Acta Botanica 2-2015.indd ACTA BOT. CROAT. 74 (2), 2015 233 Acta Bot. Croat. 74 (2), 233–244, 2015 CODEN: ABCRA 25 ISSN 0365-0588 eISSN 1847-8476 DOI: 10.1515/botcro-2015-0019 Morphological study of Chaetoceros wighamii Brightwell (Chaetocerotaceae, Bacillariophyta) from Lake Vrana, Croatia SUNČICA BOSAK1*, MARIJA GLIGORA UDOVIČ1, DIANA SARNO2 1 University of Zagreb, Faculty of Science, Department of Biology, Rooseveltov trg 6, HR-10000 Zagreb, Croatia 2 Stazione Zoologica Anton Dohrn, Villa Comunale, 80121 Napoli, Italy Abstract – Chaetoceros wighamii Brightwell is a planktonic diatom species originally described from brackish waters. Since its original description, the species has been report- ed in a wide variety of habitats, ranging from freshwater to marine. Varying descriptions exist in the taxonomic literature and several taxa have been considered as synonyms, in- cluding freshwater species Chaetoceros amanita. In this study we provide morphological and ultrastructural information on a cultured strain isolated from freshwater sample col- lected in the Lake Vrana (Vransko jezero) in Croatia, in April 2011. The cells form short and robust chains with very narrow apertures, often partially occluded by silica mem- branes. Other distinctive features observable in light microscopy are the shape and orien- tation of the setae which are very long, straight and robust, diverging in various directions from the chain axis and the single parietal chloroplast extending from valve to valve. Dis- tinct ultrastructural characteristics are the absence of processes either in intercalary or ter- minal valves and the ornamentation of the valve face with densely distributed ribs spread- ing from an irregular eccentric hyaline area without a clearly defi ned annulus. The outer surface of the terminal valve is ornamented with small spines and setae are composed of fl at longitudinal fi laments interconnected with short bars and ornamented with small spines tightly arranged around the setae. Our description agrees well with that reported for the freshwater morphotypes of C. wighamii (syn. C. amanita) and contributes for a reli- able distinction of this intriguing taxon from similar morphotypes. This fi nding supports the interpretation of Chaetoceros wighamii as a freshwater/brackish species and represents the fi rst report of a Chaetoceros species in lacustrine environment in Croatia and possibly in any Central European habitats. Key words: Chaetoceros, diatom taxonomy, Lake Vrana, morphology * Corresponding author, e-mail: suncica.bosak@biol.pmf.hr BOSAK S., GLIGORA UDOVIČ M., SARNO D. 234 ACTA BOT. CROAT. 74 (2), 2015 Introduction Chaetoceros Ehrenberg is one of the most species-rich genera among planktonic dia- toms, including approximately 170 species (VANLANDINGHAM 1968). This genus has been the subject of a number of taxonomical investigations due to its ecological relevance, ex- traordinary morphological diversity and complexity (HUSTEDT 1930, RINES and HARGRAVES 1988, HERNÁNDEZ-BECERRIL 1996, JENSEN and MOESTRUP 1998). Although the species are primarily marine a few taxa occur at very low salinities in estuaries and freshwater lakes, e.g. the small, often solitary and lightly silicifi ed species Chaetoceros muelleri Lemmer- mann or the more robust C. elmorei Boyer (RUSHFORTH and JOHANSEN 1986). The taxonomy and morphology of the most frequently occurring species Chaetoceros muelleri and similar non-marine taxa has been the subject of a revision in which two distinct varieties were rec- ognized (JOHANSEN and RUSFORTH 1985): the nominate variety C. muelleri var. muelleri and C. muelleri var. subsalsum (Lemmermann) Johansen et Rushforth. Chaetoceros wighamii Brightwell was originally described (BRIGHTWELL 1856) shortly after the genus Chaetoceros was established (EHRENBERG 1844), with special attention to the distinctive spore morphology, and a quite vague illustration of the chain features. The description leaves no doubts on the continental nature of the type locality: Chaetoceros wighamii »occurred in a gathering made from a dirty ditch of brackish water at the back of a small public-house, called The Burney Arms, which is marked on the Ordnance maps« in a