raphe system relevant to the Eunotia species is also suggested.
Size ranges were based on measurements typically several, sometimes single valves as all species were found in very limited numbers. Therefore the size ranges from the relevant literature were included in the species description.
In many diatom species with bipolar symmetry including Eunotia different morphometric data present at different valve parts, e.g. width, striae density in 10 µm etc., their dimension can be helpful in the species correct identification.
Central valve part -valve part on both sides from the transapical axis where the measuring parameter has different value comparing with other valve parts (Fig. 3a, between the arrows).
Middle valve part -valve part between central part and the beginning of valve pole (Fig. 3a, between the upper and double arrows).
Valve pole -distal valve part from its narrowest part or from the beginning of a valve narrowing to the distal end (Fig. 3a, from the double arrow to distal end of a valve).
For the algae flora of Ukraine a 'very rare species' is defined as one recorded in 1-5 localities and 'rare species' -in < 10 localities within the country territory (Palamar '-Mordvintseva et al. 1998). A 'rare species in the world flora' is defined here as one recorded in fewer than 10 localities and restricted to 1-2 continents.

c5-7
the data for the central valve part. m9-12 the data for the middle valve part. p18-20 the data for the valve pole. * species recorded for the first time in Ukraine. rare species in the world flora.

Morphology and refined terminology
The revised definitions suggested here for some widely used terms and new ones are grounded on the concept of functional morphology of the diatom frustule (Bukhtiyarova 2009a, 2019) which includes a number of theses, in particular the division of all diatom frustule structures on the basic elements and functional units. Thus, this concept allows us to describe any morphological structure on a common universal basis. Because physical-chemical properties of any material depend on the size of the particles it is compounded from, it was suggested to introduce a size scale in all the definitions of the diatom frustule structures (Bukhtiyarova 2009a).
The basic element of the diatom frustule (db-element) is a morphologically detached, homogeneous part of the frustule that possesses special physical-chemical features and provides primary basis for the frustule hierarchical construction. They belong to db-elemets of the diatom frustule as different apertures and cavities in its thickness, regularly repeated and unique silica microelemets (Bukhtiyarova 2009a: figs 1-5).
Morph of the diatom frustule (df-morph) is compound structural unit of the diatom frustule that is constructed of db-elements and/or structural units of lower orders, realizes particular functions in the diatom organism and has its own evolution (Bukhtiyarova 2009a).
The refined definitions capture raphe system as a functional unit of the diatom frustule, its different db-elements and peculiar properties relevant to Eunotia species. For the first time different raphe system types are grounded.
Raphe system -a unique compound micro df-morph of second (first) order in the diatom frustule with bipolar symmetry that consists of one or two slits, which penetrate the valve thickness, and may include additional df-morphs (central nodule, helictoglossae, tube) and/or db-elements (terminal fissures, central pores and others). One of the functions of the raphe system is active moving of the diatom unicellular organism. For the species that have an attached mode of life other functions can be performed, e.g. an attachment to substrate or between neighboring cells in a colony.
Raphe slit -a unique micro db-element in the shape of uniformly narrowed through opening of different profile into the valve thickness and noticeable length relative to the valve length.
Central raphe pore -a unique micro db-element, through hole with usually a different shape on the inner and outer valve surfaces at the proximal end of the raphe slit (Fig. 3b, arrow).
Terminal raphe fissure (tr-fissure) -a unique micro db-element in the shape of uniformly narrowed non-through notch continuing distal end of the raphe slit on external valve surface only (Figs 3e, f ).
Lacuna -a unique micro or nano-db-element, non-through hollow of different shape and location on outer or inner valve surface.
In the genus Eunotia the lacunae of the raphe terminal fissures (rtf-lacuna) usually have a dish or funnel-like shape of about 100 nm in diameter (Figs 3f, 4a, 5a, 6a, 8a, double arrows).
Helictoglossa -a unique siliceous hyaline micro df-morph of first order on the internal valve surface usually in the shape of truncated cylinder or compressed (relative to the raphe slit) asymmetric frustum with smoothly roused up side bearing fissure at the distal end of the raphe slit and abruptly roused opposite side (Figs 13a,29,30a,arrows). Any functions of helictoglossae are unstudied, yet their morphology and position allow to suggest that they work like a stopper and may regulate a mucilage mass length of uniform shape that enters into the raphe slit from inside and goes out from the diatom frustule outside.
Helictoglossa fissure (h-fissure) -a unique micro db-element in the shape of a uniformly narrowed short non-through notch on helictoglossa continuing distal end of the raphe slit on internal valve surface only.
