OPCE-STR.vp Acta Bot. Croat. 68 (1), 11–27, 2009 CODEN: ABCRA 25 ISSN 0365–0588 Phytoplankton composition of the Ebro River estuary, Spain MARÍA DEL CARMEN PÉREZ1*, NORA I. MAIDANA2, AUGUSTO COMAS3 1 Universidad Politécnica de Valencia, Av. Naranjos s/n°, 46022 Valencia, España 2 Dpto. de Biodiversidad y Biología Experimental, Universidad de Buenos Aires, Ciudad Universitaria, Pab. II, 4° piso, C1428EHA Buenos Aires, Argentina 3 Centro de Estudios Ambientales, Apdo. 202, 55100 Cienfuegos, Cuba The composition of phytoplankton in the Ebro River estuary (Spain) was analyzed at six sampling stations in April, July and October 1999 and February 2000, based on plankton net and bottle samples. A total of 304 taxa belonging to 13 classes were identified. Bacillariophyceae and Chlorophyceae were the most important groups. The diatom gen- era Nitzschia and Navicula provided most species (25 and 17, respectively). Thalassiosira duostra is recorded for the first time for this ecosystem. We propose the following new combinations: Monactinus simplex (Meyen) Corda var. echinulatum (Wittrock) comb. nov. and Monactinus simplex (Meyen) Corda var. sturmii (Reinsch) comb. nov. Keywords: phytoplankton, estuary, taxonomy, Ebro, Spain. Introduction Estuaries are highly productive systems in which nutrients are supplied from the land (DYER 1973, MC LUSKY 1989). Phytoplankton study in the Ebro River was earlier concen- trated on seasonality (SABATER and MUÑOZ (1990), small Cyclotella species (SABATER and KLEE 1990), and Chlorophyceae (PÉREZ et al. 2002, COMAS et al. 2006). Research into phytoplankton has been performed in the Ebro delta (COMIN 1984) and near bays (LÓPEZ and ARTÉ 1973; DELGADO 1987; DELGADO et al. 1990, 1995, 1996). In the framework of the PIONEER European Project, seasonal collecting data cam- paigns were undertaken during 1999–2000 to understand the impact of nutrients in the lower Ebro River and in its plume. In this study we show the results of phytoplankton com- position at six collecting sites from April 1999 to February 2000. ACTA BOT. CROAT. 68 (1), 2009 11 * Corresponding author, e-mail: perez.baliero@gmail.com U:\ACTA BOTANICA\Acta-Botan 1-09\Perez.vp 22. travanj 2009 10:59:58 Color profile: Disabled Composite 150 lpi at 45 degrees Materials and methods Study area The Ebro River is 960 km long and is one of the biggest Spanish rivers discharging in the Mediterranean Sea (Fig. 1). The Ebro estuary is classified as a »salt wedge estuary« or »type 4« after Hansen-Rattray’s classification (IBAÑEZ et al. 1997). It has a strong and clearly marked halocline due to the low tidal range. Circulation is primarily affected by the river discharge. The annual discharge (424 m 3 s –1 mean) is smoothed by the presence of about 170 dams (IBAÑEZ et al. 1999). In particular, the Mequinenza and Riba-Roja dams, located 100 km upstream from the mouth, have an important regulatory effect over the discharge in the Delta. Furthermore, these dams condition the suspended inert solids concentration (usually low) and the phytoplankton populations of the water. In the Ebro delta, rice crops and cultivated fields alternate with coastal lagoons. The re- gion has a dry Mediterranean climate with a mean annual temperature around 16–17 °C, with around 550 mm of precipitation per year (MARTINEZ et al. 1999). Samplings Four sampling campaigns on the Ebro estuary, April, July and October 1999 and Febru- ary 2000, were performed within the framework of the European PIONEER Project. Elec- tric conductivity, temperature and pH were measured in situ with a multiparametric sound- ing Hydrolab Surveyor 3. Phytoplankton samples for qualitative analyses were collected 12 ACTA BOT. CROAT. 68 (1), 2009 PÉREZ M. C., MAIDANA N. I., COMAS A. 295000 300000 305000 310000 315000 320000 4495000 4500000 4505000 4510000 4515000 4520000 R1 R2R3 R4 R5 R6 Mediterranean Sea Spain Africa Fig. 1. Location of sampling stations in the Ebro Estuary. U:\ACTA BOTANICA\Acta-Botan 1-09\Perez.vp 22. travanj 2009 10:59:59 Color profile: Disabled Composite 150 lpi at 45 degrees with bottles at different depths trying to cover the whole water column and with 20μm mesh plankton net by horizontal tows in the centre of the stream at every sampling station (Fig. 1). All samples were fixed in situ with glutaraldehyde (2% final concentration) ac- cording to SOURNIA (1978), and they were deposited at the Laboratorio de Tecnologías del Medio Ambiente of the UPV, Valencia, Spain. Detailed examinations and drawings of the material were made with Leitz Laborlux and Nikon Optiphot light microscopes with phase contrast optics. Part of each sample was prepared according to conventional methodology for the qualitative study of diatoms (HASLE and FRYXELL 1970). Observations were made with a JEOL JSM 6300 scanning electron microscope (SEM). For cryo-SEM analysis a drop of concentrated sample was placed on an isopore membrane-filter GTTP Millipore. The sample was frozen with liquid N2, coated with gold and examined using a Jeol JSM-5410 scanning microscope equipped with cryo-station. According to new results based