SPECIES OF THE DIATOM GENUS CRATICULA GRUNOW (BACILLARIOPHYCEAE) FROM MACEDONIA

The species of the genus Craticula are widely distributed in Europe, mostly occurring in brackish, saline to heavily polluted waters. Some of the species are considered as being most tolerant to pollution. Recently, detailed observations of this genus in Macedonia have been performed. During this study, 15 taxa have been recorded. One of the species (Craticula simplex) was observed with three different morphotypes, which can be distinguished by the valve size and shape. Craticula cuspidata and C. ambigua were the most frequently recorded species in various habitats, while species C. halophila, C. germainii and C. fumantii were observed only on a single locality. The highest diversity of Craticula was observed in temporary ponds on halomorphic soils and mineral springs in Eastern Macedonia.


INTRODUCTION
The genus Craticula Grunow was described based on the species C. perrotettii Grunow [1,p. 20,Fig. 1: 21] and was forgotten for a long time until Mann & Stickle [2] re-established the genus and provided a more detailed description based on ultrastructural and sexual characteristics. Most of the members of this genus were previously included in Navicula Bory section Orthostichae (Cleve [3], Hustedt [4]). One of the prominent features of this genus is the presence of "craticula"inner valves produced during resting spore formation. Such inner valves might be produced as a result of elevated salt concentrations or desiccation (Schmid [5,6]). The inner valves were referred as "heribaudii" valves and they differ by several ultrastructural features from the normal valves. However, "heribaudii" valves are so far observed only in few members of the genus Craticula.
The genus is characterized by cells with two plastids with lenticular pyrenoids that are usually associated with a cytoplasmic bridge holding the nucleus (Cox [7]). The raphe is filiform and can be located externally on thickened conopeum or on valve face (Morales & Le [8]). Internally the raphe branches are positioned on elevated sternum. The striae are composed of a single row of round or apically elongated areolae. Internally the areolae are covered by hymen. In some species areolae are separated by strongly thickened frets that appear as longitudinal ribs. In most of the species, striae are parallel to weakly radiate in the mid-valve, becoming slightly convergent toward the valve apices.
The genus is widely distributed in various habitats, from acidic oligotrophic waters to alkaline, (hyper)saline, (hyper)eutrophic and heavily polluted waters. Some of the members of the genus are considered as one of the most tolerant species to organic pollution (Lange-Bertalot [9,10]). High diversity of the genus was noticed in alkaline, slightly saline and warm (even temporary) water habitats (Lange-Bertalot [10], Lange-Bertalot et al. [11]). Craticula was observed frequently in the Tropics (Morales et al. 12, Rumrich et al. [13]), but also on Antarctica (Van de Vijver et al. [14]; Sabbe et al. [15]).

EXPERIMENTAL SECTION
More than 6.000 samples observed in this study have been collected during various field campaigns, starting from 1995 until present. Most of the samples were collected within the period June-August. Samples from the various habitats include: sediments at various depths (0.5-50 m) from Ohrid, Prespa and Dojran lakes, sediments, stones and macrophytes from rivers, small springs, streams and rivulets, high altitude glacial and nonglacial lakes, ponds and pools of various size, peat bogs, fens and mires. The altitudinal range at the different sampling sites varies between ca. 200-2500 m a.s.l. The samples with higher abundance of Craticula species are listed in Table 1. Diatom samples were cleaned by acid digestion using K2MnO4/HCl, and permanent slides were mounted in Naphrax ® . Photomicrographs were taken with a Nikon E-80i, a digital Nikon Coolpix 600. For scanning electron microscope (SEM) analyses, cleaned material was dried onto aluminum stubs and coated with gold/palladium using a sputter coater. SEM micrographs were produced with a Cambridge Instrument S4 Steroscan electron microscope operated at 5 kV. Slides are deposited in the Macedonian National Diatom Collection (MKNDC), Institute of Biology, Skopje, Macedonia (holotype), and the Friedrich Hustedt Centre for Diatom Research (BRM) in Bremerhaven, Germany (isotype).

RESULTS AND DISCUSSION
During the observations of the genus Craticula in Macedonia, 13 species in total have been recorded. One of the species, C. simplex (Krasske) Levkov comb. nov. was present with three morphotypes which differ with respect to the valve outline and size. However, we still consider these populations as a part of the morphological variation of a single species. Few valves with similar numerical features as C. acidoclinata Lange-Bertalot & Metzeltin were observed in glacial Lake Karanikoličko on Šara Mountain, but they differ from the type population with respect to the shape of the valve apices.

