Mucilaginous species of Thalassiosira Cleve emend . Hasle ( Diatomeae ) in South Brazilian waters

Mucilaginous species of Th alassiosira Cleve emend. Hasle (Diatomeae) in South Brazilian waters). Th alassiosira is a large diatom genus intensively studied since the 1970s, being well represented in diverse marine environments worldwide. Despite the long Brazilian coastline, about 8,500km, few taxonomic works have been performed to investigate the morphology of Th alassiosira species using electron microscope techniques. In this paper the mucilaginous species Th alassiosira diporocyclus, T. mala and T. minuscula were examined in light and electron microscopes, based on material gathered from South Brazilian waters, to record their frustule morphological variability. Some unrecorded fi ndings related to the cribra structure and the cingulum bands are shown. T. mala has central areolae bearing fewer cribral pores than those located in the marginal region. In addition, the valve metrics of T. diporocyclus and T. minuscula are extended in relation to that reported in the current literature. Th e two species are new records for Brazilian waters, and T. diporocyclus is a new one in the Western Atlantic Ocean.


Introduction
Since the seminal studies of Helmcke & Krieger (1953;1954) and Hasle and Fryxell (Hasle 1972a;Hasle & Syvertsen 1997 and references therein) using electron microscopy to unveil the fine structure of valve and cingulum of Th alassiosira Cleve, much knowledge has been gathered about genus morphology.For the time being, the genus Th alassiosira encompasses more than 150 species, and many more species surely are to be described in diverse marine areas worldwide.Surprisingly, the extensive Brazilian coastline, about 8,500 km, has been relatively poorly studied regarding the taxonomy of Thalassiosira.Even in South Brazil, where many investigations focusing on diatom taxonomy have been carried out, only a few works are available for Th alassiosira using electron microscopy (Torgan & Santos 2006, 2007;Garcia & Odebrecht 2099a, 2009b).Previous contributions relied on light microscope observations, generating basic surveys and checklists (Moreira Filho et al. 1990).Th ese authors listed 12 species and 2 varieties in South Brazil.Further, Procopiak et al. (2006) reviewed the studies on the diatoms of Paraná coastal waters spanning from 1918 to 2005, and recorded 20 species and 5 varieties, all of them identified through light microscopy.More recently, Garcia & Odebrecht (2009b) recorded 17 species in Rio Grande Sul.
Ecological studies in the shelf waters off Paraná state, South Brazil, pointed out the signifi cant contribution of Th alassiosira to the phytoplankton (Brandini & Fernandes 1996;Brandini et al. 1997;2007;Fernandes & Brandini 2004).Net plankton material of Paraná and additional samples from Santa Catarina and Rio Grande do Sul states were made available to start a survey on Thalassiosira species, examining their composition and frustule morphology using electron microscopy.
Th e high species richness found in the study area is related with oceanographic features such as the dominance of Shelf Water, a water mass fl owing southwestward resulting from mixing of the warm, salty and nutrient poor Tropical Water and the Coastal Water, nutrient rich and aff ected by land drainage.During summer, the deeper cold South Atlantic Central Water (SACW) mix with the Tropical Water over the shelf, eventually reaching the photic zone and promoting phytoplankton growth (Brandini 2006).In austral autumn and winter, the nutrient rich colder Subantarctic Water, fl owing northeastward carried by the Malvinas Current, mixes with the Shelf Water and transports northward temperate diatom species from Argentina and Uruguay (Fernandes & Brandini 2004).Th us far, about 58 Th alassiosira species were reported for Argentinean waters, some of them abundant in the plankton (Lange 1985;Vouilloud 2003).Th e role of the Malvinas Current in periodically changing the species richness along the Paraná coast is evident.Indeed, species typical of cold water like Th alassiosira nodulolineata, T. poroseriata and T. tumida have been detected in previous ecological studies (Fernandes & Brandini 2004), all of them regular constituents of Argentinean plankton (Lange 1985;Olguin et al. 2006).Our sampling grid covered all the seasonal oceanographic variability described above, in the Paraná shelf waters.
In this fi rst report we describe three abundant small species of Th alassiosira from South Brazilian shelf waters, aiming to record their morphological variability and to compare their metric data with material from other marine environments based on the current literature.

Material and methods
Field samples were collected monthly from August 1997 to March 1999 in fi ve stations located 10-15km apart along the Paraná coastal shelf (25°42'-25°50'S; 48°27'-48°55'W), with water depth between 10 and 50 meters.Vertical net hauls were carried out from bottom to surface in each station using a plankton net of 40 μm mesh size.Formaldehyde was immediately added to the samples, reaching 2% fi nal concentration.Additional samples were examined from material collected in Florianópolis (27°37'51.97"S;°22'25.83"W),Santa Catarina state, and in shelf waters off Rio Grande do Sul state (30°12'40.10"S;49° 8'13.91"W)during the Brazilian Antarctic Expedition PROANTAR XIII in 1994.
In the laboratory, samples were submitted to the Hasle & Fryxell (1970) technique for cleaning the frustules and examination in light and electron microscopes.About 280 permanent slides for light microscope (LM) were prepared with Naphrax (r=1.74) as mounting medium.Scanning electron microscopy (SEM) preparations were made by pipeting some drops of samples directly over the aluminum stubs, and brought to a JEOL JSM 6360LV microscope for observations at 10-15 Kv acceleration voltages and a working distance of 8 millimeters.Samples containing high numbers of valves were prepared for transmission electron microscope (TEM) examination.A small drop was put in nickel mesh 200 grids covered with Formvar and Carbon, and air dried.The JEOL JEM 1200EXII microscope was used at 80 Kv acceleration voltage.