on cytological studies and gene sequence analysis, the traditional Chlorophyceae is divided into different classes (MELKONIAN 1983, MATTOX and STEWART 1984, GRAHAM and WILCOX 2000); but, not all nomenclatural changes have been published, and therefore we are still using Chlorophyceae in the traditional sense. We ac- cept new concept, the separation of Scenedesmus Meyen in two genera: Desmodesmus and Scenedesmus s.str. (AN et al. 1999, HEGEWALD 2000). According to HEGEWALD in BUCHHEIM et al. (2005) based on inferences from rDNA data, Pediastrum Meyen could be divided into 5 independent genera. We follow this con- cept in this paper, with the exception of P. willei (Fig. 6-a), because molecular genetic data about this taxon are not yet available. In the same way, the taxonomy of Bacillariophyceae at species, genus and higher levels, has been undergoing deep changes in the last decades (ROUND et al. 1990, MEDLIN and KACZMARSKA 2004, among others), introducing many changes and also leaving a high amount of incertitude that has to be settled, and therefore in this paper we use the criteria of ROUND et al. (1990). Results The water pH ranged between 7.86 and 8.55 (mean value 8.2), surface temperature be- tween 9.81 and 27.26 °C (mean value 18.5) and the conductivity between 928 and 9071 μS cm –1 (mean value 3777). The surface salinity was measured on July 12 th and October 5 th 1999 and varied between 2.3 and 5.1. Three hundred and four taxa included in 13 algal Classes were identified (Tab. 1, Fig. 2): Bacillariophyceae 175, Chlorophyceae 75, Chrysophyceae 2, Cryptophyceae 2, Cyano- bacteria 12, Dinophyceae 17, Dictyochophyceae 2, Ebriidae 1, Euglenophyceae 9, Prasino- phyceae 1, Prymnesiophyceae 2, Xanthophyceae 2 and Zygnemaphyceae 4. Considering the species numbers, Bacillariophyceae and Chlorophyceae were the most important classes with 57 % and 25 % of the recorded species, respectively (Fig. 2). The genera Navicula sensu stricto and Nitzschia presented the highest number of species, 25 and 17 species, respectively (Tab. 1). From the total of 149 observed genera, Pediastrum, Pseudo- pediastrum, Monactinus, Stauridium and Monoraphidium (Chlorophyceae), Cryptomonas ACTA BOT. CROAT. 68 (1), 2009 13 PHYTOPLANKTON OF THE EBRO ESTUARY U:\ACTA BOTANICA\Acta-Botan 1-09\Perez.vp 22. travanj 2009 11:00:00 Color profile: Disabled Composite 150 lpi at 45 degrees 14 ACTA BOT. CROAT. 68 (1), 2009 PÉREZ M. C., MAIDANA N. I., COMAS A. Tab. 1. List of algae species observed in the Ebro Estuary (1999–2000). * diatoms observed in only one sample. BACILLARIOPHYCEAE Achnanthes cf. amoena Hustedt Achnanthidium minutissimum (Kützing) Czarnecki Achnanthidium sp. Actinocyclus normanii (Gregory) Hustedt Amphora copulata (Kützing) Schoeman et Archibald A. cf. normanii Rabenhorst A. ovalis (Kützing) Kützing A. pediculus (Kützing) Grunow in Schmidt et al. A. veneta Kützing Asterionella formosa Hassall Asterionellopsis glacialis (Castracane) F.E. Round Aulacoseira ambigua (Grunow) Simonsen A. granulata var. granulata (Ehrenberg) Simonsen A. granulata var. angustissima (Otto Müller) Simonsen A. cf. nyassensis (Otto Müller) Simonsen Bacillaria paradoxa Gmelin in Linneaeus Bacteriastrum cf. elongatum Cleve B. delicatulum Cleve B. hyalinum Lauder Caloneis amphisbaena (Bory) Cleve *C. bacillum (Grunow) Cleve Cerataulina cf. pelagica (Cleve) Hendey Chaetoceros cf. laciniosus Schütt Ch. cf. pseudocurvisetus Mangin Ch. didymus Ehrenberg Ch. muellerii Lemmermann Chaetoceros sp. Cocconeis pediculus Ehrenberg C. placentula var. euglypta (Ehrenberg) Grunow C. placentula var. lineata (Ehrenberg) Van Heurck *C. scutellum Ehrenberg Cocconeis sp. Corethron criophyllum Castracane Craticula cuspidata (Kützing) Mann in Round, Crawford et Mann *C. molestiformis (F. Hustedt) S. Mayama Ctenophora pulchella (Ralfs ex Kuetzing) Williams et Round Cyclostephanos dubius (Fricke) Round C. invisitatus (Hohn et Hellerman) Theriot, Stoermer et Hakansson Cyclotella atomus Hustedt C. choctawhatcheeana Prasad C. meduanae Germain C. meneghiniana Kützing C. ocellata Pantocsek *C. aff. polymorpha Meyer et Håkansson C. rossii Håkansson Cylindrotheca closterium (Ehr.) Reimann et Lewin *Cymatopleura elliptica (Brebisson ex Kutzing) W. Smith C. solea (Brebisson in Brebisson et Godey) W. Smith Cymbella helvetica Kützing C. tumida (Brébisson in Kützing) Van Heurck Diadesmis confervacea Kützing Diatoma moniliformis Kützing D. tenue Agardh D. vulgaris Bory Diatoma sp. Discostella glomerata (Bachmann) Houk et Klee D. stelligeroides (Hustedt) Houk et Klee D. wolterecki (Hustedt) Houk et Klee Ditylum brightwellii (T. West) Grunow Encyonema minutum (Hilse in Rabenhorst) Mann in Round, Crawford et Mann E. prostratum (Berk.) Kütz. *Encyonopsis microcephalum (Grunow) Krammer *Entomoneis paludosa (W. Smith) Reimer in Patrick et Reimer Eolimna minima (Grunow in Van Heurck) H. Lange-Bertalot in G. Moser, H. Lange-Bertalot et D. Metzeltin E. subminuscula (Manguin) G. Moser, H. Lange-Bertalot et D. Metzeltin Eucampia zodiacus Ehrenberg U:\ACTA BOTANICA\Acta-Botan 1-09\Perez.vp 16. travanj 2009 8:41:19 Color profile: Disabled Composite 150 lpi at 45 degrees ACTA BOT. CROAT. 