Craticula perrotettii Grunow
( Valve morphology, SEM (Figs 2: 1-3): Raphe branches are located on strongly thickened conopeum with equal width throughout. Central pores are dilated and slightly unilaterally deflected. Transapical striae are uniseriate composed by areola with variable size and shape. Areolae near the axial and central area have small round apertures, while the rest are apically elongated with slit-like external openings. Longitudinal frets are well developed and forming grooves in which areola are located.
Comment: The conopeum and longitudinal frets present in the specimen from Lake Dojran are narrower and not strongly pronounced as in the specimen depicted in Lange-Bertalot [10,.
Similar species: In general, C. perrotettii has a unique set of characters (valve size, shape and presence of strongly thickened longitudinal frets) that clearly separates it from other Craticula species. Craticula pampeana (Frenguelli) Lange-Bertalot has similar valve size, but different valve shape, lanceolate with broadly rounded apices (Frenguelli [30]). Craticula cuspidata has similar valve shape and longitudinal frets, however it can be clearly differentiated by its smaller valves (lenght 82-122 μm, width 21.0-27.5 μm).
Distribution in Macedonia: This species was observed only in Lake Dojran. According to Lange-Bertalot [10, p. 117] this species is rare in Europe and it was observed only in Southern Italy.
Ecology: Craticula perrotettii occurs in eutrophic to hypereutrophic habitats on organic sediment. This is а tropical/subtropical species preferring habitats with higher temperature [10]. Valve morphology, SEM (Figs 5: 1-5): The valve surface is ornamented with narrow and slightly thickened longitudinal frets ( Fig. 5: 4). Raphe is located on a narrow, strongly thickened sternum ( Fig. 5: 1). External proximal raphe endings are short, expanded into central pores and slightly unilaterally deflected (Fig. 5: 4). Distally, raphe endings are long, hooked and continuing onto the mantle ( Fig. 5: 2). Striae are uniseriate composed of narrow, apically elongated slit-like areolae, except near the central area where areolae have round foramina ( Fig. 5: 4). Internally, central nodule is slightly raised (Fig. 5: 5). Raphe branches are straight, located on inwardly elevated sternum. Raphe proximally terminates with short, unilaterally weakly deflected endings, while distally with well developed helictoglossa. Areolae are occluded by hymenes, located inside each areola ( Fig. 5 Ecology: This species was observed in wide spectrum of habitats: oligotrophic to eutrophic and hyper eutrophic lakes, oligotrophic to mesotrophic rivers, slightly polluted to highly polluted rivers. It occurs on fine inorganic or organic sediments. Comment: Craticula aff. acidoclinata resembles C. cuspidata, C. acidoclinata and C. nonambigua Lange-Bertalot, Cavacini, Tagliaventi & Alfinito. In general, C. aff. acidoclinata shares the morphological features of C. cuspidata and might represent a smaller form of C. cuspidata. However, most of the records of C. cuspidata in the available literature show larger cells with width 24-35 μm and lower striae density (11-15 in 10 μm) in contrast to C. aff. acidoclinata. Also C. cuspidata is usually observed in the eutrophic lotic and lentic waters, while the observed taxon in this study (C. aff. acidoclinata) was recorded in an oligotrophic, slightly acidic glacial lake. From aspect of the ecology, this species is similar to C. acidoclinata. Also, similarities in the morphology can be observed with the specimen depicted in Lange-Bertalot [10, Fig. 87: 2] from the type population.

Distribution in Macedonia:
This taxon was observed only in glacial Lake Karanikoličko on Šara Mountain.
Ecology: The locality where this taxon was observed represents an oligotrophic lake, circumneutral to slightly acidic, with low mineral content.

