Marine benthic dinoflagellates

Transcription

1 Kleine Senckenberg-Reihe Mona Hoppenrath, Shauna A. Murray, Nicolas Chomérat, Takeo Horiguchi Marine benthic dinoflagellates unveiling their worldwide biodiversity Schweizerbart

2 Mona Hoppenrath, Shauna A. Murray, Nicolas Chomérat, Takeo Horiguchi Marine benthic dinoflagellates unveiling their worldwide biodiversity Kleine Senckenberg-Reihe 54

3 Greetings At present fewer than two million species are known to inhabit the biosphere, but experts estimate that between 5 to 50 times as many species are actually living on our planet. The relatively unexplored deep sea is fascinating for the public by its unknown biodiversity. But there is no need to search those far reaches to discover new species, they can be found right in front of the door. To understand marine habitats, a lot of effort has been put into phytoplankton inventories worldwide. In particular, Harmful Algal Blooms (HABs) caused by diverse dinoflagellate taxa, are a major, socially and economically relevant field of research. In recent years the importance of benthic HABs is increasingly recognized because of the impact of ciguatera, which is the most important food borne disease of non-bacterial origin in the world and is caused by benthic dinoflagellate species. Benthic dinoflagellates are understudied, and the known species diversity has nearly doubled in the past 15 years, with new taxa discovered every year including new genera. This book is the first comprehensive summary of their worldwide biodiversity and biogeography, covering a total of 189 species in 45 genera. With its excellent illustrations it will certainly help to identify and monitor these species and to assess potential risks of HABs causes by some of them. Hopefully, this book will also broaden the awareness of these fascinating, tiny, single-celled marine organisms and motivate students to study them. The authors, who are among the very few expert taxonomists for these dinoflagellates (responsible for over a third of the taxon descriptions), illustrate through their long-term research that systematics and compiling inventories of life is a demanding and complex science requiring many years of experience and patience as well as advanced laboratory techniques. My congratulations go to the four authors of this timely and important monograph, which certainly will serve as a standard work for many years to come. Senckenberg is proud to have supported this great project. Volker Mosbrugger Senckenberg Gesellschaft für Naturforschung 4 Greetings

4 Foreword It is a pleasure to introduce Marine benthic dinoflagellates unveiling their worldwide biodiversity. The complicated taxonomy of benthic dinoflagellates is summarized using the most recent information from combinations of detailed microscopic observations, genetic approaches and careful, patient field studies. This work provides new and useful clues on the biogeography, systematics and ecology of this group, including some of the organisms causing harmful outbreaks, and concurrently highlights the unresolved difficulties and challenges for the thorough comprehension of the benthic dinoflagellates. This effort benefitted not only from recent technological advances, but especially, from the youth and diversity (from Germany, France, Australia and Japan) of the co-authors. The expertise of these young, motivated researchers holds much promise for the future of dinoflagellate taxonomy. At the beginning of the XXIst century, taxonomy is essential not only to establishing the worldwide biodiversity of benthic dinoflagellates, but to identify particular harmful taxa. Indeed, a main aim of this effort is to help monitoring programs prevent and mitigate the consequences of harmful events affecting human and ecosystem health. Finally, the thorough treatment of the benthic dinoflagellates provided in this book constitutes a solid basis for future studies on the structure and dynamics of benthic dinoflagellate communities. This publication is especially timely because it comes to press in the spring of 2014, ten years after Professor Ramon Margalef passed away. Margalef would be particularly delighted reading it given his special admiration for dinoflagellates, as he clearly expressed in his contribution to the VIIIth Conference on Harmful Algae held in Vigo on 1997: Dinoflagellates are admirable in their organization and behaviour (Margalef 1997). This book provides excellent images of this wonder of nature. The high quality and resolution of the microphotographs illustrate what it would be defined in Margalef s terms as a comprehensive dictionary of benthic dinoflagel- Foreword 5

5 lates or using the author s words, the unveiled worldwide biodiversity, of this group. As was Margalef, we are certain the authors have experienced the pleasure of observing nature and the major gratification will be to communicate the fruits of the long hours of meticulous and inspired work. More importantly, this book will introduce scientists to the beauty, complexity, and importance of dinoflagellates for generations to come. We congratulate M. Hoppenrath, S.A. Murray, N. Chomérat and T. Horiguchi on their fine publication. It is certain this volume will be a success and we hope that it will not be the last joint effort to bring the heretofore neglected benthic dinoflagellates to the forefront. Elisa Berdalet, Raphael Kudela, Patricia A. Tester February Foreword

6 Contents Greetings Foreword Contents Acknowledgements I. Introduction II. Materials & Methods Habitats Sampling Extraction = separation from the substrate 19 Fixation and Electron Microscopy (EM) Culturing Quantification III. Taxonomy Adenoides Alexandrium Amphidiniella Amphidiniopsis Amphidinium Ankistrodinium Apicoporus Biecheleria Bispinodinium Bysmatrum Cabra Coolia Dinothrix Durinskia Contents 7

7 Galeidinium Gambierdiscus Glenodinium Gymnodinium Gyrodinium Halostylodinium Herdmania Heterocapsa Katodinium Moestrupia Ostreopsis Peridinium partim = new genus Pileidinium Plagiodinium Planodinium Polykrikos Prorocentrum Pseudothecadinium Pyramidodinium Rhinodinium Roscoffia Sabulodinium Scrippsiella Sinophysis Spiniferodinium Stylodinium Symbiodinium spp Testudodinium Thecadinium Togula Vulcanodinium IV. Phylogeny and systematics Phylogeny of the morphological adaptations Amphidinium Amphidiniopsis, Archaeperidinium, Herdmania Peridiniales Contents

8 Cabra, Rhinodinium, Roscoffia Podolampadaceae Coolia, Gambierdiscus, Ostreopsis Gonyaulacales Prorocentrum & Adenoides Sinophysis & Sabulodinium Dinotoms Dinothrix, Durinskia, Galeidinium, Gymnodinium quadrilobatum, Peridinium quinquecorne Dinoflagellate taxa with cryptophyte-(klepto)chloroplasts The phytodinialean dinoflagellates ( Phytodiniales ) V. Biogeography VI. Ecology Attachment Life cycles Tide pools Vertical migration Blooms Spatial distribution Temporal distribution Quantitative Data VII. Toxins of benthic dinoflagellates and benthic harmful algal blooms Introduction Gambierdiscus Ostreopsis Coolia Prorocentrum Amphidinium Alexandrium Vulcanodinium References Taxonomic index Useful web pages Picture credits Authors Addresses Contents 9

9 I. Introduction The first studies of dinoflagellates inhabiting in sandy sediments were conducted early last century (Kofoid and Swezy 1921, E.C. Herdman 1922, 1924a, b, Balech 1956), however, few studies were conducted in the decades after these. Further investigations started in the 1980s (e.g. Saunders and Dodge 1984, Larsen 1985, Dodge and Lewis 1986, Horiguchi and Pienaar 1988a, Horiguchi 1995, Faust 1995). Faust and Horiguchi had a continuous interest in benthic dinoflagellates, exploring mangrove and coral reef habitats and tide pools (e.g. Faust 1993a, b, 1997, 1999, Horiguchi and Chihara 1983a, 1988, Horiguchi and Pienaar 1994a, Horiguchi et al. 2000, 2011, 2012). Comprehensive studies of sand habitats occurred in the 2000s (Hoppenrath 2000b, Murray 2003, Tamura 2005, Mohammad-Noor et al. 2007b, Al-Yamani and Saburova 2010). These studies showed that a species composition quite distinct from planktonic habitats was present in benthic habitats. Less than 10% of the about 2000 described extant dinoflagellate species appear to be benthic (Taylor et al. 2008). They occur in different types of habitats (see chapter II) and appear to be adapted to a benthic life style in their morphology, in their behavior, and some also in their life cycles (see ecology chapter VI). Some taxa are known to produce toxins impacting humans, particularly those occurring in tropical and subtropical regions (see chapter VII), which has caused an increase in research interest in benthic dino12 I. Introduction flagellates. The study of harmful benthic dinoflagellates started in late 1970s with the discovery that a benthic species, later named Gambierdiscus toxicus, was thought to be responsible for ciguatera fish poisoning, a type of human poisoning linked to the consumption of certain species of tropical reef fish (Yasumoto et al. 1977). As ciguatera fish poisoning incidences are increasing, and the distribution of toxin producing benthic taxa seems to expand, an understanding of the species diversity and their identification is becoming more and more important. Blooms of harmful benthic dinoflagellates can cause serious human and environmental health problems. Recently the potentially toxic species have been subject of intense research activities (e.g. Litaker et al. 2009, Laza-Martínez et al. 2011; reviews: Parsons et al. 2012, Hoppenrath et al. 2013a). The lack of comprehensive taxonomic investigations of benthic dinoflagellates complicates progress in our understanding of their biodiversity, biogeography and ecology, and motivated us to compile current information into this book. One hundred and eighty-nine species belonging to 45 genera are described and their known distribution recorded herein. The distribution section for the species lists the references in the following order: Arctic Ocean, North Atlantic (e.g., UK, North Sea, France, Spain, Portugal, east USA, Gulf of Mexico, Caribbean Sea), South Atlantic (e.g., Cape Town, South Africa), Mediterranean Sea,