68 (1), 2009 15 PHYTOPLANKTON OF THE EBRO ESTUARY Fallacia cf. insociabilis (Krasske) Mann in Round, Crawford et Mann *F. subhamulata (Grunow in Van Heurck) Mann in Round, Crawford et Mann Fistulifera pelliculosa (Brebisson) Lange-Bertalot Fragilaria capucina Desmaziéres F. cf. constricta Ehrenberg F. crotonensis Kitton Gomphonema olivaceum (Hornemann) Ehrenberg G. parvulum (Kützing) Kützing Gomphonema sp. Gomphosphenia lingulatiformis (H. Lange-Bertalot et E. Reichardt) H. Lange-Bertalot (=Gomphonema lingulatiforme) Guinardia striata (Stolterfoth) Hasle Gyrosigma acuminatum (Kützing) Rabenhorst G. attenuatum (Kützing) Cleve G. sciotense (Sulliv. et Wormley) Cl. KS: Curtis Hemiaulus sinensis Greville Karayevia clevei (Grunow in Cleve et Grunow) Round et Bukhtiyarova Leptocylindrus danicus Cleve L. minimus Gran Lithodesmium cf. undulatum Ehrenberg Luticola goeppertiana (Bleisch in Rabenhorst) Mann in Round, Crawford et Mann L. mutica var. ventricosa (Kützing) Hamilton in Hamilton et al. *L. nivalis (Ehrenberg) Mann in Round, Crawford et Mann Melosira varians C. A. Agardh Navicula antonii Lange-Bertalot in Rumrich et al. (=N. menisculus var. grunowii) N. capitatoradiata Germain N. cryptotenella Lange-Bertalot N. erifuga Lange-Bertalot N. gregaria Donkin N. cf. hambergii Hustedt N. lanceolata (Agardh) Kützing N. phyllepta Kützing N. recens (Lange-Bertalot) Lange-Bertalot N. rhynchocephala Kützing N. rostellata Kützing *N. salinarum Grunow *N. schroeteri Meister N. tripunctata (O. F. Müller) Bory N. veneta Kützing N. viridula (Kützing) Ehrenberg Navicula sp. Nitzschia acicularis (Kützing) W. Smith N. amphibia Grunow N. angustatula Lange-Bertalot *N. brevissima Grunow N. cf. capitellata Hustedt N. cf. rautenbachiae Cholnocky N. constricta (Gregory) Grunow N. dissipata (Kützing) Grunow *N. dubia W. Smith N. filiformis (W. Smith) Van Heurck *N. fonticola Grunow N. frustulum (Kützing) Grunow N. gracilis Hantzsch N. inconspicua Grunow *N. intermedia Hantzsch N. linearis (Agardh) W. Smith N. longisima var reversa Grunow N. microcephala Grunow N. palea (Kützing) W. Smith N. recta Hantzsch *N. sigma (Kützing) W. Smith N. sigmoidea (Nitzsch) W. Smith *N. sinuata var. tabellaria (Grunow) Grunow N. sublinearis Hustedt Nitzschia sp. Odontella mobiliensis Bailey *Planothidium ellipticum (Cleve) Round et Bukhtiyarova P. rostratum (Østrup) Round et Bukhtiyarova Pleurosigma sp. Pleurosira laevis (Ehrenberg) Compère Proboscia alata (Brightwell) Sundström Psammothidium subatomoides (Hustedt) Bukhtiyarova et Round Pseudo-nitzschia cf. pseudodelicatissima (Hasle) Hasle Tab. 1. – continued U:\ACTA BOTANICA\Acta-Botan 1-09\Perez.vp 16. travanj 2009 8:41:19 Color profile: Disabled Composite 150 lpi at 45 degrees 16 ACTA BOT. CROAT. 68 (1), 2009 PÉREZ M. C., MAIDANA N. I., COMAS A. Pseudo-nitzschia sp. Pseudosolenia calcar-avis (Schultze) Sundstrom Pseudostaurosira brevistriata (Grunow in Van Heurck) Williams et Round Puncticulata bodanica (Grunow in Schneider) Håkansson *P. radiosa (Lemmermann) Håkansson Reimeria uniseriata Sala, Guerrero et Ferrario Rhizosolenia sp. Rhoicosphaenia abbreviata (C. Agardh) Lange-Bertalot Sellaphora pupula (Kützing) Mereschkowsky Seminavis cf. gracilenta (A. Grunow ex A. Schmidt) DG Mann Skeletonema cf..costatum (Grev.) Cleve S. potamos (Weber) Hasle Staurosira construens var. subsalina (Hustedt) Bukhtiyarova S. construens var. venter (Ehrenb.) P. B. Hamilton in Hamilton, Poulin, Charles et Angell Stephanodiscus hantzschii Grunow S. hantzschii Morphotype »tenuis« (Hustedt) Håkansson et Stoermer S. parvus Stoermer et Håkansson S. rotula (Kützing) Hendey Surirella brebissonii var. kuetzingii Krammer et Lange-Bertalot *S. linearis var. helvetica (Brun) F. Meister *S. minuta (Brun) F. Meister Tabularia fasciculata (C. Agardh) D. M. Williams et Round T. cf. tabulata (C. A. Agardh) Snoeijs Thalassionema nitzschioides (Grunow) Mereschkowsky Thalassiosira baltica (Grunow in Cleve et Grunow) Ostenfeld T. duostra Pienaar in Pienaar et Pieterse T. cf. faurii (Gasse) Hasle T. pseudonana Hasle et Heimdal T. cf. visurgis Hustedt T. weissflogii (Grunow) G. Fryxell et Hasle Thalassiosira sp. 1 Thalassiosira sp. 2 Tryblionella hungarica (Grunow) D. G. Mann *T. suevica Grunow (=N. levidensis var. salinarum Grunow) Ulnaria acus (Kützing) Aboal U. ulna (Nitzsch) P. Compère CHLOROPHYCEAE s. l. Chlorococcales s. l. Actinastrum hantzschii var. hantzschii Lagerh. A. hantzschii var. subtile Wolosz. Ankyra ancora (G.M. Smith) Fott Botryococcus cf. braunii Kütz. Chlorotetraedron incus (Teil.) Kom. et Kovac. Closteriopsis longissima (Lemm.) Lemm. Coelastrum indicum Turn. C. microporum Näg. C. pseudomicroporum Kors. C. reticulatum var. polychordum Kors. Coenococcus fottii Hind. Crucigenia smithii (Bourr. et Manguin) Kom. C. tetrapedia (Kirchn.) W. et G.S. West Crucigeniella cf. pulchra (W. et G.S. West) Kom. Desmodesmus abundans (Kirchn.) Hegew. D. armatus s.l. (Chod.) Hegew. D. brasiliensis (Bohl.) Hegew. D. communis (Hegew.) Hegew. D. denticulatus var. fenestratus (Teil) Hegew. D. intermedius (Chod.) Hegew. D. maximus (Meyen) Hegew. D. opoliensis (Richter) An et al. D. pannonicus (Hortob.) Hegew. D. spinosus (Chod.) Hegew. Dictyosphaerium ehrenbergianum Näg. D. tetrachotomum Printz Dictyosphaerium sp. Golenkinia radiata Chod. Kirchneriella obesa (W. West) Schmidle Lagerheimia ciliata (Lagerh.) Chod. L. subsalsa Lemm. Micractinium pusillum Fres. Monactinus simplex var. simplex (Meyen) Corda M. simplex var. sturmii (Reinsch) comb. nov. Tab. 1. – continued U:\ACTA BOTANICA\Acta-Botan 1-09\Perez.vp 16. travanj 2009 8:41:19 Color profile: Disabled Composite 150 lpi at 45 degrees ACTA BOT. CROAT. 