sample rich in brackish/freshwater diatom taxa such as Campylodiscus clypeus and one or more Mastogloia species (BRIGHTWELL 1856, p. 108). The species Chaetoceros amanita Cleve Euler (CLEVE-EULER 1915) was established at a later date based on the description of fossil resting spores, characterized by a spiny dome- shaped primary valve and a truncate secondary valve, which were similar to resting spores described by Brightwell for C. wighamii. KACZMARSKA et al. (1985) provided the fi rst de- tailed description of the vegetative forms of C. amanita from the Blue Lake Spring, Utah, USA, and discussed the similarity and the possible conspecifi city of C. amanita and C. wighamii, that was already considered in previous studies (KOLBE and KRIEGER 1942, CLEVE-EULER 1951). Over the years, C. wighamii has been reported in a wide variety of habitats, ranging from freshwater (SANCHEZ CASTILLO et al. 1992) to marine (CUPP 1943, SNOEIJS 1993, JENSEN and MOESTRUP 1998, HAECKY et al. 1998, BÉRARD-THERRIAULT et al. 1999) and was consid- ered as the valid synonym of different marine species, including C. bottnicus Cleve, C. bi- concavum Gran, C. caspicum Ostenfeld (HUSTEDT 1930, VANLANDINGHAM 1968, HASLE and SYVERTSEN 1997) and C. fallax Proschkina Lavrenko (JENSEN and MOESTRUP 1998). SANCHEZ CASTILLO et al. (1992) discussed the identity of C. wighamii in relation with the taxa traditionally associated to it, i.e. C. amanita, C. bottnicus, C. biconcavum and C. cap- sicum. The authors compared the original descriptions and material from different areas, including the type locality of C. wighamii, and recognized the existence of two different species: 1) C. wighamii, which corresponded to specimens from brackish/freshwater envi- ronments and has as the only valid synonym C. amanita, and 2) C. bottnicus, originally described from the Baltic Sea (AURIVILLIUS 1896), which included most of the examined marine morphotypes. This view has been disregarded in some of the following studies (HASLE and Syvertsen 1997, JENSEN and MOESTRUP 1998) or partially considered in other investigations where the name C. wighamii was retained but a possible misinterpretation was taken into account (SNOEIJS 1993, BÉRARD-THERRIAULT et al. 1999). THE MORPHOLOGY OF CHAETOCEROS WIGHAMII FROM LAKE VRANA ACTA BOT. CROAT. 74 (2), 2015 235 As a result of this complicated taxonomic history, C. wighamii has been often perceived as a highly variable species (HUSTEDT 1930, CUPP 1943, RINES and HARGRAVES 1988, JENSEN and MOESTRUP 1998), whereas there are indications (SANCHEZ CASTILLO et al. 1992) that some of the morphological descriptions, synonyms and reports from different habitats, refer indeed to morphologically similar but yet distinct species. Possible misidentifi cations can be primarily attributed to the lack of strongly distinctive characters in the vegetative cell morphology reported by BRIGHTWELL (1856) and to the fact that the presence of the very typical resting spores was rarely used to support a positive identifi cation of the species. In this study we investigated the morphology of a strain of C. wighamii (syn. C. amani- ta) isolated from the freshwater Lake Vrana in Croatia using light (LM), scanning electron (SEM), and transmission electron (TEM) microscopy. The aim is to provide detailed infor- mation, especially on the ultrastructural features of the frustule, allowing for a better delin- eation of this intriguing taxon and its distinction from apparently similar morphotypes. Material and methods Study area Situated in the central part of the eastern Adriatic Coast, Lake Vrana and its surround- ings were declared a nature reserve on July 21, 1999. It is the largest Croatian lake (length 13.6 km and width between 1.4 and 3.4 km). The lake area is between 2980 ha and 3020 ha and extends parallel to the coast in a northwest-southeast direction, separated from the sea by an 800–2500 m wide limestone ridge (up to 113 m above sea level). The basin is a karst drainage system in the area of Ravni Kotari with a surface area of 49400 ha. It is a polymic- tic karstic cryptodepression connected to the Adriatic Sea by a narrow channel at its south- ern side. In the northwestern section the lake is