Biodiversity of free-living flagellates in Kuwait’s intertidal sediments

Abstract Taxonomic data of free-living benthic fl agellates in Kuwait’s intertidal sediments are summarized. A full list of the species composition is presented, including distribution on diff erent sediment types, species occurrence and light micrographs for each taxon identifi ed. A total of 67 fl agellate species were identifi ed, representing six classes. Most of them are reported from Kuwait for the fi rst time. Th e most abundant and diverse species were sand-dwelling dinofl agellates (43 taxa).


Introduction
Th e marine coast of Kuwait, which extends for about 170 km, is composed of a variety of coastal habitats (Al-Yamani et al. 2004).Kuwait's coast may conveniently be divided into two primary regions, which refl ect the position of Kuwait's off shore energy zones.In the north, suspended material from the Shatt al-Arab delta has settled to form extensive intertidal mud and sand fl ats within the rather protected low-energy zone of Kuwait Bay.Intertidal sediments grade from mud in the north and west of the Bay, where limited salt marshes may occur, to medium or fi ne sand beaches at the entrance of the Bay.On the more exposed open coast south of Kuwait Bay in high-energy ar-eas, medium and coarse sand beaches extend down to the Saudi Arabian border and beyond.Within these two broad categories, however, there are several other shoreline types, which can grade into one another.Th us, all stages of mud, sandy mud, muddy sand, sand, sandy-rocky fl ats, or rocky shore can be found along the coastline of Kuwait, often in close proximity of each other (Jones 1986a, b).
Th is unique and very productive component of Kuwait's marine ecosystem is poorly studied in terms of its microbenthic community.Previous investigations of marine protists in some parts of Kuwait's coastline focused on their general productivity and/ or diversity especially for the diatom/cyanobacteria component of the microalgal community (e.g.Hendey 1970, Clayton 1986, Jones 1986b, Hoff man 1996, Al-Yamani et al. 2004, Al-Zaidan et al. 2006), whereas information on taxonomy and ecology of benthic fl agellates in Kuwait's coastal zone is totally lacking.Many of the benthic fl agellates are abundant in intertidal substrata and their contributions to benthic and shallow marine ecosystems may be signifi cant.Several species from the benthic fl agellated community are known to be potentially toxic; therefore, the study of their biology and potential toxic reactions in shallow waters is important.
Th e aim of this study was to document the biodiversity of benthic fl agellates along the Kuwaiti coastline.Additionally, their abundance and distribution on diff erent sediment types are reported.

Methods
We selected 14 localities along Kuwait's coast (Fig. 1) for our investigation.Each study site stretched for about 100 m and consisted of one or more of the following substrates: mud, sandy mud, muddy sand, or sand.In total, we collected 127 sediment samples from diff erent intertidal heights during 2005 to 2007.Using plastic tube corers, we sampled the top 3 to 4 cm of sandy sediment, or 3 to 4 mm of muddy sediment.Th ese samples were transported to the laboratory where fl agellates were separated from the sediment using Uhlig's (1964) frozen seawater method and a 110 μm mesh fi lter.Flagellates were collected in a Petri dish beneath the fi lter and examined alive with a Leica DMIL inverted microscope at ×35 to ×200 magnifi cation.For detailed observations, fl agellates were isolated by micropipette and examined with a Leica DMLM microscope or with a Carl Zeiss Axiovert 200M microscope using transmitted-light with a bright fi eld and phase contrast at ×400 to ×1000 magnifi cation.Flagellate plate patterns were made using Calcofl uor White M2R (Fritz and Triemer 1985).Th e cells were examined on an epifl uorescent (violate excitation ca 430 nm, blue emission ca 490 nm) Axiovert 200M microscope.We examined both the dorsal and ventral sides of each fl agellate, and obtained micrographs using either Axiovert 200M microscope equipped with an AxioCam HRc digital camera or a Leica DMLM microscope with a Leica DFC 320 digital camera.Cell size was measured by light microscopy using a calibrated ocular micrometer, and some morphometric measurements were obtained from micrographs using Carl Zeiss Axio Visiton 3.0 software.All observations presented here are based on light microscope investigations of freshly-collected living cells.
We used the taxonomic classifi cation scheme of Th rondsen (1997), which was a partially modifi ed classifi cation of Christensen (1962Christensen ( , 1966)).Th e recent taxonomic position of Protaspis is presented in accordance with Adl et al. (2005).Species names are listed in alphabetical order within each class (Table 1).