68 (1), 2009 17 PHYTOPLANKTON OF THE EBRO ESTUARY M. simplex var. echinulatun (Wittr.) comb. nov. Monoraphidium arcuatum (Kors.) Hind. M. contortum (Thur.) Kom-Legn. M. griffithii (Berk.) Kom-Legn. Nephrocytium schilleri (Kamm.) Com. Oocystidium ovale Kors. Oocystis lacustris Chod. O. marssonii Lemm. Pediastrum duplex var. duplex Meyen Pediastrum willei Comas et al. Planktosphaeria gelatinosa G.M. Smith Pseudopediastrum boryanum var. boryanum (Turp.) Hegew. Pseudoschroederia antillarum (Kom.) Hegew. et Schnepf P. robusta (Kors.) Hegew. et Schnepf Raphidocelis contorta (Schmidle) Marvan et al. Scenedesmus acuminatus (Lagerh.) Chod. S. arcuatus (Lemm.) Lemm. S. ellipticus Corda S. obliquus var. dimorphus (Turp.) Hansg. S. obtusus Meyen Schroederia setigera (Schröd.) Lemm. Selenastrum bibraianum Reinsch. S. gracile Reinsch. Siderocelis ornata (Fott) Fott Stauridium tetras (Ehrenb.) Hegew. Tetrachlorella alternans (G.M. Smith) Kors. Tetraedron caudatum (Corda) Hansg. Tetrastrum komarekii Hind. T. staurogeniaeforme (Schröd.) Lemm. Treubaria triappendiculata Bern. Westella botryoides De Wild O. Ulotrichales s. l. Elakatothrix genevensis (Reverdin) Hind. E. subacuta Kor{. Klebsormidium subtile (Kütz.) Silva et al. Koliella cf. spiculiformis (Vischer) Hind. Planctonema lauterbornii Schmidle O. Volvocales Chlamydomonas sp. Eudorina elegans Ehrenb. Gonium pectorale O. F. Müller Pandorina morum (Müller) Bory Pseudoscourfieldia marina (Throndsen) Manton CHRYSOPHYCEAE Dinobryon sp. Mallomonas sp. CRYPTOPHYCEAE Cryptomonas sp. Rhodomonas sp. CYANOPHYCEAE Aphanizomenon flos-aquae (L.) Ralfs ex Bornet et Flahault Coelomoron pusillum (Van Goor) Kom. Chroococcus limneticus Lemm. C. cf. microscopicus Kom-Legn. et Cronberg Cyanobium sp. Geitlerinema splendidum (Grez. ex Gom.) Anagn. et Kom. Merismopedia sp. Microcystis aeruginosa Kütz. Planktolyngbya cf. brevicellularis Cronberg et Kom. Planktothrix isothrix (Skuja) Kom. et Komark. Pseudanabaena sp. Spirulina major Kütz. ex Gom. DINOPHYCEAE Ceratium candelabrum (Ehrenb.) Stein C. furca (Ehrenb.) Claparède et Lachmann C. fusus (Ehrenb.) Duj. C. hirundinella (O.F. Müller) Schrank C. pentagonum Gourr. C. tripos (O.F. Müller) Nitzsch Dinophysis sacculus Stein Dinophysis sp. Gymnodinium sp. Peridinium sp. Podolampas sp. Prorocentrum micans Ehrenb. Prorocentrum sp. 1 Protoperidinium cf. conicum (Gran) Balech Tab. 1. – continued U:\ACTA BOTANICA\Acta-Botan 1-09\Perez.vp 22. travanj 2009 11:00:00 Color profile: Disabled Composite 150 lpi at 45 degrees (Cryptophyceae), and Cyclotella (Bacillariophyceae) (Figs. 3: a–d) were very frequent in our samples. We identified 175 diatoms belonging to 69 genera (25 centrics and 44 pennates). Centric diatoms (Figs. 3, 4, 5) were less numerous than pennate. A high percentage of the identified species (82.3%) are known from non-marine habitats and only 34.8% of diatoms were planktonic. Twelve species: Amphora pediculus, Bacillaria paradoxa, Cocconeis placentula var. euglypta, Cyclostephanos dubius (Fig. 4-a), Cyclotella atomus, C. meduanae (Figs. 3: a–b), C. meneghiniana, Nitzschia palea, Skeletonema potamos (Fig. 4-b), Stephanodiscus rotula (Fig. 4-e), Thalassiosira weissflogii (Fig. 5-b) and Ulnaria ulna were present in all the stud- 18 ACTA BOT. CROAT. 68 (1), 2009 PÉREZ M. C., MAIDANA N. I., COMAS A. P. cf. depressum (Bailey) Balech Protoperidinium sp. 1 Scrippsiella trochoidea (Stein) Balech DICTYOCHOPHYCEAE Dictyocha crux Ehrenb. D. fibula Ehrenb. EBRIIDEA Hermesinum adriaticum Zacharias EUGLENOPHYCEAE Euglena spirogyra Ehrenb. E. tripteris (Duj.) Klebs Euglena sp. Eutreptiella sp. Lepocinclis texta (Duj.) Lemm. emend. Conrad Lepocinclis sp. Phacus acuminatus Stokes P. tortus Lemm. Trachelomonas sp. PRASINOPHYCEAE Pyramimonas sp. PRYMNESIOPHYCEAE Periphyllophora mirabilis (Schiller) Kamptner Syracosphaera sp. XANTHOPHYCEAE Goniochloris cf. muticum (A. Braun) Fott Pseudostaurastrum hastatum (Reinsch) Chod. ZYGNEMAPHYCEAE Closterium sp. Cosmarium sp. Staurastrum gracile var. gracile Ralfs S. smithii (G.M. Smith) Teiling Tab. 1. – continued BACILLARIOPHYCEAE 57.6% CYANOBACTERIA 3.9% DINOPHYCEAE 5.6% CHLOROPHYCEAE 24.7% EUGLENOPHYCEAE 3% ZYGNEMAPHYCEAE 1.3% OTHER CLASSES 3.9% Fig. 2. Relative contribution of species among classes to total number of phytoplankton species in the Ebro Estuary (1999–2000). U:\ACTA BOTANICA\Acta-Botan 1-09\Perez.vp 22. travanj 2009 11:00:00 Color profile: Disabled Composite 150 lpi at 45 degrees ied samples, while 22 diatoms were found in only one sample during the studied period (Tab. 1). Thalassiosira duostra (Figs. 5 c–f) is recorded for the first time in this ecosystem. The present paper reports four new Chlorococcales s.l. and three Ulotrichales s.l. The new findings are: Botryococcus cf. braunii, Lagerheimia ciliata, Lagerheimia subsalsa, Westella botryoides and the Ulotrichales s.l: Planctonema lauterbornii, Klebsormidium subtile and Koliella cf. spiculiformis. We found two different varieties of Pediastrum sim- ACTA BOT. CROAT. 