10 Arabian/Persian Gulf, Indian Ocean (e.g., Viet Nam, Malaysia, West Australia, South Africa), North Pacific (e.g., Sea of Japan, Korea, Japan, BC Canada, California), South Pacific (e.g., East Australia, New Caledonia, French Polynesia, New Zealand). It is the first comprehensive treatise on the group, and it is our intention that it will facilitate further studies. The classification of dinoflagellates is currently changing and is far from being settled, with the discovery of new species and genera, and the rearrangements of systematic entities. Many benthic dinoflagellate genera have unusual morphologies and appear to be not closely related to known planktonic taxa, and molecular phylogenetic analyses frequently show low statistical support for any relationship (see chapter IV). They show unique thecal plate arrangements when compared to planktonic species, e.g. Adenoides, Amphidiniella, Cabra, Planodinium, Rhinodinium, Sabulodinium (see taxonomy chapter III). Therefore, no higher classification was used in this book and the genera (and species within a genus) are presented in alphabetical order. No keys were provided but information about similar species with which a taxon can be confused is given. A good introduction to dinoflagellates is the Tree of Life web project page ( tolweb.org/dinoflagellates/2445). Summaries of main dinoflagellate characteristics were published in Hoppenrath et al. (2009a, 2013a) and of their diversity in F. J. R. Taylor et al. (2008). The cell orientation is explained in figure 1. For the thecal plate designation, the Kofoid system as modified and described in Fensome et al. (1993) was followed (Fig. 2). Some benthic taxa have thecal tabulations difficult to interpret and sometimes different designations (plate formulae) have been published for one taxon. As our understanding of the morphological and genetic diversity of dinoflagellates has increased in recent years, some original descriptions of species may no longer be adequate to identify a taxon. Cryptic species diversity has been detected already (Murray et al. 2012), and it is highly likely that further cryptic species will be found. Furthermore, some old taxonomic concepts are no longer valid. For example within the unarmoured (athecate, naked) dinoflagellate genera some genus delimitations are unsatisfactory, and reclassification is still ongoing. For instance, the genera Amphidinium, Gymnodinium, and Gyrodinium were redefined (Daugbjerg et al. 2000, Flø Jørgensen et al. 2004a, Murray et al. 2004). As a consequence of the redefinitions many species can no longer be classified in the genera, need reinvestigation, reclassification or classification within new genera. For practical reasons and not to make them nameless, the old generic names were used in this book, and the genera were separated into sensu stricto (s.s.) and sensu lato (s.l.) species. Dinoflagellates are protists that historically have been treated in accordance with the International Code of Botanical Nomenclature (ICBN) and the International Code of Zoological Nomenclature (ICZN) ambiregnal taxa. It has been agreed on solely applying the botanical code for dinoflagellates in future, and we here follow the latest version of the International Code of Nomenclature (ICN) for algae, fungi, and plants the Melbourne Code (McNeill et al. 2012). Some species epithets (names) have been corrected, following article 32.2: Names or epithets published with an improper Latin termination but otherwise in accordance with this Code are regarded as validly published; they are to be changed to accord with Art , 21, 23, and 24, withi. Introduction 13

11 Fig. 1: Cell orientation. A D: Dinokont cells. A, B: Dorsoventrally flattened cell. C, D: Laterally flattened cell. E: Prorocentroids, desmokont cell. 14 I. Introduction

12 Fig. 2: Kofoid system of thecal plate designation. out change of the author citation or date (see also Art ). For the holotype designation in many published (past) new dinoflagellate species descriptions article 40.5 applied and still applies: For the purpose of Art. 40, the type of a name of a new species or infraspecific taxon of microscopic algae or microfungi (fossils excepted: see Art. 8.5) may be an effectively published illustration if there are technical difficulties of preservation or if it is impossible to preserve a specimen that would show the features attributed to the taxon by the author of the name. I. Introduction 15

13 III. Taxonomy Adenoides [Aden: gland; eidos: sight neutral] Adenoides Balech Publication: Balech, 1956, Revue Algologique 2, pp , Figs 1 8. Type species: A. eludens (Herdman) Balech. Plate formula: APC 4 6c 4s 5 5p 1 or APC 4 6c 5s 5 3p 2. Description: Thecate genus with laterally flattened cells with a minute, depressed and scarcely visible epitheca. Shallow cingulum without displacement almost at the anterior cell end. No precingular plate series. Remarks: A taxonomic problem with the original description of the type species has been discussed in detail in Hoppenrath et al. (2003, pp. 385, 389) who reinvestigated and revised the description of A. eludens. Whether a second Adenoides species, described by Herdman (1922) as Amphidinium species and transferred to Adenoides by Dodge (1982; without own observations), really exists, is not clear. Because of this uncertainty it has not been included herein. Adenoides eludens (Herdman) Balech Publication: Balech, 1956, Revue Algologique 2, p. 30. Basionym: Amphidinium eludens E.C. Herdman; Herdman 1922, Proceedings and Transactions of the Liverpool Biological Society 36, pp (26), Figs 1, (2). 22 III. Taxonomy Adenoides Illustrations: F i g s 5, 6. Size: µm long, µm deep. Plate formula: APC 4 6c 4s 5 5p 1 or APC 4 6c 5s 5 3p 2. Chloroplasts: Two lobed brown peridininchloroplasts. Description: Round to oval, asymmetrical, laterally flattened cells with minute depressed and scarcely visible epitheca. The hypotheca is longer dorsally than ventrally. Smooth thecal plates with pores. Shallow cingulum without displacement almost at the anterior cell end. Short and slightly depressed sulcus with one flagellar pore located in the anterior third of the cell. No precingular plate series. Two conspicuous large pores at the dorsal posterior end. Two striking pyrenoids visible as rings because of the starch sheaths. Nucleus in the lower dorsal hyposome half. Distribution: Sandy sediments. Port Erin, Isle of Man, UK (Herdman 1922); North Sutherland, Scotland, UK (Dodge 1989); North German Wadden Sea, Germany (Hoppenrath 2000b, Hoppenrath et al. 2003); Normandy, France (Paulmier 1992); Roscoff, Brittany, France (Balech 1956, Dodge and Lewis 1986); Elba, Italy (Hoppenrath unpubl. obs.); Arabian Gulf, Kuwait (Saburova et al. 2009, Al-Yamani and Saburova 2010); Sea of Japan, Russia (Konovalova and Se-

14 Fig. 5: Adenoides eludens. A C: Different focal planes, note the ring-like starch sheath around the pyrenoid (arrow); p = pusule, n = nucleus. Scale bars: 10 µm. Fig. 6: Adenoides eludens. A: Left lateral view. B, C: Right lateral view; note the thecal pores in C. D H: Drawings of the plate pattern. D: Left lateral. E: Right lateral. F: Dorsal. G: Ventral. H: Epitheca, cingulum and sulcus. Scale bars: 10 µm. III. Taxonomy Adenoides 23

15 Fig. 8: Amphidiniella sedentaria. A: Ventral view. B: Mid cell focus showing the pusule (p) and the nucleus (n). C: Note the starch ring around the pyrenoid (arrow). D: Note the pyrenoid (arrow) and food body (fb). Scale bars: 10 µm. Fig. 9: Amphidiniella sedentaria. A: Dorsal view, SEM (photo by N. Borchhardt), scale bar: 5 µm. B D: Drawings of the plate pattern, modified after Horiguchi (1995). B: Ventral. C: Apical, epitheca. D: Antapical, hypotheca and part of the sulcus. the middle of the right cell half. Nucleus in the posterior part of the cell. Smooth thecal plates with pores, except for the first apical intercalary plate (1a) with strong ornamentation. Relatively large bean-shaped apical pore plate with slit-like apical pore covered by a lip-shaped projection. Ventral pore adjacent to the first apical plate (1 ) and in contact with plates 4 and III. Taxonomy Amphidiniella Similar species: Murray (2003) recorded a similar species with apical hook under the name Amphidiniella sp 1. This taxon was also discovered in Germany, France, Italy and Kuwait (Hoppenrath, Chomérat and Saburova unpubl. data) and it seems not to be related to Amphidiniella (new genus description in preparation). Remarks: In culture the species attaches,