Craticula sardiniana Bahls (Figs 7: 1-3)
Valve morphology, LM: Valves are lanceolate gradually tapering towards apices. Apices weakly protracted and rounded. Valve length varies from 76-93 μm and valve width from 18.5-19.5 μm. Axial area is very narrow, linear. Central area is absent to slightly concave. Raphe is straight, filiform. Proximal raphe endings are expanded into central pores and slightly deflected, while distal endings are hooked and reaching the valve mantle at the apex. Transapical striae are parallel in the mid-valve, becoming slightly convergent towards the apices, 13-17 in 10 μm. Areolae are hardly visible with LM, 28-32 in 10 μm.
The differences between C. sardiniana from the type population of Bahls [34, figs 96-101], and the population from Macedonia might be noticed in the shape of valve apices. In C. sardiniana from the type population, apices are bluntly rounded and not protracted, while the population from the halomorphic soils is characterized by weakly protracted and narrower apices. However, the valve shape of the Nomenclatural synonym: Navicula cuspidata var. ambigua (Ehrenberg) Cleve [36, p. 110] Valve morphology, LM (Figs 8: 1-12): Valves are broadly elliptic, elliptic-lanceolate to lanceolate to with abruptly protracted and subcapitate apices. Valve margins are with "shoulders" near the valve apices. Valve length varies from 47-77 μm, valve width 14.5-20.0 μm. Axial area is very narrow, linear. Central area is absent to slightly wider than axial area. Raphe is straight, filiform. Proximal raphe endings are expanded into central pores and slightly deflected, while distal endings are hooked and reaching the valve mantle at the apex. Transapical striae slightly radiate in the midvalve, becoming slightly convergent towards the apices, 14-17 in 10 μm. Areolae are visible with LM, 26-32 in 10 μm.
Ecology: This species was observed in various habitats starting from deep oligotrophic Lake Ohrid, mesotrophic Lake Prespa, the eutrophic River Bregalnica and Lake Dojran. It usually occurs in epipelic diatom assemblages on organic or fine inorganic sediments. Valve morphology, LM: Valves are rhombic-lanceolate to elliptic-lanceolate with abruptly protracted and sub-capitate apices. Valve margin is gradually narrowing towards the apices in the larger and medium-sized specimens, while in smaller specimens (Figs 11: 6-10) "shoulders" might be present. Valve length varies from 49-66 μm, valve width 12.5-18.0 μm. Axial area is very narrow, linear. Central area is absent to slightly wider than axial area. Raphe is straight, filiform. Proximal raphe endings are expanded into central pores and slightly deflected, while distal endings are hooked and reaching the valve mantle at the apex. Transapical striae radiate in the mid-valve, becoming slightly convergent towards the apices, 14 . Craticula nonambigua and C. ambigua can be hardly differentiated by the valve size. The main difference is that the valve margins in C. ambigua have "shoulders" near the apices. Craticula lange-bertalotii has wider valves (width = 21-25 μm) with slightly undulated margins. Craticula fumantii has narrower, lanceolate valves (not rhombic-lanceolate or ellipticlanceolate as in C. nonambigua).
Distribution in Macedonia: During this study, C. nonambigua was observed only in a small, temporary pond on halomorphic soils, Ovče Pole, near the town of town of Sveti Nikole. However, some other records of C. ambigua in Macedonia might belong to this species or to C. fumantii.
Ecology: Not precisely known. During this study, C. nonambigua was observed in an alkaline dystrophic pond and channel on halomorphic soils with high electrolyte content (high conductivity).  (Figs 14: 1, 2). Raphe is located on a moderately wide, strongly thickened sternum, slightly expanded in the central area ( Fig. 14: 2). External proximal raphe endings are short, expanded into central pores and slightly unilaterally deflected ( Fig. 14: 2). Distally, raphe endings are long, hooked and continuing onto the mantle (Fig.  14: 3). Striae are strongly radiate, uniseriate, composed mainly of round areolae. Areolae near the raphe sternum, and especially around the central area have larger, slightly elongated to ovate foramina ( Fig. 14: 2). Internally, central nodule is slightly raised. Raphe branches are straight located on inwardly strongly elevated sternum. Raphe proximally terminates with short, simple endings, while distally with well developed helictoglossa. Areolae are covered by hymenes, located inside each areola (figure not shown).