68 (1), 2009 19 PHYTOPLANKTON OF THE EBRO ESTUARY Fig. 3. SEM micrographs of Cyclotella and Discostella. a–b – Cyclotella meduanae (a – external view, b – internal view), c – C. rossii, d – C. aff. polymorpha, e – Discostella pseudo- stelligera, f – D. woltereckii U:\ACTA BOTANICA\Acta-Botan 1-09\Perez.vp 16. travanj 2009 8:41:21 Color profile: Disabled Composite 150 lpi at 45 degrees plex (var. sturmii and var. echinulatum), which should be transferred to Monactinus sim- plex (Meyen) Corda (Chlorococcales s.l.) Due to Cyanobacteria, a population probably belonging to Cyanobium was observed. This species was a representative of the picophytoplankton fraction for the whole of the study period. Among Dinophyceae, several Ceratium and Protoperidinium marine species were ob- served near the river mouth (stations 1 and 2, Fig. 1). With respect to potentially harmful species, during this period were observed the dinoflagellates Dinophysis sacculus and Prorocentrum cf. micans (Fig. 6 b–c) in the samples closest to the sea. 20 ACTA BOT. CROAT. 68 (1), 2009 PÉREZ M. C., MAIDANA N. I., COMAS A. Fig. 4. SEM micrographs of Cyclostephanos, Skeletonema and Stephanodiscus. a – Cycloste- phanos dubius, b – Skeletonema potamos, c – Stephanodiscus hantzschii Morphotype »hantzschii«, d – St. parvus, e – St. rotula, f – St. hantzschii Morphotype »tenuis« U:\ACTA BOTANICA\Acta-Botan 1-09\Perez.vp 16. travanj 2009 8:41:26 Color profile: Disabled Composite 150 lpi at 45 degrees Discussion The taxonomic inventory and quantitative description of organisms are important pre- requisites for understanding the status and functioning of ecosystems (JOHN 1994). The high species richness of Chlorophyceae and Bacillariophyceae in the lower Ebro River (75 and 175 taxa respectively) may be interpreted as typical of freshwater ecosystems. When compared to other regional ecosystems, the species richness of Chlorophyceae in the Ebro estuary is evident (SABATER 1990, MONTESANTO et al. 2000). ACTA BOT. CROAT. 68 (1), 2009 21 PHYTOPLANKTON OF THE EBRO ESTUARY Fig. 5. SEM micrographs of Thalassiosira. a – Thalassiosira pseudonana, b – T. weissflogii, c – f – T. duostra (c–e – external views with different arrangements of the central groups of fulto- portulae, f – internal view) U:\ACTA BOTANICA\Acta-Botan 1-09\Perez.vp 16. travanj 2009 8:41:31 Color profile: Disabled Composite 150 lpi at 45 degrees With respect to Chlorococcales, this in its present concept comprises only the family Chlorococcaceae and is transferred to Chlamydophyceae (ETTL and GÄRTNER 1988), how- ever several other genera have been replaced in Sphaeropleales (HEGEWALD and HANAGATA 2000) or in the quite different class of Trebouxiophyceae (FRIEDL 1995, HEPPERLE et al. 2000, WOLF et al. 2002). Monactinus simplex (Meyen) Corda (= Pediastrum simplex Meyen) is more widely dis- tributed in warm regions, but in recent years the area is a little more enlarged. According to the last monograph on the genus Pediastrum (KOMÁREK and JANKOVSKÁ 2001) few taxo- nomical varieties were accepted, p.i. the vars. echinulatum Wittr and sturmii (Reinsch) Wolle. In this study both taxa are very well represented, and, since they have not been yet transferred to Monactinus we propose here the following new combinations: Monactinus simplex (Meyen) Corda var. echinulatum (Wittrock) comb. nov. (Basionym: Pediastrum simplex var. echinulatum Wittrock in Wittrock, V. et Nordsted, O., Alg. Aq. Dulc. Exsicc. Praec. Scand. Fasc. 5, No. 235, 1833). A variety characterized by cell walls ornamented by prominent granules and coenobia usually with small and irreg- ular holes (Fig. 7a). Monactinus simplex (Meyen) Corda var. sturmii (Reinsch) comb. nov. (Basionym: Pediastrum sturmii Reinsch, Die Alg. Fl. mittl. Theil. Frank., p. 90, Tab. 7: 1, 1867.) is characterized by the lateral sides of lobes in old coenobia distinctly convex and cell wall with very fine and regular granulations (Fig. 7-b). 22 ACTA BOT. CROAT. 