16 living mainly motionless as film but also can swim quite rapidly (Horiguchi 1995). Distribution: Sandy beaches, intertidal sand flats or on the surface of dead coral. Elba, Italy (Borchhardt and Hoppenrath unpubl. obs.); Palm Beach, Kwazulu-Natal, South Af- rica (Horiguchi 1995); Shark Bay, Australia (AlQassab et al. 2002); Sesoko Beach and Ondo, Okinawa, Japan (Horiguchi 1995, Horiguchi unpubl. obs.); Sydney, Australia (Murray 2003). References: Al-Qassab et al. (2002), Murray (2003). Amphidiniopsis [Amphidinium; opsis: aspect feminine] Amphidiniopsis Wołoszyńska Publication: Wołoszyńska, 1928, Archives d Hydrobiologie et d Ichtyologie 3, p Type species: A. kofoidii Wołoszyńska. Plate formula: (original description); APC a c 3 5s 5 2 (today). Description: Thecate genus with cells having a smaller epitheca and a large hypotheca. Currently the genus is characterized by an ascending cingulum, a distinctive curved sulcus and hypothecal plate pattern (Hoppenrath et al. 2009b). Species are laterally or dorsoventrally flattened, with a complete or incomplete cingulum, and with or without an apical hook. Diverse cell morphologies have been described and morphological variability is known (e.g. Selina and Hoppenrath 2008). Three major subgroups can be recognized: (1) laterally flattened species with complete cingulum, (2) dorsoventrally flattened species with complete cingulum, sulcus positioned in the middle of the cell, no apical hook, and one or two anterior intercalary plates, and (3) dorsoventrally flattened species with complete or incomplete cingulum, sulcus positioned in the middle of the cell (the deepened part of the sulcus can be shifted to the left side), with an apical hook pointing to the left, and three anterior intercalary plates (Hoppenrath et al. 2012b). Group 1: A. arenaria, A. dentata, A. galericulata, A. kofoidii, A. sibbaldii. Group 2: A. aculeata, A. hexagona, A. hirsuta, A. konovalovae, A. striata, A. swedmarkii (A. rotundata but with shifted sulcus and three anterior intercalary plates?). Group 3: A. korewalensis, A. pectinaria, A. uroensis (A. cristata but with only one anterior intercalary plate? A. dragescoi and A. rotundata but without apical hook?). All currently known species are heterotrophic, benthic (sand-dwelling) and marine, except of one freshwater species (A. sibbaldii). Remarks: The history of records, nomenclatural changes and classification schemes of the species were summarized in Hoppenrath (2000f) and Hoppenrath et al. (2009b). A revision of the genus is needed and it is possible that it contains several subgenera or that it is polyphyletic (e.g. Hoppenrath et al. 2012b). Herdmania seems to be closely related to Amphidiniopsis (Yamaguchi et al. 2011a, Hoppenrath et al. 2012b). Amphidiniopsis aculeata Hoppenrath, Koeman et Leander Publication: Hoppenrath et al., 2009b, Marine Biodiversity 39, pp. 4 6, Figs 1 2, 3A. Illustrations: Figs 11A C. Size: µm long, µm wide. Plate formula: APC 4 2a 7 3c 5s 5 2. Chloroplasts: none. III. Taxonomy Amphidiniopsis 27

17 Fig. 10: Amphidiniopsis spp. (laterally flattened species), drawings of the plate pattern. A C: A. arenaria. D F: A. dentata. G I: A. galericulata. J M: A. kofoidii (selected drawings from the original description, Wołoszyńska 1928). A, D, G, J: Left lateral. B, E, H, K: Right lateral. C, F, I, M: Apical, epitheca. L: Ventral. 28 III. Taxonomy Amphidiniopsis

18 Fig. 11: Amphidiniopsis spp. (dorsoventrally flattened species), drawings of the plate pattern. A C: A. aculeata. D F: A. hexagona. G I: A. hirsuta. J L: A. konovalovae. A, D, G, J: Ventral. B, E, H, K: Dorsal. C, F, I, L: Apical, epitheca. III. Taxonomy Amphidiniopsis 29

19 cell side, covering the apical pore. Cingulum ascending about two cingulum widths. Characteristically curved sulcus with its deepened part slightly shifted to the left. Nucleus in the hyposome centre. Thecal plates ornamented with small/short spines and pores. Several irregular antapical spines, the most lateral ones being the largest. Three anterior intercalary plates. Third postcingular plate (3 ) medium sized, symmetric, pentagonal and distinctively pointed posterior. Similar species: Amphidiniopsis cristata, A. korewalensis, A. pectinaria. All dorsoventrally flattened species can be differentiated by the shape and size of plate 3, the shape of plate 1a, the connection or separation between plate 2 and the sulcus, the size of plate 3, the thecal ornamentation, the presence or absence of antapical projections, and the presence or absence of an apical hook. Distribution: Sandy sediments. Sea of Japan, Russia (Konovalova and Selina 2010, Selina and Hoppenrath 2013); Uro, Kagawa, Japan (Toriumi et al. 2002); Boundary Bay, BC, Canada (Hoppenrath unpubl. obs.). References: Hoppenrath et al. (2012b), Selina and Hoppenrath (2013). Amphidiniopsis yoshimatsui Hoppenrath sp. nov. Publication: Yoshimatsu et al., 2000, Phycological Research 48, p. 108, Figs 1 9. Holotype: Fig. 1 in Yoshimatsu et al. (2000). Iconotype (herein): Figs 12 G I. Synonym: Amphidiniopsis swedmarkii sensu Yoshimatsu et al. (2000). Type locality: sandy beach, Yashima Bay, Kagawa Prefecture. Size: µm long, µm wide. Plate formula: APC 4 2a 7 3c 4s 5 2 (Hoppenrath et al. 2009b). 40 III. Taxonomy Amphidiniopsis Chloroplasts: none. Description: Broad oval ( barrel -shaped, posterior truncated), dorsoventrally flattened cells with a small cap-like epitheca narrower than the hypotheca. Cingulum ascending about one cingulum width, consisting of three plates. Characteristically curved sulcus in the middle of the hypotheca, consisting of four plates (new interpretation by Hoppenrath et al. 2009b of the provided SEM images by Yoshimatsu et al. 2000). Nucleus most likely in the lower left lateral hyposome half (not cleary discernable in the LM images provided by Yoshimatsu et al. (2000, Figs 3, 4). Thecal plates ornamented with irregular small projections (nipple-like). Two apical intercalary plates. Third postcingular plate (3 ) medium sized, nearly symmetric, pentagonal (appears nearly square) and very slightly pointed posterior. No antapical processes. A more detailed description in Yoshimatsu et al. (2000). Diagnosis: Amphidiniopsis yoshimatsui can be distinguished from the other dorsoventrally flattened species by the following features: (1) relatively narrow first postcingular plate (1 ), causing the sutures between plates 1, 2 and 1 to be visible in ventral cell view; (2) very distinctive size and shape of the third postcingular plate (3 ); (3) hexagonal first anterior intercalary plate (1a); (4) connection between the second apical plate (2 ) and the sulcus; (5) no apical hook; (6) no antapical projections (spines). It can be distinguished from the laterally flattened species by its dorsoventral flattening. Similar species: Amphidiniopsis aculeata, A. hexagona, A. hirsuta, A. konovalovae, A. striata, A. swedmarkii. All dorsoventrally flattened species can be differentiated by the shape and size of plate 3, the shape