Results and discussion
Flagellated inhabitants of Kuwait's intertidal sediments were very diverse and mostly composed of sand-dwelling dinofl agellates, euglenids and cryptomonads.We identifi ed a total of 67 fl agellate species (Table 1, Figs 2-7); most of them being reported from Kuwait from the fi rst time.Table 1.List of species encountered during the survey of free-living fl agellates associated with intertidal sediments along the coastline of Kuwait, 2005Kuwait, -2007.Abbreviations for substrate are as follows: 'M'mud, 'MS' -muddy sand, 'S' -sand, 'P' -small saline ponds, 'MA' -macroalgae; categories of occurrence are: 'C' -common, 'F' -frequent, 'R' -rare; light microphotographs: fi gure numbers are followed by the numbers of the photographs.Th e diversity of the fl agellated group was mainly due to sand-dwelling dinofl agellates and euglenids.Amphidinium, with 17 species, was among the most abundant and diverse sand-dwelling dinofl agellate genera.Within this genus, the large, unarmored A. scissum was widely distributed throughout the year and may be the most abundant on Kuwait's intertidal sand fl ats.Th is species was occasionally found in other habitats.
A limited number of benthic dinofl agellates are potentially harmful, as they are capable of producing toxins, which may result in an intoxication of the marine environment.Among taxa of sand-dwelling dinofl agellates recorded in Kuwait were eight species that must be considered potentially harmful.Prorocentrum concavum      has been reported to produce three diol esters of okadaic acid and ichthyotoxin (Yasumoto et al. 1987, Hu et al. 1993).Prorocentrum lima may produce okaidic acid and dinophysistoxins, which may cause diarrhetic shellfi sh poisoning (Murakami et al. 1982, Tindall et al. 1984, Torigoe et al. 1988, Lee et al. 1989, Hu et al. 1993).Prorocentrum rhathymum may produce water-soluble fast-acting toxins and hemolytic eff ects (Nakajima et al. 1981, Tindall et al. 1989).In the summer of 1999, a bloom of P. rhathymum (reported as P. mexicanum) caused a massive fi sh kill in Kuwait Bay (Al-Yamani et al. 2004).Coolia monotis is known to produce cooliatoxin, which is presumably related to yessotoxin (Holmes et al. 1995).It may be involved in ciguatera (Tindall and Morton 1998).Haemolysins, compounds toxic to fi sh, have been isolated from Amphidinium carterae (Yasumoto et al. 1987).Amphidinium gibbosum may produce cytotoxic metabolites, the most potent of which (caribenolide I) had anti-tumor eff ects (Bauer et al. 1994, Bauer et al. 1995a, b, Maranda and Shimizu 1996).Haemolytic and antifungal properties (amphidinols) are reported from A. operculatum; it may also be toxic to fi sh (Yasumoto et al. 1987).Peridinium quinquecorne can cause anoxia and fi sh kills, if occurring with very high cell densities (Fukuyo et al. 1990).Th ese potentially harmful dinofl agellates were present in Kuwait's benthic microalgal community during 2005 to 2007, but never occurred in great numbers.Euglenids comprised auto-and heterotrophic species.Among them, Anisonema acinus was the most common and widespread species in Kuwait's muddy and sandy sediments.Petalomonas minor and Urceolus sabulosus were mainly recorded in intertidal mudfl ats of Kuwait Bay, where they can be quite abundant.
Th e highest species diversity of fl agellates in Kuwait's soft sediments was associated with the southern intertidal sand fl ats.Together with benthic diatoms and cyanobacteria, autotrophic fl agellates are likely to be among the most important contributors to primary productivity in the intertidal zone.Heterotrophic species may play an important role in intertidal food webs, consuming even large diatoms and other fl agellates.

Figure 1 .
Figure 1.Area of investigation.A Arabian Gulf, with inset showing the greater region in which the sampling area was located B Map of Kuwait showing sampling sites (sites located within low-energy and high-energy zones are labeled with black dots and black squares, respectively).