between 0.5 and 1 m deep, and 4–6 m in the southeast. The fl ow of water in the lake is infl uenced by conduction waves caused by the south-eastern and north-westerly winds. The channel Prosika (length 800 m, width 4 m, depth 5–6 m) connects the lake with the Adriatic Sea. The channel is used for drainage and was constructed in 1895 but was subsequently expanded, deepened and regulated. The ex- tensive fl oodplain around Lake Vrana is a natural marsh with high biodiversity. The physico-chemical parameters presented in Table 1 were measured approximately bimonthly from February to October 2011 (N = 7) at a single station, situated in the central part of the lake (43°54’27’’N, 15°33’57’’E). The observed ranges of values agree well with the results of previous investigations (GLIGORA et al. 2007), indicating considerable infl u- ence of the Mediterranean climate conditions both on the nutrient levels and on tempera- ture. During 2011 the salinity in the lake ranged between 2.4 and 6.5 psu, in April 2011, when the diatom strain was isolated in culture the salinity was 2.9 psu. Strain isolation, culture condititons and morphological analyses The water sample was collected at the surface (0.5 m depth) of the lake on April 26, 2011 and examined fresh upon the arrival at the laboratory, in a plastic Petri dish. The indi- vidual cell chains were isolated using a Pasteur micropipette and an inverted Olympus CKX41 light microscope (Olympus, Tokyo, Japan) equipped with bright-fi eld optics and phase contrast. The chains were fi rst placed in sterile 35-mm Falcon polystyrene tissue cul- ture dish (model 353001 BD Labware, Le Pont de Claix, France) fi lled with ca. 2 mL F/2 BOSAK S., GLIGORA UDOVIČ M., SARNO D. 236 ACTA BOT. CROAT. 74 (2), 2015 medium (GUILLARD 1975) and observed each day during the fi rst week for growth and con- ditions of the cells. The culturing medium was prepared using Guillard’s (F/2) marine water enrichment solution (Sigma-Aldrich) according to the manufacturer instructions. The water used for medium preparation was collected from the Lake Vrana and sterilized by fi ltration through 0.2-μm Nucleopore fi lters. When the cell density was high enough the established clonal culture was transferred from a dish to 70 mL Falcon polystyrene cell culture fl asks (model 353082; BD Biosciences, Le Pont De Claix, France) fi lled with 30 – 40 mL of F/2 medium. The strain designated PMF M1 was maintained in a growth culture chamber under cool white (40 W) fl uorescent light (30 μmol photons m−2 s−1) at a room temperature of 20 °C in a 16 h:8 h light:dark cycle and sub-cultured every 1–2 weeks. The material for morphologi- cal studies was obtained in the exponential growth phase within the fi rst month after estab- lishment of the culture. For LM observations, a Zeiss Axiophot microscope (Carl Zeiss, Oberkochen, Germa- ny), equipped with bright-fi eld, phase contrast and Nomarski differential interference con- trast (DIC) optics was used. Light micrographs were taken with a Zeiss AxioCam MRc digital camera and processed with an AxioVision 4.8.2 digital image processing software. The culture material was subjected to cleaning treatment in order to remove the organic matter from diatom frustules. The sample was fi rst desalinized by rinsing with distilled wa- ter, treated with strong acids (1:1:4; sample:H2SO4:HNO3), boiled for a few seconds and then washed again with distilled water. For SEM observations, the cleaned material was fi ltered and air dried on 3-μm Nucleopore polycarbonate fi lter (Nucleopore, Pleasanton, Tab. 1. Physico-chemical variables in Lake Vrana from February to October 2011. MIN – minimum, MAX – maximum, AVG – average, STDEV – standard deviation. Number of sampling = 7. The values measured in April 2011 when the strain of Chaetoceros wighamii was isolated in culture are presented separately. Variable MIN MAX AVG STDV April 2011 Temperature (°C) 2.8 25.6 17.6 8.1 13.9 pH 7.9 9.0 8.5 0.4 7.98 Conductivity (μS cm–1) 3870 8760 5434 1797 4090 Total suspended solids (mg L–1) 3.6 14 8.6 3.8 14 Alkalinity m-value (mg CaCO3 L–1) 66 196 121 61 196 Hardness (mg CaCO3 L–1) 422 1242 706 279 689 O2 (mg L–1) 7.8 12.9 9.5 1.8 9.1 Saturation O2 (%) 83 119 97 13 88.3 NH4+ (mg L–1) <0.010 0.129 0.047 0.041 0.031 NO2– (mg L–1) 0.002 0.016 0.007 0.005 0.016 NO3– (mg L–1) 0.031 1.980 0.722 0.790 1.340 Total inorganic N (mg L–1) 0.064 2.004 0.769 0.805 1.387 Organic N (mg L–1) 0.215 0.328 0.274 0.035 0.268 PO4 (mg L–1) 0.008 0.099 0.034 0.039 0.008 Total P (mg L–1) 0.021 0.233 0.084 0.070 0.067 THE MORPHOLOGY OF CHAETOCEROS WIGHAMII FROM LAKE VRANA ACTA BOT. CROAT. 