20 of plate 1a, the connection or separation between plate 2 and the sulcus, the size of plate 3, the thecal ornamentation, the presence or absence of antapical projections, and the presence or absence of an apical hook. Distribution: Sandy sediments. Kagawa, Japan (Yoshimatsu et al. 2000). References: Hoppenrath et al. (2009b, 2012b). Amphidinium [amphi: around, on both sides; dino neutral] Amphidinium Claparède et Lachmann Publication: Claparède and Lachmann, 1859, Mémoires de l Institut National Genevois 6, p Type species: A. operculatum Claparède et Lachmann. Description: Athecate genus of generally dorsoventrally flattened cells with a minute, crescent-shaped or triangular, usually left-deflected epicone. Remarks: This genus was revised, and as now circumscribed, includes only those species with a minute, triangular or left-deflected epicone (Flø Jørgensen et al. 2004a, Murray et al. 2004), rather than species in which the epicone constitutes 1/3 or less of the cell length. Amphidinium appears to be one of the earliest evolving dinoflagellate lineages (Orr et al. 2012). Species that do not fit the criteria for the genus as redefined (Flø Jørgensen et al. 2004a, Murray et al. 2004), but have not yet been investigated to determine the generic affinities, are included here as Amphidinium sensu lato. Amphidinium sensu stricto: Amphidinium bipes E.C. Herdman Publication: Herdman, 1924b, Proceedings and Transactions of the Liverpool Biological Society 38, p. 78, Fig. 19. Illustrations: Fig. 14 A. Size: µm long, µm wide, L:W Chloroplasts: none. Description: Cell oval to oblong in ventral view, dorsoventrally flattened. Hypocone with a strong antapical notch, creating a distinctly bilobed appearance. Epicone small, triangular in ventral view, slightly deflected to the left. A groove extends onto the epicone, almost reaching the apex. Nucleus rounded, approximately 10 µm, in the hypocone. Distribution: Sandy sediments. UK (Herdman 1924b, Dodge 1982); Danish Wadden Sea (Larsen 1985); Germany (Hoppenrath 2000b); France (Paulmier 1992); Elba, Italy (Hoppenrath unpubl. obs.); Seto Inland Sea, Japan (Ono et al. 1999); Canada (Baillie 1971). Amphidinium carterae Hulburt Publication: Hulburt, 1957, Biological Bulletin Marine Biological Laboratory Woods Hole 112, p Basionym: Carter (1937, Figs 12 15, as A. klebsi). Synonym: Amphidinium eilatensis Lee is morphologically indistinguishable from A. carterae (Lee et al. 2003a). Partial large subunit ribosomal RNA sequence data show that it corresponds to Genotype 2 of A. carterae (Murray et al. 2004). Illustrations: Fig. 14 B, 15 A, B. III. Taxonomy Amphidinium 41

21 Fig. 14: Amphidinium sensu stricto species. A: A. bipes. B: A. carterae. C, D: A. cupulatisquama. E H: A. gibbosum. I K: A. herdmanii. Scale bars: 10 µm. 42 III. Taxonomy Amphidinium

22 Fig. 15: Amphidinium sensu stricto species. A, B: A. carterae. C, D: A. massartii. B, D: Epifluorescence showing the chloroplast morphologies of the species. C: photo curtesy of M. Tamura. Scale bars: 10 µm. III. Taxonomy Amphidinium 43

23 Bispinodinium [bis: twice; spina: spine; dino neutral] Bispinodinium Yamada, Terada, Tanaka et Horiguchi Publication: Yamada et al., 2013, Journal of Phycology 49, 558. Type species: B. angelaceum Yamada, Terada, Tanaka et Horiguchi. Plate formula: athecate taxon. Acrobase: magnifying glass-shaped, consisting of linear part extending from the sulcus and circular part at the centre of the epicone. Description: Amphidinium (sensu lato)-like athecate photosynthetic dinoflagellate. Cells composed of a short epicone and a large hypocone. Cingulum completely encircling the cell. Acrobase shaped like a magnifying glass: Spinoid apparatus (pair of long fibrous structures) extending from the circular apical groove to the vicinity of the nucleus. Bispinodinium angelaceum Yamada, Terada, Tanaka et Horiguchi Publication: Yamada et al., 2013, Journal of Phycology 49, p. 558, Figs 1 7. Illustrations: Fig. 23. Size: µm long, µm wide. Acrobase: magnifying glass-shaped. Chloroplasts: Two brownish-yellow peridininchloroplasts, with many elongated parallel chloroplast lobes at right angles to the cingulum. Description: As for the genus. Cells oblong in ventral view and dorsoventrally flattened, almost axi-symmetric (laterally symmetri- 62 III. Taxonomy Bispinodinium cal) along with the sulcus; cingulum located at about one third of the total cell length from the anterior of the cell (ratio of ), and both extremities of the cingulum curved slightly downward at the junction with the sulcus. Sulcus straight, narrow and reaching the antapex. Nucleus spherical and located in the center of the hypocone. Chloroplasts of peridinin-type, brownish-yellow in colour, with many elongated parallel chloroplast lobes at right angles to the cingulum. Two lateral pyrenoids located on the right and left side of the hypocone, respectively. Similar species: Amphidinium latum Lebour is most similar to Bispinodinium angelaceum in general cell morphology. A. latum can be distinguished in having kleptochloroplasts of cryptophyte origin, not permanent chloroplasts (Horiguchi and Pienaar 1992) and in the shape of the acrobase (Murray and Patterson 2002b). Remarks: The species was collected from sand samples obtained from the seafloor at a depth of 36 m in subtropical Japan. In the undisturbed conditions it spends most of the time as a quiescent cell, embedded in a mucus matrix. In this state, the cell stands up attached by its posterior end to the bottom of the culture vessel. Swimming cells move very slowly and in a zig-zag manner. Among known dinoflagellates the spinoid apparatus is unique to this species. Distribution: off Mageshima Island, Kagoshima Prefecture, Japan (Yamada et al. 2013).

24 Fig. 23: Bispinodinium angelaceum. A: Ventral view showing the cingulum and sulcus. B: Ventral episomal view showing part of the acrobase. C: Mid cell focus, note the spinoid apparatus and the round nucleus. D: Chloroplasts in epifluorescence microscopy. Scale bars: 10 µm. III. Taxonomy Bispinodinium 63

25 pore has a crest and the canal plate is long. Separation of plates 2a and 3a by the third apical plate (3 ). Pentagonal and asymmetric first apical plate (1 ). Cingulum descending about one cingulum width. Deep and narrow sulcus with one sulcal list formed by plate 1. Reticulated thecal ornamentation. Gelatinous apical stalk. Numerous oval to rod-shaped, radially arranged chloroplasts. Stigma in the midsulcal area. Nucleus in the cell centre. Similar species: Other Bysmatrum species, see above (B. arenicola). Remarks: The species can form blooms. Distribution: In marine and estuarine habitats in sandy sediments or associated with coral rubble or floating detritus. Roscoff, France (Dodge and Lewis 1986); Sarasoa Bay, Florida, USA (Steidinger and Balech 1977); Caribbean Sea, Belize (Faust 1996); Elba, Italy (Hoppenrath unpubl. obs.)?; Porto-Lagos, North Aegean Sea, Greece (Gottschling et al. 2012); Aral Sea (Ostenfeld 1908); Oshigaki Island, East China Sea and Iriomote Island, Japan (Faust 1996). References: Faust (1996); Gottschling et al. (2012); Steidinger and Balech (1977). Bysmatrum teres Murray, Hoppenrath, Larsen et Patterson Publication: Murray et al., 2006a, Phycologia 45, 162, Illustrations: Figs 24 C, D, 27 A D. Size: µm long, µm wide. Plate formula: APC 4 3a 7 6c 4s 5 2. Chloroplasts: many golden-brown peridininchloroplasts. Description: Olique ellipsoidal dorsoventrally flattened cells with epitheca smaller than hypotheca. Large teardrop to nearly triangular apical pore complex with collar. Cingulum strongly descending about times the cingulum width. Plates 2a and 3a widely separated. Narrow and deep sulcus with left sulcal list pointing posteriorly. The two antapical plates appear as a continuation of the sulcus. Thecal plates are smooth with scattered pores. Many rod-shaped chloroplasts. Nucleus in the cell centre. Similar species: Other Bysmatrum species, see above (B. arenicola). Distribution: Sandy sediments. Arabian Gulf, Kuwait (Saburova et al. 2009, Al-Yamani and Saburova 2010); Town Beach, Broome, NW Australia (Murray et al. 2006a). Cabra [Australian dialectal word feminine] Cabra Murray et Patterson, emend. Chomérat, Couté et Nézan Publication: Murray and Patterson, 2004, European Journal of Phycology 39, p Emendation: Chomérat et al., 2010a, Marine Biodiversity 40, pp Type species: C. matta Murray et Patterson. Plate formula: Po Pt 3 1a 5 3c?s 5 1. Description: Heterotrophic thecate genus with cells more or less pentagonal and irregular in lateral view. Cells are strongly 70 III. Taxonomy Cabra compressed and asymmetric, appearing different from the left as compared to the right side. The cingulum is markedly ascending: it is oblique in left lateral view while it is straight and located near the apex in the right view. It appears to be incomplete but the c3 plate possesses a narrow extension located ventrally between plates 4 and 5 and reaching the sulcus. Three prominent pointed flanges are present on the antapical side (on the 1, 5