Craticula fumantii
Similar species: Craticula ambigua, C. nonambigua and C. johnstoniae Bahls [34,. Craticula ambigua and C. nonambigua appear very similar to C. fumantii. Although Lange-Bertalot et al. [11, p. 34] stated that there is a difference in the striae density between C. ambigua and C. fumantii, in this study that cannot be confirmed. Differentiation between these three species can be made in combination of valve shape and size. Craticula ambigua and C. nonambigua are wider (14.5-20.0 μm and 12.5-18.0 μm respectively) with elliptic-lanceolate to rhombic-lanceolate valve outline. Craticula johnstoniae has similar valve outline as C. fumantii, but the valves are larger (length 68-113 μm, width 15.6-19.5 μm).
Distribution in Macedonia: Craticula fumantii has been observed on two localities in Macedonia: Lake Karanikoličko on Šara Mountain and the River Buturica, Mariovo.
Ecology: Not precisely known. In Macedonia it was observed in an oligotrophic lake and river, slightly acidic to circumneutral, with low mineral content. The species is rare in the sample and SEM observations of the valve exterior were not possible and only valve interior was observed. Internally, central nodule is distinct, elliptical and slightly raised (Fig.  14: 5). Raphe branches are straight located on inwardly strongly elevated sternum (Fig. 14: 4). Raphe proximally terminates with short, slightly deflected endings, while distally with well developed helictoglossa (Fig. 14: 4). Striae are strongly radiate, uniseriate, composed mainly of round areolae. Areolae probably are covered by hymenes, but due to the cleaning process, hymens are corroded. Areolae have slightly transapically elongated internal foramina (Fig. 14: 4). Comment: The population of C. germainii from Macedonia is characterized by smaller valves compared to the type population (length 42-58 μm, width 11.5-13.5 μm). Additionally, difference in the striae orientation between this and the type population can be noticed. The striae in the type population are more or less equally spaced, are parallel to slightly radiate throughout the valve, while the observed valves in this study have more distantly spaced striae in the mid-valve and distinctly radiate striae. No SEM images are available for C. germainii, so ultrastructural features cannot be compared. In this moment we consider this population as conspecific with C. germainii, but further studies might suggest their separation into two taxa.
Similar species: Craticula germainii has unique set of characters (valve shape, striae orientation) that make its identification simple and cannot be confused with other species. Craticula simplex (Krasske) Levkov comb. nov. and C. accomodiformis Lange-Bertalot have comparable valve size, but they significantly differ in valve shape.
Distribution in Macedonia: Craticula germainii was observed only in a small, temporary pond on halomorphic soils, Ovče Pole, near the town of Sveti Nikole.
Ecology: Craticula germainii prefers alkaline dystrophic waters with high electrolyte content (high conductivity) such as temporary ponds and alkaline ferns. Distribution in Macedonia: Craticula minusculoides was observed only in a small, temporary pond on halomorphic soils, Ovče Pole, near the town of Sveti Nikole. It is extremely rare in the sample.
Ecology: Not precisely known. According to Lange-Bertalot [10, p. 115] it occurs in electrolyte rich, eutrophic, α-β mesosaprobic waters. The locality where it was observed is characterized by slightly alkaline water (pH = 8.3) with high conductivity (1300 μS•cm -1 ). Nomenclatural synonym: Navicula halophila (Grunow) Cleve [36, p. 109] Valve morphology, LM: Valves are rhombic to rhombic-lanceolate with acute to slightly protracted apices. Valve length varies from 27-54 μm, valve width 9.0-12.0 μm. Axial area is very narrow, linear. Central area is absent with same width as axial areal. Raphe is straight, filiform. Proximal raphe endings are expanded into central pores and slightly deflected, while distal endings are hooked and reaching the valve mantle at the apex. Transapical striae are parallel to slightly radiate in the mid-valve becoming strongly convergent towards the apices, 17-19 in 10 μm. Areolae are hardly visible with LM, c. 35 in 10 μm.

Craticula accomodiformis Lange-Bertalot
Similar species: Craticula halophila can be hardly confused with other species, since it has characteristic valve shape that makes its identification simple. Craticula accomodiformis Lange-Bertalot has similar size, but it has ellipticlanceolate valves with shortly rostrate apices.