74 (2), 2015 237 CA), mounted on a stub, sputter coated with gold and examined in a JEOL JSM-6500F SEM (JEOL-USA Inc., Peabody, MA, USA). TEM observations were made by deposition of cleaned and rinsed material on Formvar/carbon coated 200-mesh nickel grids and exam- ined in a LEO 912AB TEM (LEO, Oberkochen, Germany). Preserved material and TEM grids are available from the authors upon request. The general diatom terminology used for the morphological descriptions follows pro- posals recommended by ANONYMOUS (1975), ROSS et al. (1979), ROUND et al. (1990), HASLE and SYVERTSEN (1997). The specifi c terminology for Chaetoceros genus follows EVENSEN and HASLE (1975), RINES and HARGRAVES (1988) and KOOISTRA et al. (2010). Results Chaetoceros wighamii Brightwell 1856: 108, pl. 7: fi gs 19–36 described as Chaetoceros amanita Cleve Euler in KACZMARSKA et al. (1985), RUSHFORTH and JOHANSEN (1986), RINES and HARGRAVES (1988) and as C. wighamii in SANCHEZ CASTIL- LO et al. (1992). Light microscopy observations Chains are straight, robust and usually short, composed of 4–7 cells. Cells are elliptical in valve view and rectangular in girdle view with the pervalvar axis often shorter (8–15 μm) than the apical axis (14–26 μm). Each cell contains a single large chloroplast in the shape of a parietal plate extending from valve to valve (Figs. 2B–C). The valve face is fl at, sometimes with a central infl ation which is usually more visible on the terminal valves. No process was visible in either intercalary or terminal valves on > 30 observed chains. Valve mantle is low with a very slight constriction near the abvalvar margin. The girdle is usually equi-dimensional in height with the mantle. Valves of adjacent cells touch at the corners. Apertures are slit-shaped and very narrow, partially occluded by silica membranes which are sometimes visible in LM (not shown). Intercalary setae originate from the valve apices Fig. 1. Position of sampling site at Lake Vrana (Croatia). BOSAK S., GLIGORA UDOVIČ M., SARNO D. 238 ACTA BOT. CROAT. 74 (2), 2015 and cross immediately at chain margin, without basal part (Figs. 2B–C). Setae lack chloro- plasts. Intercalary setae are very long (up to 300 μm), straight, and stiff diverging in various directions, some perpendicular but more often they are oriented at a variable angle to the chain axis (Fig. 2A). In most cases sibling setae diverge from each other at an angle of 90° belonging to Brunel Group III (Fig. 2D), but in the same chain they can also diverge equal- ly from the apical plane at an angle of 20–30° conforming to Brunel Group II. Terminal se- tae have the same morphology as intercalary ones but they are oriented more parallel to the chain axis, diverging in a broad U- or V-shaped curve towards the end of the chain (Fig. 2A). Electron microscopy observations The valve is ornamented with very densely distributed anastomosing ribs spreading from an irregularly shaped hyaline area positioned slightly eccentrically on the valve face (Figs. 3B–C). The valve face is not perforated by poroids. The outer surface of the terminal valves is covered by minute spines (Fig. 3F). The process was not observed on ten interca- lary and terminal valves examined with EM (Figs. 3A–E). In all valves the marginal ridge between the valve face and the valve mantle is ornamented with a high hyaline rim (Fig. 3E). In intercalary valves, the aperture between sibling cells is partially occluded by silica membranes appearing to project from the hyaline rim (Fig. 3A). The setae are circular in Fig. 2. Light micrographs of Chaetoceros wighamii, strain PMF M1. A) The complete chain show- ing orientation of the setae. B) Cells with one parietal chloroplast per cell extending from valve to valve. C) Four-celled chain. D) Sibling valves in valve view with setae diverging from each other at an angle of 90°, belonging to Brunel Group III. Scale bars: A = 50 μm; B–D = 10 μm. THE MORPHOLOGY OF CHAETOCEROS WIGHAMII FROM LAKE VRANA ACTA BOT. CROAT. 