68 (1), 2009 PÉREZ M. C., MAIDANA N. I., COMAS A. Fig. 6. SEM micrographs Pediastrum, Dinophysis and Prorocentrum. a – Pediastrum willei, b – Dinophysis sacculus, c – Prorocentrum cf. micans U:\ACTA BOTANICA\Acta-Botan 1-09\Perez.vp 22. travanj 2009 11:00:12 Color profile: Disabled Composite 150 lpi at 45 degrees The genera Scenedesmus and Desmodesmus are represented by 5 and 11 species, re- spectively, being two of the richest genera (PÉREZ et al. 2002), resembling the situation in the Danube River (SCHMIDT et al. 1994, KISS and SCHMIDT 1998). On the other hand, the number of Chlorophyceae found in our study is higher (71 taxa) in comparison with the number found by SABATER and MUÑOZ (1990) between 1986 and 1987 in four sites located along the last 60 km of the river Ebro (41 taxa). It could indicate a higher trophic level. In our results, as compared with those of SABATER and MUÑOZ (1990), 18 Chloro- phyceae species were not observed in our samples. These authors observed that diatoms and green algae were the most abundant algae. They concluded that the phytoplankton dy- namics of the Ebro River is based upon two major factors: water flow and water salinity. MONTESANTO et al. (2000) recorded a total of 122 taxa of suspended algae in the Aliakmon River in Greece between February 1995 and January 1996. These authors ob- served that the two most important groups were Chlorophyta with 43.4% of total species number (53 representatives) and Bacillariophyceae with 19.7% containing a great number of tychoplanktonic species either from the phytobenthos or the littoral zone. On the other hand, VILI^I] et al. (2000) observed that the marine diatoms and dinoflagellates provided the dominant phytoplankton groups in the eastern Adriatic Zrmanja River Estuary (Croatia). As was expected, diatoms were represented by more non-marine (80%) than marine (20%) taxa. Of the non marine diatoms, pennates were well represented with a high number of species (124) while centrics had the lower species numbers (16). On the other hand, ma- rine diatoms were represented by similar numbers of centrics and pennates (20 and 15, re- spectively). Among centrics, Cyclotella species are not easy to distinguish from each other due to their high degree of polymorphism (HÅKANSSON 2002, RIVERA et al. 2003) and the possible convergence of some ultrastructural features and they were usually included in species complex such is the case of some taxa recorded for the Ebro River. Cyclotella rossii (Fig. 3-c) was present in all sampling sites along the study period. It clearly differs from C. ocellata and other related species due to the regularly aligned series of pores at the central part of the valves and by the marginal striae with similar length. ACTA BOT. CROAT. 68 (1), 2009 23 PHYTOPLANKTON OF THE EBRO ESTUARY Fig. 7. Light microphotographs of Monactinus simplex. a – Monactinus simplex var. echinulatum, b – Monactinus simplex var. sturmii. U:\ACTA BOTANICA\Acta-Botan 1-09\Perez.vp 22. travanj 2009 11:00:12 Color profile: Disabled Composite 150 lpi at 45 degrees Cyclotella aff. polymorpha (Fig. 3-d) was found only in station R5 and was represented by only few valves that could be distinguished from C. rossii by the irregular length of the marginal striae and from C. ocellata mainly by the radially oriented rows of puncta or orbiculi in the central part of the valve. Although C. ocellata provides a variable Cyclotella ocellata-krammeri-rossii complex (KNIE and HÜBENER 2007), the differentiating features of C. rossii is that the valves are the regular length of the marginal striae. We agree with KNIE and HÜBENER (2007) and GENKAL and POPOVSKAYA (2008) about the need to perform further investigations into the autoecology as well as DNA sequence analyses to clarify the taxonomic status of this species, also including C. polymorpha in this group of taxa. Stephanodiscus rotula (Fig. 4-e) and S. parvus (Fig. 4-d) were present in all the sampling stations. S. rotula should not be confused with other related species, such as S. alpinus be- cause of the valves have the central fultoportulae in heterotopic position (according to HÅKANSSON 2002). On the other hand, S. parvus (Fig. 4-d) differs from the very closely re- lated S. minutulus because it always has the single central fultoportula in an isotopic position. Remarkable in the Ebro River samples was the presence of Thalassiosira duostra (Fig. 5: c–f), a diatom with a scarcely known geographical distribution. It was mentioned for South Africa (the type locality), and found in some European rivers and ponds and in a res- ervoir in Brazil (PIENAAR and PIETERSE 1990, TORGAN et al. 2004). The Ebro material agrees in every respect with the morphological characteristics of the species. The higher relative abundance of benthic diatoms in February 2000 was probably re- lated to rainfall occurring in the catchment area during the previous days to sampling. SABATER (1990) observed a great influence of diatom benthic species in the phytoplankton of the medium-sized Mediterranean river the Ter (Spain). Previous studies dealing with the phytoplankton composition in the Ebro River, re- ported 59 freshwater diatoms (SABATER and KLEE 1990, SABATER and MUÑOZ 1990), which is lower number in comparison to 137 freshwater diatoms identified in this study. The absence of marine taxa in those previous studies could be explained by the differ- ences in sampling methodologies, i.e. the sub-superficial water samples in SABATER and KLEE (1990) and in SABATER and MUÑOZ (1990) as against the. covering of all the water col- umn in this study including the salt wedge. The picocyanobacteria were represented by an important population of Cyanobium sp. and its distribution and abundance