Distribution in Macedonia:
So far, C. halophila was observed only in temporary ponds on Slan Dol. It was reported from Lake Dojran, but this data was not confirmed during this study.
Comment: The observed species has similar characteristics as Navicula simplex Krasske [43, Fig. 2: 33]. In the past, this taxon was recognized as a separate and distinct species by Hustedt [44, p. 296, fig. 500 [14, figs 19-36]). Craticula simplex can be differentiated from C. guaykuruorum by the shape of valve apices (narrow and weakly protracted), stria density (21-23 in 10 μm) and orientation (parallel in C. guaykuruorum) as well as by the absence of longitudinal frets. Craticula elkab has comparable valve size and shape with morphotype 3 (Figs 18: 1-10), but it differs with the shape of the valve apices (narrowly rounded to weakly protracted especially in larger specimens) and striae density (22-26 in 10 μm). Craticula riparia var. riparia is characterized by larger valves (35-50 μm long, 8.0-10.5 μm wide) with 15-18 striae in 10 μm which are parallel through. Craticula riparia var. mollenhaueri Lange-Bertalot [9, figs 70: 10-13) has comparable valve size as C. simplex, but can be differentiated by the presence of strongly silicified raphe sternum and striae orientation (parallel in C. riparia var. mollenhaueri). Probably the most similar species to C. simplex is C. antarctica. The latter species is characterized by lanceolate valves with (sub) capitate apices, 23.5-36.0 μm long and 6.2-8.0 μm wide with 17-22 striae in 10 μm which are radiate in the middle, becoming convergent towards apices. Slight difference between these two species can be noticed in the valve shape (rhombic lanceolate vs. elliptic lanceolate in C. antarctica) and valve apices (rostrate vs. capitate C. antarctica). Difference can be noticed also in ecology: Craticula antarctica was observed in a cold lake on Antarctica, while C. simplex was observed in thermo-mineral springs and warm temporary ponds on halomorphic soils. Distribution in Macedonia: Craticula simplex was observed on several localities in Macedonia: halomorphic soils in Sveti Nikole and Gladno Pole, the thermo-mineral springs near the villages Gabrovo, Stamer and Katlanovo, temporary ponds in Slan Dol and mining Lake Usje.
Ecology: Eutraphentic species, it was observed in the most polluted sites in rivers. In the River Vodenišnica (a heavy polluted river with communal and industrial waste waters) is one of the dominant species, together with C. subminuscula Externally, areolae are covered with hymens. Inter-nally, central nodule is slightly inwardly elevated ( Fig. 20: 6). Raphe branches are straight located on inwardly weakly elevated sternum (Fig. 20: 6). Raphe proximally terminates with short and distinctly deflected endings, while distally with slightly developed helictoglossa. Areolae covered by hymenes, located inside each areola towards the outer valve surface (Fig. 20: 6).
Similar species: Craticula molestiformis can be confused with C. subminuscula. Both species have similar valve size and valve shape, but can be differentiated by the stria density Distribution in Macedonia: Craticula molestiformis was observed only in the reservoir Ratevsko. It is possible to have broader distribution in Macedonia, but very likely it is overlooked or misidentified with other small-celled species.
Ecology: According to Lange-Bertalot [10] C. molestiformis prefers electrolyte rich, often heavily polluted waters. However, during this study it was observed in the oligo-to mesotrophic reservoir and Lake Vevčansko on Mountain Jablanica. Similar species: As mentioned above, C. subminuscula can be confused with C. molestiformis, but both species can be easily differentiated by the raphe morphology, stria orientation and density.

Distribution in Macedonia:
The rivers: Vodenišnica, Strumica, Bregalnica and Vardar. Probably it has wider distribution in polluted rivers, but very likely it is neglected or confused with other small-celled species. Hustedt [17] recorded this species from Lake Ohrid under N. demissa. However, during the recent observations of the flora of Lake Ohrid (Levkov et al. [52]) and this study, N. demissa (or C. subminuscula) was not recorded.
Ecology: Craticula subminuscula is one of the most tolerant species to organic pollution. During this study, this species was observed with high abundance in two of the most polluted rivers: Vodenišnica and Bregalnica.

CONCLUSIONS
The Republic of Macedonia is characterized by presence of various water habitats which host highly diverse diatom flora. Recently more attention was paid to the so called "extreme habitats". Those habitats include thermo-mineral springs, temporarily wet rocks, hypersaline lakes, wet halomorphic soils, ice layers and caves. Such habitats are unfavourable for most of the algae, but on the other hand, they are inhabited by specialized organisms which are adapted to numerous fluctuating and extremely adverse physico-chemical conditions. During this study the highest diversity of Craticula was observed exactly in such extreme habitats. A large number of species were observed on the halomorphic soils near Gladno Pole and Slan Dol. However, these regions are most intensively utilized for agriculture and farming which have a significant impact on their surface area and degradation. Moreover, the scenarios for climate change of the region of Gladno Pole suggest decrease of precipitation and increase of air temperature. Such alterations will have an influence on environment and the functioning of habitats. Therefore the distribution of the remarkable diatom species inhabiting these habitats is under threat.                     Distally raphe fissures strongly deflected, not continuing on the valve mantle. Proximally raphe fissures terminate with small central pores, slightly deflected on the same side. Areolae with small, round foramina. 5, 6. Internal view.