74 (2), 2015 239 cross-section, 0.6–0.8 μm in diameter, composed of fl at, thick longitudinal fi laments ar- ranged in spiral pattern around the setae length and interconnected by very short transverse bars. The space between bars corresponds to a spiral row of minute poroids. The fi laments are ornamented with spines (3 per 1 μm) arranged in a helicoidal pattern around the setae. (Figs. 3G–H). Discussion A combination of morphological and ultrastructural characters allows for the ascription of the morphotype from Lake Vrana to C. wighamii Brightwell (syn. C. amanita) sensu SANCHEZ CASTILLO et al. (1992). The robust colonies have very long, stiff and straight inter- calary setae projecting in various directions from the chain axis and terminal setae oriented more parallel to the chain axis, diverging in a broad U or V- shaped curve towards the end of the chain. The apertures between sibling valves are partially occluded by a silica mem- brane which is sometimes visible in LM. The partial sealing of the apertures was consid- ered peculiar for C. wighamii syn. C. amanita (KACZMARSKA et al. 1985, RUSHFORTH and JOHANSEN 1986), but was not described in C. wighamii from Lake Chica (SANCHEZ CASTILLO et al. 1992) where apparently only terminal valves were observed by electron microscopy. A silica membrane partially occluding the apertures is not unique for C. wighamii as a simi- lar structure (silica wall) has been observed in other very different species such as C. de- cipiens (EVENSEN and HASLE 1975) and C. diversus (HERNÁNDEZ-BECERRIL 1996). Fig. 3. SEM (A, D, E, G) and TEM (B, C, F, H) micrographs of Chaetoceros wighamii, strain PMF M1. A) Two sibling valves with aperture partially occluded by silica. B) Two overlapped sibling intercalary valves showing the valve pattern with anastomosing ribs. C) Detail of the valve with the irregularly shaped hyaline eccentric area. D) Internal view of the terminal valve. E) Terminal valve in girdle view showing the hyaline rim extending from the marginal ridge (arrow). F) Detail of the terminal valve with minute spines and the structure probably corresponding to the girdle band. G) Detail of a seta with spirally positioned spines. H) De- tail of a seta. Scale bars: A, B, D, E = 5 μm; C, G, H = 1 μm; F = 0.5 μm. BOSAK S., GLIGORA UDOVIČ M., SARNO D. 240 ACTA BOT. CROAT. 74 (2), 2015 A rather distinctive feature in our material is the absence of any processes and central annulus in either intercalary or terminal valves. A process is present on terminal valves in most of the species in the subgenus Hyalochaete, and generally on every valve in the sub- genus Chaetoceros (EVENSEN and HASLE 1975, HERNÁNDEZ-BECERRIL 1996). The lack of pro- cesses was recognized as a common feature in several unicellular inland taxa which are nowadays considered as synonyms of C. muelleri var. subsalsum (Lemmermann) Johansen & Rushforth (REINKE 1984, JOHANSEN and RUSFORTH 1985) and was already reported in specimens from freshwater fi eld material of C. wighamii (KACZMARSKA et al. 1985 as C. amanita, SANCHEZ CASTILLO et al. 1992). KACZMARSKA et al. (1985) were not able to confi rm the presence of any process after examination of 200 valves but suggested that a rimopor- tula may be produced under some circumstances. Our observation in cultivated material, although based on a limited number of examined valves, constitutes a further evidence of the lack of the process in the freshwater specimens of C. wighamii. The presence of the central process bearing a long external tube was illustrated in cul- tures of C. wighamii from the Baltic Sea (syn. C. bottnicus, C. biconcavum, C. capsicum and C. fallax, JENSEN and MOESTRUP 1998). The projection is clearly visible in LM illustra- tions and sometimes is present in sibling cells, probably as a result of impending chain divi- sion with the process-bearing intercalary valves becoming terminal valves. The Baltic mor- photype appears rather similar to the croatian morphotype as regarding the very variable