Symmetric raphe system -type of raphe system with straight equal length of the slits and similar accompanied db-elements that are located symmetrically relatively both to the longitudinal and transapical axes of bipolar frustule. Examples of this type of raphe system can be found among species of Cavinula D.G. Mann & Stickle in Round et al. (1990), Hippodonta , Navicula Bory (1822), Psammothidium Bukht. & Round (1996) and other diatom genera.
Mirror-symmetric raphe system -type of raphe system with equal length and same shaped raphe slits, similar accompanied db-elements that all together are located mirror symmetrically relative to the transapical axis or/and in girdle view of the diatom frustule. Examples of this type of raphe system can be found in species of Amphora Ehrenb. ex Kütz. (1844), Cymbella Agardh (1830), Eunotia and other genera.
Mantle-offset raphe system (Lat.) -type of raphe system which partially or completely disposes on the valve mantle. This type of raphe system characterizes the genus Eunotia.
Brevisslit raphe system (Lat.) -type of raphe system with the slits which disposes only along part of valve length and absent on the rest of it. The examples of this raphe system type can be found in the genera Actinella F.W. Lewis (1864), Eunotia, Rhoicospenia Grunow (1860) and in others.
Thus, species of the genus Eunotia possess of mirror-symmetric, mantle-offset, brevisslit raphe system.
Basal striae -type of striae in which the areolae and all additional db-elements accompanying them occupy interstria height in whole (Bukhtiyarova 2015: figs 2, 10-15), or by other words, the valve thickness completely.
The terms proportional, packed, distant kinds of striae have been defined on the ratio between stria and interstria areas (Bukhtiyarova 2015).
Frustule bi-symmetric, bipolar, biraphid with mirror-symmetric, mantle-offset, brevisslit type of raphe. Valves dorsiventral, with undulate dorsal margin and weak depression in its central part, slightly concave ventral side and subcapitate broad rounded poles. Striae basal, uniserial, distant, denser at the poles. Areolae small with round outer foramina (Fig. 28a). Raphe system consists of two short filiform slits on ventral valve mantle, distal ends of slits finish on external valve surface on ventral pole corners by small round pore outers (Fig. 28a) connected with small helictoglossae (Fig. 29); tr-fissures absent.
Ecology. Freshwater epiphytic species, often collected on different Sphagnum species, occurs in acidic (pH 5.5-5.6), oligotrophic waters with low electric conductivity and buffered by humic acids. The specimens from Type population were collected at 11-13 °C (Kulikovskiy et al. 2010b).
Frustule bi-symmetric, bipolar, biraphid with mirror-symmetric, mantle-offset, brevisslit type of raphe. Valves slightly dorsiventral, linear with weakly convex dorsal margin, concave ventral margin and protracted rounded poles. The mantle's height is equal to about 0.5 of valve width, abruptly perpendicular to the valve surface (see Costa 2015: pl. 28/figs 2, 4). Striae basal, uniserial, distant, uniformly spaced along the valve and compacted at the poles, uninterrupted on dorsal mantle/valve junction and interrupted by sternum on ventral mantle; on dorsal mantle short intercalar striae present (see Costa 2015: pl. 28/figs 2, 4). Areolae small with round outer foramina. Raphe system consists of two short filiform slits on ventral hyaline part of mantle, distal ends of the slits turned to the valve centre under right angle and finish on external valve surface in ventral corner of the poles by small round pores (see Costa 2015: pl. 28/figs 1, 2) connected with helictoglossae; tr-fissures absent.
Distribution Frustule bi-symmetric, bipolar, biraphid with mirror-symmetric, mantle-offset, brevisslit type of raphe. Valves dorsiventral, uniform in width, with two very weak undulations on dorsal margin, weakly concave ventral margin and protracted broadly rounded poles slightly deflected to dorsal side. Striae basal, uniserial, distant, evenly spaced. Areolae small with round foramina (Fig. 12a). Raphe system consists of two short filiform slits on ventral valve mantle; helictoglossae average in size; one rimoportula present at the middle of pole (Fig. 12a).
Ecology. Freshwater species, epiphytic on the moss, at an altitude of about 2300 m a.s. (Pavlov and Levkov 2013). Comments. Raphe system has not been studied from outer valve surface in SEM but this species certainly does not have tr-fissures which are always situated on a hya- line field (Fig. 12a) and distal ends of the raphe slits finish on ventral valve margin at the poles, similar to E. dorofeyukiae (Fig. 27a).
Most specimens in the population from North Macedonia differ through having much narrower poles (Pavlov and Levkov 2013: pl. 13/figs 3-6) than in the type population.