26 Fig. 28: Cabra spp. drawings. A C: C. aremorica. D F: C. matta. G I: C. reticulata. A, D, G: Right lateral views. B, E, H: Left lateral views. C, F, I: Apical views, epithea. Scale bars: 10 µm. and 1 plates), while one is located on the anterior dorsal margin of the 3 plate. The thecal ornamentation differs among species. Pores of two sizes are present. A particular area of dense pores or small depressions is present on the 1 plate, close to the sulcus. The nucleus is dorsal and cells often contain food-bodies. III. Taxonomy Cabra 71

27 Fig. 29: Cabra spp. LM and SEM. A C: C. aremorica. D F: C. matta. G I: C. reticulata. A, D, G: Light microscopy. B, E, H: Right lateral views in SEM. C, F, I: Left lateral views in SEM. Scale bars: 10 µm. Fig. 30: Cabra spp., theca details shown in SEM. A F: C. aremorica. G, H: C. matta. I, J: C. reticulata. A: Right lateral to ventral view. B: Dorsal to right lateral view. C: Antapical to left lateral view. D: Apical to left lateral view. E: Ventral epitheca half. F: Detail of the apical 72 III. Taxonomy Cabra

28 pore complex and the surrounding plates. G: Apical to left lateral view. H: Detail of the apical pore complex. I: Apical to left lateral view. J: Detail of the apical pore complex. Scale bars: 10 µm in A D, G, I, 5 µm in E, and 2 µm in F, H, J. III. Taxonomy Cabra 73

29 Plagiodinium belizeanum Faust et Balech Publication: Faust and Balech, 1993, Journal of Phycology 29, pp , Figs Illustrations: Fig. 59. Size: µm long, µm wide, 7 9 µm deep. Plate formula: (Po) 5 0 5c 5s 5 1. Chloroplasts: Many(?), colour not described. Description: Oblong to oval laterally flattened thecate cells with extremely small finger-like, dorsoventrally inclined epitheca (descending ventrally). Complete cingulum without displacement. Sulcus relatively short or about half the cell length? Nucleus located posteriorly. Smooth thecal plates with pores. No precingular plate series. The presence of an apical pore (plate) was not reliably demonstrated in the original description. Similar species: Superficially resembling athecate Amphidinium s.s. species. Remarks: The taxon is in need of reinvestigation. Distribution: On floating detritus, coral rubble and sediment in mangrove habitats. Twin Cays, Beliz (Faust and Balech 1993); Malaysia (Mohammad-Noor pers. comm.). Planodinium [planus: even, flat; dino neutral] Planodinium Saunders et Dodge Publications: Saunders and Dodge, 1984, Protistologica 20, pp. 275, 278. Type species: P. striatum Saunders et Dodge. Plate formula: 3 1a 8 7?c 4?s 5 1. Description: Thecate genus with laterally flattened, oval cells with a truncated apex. Very small epitheca, only about one eighth to one tenth of the cell length. Planodinium striatum Saunders et Dodge Publication: Saunders and Dodge, 1984, Protistologica 20, pp. 278, Figs Synonym: Thecadinium petasatum partim sensu Baillie Illustrations: Fig. 60. Size: µm long, µm deep. Plate formula: 3 1a 8 7?c 4?s 5 1. Chloroplasts: none. Description: Strongly laterally flattened, more or less rectangular cells with slightly longer dorsal side. Small cap-like or domed epitheca, only about one eighth to one tenth of the cell length. No apical pore. Thecal plates ornamented with ridges, mainly longitudinal ones on the hypotheca, and larger pores surrounded by a ring of smaller pores. Additionally small pimples on the epithecal plates. Slightly descending (about one cingulum width) deep cingulum. Sulcus displaced on the left lateral side, not reaching the antapex. A narrow Fig. 60: Planodinium striatum. A, B: Same cell in different focal planes. A: Left lateral view. C: Mid cell focus, note the red food body (photo by S. Sparmann). D F: Drawings of the plate pattern. D: Left lateral view. E: Right lateral view. F: Apical view, epitheca and cingulum. G, H: SEMs showing the thecal plate ornamentation. G: Left lateral view. H: Right lateral view. Scale bars: 10 µm. 130 III. Taxonomy Planodinium

30 III. Taxonomy Planodinium 131

31 present. Gametes have only four zooids and one nucleus. Similar species: Polykrikos herdmaniae without chloroplasts. Remarks: Hyaline vegetative (division?) cysts have been observed. Distribution: Sandy sediments. Port Erin, Isle of Man, UK (Herdman 1922, 1924a); North Sutherland, Scotland, UK (Dodge 1989); North German Wadden Sea, Germany (Hoppenrath 2000b); Normandy, France (Paulmier 1992); Roscoff, Brittany, France (Balech 1956); Brittany, France (Dragesco 1965); Salt Pond, Woods Hole, USA (Hulburt 1957); Boundary Bay, BC, Canada (Hoppenrath and Leander 2007b). References: Baillie (1971), Balech (1956), Dodge (1982, 1989), Dragesco (1965), Herdman (1922, 1924a), Hulburt (1957), Lebour (1925), Paulmier (1992), Schiller (1933). Prorocentrum [pro: in front; kentron: goad neutral] Prorocentrum Ehrenberg Publication: Ehrenberg, 1834, Abhandlungen der Physikalisch-Mathematischen Klasse der Königlichen Akademie der Wissenschaften zu Berlin Type species: P. micans Ehrenberg. Synonyms: Dinopyxis Stein, Exuviaella Cienkowski, Postprorocentrum Gourret. Plate formula: The theca consists of two major plates (right and left) that meet in a sagittal suture and an apical cluster of 5 to 14 tiny platelets around two pores in the periflagellar area (Faust et al. 1999, Fensome et al. 1993). Labeling or naming of the platelets has been proposed by Hoppenrath et al. (2013a), see Fig. 63. Chloroplasts: All known species have goldenbrown peridinin-chloroplasts. Description: Two flagella arise from one pore, the flagellar pore (desmokont flagellation). The second pore, the accessory pore (syn.: auxiliary or apical pore), is associated with mucocysts. Cell division is by desmoschisis with the division line along the left plate sagittal suture and the periflagellar platelets separating together with the right plate. Remarks: About 80 species have been de134 III. Taxonomy Prorocentrum scribed, of which 29 are benthic. At least nine species of the genus Prorocentrum have been shown to produce toxins (see chapter about toxic taxa, p. 226). Instances of harmful algal blooms resulting from benthic species of Prorocentrum are rare. The species inhabit the interstitial spaces of marine sediments, and they live epiphytically on macroalgal surfaces, floating detritus and corals. For a more detailed review, including proposals for a unified terminology that will be used herein, see Hoppenrath et al. (2013a). Prorocentrum belizeanum Faust Publication: Faust, 1993b, Journal of Phycology 29, p. 101, Figs Illustrations: Fig. 62. Size: µm long, µm wide. Platelet formula: ? 8. Description: Round to oval cells with central pyrenoid (starch ring). Reticulate-foveate thecal surface with scattered pores and marginal row of pores. The periflagellar area is wide V-shaped, with collar, thick flange, and platelet lists adjacent to the pores. Three deep depressions on platelet 1. The number of periflagellar platelets is