orientation of the setae, but looks different for the more delicate aspect and the wider aper- tures of the chains. As noted by SNOEIJS (1993), detailed studies on the species from the Baltic Sea are required to reveal if it corresponds to C. wighamii or to C. bottnicus, which was indeed considered as a synonym of C. wighamii by JENSEN and MOESTRUP (1998). The absence of a distinct annulus is not common among Hyalochaete species that gen- erally have a thickened annulus surrounding the hyaline area from which the dichotomous- ly branching ribs extend towards the valve margin (EVENSEN and HASLE 1975, HERNÁNDEZ- BECERRIL 1996). In C. wighamii, the valve face shows a peculiar ornamentation pattern with densely distributed ribs spreading from an irregularly shaped hyaline area. KACZMARSKA et al. (1985) described a similar pattern as »composed of anastomosing areas of light silifi ca- tion« probably referring to the hyaline area between the ribs. The lack of a clearly defi ned annulus was also observed in the freshwater species Chaetoceros muelleri var. subsalsum (JOHANSEN and RUSHFORTH 1985) but the ribs are thicker and do not diverge in such a dense branching pattern as in C. wighamii. The presence of small spines or protrusions on the outer valve surface in C. wighamii was already noticed in previous studies (KACZMARSKA et al. 1985 as C. amanita, SANCHEZ CASTILLO et al. 1992). The seta ultrastructure has been considered as useful taxonomic character used in spe- cies delineation in Chaetoceros since the fi rst EM studies were published (EVENSEN and HASLE 1975). This idea was recently developed further by LEE et al. (2014) who proposed the use of several setae characters as the identifi cation criteria in the subgenus Chaetoceros, which includes species with thick and chloroplasts containing setae. Chaetoceros wighamii belongs to the subgenus Hyalochaete which comprises species possessing relatively thin setae without chloroplasts. In C. wighamii, the setae are composed of long longitudinal fi la- ments, which are arranged in a spiral pattern around the seta axis and are connected by short transverse bars. The arrangement of longitudinal fi laments and transverse bars results in a reticulate pattern when setae are observed in TEM (KACZMARSKA et al. 1985). The fi la- ments are adorned with somewhat strong small spines in a helicoidal arrangement. The THE MORPHOLOGY OF CHAETOCEROS WIGHAMII FROM LAKE VRANA ACTA BOT. CROAT. 74 (2), 2015 241 same pattern was previously illustrated in C. wighamii (KACZMARSKA et al. 1985 as C. ama- nita, SANCHEZ CASTILLO et al. 1992). Generally, Hyalochaete species possess setae with spines, with the exception of C. vixvisibilis (HERNÁNDEZ-BECERRIL et al. 2010), but their size and pattern shows differences among species (HERNÁNDEZ-BECERRIL 1996, KOOISTRA et al. 2010). Spines adorning setae in C. wighamii are tightly arranged around the axis and appear slightly longer and more silicifi ed compared to the spines present in other species, as, for example, Chaetoceros curvisetus or C. lauderi (KOOISTRA et al. 2010, EVENSEN and HASLE 1975).The details on chloroplast number, shape and position have not been known previ- ously in Chaetoceros wighamii syn C. amanita, as the previous observations were based mostly on cleaned material (KACZMARSKA et al. 1985, SANCHEZ CASTILLO et al. 1992). Ac- cording to our observations on live material, Chaetoceros wighamii has one chloroplast, which is plate-like or slightly lobed, situated parietally within the cell but extending from valve to valve, differently from, for example, C. affi nis which has the single parietal plastid positioned around the girdle (RINES and HARGRAVES 1988). The apical length size was considered by CLEVE-EULER (1951) and later adopted by KACZMARSKA et al. (1985) as one of the characters considered on which their specimens were designated as C. amanita and not as C. wighamii. The authors compared their mea- surements with the data on apical length reported for marine morphotypes named C. wighamii by HUSTEDT (1930) and CUPP (1943) which range between 7 and 18 μm, these are considerably smaller than the C. wighamii syn C. amanita range. However, the morphotype illustrated and