Very rare species occurs only in Europe in three localities in low abundance. Frustule bi-symmetric, bipolar, biraphid with mirror-symmetric, mantle-offset, brevisslit type of raphe. Valves dorsiventral, uniform in width, arcuate, with rounded poles. Striae basal, uniserial, distant, evenly spaced (Figs 7, 8, 8a). Areolae small with round outer foramina. Raphe system consists of two short filiform slits on ventral valve mantle; tr-fissures curved on the valve surface, pass along four striae on the middle of valve and end up by round lacunae (Fig. 8a).

Eunotia neocompacta S. Mayama in Mayama and
Mayama 1997: length 18-57 µm, width 3.5-5 µm, striae density 20-22 in 10 µm. Frustule bi-symmetric, bipolar, biraphid with mirror-symmetric, mantle-offset, brevisslit type of raphe. Valves dorsiventral, weakly arcuate, uniform in width, with truncated poles strongly deflected to dorsal side. Striae basal, uniserial, distant, evenly spaced (Fig. 30a). Areolae small with round outer foramina. Raphe system consists of two short filiform slits which are straight on ventral valve mantle and widely rounded at valve poles, distal ends of the slits finish on external valve surface on the middle of the poles by small round pores connected with helictoglossae of average size ( Comments. In some publications the illustrations of this species are not uniform in valve outline, therefore only those microphotos which correspond to the species concept in Mayama (1997) are cited in the present paper. For instance, the specimen in Bouchard et al. (2018: pl. 1/ fig. 6) has arcuate valve and longer poles therefore does not match to E. neocompacta sensu stricto.
Frustule bi-symmetric, bipolar, biraphid with mirror-symmetric, mantle-offset, brevisslit type of raphe. Valves dorsiventral, with strongly convex dorsal and weakly concave ventral margins, gradually narrowed to rostrate poles that are about perpendicular to the valve margins. Striae basal, uniserial, distant, irregularly spaced. Areolae small with round outer foramina. Raphe system consists of two short filiform slits on ventral valve mantle, distal ends of the slits finish on external valve surface on about 0.3 of pole width by small round pore-like outer connected with helictoglossae (see Pavlov and Levkov 2013: pl. 18/ fig. 7); tr-fissures absent.
Ecology. Freshwater epiphytic species. Distribution. Species was recorded in most European countries and on all continents except Antarctica (M. Gury in Guiry and Guiry 2019). In Ukraine. First record in the Cheremsky Nature Reserve, tract Obkopane, ditch, epiphyton on Sphagnum sp.
Ecology Frustule bi-symmetric, bipolar, biraphid with mirror-symmetric, mantle-offset, brevisslit type of raphe. Valves dorsiventral, uniform in width, with slightly undulate dorsal margin and weak depression in its central part, usually straight ventral side, sometimes with weak central convexity (see Bílý and Marvan 1962: fig. 1), and broad rounded poles deflected to dorsal side. Striae basal, uniserial, distant, denser at the poles, irregularly spaced. Raphe system consists of two short filiform slits on ventral valve mantle, distal ends of the slits terminate at the poles about 0.3 of valve width from ventral margin.

Eunotia tetraodon
Frustule bi-symmetric, bipolar, biraphid with mirror-symmetric, mantle-offset, brevisslit type of raphe. Valves dorsiventral, with strongly convex, four-times strongly undulate dorsal and weakly concave ventral margins, gradually narrowed to the protracted poles that continue the dorsal arc of valve margin. Striae basal, uniserial, distant, irregularly spaced, on dorsal side shortened intermediate striae present. Areolae small with round outer foramina. Raphe system consists of two short filiform slits on ventral valve mantle that follow pole margin and finish on about 0.5 of pole width by small round pore (see Pavlov and Levkov 2013: pl. 26/fig. 11) connected with helictoglossae; tr-fissures absent.
Comments. In Ukraine this species is quite rare. Only five reliable records exist that were accompanied by illustrations, including this paper, and all are from Ukrainian Polissya and Carpaty. Initially this species was reported by Topachevsky and Oksiyuk (1960) as E. robusta however their illustration corresponds to E. tetraodon. The next reports came almost 60 years later (Malakhov et al. 2017, present paper).
Comments. This specimen is similar to E. mongolica in valve outline, however it differs in metric parameters and fine morphology, having shorter tr-fissures (Fig. 17a).  Kulikovskiy et al. 2016: pl. 19/figs 8, 9). However, the illustrations by Grunow (1862: p. 336, pl. 3/ figs 10a-c) show a much more arcuate valve outline that differs significantly from the literature concept.