were studied in PEREZ and CARRILLO (2005). With respect to potentially harmful dinoflagellates, we observed two interesting spe- cies: Dinophysis sacculuss (Fig. 6-b), recorded as toxic in the Mediterranean Sea by ZINGONE et al. (1998, 2006), and Prorocentrum cf. micans (Fig. 6-c), a very common spe- cies in coastal and estuarine waters (STEIDINGER and TANGEN 1996). Ebridian flagellates have only two extant species, Ebria tripartita and Hermesinum adriaticum, and this latter was observed in our study (Tab. 1) in one sample from the river mouth. This flagellate spe- cies is heterotrophic and possibly mixotrophic and is restricted to warmer waters (HAR- GRAVES 2002). Acknowledgemens The authors are greatly indebted to Prof. Dr. F. Round of the University of Bristol for his valuable comments about some diatom species, to M. Rodilla, S. Falco, I. Romero and R. Martínez for their technical assistance with sampling and to the technicians of the Elec- 24 ACTA BOT. CROAT. 68 (1), 2009 PÉREZ M. C., MAIDANA N. I., COMAS A. U:\ACTA BOTANICA\Acta-Botan 1-09\Perez.vp 22. travanj 2009 11:00:13 Color profile: Disabled Composite 150 lpi at 45 degrees tronic Microscopy Service of the UPV (Valencia) for their assistance with SEM images. We thank the reviewers, who improved the quality of the manuscript. This project has been financed by European community MAS3-CT98-0170 (DG12-VOMA): PIONEER »Prep- aration and integration of analysis tools towards operational forecast of nutrients in estu- aries of European rivers«. References AN, S. S., FRIEDL, T., HEGEWALD, E., 1999: Phylogenetic relationship of Scenedesmus and Scenedesmus-like coccoid green algae as inferred from ITS-2 rDNA sequence compari- sons. Plant Biology 1, 1–11. BUCHHEIM, M., BUCHHEIM, J., CARLSON, T., BRABAND, A., HEPPERLE, D., WOLF, M., HEGEWALD, E., 2005: Phylogeny of the Hydrodictyaceae (Chlorophyceae) inferences from rDNA data. Journal of Phycology 41, 1039–1054. COMAS GONZÁLEZ A., PÉREZ BALIERO, M. C., GONZÁLEZ DEL RÍO RAMS, J., 2006: Pedia- strum willei nom. et sp. nov. (Chlorophyta, Neochloridales) from the Ebro river (Spain) and its relations to P. muticum Kütz. sensu Brunnthaler 1915 pro parte. Algological Studies 120, 5–13. COMIN, F. A., 1984: Características físicas y químicas y fitoplancton de las lagunas costeras, Buda, Encañizada y Tancada (Delta del Ebro). Oecologia Aquatica 7, 79–162. DELGADO, M., 1987: Fitoplancton de las bahías del delta del Ebro. Investigación Pesquera 51, 517–548. DELGADO, M., ESTRADA, M., CAMP, J., FERNÁNDEZ, J. V., SANTMARTI, M., LLETI, C., 1990: Development of a toxic Alexandrium minutum Halim (Dinophyceae) bloom in the har- bour of Sant Carles de la Ràpita (Ebro Delta, northwestern Mediterranean). Scientia Marina 54, 1–7. DELGADO, M., FERNÁNDEZ, J. V., GARCÉS, E., MATAMOROS, E., CAMP, J., 1995: Prolife- ración de un dinoflagelado del género Gyrodinium en la bahía de Alfacs (Delta del Ebro) asociado a mortandad de peces. Proceedings of the 5 National Congress of Aqua- culture, Sant Carles de la Ràpita, 700–704. DELGADO, M., GARCÉS, E., CAMP, J., 1996: Growth and behaviour of Dinophysis sacculus from NW Mediterranean. In: YASUMOTO, T., OSHIMA, Y., FUKUYO, Y. (eds.), Harmful and toxic algal blooms, 261–264. IOC-UNESCO, Sendai, Japan. DYER, K., 1973: Estuaries: a physical introduction. John Wiley, London. ETTL, H., GÄRTNER, G., 1988: Chlorophyta II. Tetrasporales, Chlorococcales, Gloeoden- drales. In: ETTL, H., GERLOFF, J., HEYNIG, H., MOLLENHAUER, D. (eds.), Süsswasser- flora von Mitteleuropa, 10. G. Fischer, Stuttgart. FRIEDL, T., 1995: Inferring taxonomic positions and testing genus level assignments in coccoid green lichen algae: a phylogentic analysis of 18 S ribosomal RNA sequences from Dictyochloropsis reticulata and from members of the genus Myrmecia (Chloro- phyta, Trebouxiophyceae cl. nov.). Journal of Phycology 31, 632–639. GENKAL, S. I., POPOVSKAYA, G. I., 2008. Morphological variability of Cyclotella ocellata from Lake Khubsugul (Mongolia). Diatom Research 23, 75–91. ACTA BOT. CROAT. 68 (1), 2009 25 PHYTOPLANKTON OF THE EBRO ESTUARY U:\ACTA BOTANICA\Acta-Botan 1-09\Perez.vp 22. travanj 2009 11:00:13 Color profile: Disabled Composite 150 lpi at 45 degrees GRAHAM, L. E., WILCOX, L. W., 2000: Algae. Prentice Hall Inc. HÅKANSSON, H. 2002. A compilation and evaluation of species in the general Stephano- discus, Cyclostephanos and Cyclotella with a new genus in the family Stephano- discaceae. Diatom Research 17, 1–139. HARGRAVES, P. E., 2002: The ebridian flagellates Ebria and Hermesinum. Plankton Biol- ogy and Ecology 49, 9–16. HASLE, G. R., FRYXELL, G. A., 1970: Diatoms: cleaning and mounting for the light and electron microscopy. Transactions of the American Microscopy Society 86, 469–474. HEGEWALD, E., 2000: New combinations in the genus Desmodesmus (Chlorophyceae, Scenedesmaceae). Algological Studies 96, 1–18. HEGEWALD, E., HANAGATA, N., 2000: Phylogenetic studies on Scenedesmaceae (Chloro- phyta). Algological Studies 100, 29–49. HEPPERLE, D., HEGEWALD, E., KRIENITZ, L., 2000: Phylogenetic position of the Oocy- staceae (Chlorophyta). Journal