described by HUSTEDT (1930) is very different from the original description of C. wighamii, with characters such as delicate chains, thin setae and cells with drawn up poles, which probably correspond to a different species with marine preferences. The cell apical length measured in our study ranged between 14 and 26 μm, which is comparable to the range reported in other studies of C. wighamii syn. C. amanita: 18–40 μm, 15–20 μm, 19–31 μm, (KACZMARSKA et al. 1985, RINES and HARGRAVES 1988, SANCHEZ CASTILLO et al. 1992), respectively. One of the most distinctive characters of C. wighamii is a resting spore with primary valve covered with spines and secondary valve shaped as a truncated cone (BRIGHTWELL 1856). Unfortunately, the resting spore has not been observed in this study. However, rest- ing spores morphologically similar to those reported for C. wighamii by BRIGHTWELL (1856) and SANCHEZ CASTILLO et al. (1992) as well as by KACZMARSKA et al. (1985) and RINES and HARGRAVES (1988) as C. amanita, have been frequently found in the sediments from the Lake Vrana (Fig. 4, I. Galović, personal communication). The distinct cone-shaped secondary Fig. 4. The LM micrograph (photo by I. Galović) of a resting spore in Lake Vrana, having the spi- nose secondary valve distinctively shaped as a truncated cone considerably narrower in dia- meter than the primary valve. Scale bar = 10 μm. BOSAK S., GLIGORA UDOVIČ M., SARNO D. 242 ACTA BOT. CROAT. 74 (2), 2015 valve is a feature observed in some other freshwater Chaetoceros species, e.g. C. elmorei and C. muelleri, which however generally form smooth spores (RUSHFORTH and JOHANSEN 1986). The results of this study support the idea that the freshwater morphotypes of C. wighamii are distinct from the marine morphotypes. However, morphological and molecular studies on material from different geographical areas are required to defi nitely clarify if (1) C. wighamii is a very variable species which includes phenotypically different populations able to adapt to ecologically distinct habitats, or (2) different species have been identifi ed over time as C. wighamii. Future studies should also investigate phylogenetic relationships among different fresh- water Chaetoceros species which share similar distinctive features such as absence of pro- cess and related spore morphology. Conclusion Our observations on morphology of Chaetoceros wighamii isolated from Lake Vrana, Croatia agree well with the description reported for the freshwater morphotypes of C. wighamii (syn. C. amanita) and contribute to a better distinction of this taxon from similar species. C. wighamii is a freshwater/brackish species whereas the taxonomical affi liation of the marine synonyms should be further investigated. Additional species-specifi c ultrastruc- tural features are described, such as the particular valve ornamentation pattern and the posi- tion and shape of the chloroplast. Until now the morphotype corresponding to Chaetoceros wighamii (syn. C. amanita) including observations of both vegetative cells and resting spores has been reported from inland waters in USA (KACZMARSKA et al. 1985), Southern (SANCHEZ CASTILLO et al. 1992) and Northern Europe (BRIGHTWELL 1856). This report repre- sents the fi rst report of this species from Croatia and from Central European habitats. Acknowledgements Special thanks to Franco Iamunno (SZN, Naples, Italy) on his valuable help with elec- tron microscopy. The presented research was funded by the Ministry of Science, Education and Sport of the Republic of Croatia Projects No. 119-1191189-1228 and 119-0000000- 1229 and by the European Community – Research Infrastructure Action under the FP7 »Capacities« Specifi c Programme (Ref. ASSEMBLE grant agreement no. 227799). We are thankful to Dr. Ines Galović, Croatian Geological Institute, Zagreb, Croatia for the image of C. wighamii resting spore. References ANONYMOU S, 1975: Proposals for a standardization of diatom terminology and diagnoses. Nova Hedwigia Beiheft 53, 323–354. AURIVILL IUS, C., 1896: Das Plankton des Baltichen Meeres. Bihang till Kungliga Svenska Vetenskapsakademiens Handlingar 21 Heft 4, No. 8, 1–83. 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