The other species whose small specimens have similar valve outline to the discovered specimens was reported as Eunothia fennica (Hustedt) Lange-Bert. in Werum and Lange-Bertalot (2004) by Noga (2019: only figs 2e-g, 3c-e). However, the detail study of E. fennica (Hamilton and Siver 2010: figs 1-11, 16-29) shows a clear difference from the specimens on Figs 17-19 and the ones, cited from Noga (2019): in valve outline, presence of spines, morphonetric data. Hamilton and Siver (2010) also have underlined that E. fennica can be confused with E. paludosa, more over, the authors found both species in the same sample.
Distribution in Ukraine. The Cheremsky Nature Reserve, tract Obkopane, Lake Redychi, epiphyton on Sphagnum sp Comments. These exemplars differ from E. meridiana in valve outline, narrower valves and higher striae density.
Comments. Both Eunotia sp. 6 and Eunotia sp. 7 have similar morphology, valve width and striae density. Moreover, both exemplars were found in the same sample, which may suggest that they belong to the same species, but insufficient data does not provide a conclusion at present.

Discussion and conclusions
The genus Eunotia is one of the largest within the Order Bacillariophyta and totals 589 valid taxa. This can be considered as an evolutionary success of the genus, relevant to the frustule morphology that is well suited to the ecological conditions where the Eunotia species inhabit.
The presence of raphe system is certainly a progressive feature which has appeared in the diatom frustule evolution since the number of species bearing it exceeds significantly the ones without raphe.
The complicated morphology of Eunotia species has led to the numerous synonyms -more than 60% of taxonomic names (Guiry M in Guiry and Guiry 2019). Therefore it is a necessary task to find reliable morphological characters which can be useful in species identification and description.
The genus Eunotia possesses of mirror-symmetric, mantle-offset, brevisslit raphe system the combination of the characters in which is unique among diatom genera. At the same time different Eunotia species have peculiar details in the raphe system which belong to species rank of taxonomy: presence / absence of tr-fissures and their shape, shape of raphe slits and their position on the valve etc. ( Table 1).
The morphological analysis carried out in this study revealed that 8 of 12 studied species of Eunotia do not have terminal raphe fissures (Table 1). Instead, the distal ends of the raphe slits terminate on the outer valve surface by pore-like (Figs 3c,28a,30) or funnel holes (Fig. 3d, see Mayama 1997: fig. 31) which are connected with helictoglossae. However, in the literature those distal ends of the raphe slits were erroneously described as tr-fissures (e.g. Pavlov and Levkov 2013: p. 20, pl. 18/fig. 7;p. 43, pl. 26/ fig. 11). Eunotia krammeri Kulikovskiy et al. also has a raphe system without tr-fissures that is clearly visible on SEM illustrations both from external and internal valve surfaces (Kulikovskiy et al. 2010a: p. 102, pl. 7/figs 18, 19). Nevertheless, the authors report them in their description of this species.
In morphology of Eunotia the characters suitable for the species identification are quite restricted. Besides, the frustule ultrastructure is poorly studied even for the species which were investigated with SEM, in particularly, raphe system. For many species there is still no data on the slits form, central pores etc. In the meantime, among key diagnostic characters for the Eunotia species identification more than half include the ones which refer to the peculiarity of raphe system (Table 1). For instance, central raphe pores usually have a different shape on the inner and outer valve surfaces, however their shape on inner surface is uniform within the genera and some taxa of higher rank of taxonomy while the shape of central pores on the outer valve surface has the species rank of taxonomy (Bukhtiyarova and Pomazkina 2013).
Thus, careful application of suggested terms in future is necessary when describing new Eunotia taxa and will be helpful in correct identification of the known species.
Species of Eunotia prefer acidic, dystrophic or oligotrophic freshwater habitats, mostly of low conductivity and usually inhabit in epiphytic or epilithic hydrotopes (Alles et al. 1991, Metzeltin and Lange-Bertalot 1998, Siver et al. 2006, Cantonati and Lange-Bertalot 2011, Pavlov and Levkov 2013, Bahls et al. 2018). In Ukraine 32 species and eight varieties of Eunotia were known until this study and now 9 more species are reported for the first time. Thus, the total number of Eunotia species in Ukraine is 41, which is only 7% of the species in this genus worldwide. This is indirect evidence of insufficient investigation of the wetlands in Ukraine where Eunotia has high species richness. The findings in the present study include five species widely distributed in the world flora on most continents and seven rare species that are known from several locations, among the latter are E. genuflexa, E. jarensis and E. ruzickae, which are probably European endemics. At present, in the Cheremsky Nature Reserve, 20 species have been recorded, which is the largest number of Eunotia species in any region of Ukraine. In total 19 Eunotia species were recorded in this study together with the ones which were not identified to the species level. Given the large number of poorly studied oligotrophic lakes and bogs in the country, especially in Ukrainian Polissya, it is possible to predict the future discovery of many more Eunotia species from Ukraine.