32 not known yet, seven or eight platelets were identified from published images (Faust et al. 2008). The kidney-shaped nucleus is located posteriorly. Similar species: P. hoffmannianum, P. sabulosum, P. tropicale Remarks: These similar species have been distinguished mainly based on variability of size and ornamentation (number, size and shape of depressions), but it is now obvious that these criteria are subject to large variation. A detailed reinvestigation of all the morphotypes described as separate species is necessary to define species boundaries. The species has been shown to produce okadaic acid (OA), see also toxicity chapter VII, p Distribution: Twin Cayes, Belize (Faust 1993b); Mexican Pacific (Okolodkov and Gárate-Lizárraga 2006). References: Faust et al. (1999), Faust and Gulledge (2002), Faust et al. (2008). Prorocentrum bimaculatum Chomérat et Saburova Publication: Chomérat et al., 2012, Journal of Phycology 48, pp , Figs 2 5. Illustrations: Figs 62, 64 A. Size: µm long, µm wide. Platelet formula: 1a,b Description: Oblong oval cells with smooth thecal surface with scattered small pores and two circular areas above and below the plate centre devoid of pores. Marginal row of large pores (or deep depressions). The periflagellar area is wide V-shaped with a short collar. Nine platelets, some overlapping others. The chestnut-shaped nucleus is located posteriorly. Distribution: Sandy sediments. Arabian Gulf, Kuwait (Chomérat et al. 2012). Prorocentrum borbonicum Ten-Hage et al. Publication: Ten-Hage et al., 2000b, Phycologia 39, pp , Figs Illustrations: Figs 62, 65 A F. Size: µm long, µm wide. Platelet formula: a,b 7? 8. Description: Broad oval to ovoid cells with central pyrenoid (starch ring). Foveate thecal surface with scattered pores inside and between the depressions. Plate centre devoid of large pores. Depressions are deeper at the centre of the right plate. The periflagellar area is wide V-shaped. Eight or nine platelets. The nucleus is located posteriorly. Remarks: The species has been shown to produce unidentified neurotoxins subsequently characterized as borbotoxins (see also toxicity chapter VII, p. 228). Distribution: North Aegean Sea, Mediterranean, Greece (Aligizaki et al. 2009, Ignatiades and Gotsis-Skretas 2010); Reunion Island (Ten-Hage et al. 2000b). References: Aligizaki et al. (2009). Prorocentrum caribaeum Faust Publication: Faust, 1993b, Journal of Phycology 29, pp , Figs Illustrations: Fig. 62. Size: µm long, µm wide. Platelet formula: not worked out yet. Description: Heart-shaped, posteriorly pointed, asymmetric cells. Foveate thecal surface with characteristic pore pattern (one apical row, posterior radial rows, open circle of pores at the posterior tip). Plate centre devoid of pores. The periflagellar area is wide V-shaped, probably consisting of 7 platelets but an eighth platelet (#7) could be present (Hoppenrath et al. 2013a). Platelet 5 in contact with 8. Wing on platelet 1. Second shorter wing, more a list, at III. Taxonomy Prorocentrum 135

33 Fig. 62: Prorocentrum spp., drawings to the same scale. Cells shown from the right cell side. Pore pattern and ornamentation partly given. 136 III. Taxonomy Prorocentrum

34 Fig. 63: Prorocentrum spp., drawings to the same scale. Cells shown from the right cell side. Pore pattern and ornamentation partly given. Lower right corner: Numbering/lettering system for the platelets of the periflagellar area. ap = accessory pore, fp = flagellar pore. the most ventral platelet 4. Kidney-shaped nucleus posteriorly. Similar species: The planktonic P. micans with an apical spine instead of a wing. Remarks: The spelling of the adjective caribaeum has been altererd to be consistent with the Latin orthography Caribae insulae for caribbean islands. Distribution: Twin Cayes, Belize (Faust 1993b). Prorocentrum clipeus Hoppenrath Publication: Hoppenrath, 2000a, European Journal of Protistology 36, pp , Figs Illustrations: Figs 62, 66 A C. Size: µm long, µm wide. Platelet formula: 1a,b Description: Nearly circular cells with smooth thecal surface with no visible pore pattern III. Taxonomy Prorocentrum 137

35 VI. Ecology Benthic dinoflagellate species inhabit the interstitial spaces of marine sediments ( sand-dwelling ), and they live epiphytically on macroalgal and seagrass surfaces (i.e. phycophilic), in tide pools, and on floating detritus and corals. The majority of benthic species are sand-dwelling, rather than epiphytic (only about 40 species), in contrast to the results of Fraga et al. (2012). Benthic species can have a wide range of habitats, as Bymatrum subsalsum, a species adapted to a benthic (sand), epiphytic (macroalgae), and planktonic (lagoonal mangrove waters) life (Faust 1996), but most have been recorded only in the interstitial or as epiphytes. Knowledge about the ecology of benthic species is very limited, partly because of the difficulties in enumerating them (see below). A survey of benthic microalgae, including dinoflagellates, in coral reef sediments of the southern Great Barrier Reef indicated that they are ubiquitous, abundant and productive key components of the reef ecosystem (Heil et al. 2004). The available information about their ecophysiology, for example growth rates, nutrient uptake, and toxin production is scarce. It has been reviewed for benthic Prorocentrum by Glibert et al. (2012), Gambierdiscus and Ostreopsis by Parsons et al. (2012). The significance of parasites for species mortality is not understood yet. Only for one Prorocentrum species, P. fukuyoi, parasitic infection has been described (Leander and Hoppenrath, 2008). One hundred and twenty of the described 189 species currently known are photosynthetic. The photosynthetic species may be adapted to low-light conditions, given that 212 VI. Ecology interstitial spaces may receive less light than planktonic habitats. On the other hand, epiphytic species in shallow tropical coral reef sites may be adapted to the high light that would be expected in such sites (Fraga et al. 2012). Many species may be mixotrophic, as food bodies have been observed in some chloroplast containing species. Taxa with a diatom endosymbiont are known, see dinotoms above (chapter IV). Some species have kleptochloroplasts of cryptophyte origin, Amphidinium poecilochroum, A. latum, and Gymnodinium myriopyrenoides. The feeding modes of heterotrophic and mixotrophic species are largely unknown. A peduncle has been observed for the photosynthetic Bysmatrum arenicola (Horiguchi and Pienaar 1988a), and Amphidinium poecilochroum (Larsen 1988). Observations of diatoms which have been ingested as a whole, suggest phagotrophy for Ankistrodinium semilunatum, Katodinium glandulum, and Gyrodinium spec. Most benthic dinoflagellates are small and have (sometimes strongly) flattened cells. They can be flattened dorsoventrally (e.g., some Amphidiniopsis species), laterally (e.g., Planodinium), oblique laterally (e.g., Sinophysis), or anterior-posteriorly (e.g., some Gambierdiscus species). This may facilitate their movement in interstitial habitats, and also allow them to attach more easily to surfaces. Alternatively, it has been proposed that the flattened surfaces may act to increase nutrient uptake in oligotrophic conditions, as the surface/volume ratio is higher than in spherical cells (Fraga et al. 2012). The cells do not possess striking extensions like

36 wings, spines, or horns. Sinophysis (belonging to dinophysoid dinoflagellates, which can have very elaborate cell extensions like sulcal wings and cingular lists) has only sulcal lists tightly clung to the cell. These smooth and often flattened cell outlines are interpreted to reflect the life in the interstitial habitat. As mentioned above (chapter IV, p. 194) several benthic species seem to cover (protect?) their apical pore, a feature not obvious in planktonic species. The ecological significance of this morphological character is not understood yet. Attachment Several benthic genera/species have a dominating non-motile coccoid life cycle stage (vegetative cyst) attached to the sediment/substrate surface, see above ( Phytodiniales review in chapter IV). These are in most cases division cysts. Other taxa can attach by the means of stalks. Some species of the genus Amphidinium for example are able to very effectively attach to a surface with their posterior flagellum within milliseconds when disturbed (unpubl. obs.). Several taxa are known to produce large amounts of mucus, like some Prorocentrum (e.g. P. rhathymum), Ostreopsis or Coolia species. Production of mucus by O. lenticularis has been discussed in Fraga et al. (2012). Clouds of cells can live within a continuous mucus package of different shapes. These are most likely adaptations to the conditions in their benthic habitats that will protect them, e.g., for being dislocated by wave actions or desiccated at low tide. Life cycles There is almost no information about the life cycles of most benthic dinoflagellate species, except the Phytodiniales as mentioned above, Amphidinium klebsii (Barlow and Triemer 1988; synonym of A. steinii), Vulcanodinium rugosum (Zeng et al. 2012), and Ostreopsis cf. ovata (Bravo et al. 2012). Vulcanodinium rugosum has two modes of asexual reproduction including non-motile cells (Zeng et al. 2012), of which the ecological significance has not been understood yet. Sexual reproduction has only been described for Amphidinium klebsii (Barlow and Triemer 1988), Coolia monotis (Faust 1992) and Ostreopsis cf. ovata (Bravo et al. 2012). Cysts, formed as part of the sexual reproductive cycle ( resting cysts as known for planktonic taxa), are not yet known from benthic species. A tentative observation was made of resting cysts in a Bysmatrum subsalsum culture (Zinssmeister pers. comm.). It is known that Scrippsiella species produce calcareous resting cysts but none were recorded for S. hexapraecingular. For Prorocentrum lima and P. foraminosum resting cysts were described (Faust 1990b, Faust et al. 1999) but these findings need verification (Hoppenrath et al. 2013a). Cysts described for Ostreopsis cf. ovata are suggested to constitute an overwintering stage (Bravo et al. 2012), thus functionally being equivalent to resting cysts. Tide pools Benthic dinoflagellates also live in a special habitat: tide pools. As far as we know there are several species exclusively living in tide pools: Alexandrium hiranoi, Biecheleria natalensis, Bysmatrum gregarium, Dinothrix paradoxa, Durinskia capensis, Gymnodinium pyrenoidosum and Scrippsiella hexapraecingula. Some species can form dense blooms discolouring the water. Within the tide pool the species show a diurnal cycle (vertical migration) with a benthic non-motile stage (sinking to the bottom a few VI. Ecology 213