of Phycology 36, 590–595. IBAÑEZ, C., PONT, D., PRAT, N., 1997: Characterization of the Ebro and Rhone estuaries – a basis for defining and classifying salt wedge estuaries. Limnology and Oceanography 42, 89–101. IBAÑEZ, C., PRAT, N., CANICIO, A., CURCÖ, A., 1999: El delta del Ebro, un sistema amenazado. Bakeaz, Bilbao. JOHN, D. M., 1994: Biodiversity and conservation: an algal perspective. The Phycologist 38, 3–15. KISS, K. T., SCHMIDT, A., 1998. Changes of the Chlorophyta species in the phytoplankton of the Hungarian section of the Danube River during the last decades (1961–1997). Biologia, Bratislava 53, 509–518. KNIE, M., HÜBENER, T., 2007: Morphological variability of the Cyclotella ocellata-kram- meri-rossii complex in field samples and cultures. Proceedings of the 1 Central Euro- pean Diatom Meeting, Berlin, 83–86. KOMÁREK, J., JANKOVSKÁ, J., 2001: Review of the green algal genus Pediastrum: impli- cation for pollen-analitycal research. Bibliotheca Phycologica, J. Cramer, Stuttgart. LOPEZ, J., ARTÉ, P., 1973: Hidrografía y fitoplancton del puerto del Fangar (delta del Ebro). Investigación Pesquera 37, 17–56. MARTÍNEZ, S., RIOS, J., PINTÓ, J.,CAMPOS, A., 1999: Geografía. Ed. Teide S.A., Barce- lona. MATTOX, K. R., STEWART, K. D., 1984: A classification of the green algae. A concept based on comparative citology. In: IRVINE, D. E. G., JOHN, D. M. (eds.), Systematic of the green algae, 29–72. Academic Press, London. MC LUSKY, D., 1989: The estuarine ecosystem. Chapman, London. MEDLIN, L. K., KACZMARSKA, I., 2004: Evolution of the diatoms, 5. Morphological and cytological support for the major clades and taxonomic revision. Phycologia 43, 245–270. MELKONIAN, M., 1983: Phylum Chlorophyta. In: MARGULIS, L., CORLISS, J. O., MELKO- NIAN, M., CHAPMAN, D. J. (eds.), Handbook of Protoctistas, 597–660. Jones and Barlett, London. 26 ACTA BOT. CROAT. 68 (1), 2009 PÉREZ M. C., MAIDANA N. I., COMAS A. U:\ACTA BOTANICA\Acta-Botan 1-09\Perez.vp 22. travanj 2009 11:00:13 Color profile: Disabled Composite 150 lpi at 45 degrees MONTESANTO, B., ZILLER, S., DANIELIDIS, D., ECONOMOU-AMILLI, A., 2000: Phyto- plankton community structure in the lower reaches of a Mediterranean river (Aliak- mon, Greece). Archive für Hydrobiologie 147, 171–191. PÉREZ, M. C., COMAS, A., GONZALEZ DEL RÍO, J., SIERRA, J. P., 2002: Planktonic Chloro- phyceae from the lower Ebro River (Spain). Acta Botanica Croatica 61, 99–124. PIENAAR, C., PIETERSE, A. J. H., 1990: Thalassiosira duostra sp. nov. a new freshwater centric diatom from the Vaal River, South Africa. Diatom Research 5, 105–111. RIVERA, P., CRUCES, F., VILA, I. 2003. Cyclotella ocellata Pantocsek (Bacillariophyceae): primera cita en Chile y comentarios sobre su variabilidad morfologica. Gayana Botany 60, 123–131. ROUND, F. E., CRAWFORD, R. M., MANN, D. G., 1990: The diatoms. Biology and morphol- ogy of the genera. Cambridge Univ. Press, Cambridge. SABATER, S., 1990: Phytoplankton composition in a medium-sized mediterranean river: The Ter (Spain). Limnetica 6, 47–56. SABATER, S., KLEE, R., 1990: Observaciones sobre diatomeas centrales del fitoplancton del río Ebro, con especial interés en algunas pequeñas Cyclotella. Diatom Research 5, 141–154. SABATER, S., MUÑOZ, I., 1990: Succesional dynamics of the phytoplankton in the lower part of the river Ebro. Journal of Plankton Research 12, 573–592. SCHMIDT, A., KISS, K. T., BARTALIS, E. T., 1994: Chlorococcal algae in the phytoplankton on the Hungarian section of the River Danube in the early nineties. Biologia, Bratislava 49, 553–562. SOURNIA, A., 1978: Phytoplankton manual. Monographs on oceanographic methodology 6, UNESCO, Paris. STEIDINGER K. A., TANGEN, K., 1996: Dinoflagellates. In: TOMAS, C. R. (ed.), Identifying marine diatoms and dinoflagellates, 387–584. Academic Press, Inc., San Diego. TORGAN, L. C., HENRIQUES VIEIRA, A. A., GIROLDO, D., BAHI DOS SANTOS, C., 2004: Mor- phological irregularity and small cell size in Thalassiosira duostra maintained in cul- ture. Proceedings of the 18 International Diatom Symposium, Bristol, 407–416. VILI^I], D., KR[INI], F., BURI], Z., CAPUT, K., 2000: Taxonomic composition and abun- dance of phytoplankton in the middle reach of the karstic Zrmanja Estuary (Croatia). Acta Botanica Croatica 59, 361–374. WOLF, M., KRIENITZ, L., HEPPERLE, D., 2002: Phylogenetic position of Actinastrum hantzschii Lagerheim 1882 (Chlorophyta, Trebouxiophyceae). Algological Studies 104, 59–67. ZINGONE, A., MONTRESOR, M., MARINO, D., 1998: Morphological variability of the poten- tially toxic dinoflagellate Dinophysis sacculus (Dinophyceae) and its taxonomic rela- tionship with D. pavillardii and D. acuminata. European Journal of Phycology 33, 259–273. ZINGONE, A., SIANO, R., D’ALELIO, D., SARNO, D., 2006: Potentially toxic and harmful microalgae from coastal waters of the Campania region (Tyrrhenian Sea, Mediterra- nean Sea). Harmful Algae 5, 321–337. ACTA BOT. CROAT. 68 (1), 2009 27 PHYTOPLANKTON OF THE EBRO ESTUARY U:\ACTA BOTANICA\Acta-Botan 1-09\Perez.vp 16. travanj 2009 8:41:33 Color profile: Disabled Composite 150 lpi at 45 degrees