37 hours before the tide comes into the pools at high tide) and a motile stage swimming in the water column during low tide. During night time cell division takes place when they attached themselves at the bottom of tide pool. In the morning motile cells are released (e.g. Horiguchi and Chihara 1988). Bysmatrum arenicola, occurring only in the tide pools with sandy bottom, exhibits a slightly different behavior, see below. Vertical migration Vertical migration is not only known from tide pool species but also from Peridinium quinquecorne and Bysmatrum subsalsum. Peridinium quinquecorne has been reported from plankton and sediment samples (e.g. Horiguchi and Soto 1994, Murray 2003, Okolodkov and Gárate-Lizárraga 2006, Saburova et al. 2009), forming blooms in eutrophic shallow waters (Horstmann 1980, Madariaga et al. 1989, Trigueros et al. 2000). During incoming tide with intensive solar radiation it moved into a near surface layer of the water column and disappeared from the water body by attaching to benthic particles (Horstmann 1980). The species reacted to tidal changes and radiation (tidal clock with photic response) and had a remarkably high swimming speed (Horstmann 1980). Faust (1996) reported dense blooms in the water column and daily vertical migration to the bottom for Bysmatrum subsalsum. Vertical migration of dinoflagellates has also been recorded within the sediment. Amphidinium herdmanii showed strong tide related migration in tidal flats (Herdman 1921, 1922). Also Gymnodinium venator (as Amphidinium pellucidum) was reported to migrate in intertidal sand (Ganapati et al. 1959). Bysmatrum arenicola was found to show vertical migration in the sandy sediment of tidal pools, 214 VI. Ecology but never came up into the water column (Horiguchi and Pienaar 1988a). Cells migrated to the surface of the sand at low tide. At high tide they disappeared from the sand surface moving deeper. These vertical migrations are also known for further intertidal, benthic protists (like diatoms) and the rhythm seems to be of a tidal and diurnal nature (e.g. Palmer and Round 1967). The relative role of endogenous versus environmental control of the migratory behavior of benthic biofilm-forming protists (dinoflagellates not mentioned) was assessed by Coelho et al. (2011). Blooms Tide pool species can form dense blooms discolouring the water (Fig. 92C, D). Bysmatrum subsalsum can form dense blooms in the neritic/tidal plankton (water column) as reported by Faust (1996). Herdman (1921, 1922, 1924a, b) and Dragesco (1965) investigated dinoflagellate species occurring in discoloured sand patches, described the rhythmic appearance and disappearance of these cell accumulations and the spatial species distribution in the tidal area. Faust (1995) investigated species found in coloured sand from Belize. Fraga et al. (2012) reviewed the main ecological factors affecting the population dynamics of bloom-forming epiphytic dinoflagellates, in particular, the genera Ostreopsis and Gambierdiscus. These blooms are of special interest as they can produce harmful toxins. They found that the factors effecting benthic harmful algal blooms (BHABs) are very different to those impacting planktonic species (Fraga et al. 2012). In particular, Ostreopsis siamensis blooms seem to be highly variable and ephemeral on open coasts, due to daily change in wind and wave intensity and direction (Shears and Ross 2009). These

38 Fig. 92: Benthic dinoflagellate blooms visible as discolourations of the sediment or in the water column. A: Thecadinium kofoidii bloom on wet sandy beach sediment. B: Bysmatrum arenicola, discolouration on the sand surface in a tide pool. C: Bysmatrum sp. bloom, note the typical cloud-like mass. D: Dense Bysmatrum gregarium bloom, the cloud-like mass becomes obscure; typical tide pool dinoflagellate bloom. blooms will be most intense in wave protected areas following periods of calm sea conditions inducing stratification and strong surface water warming (Shears and Ross 2009). Granéli et al. (2011) demonstrated that Ostreopsis ovata grew faster at higher temperatures while highest tox- icities were recorded at lower temperatures. Thus a bloom triggered at higher latitudes under lower temperatures could have higher toxin levels and impacts on the environment (Granéli et al. 2011). The environmental conditions enhancing toxicity are species-specific and cannot be VI. Ecology 215

39 VII. Toxins of benthic dinoflagellates and benthic harmful algal blooms Introduction Benthic dinoflagellates produce a profusion of different toxic and bioactive chemical compounds, some of which are the cause of major benthic Harmful Algal Blooms (BHABs). These include at least thirty species of many genera, in particular: the Gonyaulacalean taxa Gambierdiscus, Ostreopsis, Coolia, and Alexandrium, unarmoured genera such as Amphidinium, and the genera Prorocentrum and Vulcanodinium. The most commonly reported seafood nonbacterial disease in the world is Ciguatera Fish Poisoning (CFP), which results in 50, ,000 cases per year worldwide (Fleming et al. 1998). This is due to the accumulation of toxins produced by species of the genus Gambierdiscus, which are common throughout tropical and warm sub-tropical benthic habitats worldwide (range now increased to temperate NSW). Symptoms can be severe in populations exposed to CFP over the long term. This syndrome disproportionately impacts some small island and developing nations, in the Pacific and Indian Ocean, as well as the Caribbean Sea, in which populations may consume reef fish as a major part of their diet. For example, studies in New Caledonia have estimated life time incidence rates of CFP of % of the population (Baumann et al. 2010, Laurent et al. 1992). Besides the 218 public health impacts in these nations, CFP can be an impediment to the development of fisheries export industries, which are often economically significant for small island nations (Skinner et al. 2011). CFP also occurs in developed regions such as North America, Australia, Japan and more recently, Europe. More than 1,400 cases are known from Australia in the period , including two fatalities (Gillespie et al. 1986, Hamilton et al. 2010, Stewart et al. 2010). In the last 10 years, the first possible incidences of CFP from fish caught in Madeira and the Canary Islands (Gouveia et al. 2009, PerezArellano et al. 2005, Kaufmann et al. 2013), as well as the identification of Gambierdiscus species in the Mediterranean Sea, have been reported (Aligizaki and Nikolaidis 2008, Holland et al. 2013). The possibility that species of Gambierdiscus may be expanding their ranges in sub-tropical waters has been suggested (Aligizaki et al. 2008a). In the United States, CFP is endemic to states and territories such as Florida, Hawaii, Puerto Rico, Guam, and the US Virgin Islands, with incidence rates varying widely (Dickey and Plakas 2010). In Japan, many outbreaks of CFP are known from southern Kyushu and Okinawa, including one well documented incident in Kagoshima Prefecture in 2008 (Oshiro et al. 2009, 2011), and Gambierdiscus species are widely distributed (Kuno et al. 2010, Nishimura et al. 2013). VII. Toxins of benthic dinoflagellates and benthic harmful algal blooms

40 In the past 10 years, BHABs have begun to be documented increasingly frequently in several parts of the world. Species of the genus Ostreopsis are implicated as the cause of extensive BHABs producing palytoxins (PTX), one of the most potent toxins yet investigated. PTXs can cause respiratory and other health problems when inhaled as toxin aerosols, and have had impacts on the tourism industry in some Mediterranean countries, most notably Italy and France. PTXs can also accumulate in fish and shellfish, similarly to other marine biotoxins produced by planktonic species. While less commonly associated with BHAB events, several other genera of benthic dinoflagellates produce toxins and can occasionally cause fish kills, damage growing shellfish, or accumulate in seafood. Many of these species, such as the Okadaic Acid producing species of Prorocentrum, are often associated with sub-tropical or tropical habitats more commonly than temperate habitats. However some Okadaic Acid producing species, such as Prorocentrum lima, are cosmopolitan and common in temperate to tropical habitats. As climate change impacts are increasingly felt in many parts of the world, the question of range expansion and increased HAB events due to these species will be an important issue to address (Hallegraeff 2010). Most recently, new methods have been developed to monitor for BHAB producing species, including methods based on quantitative real time PCR detection. The advantage of these methods is that genera in which species identification can be very difficult using light microscopy, for example, Ostreopsis and Gambierdiscus, can be detected at clade level. Examples include assays targeting regions of the ribosomal RNA domain in the species Gambierdiscus belizeanus, G. caribaeus, G. carpenteri, G. carolinianus, and G. ruetzleri (Vandersea et al. 2012). Assays have also been developed for the quantification of Ostreopsis cf. ovata (Perini et al. 2011). Gambierdiscus Species of Gambierdiscus produce the potent neurotoxins Ciguatoxins (CTX) and Maitotoxins (MTX) and their analogues (Yasumoto et al. 1977, Yasumoto 2005). Within vector fish and invertebrate species, the toxins can be modified to form chemical congeners whose toxicity can vary significantly (Lehane and Lewis 2000), and more than 50 different congeners of CTX are known (Yasumoto 2001). CTX analogues can accumulate through the food chain, from small fish grazing on the coral reefs into the organs of larger predators (Yasumoto et al. 1977), and lead to Ciguatera Fish Poisoning (CFP) in humans who consume the fish. MTX is the largest non-protein, non-polysaccharide compound discovered to date. Little is known about whether MTX may accumulate in the flesh of fish. There are indications that it may be able to accumulate when fish are exposed to Gambierdiscus species in sufficient quantities (Kohli et al. 2012). The role of MTX in CFP is uncertain (Holmes and Lewis 1994), and needs to be reexamined. People suffering from CFP have been reported to display an extensive and complex range of gastro-intestinal, cardio-vascular and neurological symptoms (reviewed in Friedman et al. 2008). Neurological symptoms including paraesthesias (= pins and needles ) of the extremities and circum-oral region, and cold allodynia (= pain due to a stimulus that does not normally cause pain) have been found to be the most common symptoms in one review of 3,000 CFP cases (Bagnis et al. 1985b, > 87 % of cases). Arthralgia (= joint pain) and myalgia VII. Toxins of benthic dinoflagellates and benthic harmful algal blooms 219

41 Compound name Gambierdiscus species and strain Origin Type of compound ciguatoxins (CTXs), Maitoxins (MTXs) G. polynesiensis French Polynesia CTXs, CTX-3C, -3B, -4A, -4B and M-seco-CTX-3C G. toxicus French Polynesia, Vietnam, Rangiroa Atoll CTXs?, P-CTX-3C G. australes French Polynesia, Cook Islands, Hawaii MTXs, CTXs?, 2,3-dihydroxy PCTX-3C G. belizeanus French Polynesia MTXs?, 2,3-dihydroxy PCTX-3C G. caribaeus Caribbean sites MTXs? G. carolinianus North Carolina, Florida, Puerto Rico MTXs? G. carpenteri Guam, Belize, Hawaii MTXs? G. ribotype 2 Martinique, St Thomas MTXs? G. ruetzleri Belize, North Carolina MTXs? G. excentricus Canary Islands CTXs, MTXs Table 2: Known toxins associated with species of Gambierdiscus, identified via genetics and/or SEM. Abbreviations: MBA- Mouse Bioassay, RBA- Receptor binding Assay, HELAHuman erythrocyte lysis assay, NCBA- Neuro-2a cell binding assay. 220 VII. Toxins of benthic dinoflagellates and benthic harmful algal blooms

42 Toxicity studies References LC-MS positive for CTXs, HPLC, MBA positive Chinain et al MBA negative for CTXs, RBA positive for CTXs, LC-MS positive for CTXs, LC-MS positive for CTXs Chinain et al. 1999; Chinain et al. 2010; Roeder et al. 2010; Yogi et al MBA positive for CTXs, LC-MS negative for CTXs, positive for MTXs, LC-MS positive for CTXs Chinain et al. 2010; Rhodes et al. 2010a; Kohli et al. 2012; Nishimura et al RBA positive, HELA positive for MTXs, LCMS positive for CTXs Chinain et al. 2010; Holland et al. 2013; Roeder et al HELA positive for MTXs Holland et al HELA positive for MTXs Holland et al HELA positive for MTXs Holland et al HELA positive for MTXs Holland et al HELA positive for MTXs Holland et al NCBA positive for CTXs and MTXs Fraga et al VII. Toxins of benthic dinoflagellates and benthic harmful algal blooms 221

43 Zhou J. and Fritz L Ultrastructure of two marine dinoflagellates, Prorocentrum lima and Prorocentrum maculosum. Phycologia 32: Zhou J. and Fritz L Okadaic acid antibody localizes to chloroplasts in the DSP-toxin-producing dinoflagellates Prorocentrum lima and Prorocentrum maculosum. Phycologia 33: Taxonomic index Names printed in bold are the valid names of the species dealt with in this book. Names printed in red are potentially toxic (toxin-producing) species. A Adenoides 13, 22, 23, 194, 197 Adenoides eludens 22, 23, 197 Alexandrium 24, 25, 196, 213, 218, 227, 233 Alexandrium hiranoi 24, 25, 213, 227, 233 Alexandrium pseudogonyaulax 25 Amphidiniella 13, 25, 26, 194, 196 Amphidiniella sedentaria 25, 26 Amphidiniopsis 27, 194, 195, 196, 210, 212 Amphidiniopsis aculeata 27, 29, 32, 33, 34, 36, 37, 40 Amphidiniopsis arenaria 27, 28, 30, 31, 32, 33, 36 Amphidiniopsis cristata 27, 30, 34, 35, 39, 40 Amphidiniopsis dentata 27, 28, 30, 31, 33, 36 Amphidiniopsis dragescoi 27, 31, 39, 194, 196 Amphidiniopsis galericulata 27, 28, 30, 31, 32, 33, 210 Amphidiniopsis hexagona 27, 29, 30, 32, 33, 34, 36, 37, 40 Amphidiniopsis hirsuta 27, 29, 30, 32, 33, 34, 36, 37, 40 Amphidiniopsis kofoidii 27, 28, 30, 31, 32, 33, 36 Amphidiniopsis konovalovae 27, 29, 30, 32, 33, 34, 36, 37, 40, 210 Amphidiniopsis korewalensis 27, 31, 34, 35, 39, 40, 210 Amphidiniopsis pectinaria 27, 31, 34, 35, 39, References Amphidiniopsis rotundata 27, 35, 38, 196 Amphidiniopsis sibbaldii 27, 36 Amphidiniopsis striata 27, 30, 32, 33, 34, 36, 37, 38, 40 Amphidiniopsis swedmarkii 27, 30, 32, 33, 34, 36, 37, 38, 40 Amphidiniopsis urnaeformis 37 Amphidiniopsis uroensis 27, 31, 34, 35, 37, 39 Amphidiniopsis yoshimatsui 38, 40 Amphidinium 13, 22, 41, 180, 193, 195, 201, 211, 213, 218, 227, 230 Amphidinium asymmetricum 188 Amphidinium asymmetricum var. britannicum 188 Amphidinium asymmetricum var. compactum 189, 190 Amphidinium bipes 41, 42 Amphidinium boekhoutensis 45, 47 Amphidinium boggayum 51 Amphidinium britannicum 188, 190 Amphidinium carterae 41, 42, 43, 47, 195, 211, 227, 230 Amphidinium corpulentum 51, 53, 98, 190 Amphidinium corrugatum 178 Amphidinium cupulatisquama 42, 44, 45, 50, 210 Amphidinium dentatum 55 Amphidinium eilatensis 41 Amphidinium elegans 48

44 Dinoflagellates are important primary producers, symbionts, but, at the same time, also consumers and parasites. The species composition in benthic habitats is quite distinct from planktonic habitats. The lack of comprehensive taxonomic studies of these taxa has complicated our progress in understanding dinoflagellate biodiversity, biogeography, and ecology. In recent years, benthic harmful algal blooms have attracted increasing interest because of the impact of ciguatera, the most important food-borne disease of nonbacterial origin worldwide, which is caused by benthic dinoflagellate species. These taxa seem to have widened their distribution lately. This book summarizes the knowledge about the currently known benthic dinoflagellate species for the first time. It presents the first comprehensive identification help for benthic dinoflagellates and is a basic contribution to improve monitoring efforts worldwide. About 190 species in 45 genera are presented, illustrated with more than 200 color images, about 150 scanning electron micrographs, and more than 250 drawings.