Th alassiosira minuscula
Light Microscope: Cells cylindrical with many parietal ellipsoid chloroplasts.Th e cells are enclosed in circular pads of mucilage joined together to form large colonies (Fig. 24-28).Valve circular, valve diameter 16-23 μm.Surface areolated; areolae barely perceptible even in phase contrast microscopy.Striae pattern fasciculate, almost indistinct (Fig. 29-32).One ring of fultoportulae is present at the marginal area (Fig. 29).Rimoportula is large and elongate, marginal, but discreetly displaced towards the valve center in relation to the fultoportulae ring (Fig. 30-31).One or two fultoportulae are placed nearer the rimoportula than the others (Fig 31).One single fultoportula can be hardly seen at the valve center (Fig 31).

Th alassiosira mala
Light Microscope: Cells joined in dense mucilaginous colonies.Valves circular, valvar diameter 3-12μm.Valvar surface areolated; areolae faintly noticeable (Fig. 48-49).One marginal ring of fultoportulae.An almost undistinguishable fultoportula is located at the middle distance between the margin and the center, and always aligned with the rimoportula.Rimoportula larger than the fultoportulae and marginal placed between the ring of fultoportulae (Fig. 48).

Discussion
Th e material studied did not reveal too much discrepancy regarding valve morphology when compared to data from the literature (Tables 1, 2, 3).T. diporocyclus and T. minuscula had the number of marginal areolae in 10 μm increased from 32-36 to 39-41, and from 32-39 to 36-45, respectively (Hasle 1972b;Rivera 1981).T. mala showed the greatest variability in the number of cribral pores per areola, especially the small frustules, in which the central areolae have as few as two pores while the marginal region 8-16 pores.Th e pictures consulted in the literature listed in Table 3 did not allow us to record the number and possible shift ing of cribral pores in T. mala from other world regions.
When viewed in light microscope T. diporocyclus, newly recorded for the Western Atlantic Ocean, may be easily confounded with Thalassiosira subtilis (Ostenfeld) Gran.Th is species has the rimoportula slightly displaced to the valve center regarding the marginal ring of fultoportulae (see illustrations of Hasle 1972b andHallegraeff 1984), that is, not aligned as in T. diporocyclus.Th ere is a higher number of fultoportulae scattered on the valve surface of T. subtilis than in T. diporocyclus, in which they are organized in two rings (Hasle 1972b; this study).On the other hand, this distinction is weakened as the valve size diminishes for the areolae of T. subtilis become arranged in rings.In such a circumstance, rimoportula position is the best criterion to diff erentiate the two species.Additionally, the external opening of T. subtilis is elongate and gives rise to a tube, while subrectangular and, as far is known, lacking external tube, in T. diporocyclus.Comparing T. diporocyclus with T. minuscula, the latter has one ring of marginal fultoportulae, not two like in T. diporocyclus; *: Described as forming sectors or radial, but author's illustrations show fasciculate pattern sensu Johansen & Fryxell (1985).and there is one or two fultoportulae closely associated with the rimoportula of T. minuscula, a feature not observed in T. diporocyclus.Rivera (1981) documented a long external tube in the rimoportula of T. minuscula.In our material, the tube was detected only in one valve, illustrated in Figure 38.
T. diporocyclus and T. mala are new records for Brazilian waters, even though they were abundant during the study off Parana (F.P. Brandini, unpublished data) besides T. minuscula.Their small dimensions could explain the absence of previous records, but T. diporocyclus and T. minuscula occur in dense gelatinous colonies, which should make them easy to detect during routine microscope counting or identifi cation.Previous researchers collected material only in shallow waters off Paraná, mainly in estuarine areas, where T. diporocyclus and T. mala were not detected (Fernandes & Brandini 2004, Procopiak et al. 2006, Lehmkuhl et al., 2010).On the other hand, lugol preserved samples may destroy the mucilage, and the species would be named or counted as "unidentifi ed centrics" or "Th alassiosira spp.". (e.g.Brandini & Fernandes 1996;Fernandes & Brandini 2004).Once these small diatoms have been reliably described, their identifi cation during routine plankton surveys will be greatly improved and increase the resolution of ecologically oriented projects.*: Described as radial, but author's illustrations show fasciculate pattern sensu Johansen & Fryxell (1985).To the south of our study area, T. mala and T. minuscula were already reported in Argentinean waters, reaching high density in late winter and early spring (Gayoso 1989;Ferrario & Sar 1988;Sar et al. 2001;2002;Vouilloud 2003).In our material from Paraná state, these species were abundant in late spring, when they comprised the main phytoplankton biomass.The spring maximum accompanied the overall intensive growth of diatoms due to the seasonal upwelling of SACW cold nutrient rich waters off Paraná coast from November to February (Brandini et al. 2006).Other authors also recorded an increase in diatom biomass during enrichment events, especially in spring and summer (e. g.Licea 1992;Aké-Castillo et al. 1999).
T. mala is a potentially harmful microalgae inhabiting coastal regions (Takano 1976).It presumably caused economical losses in Tokyo bay, Japan due to intense mucilage production and consequent obstruction of the fi ltering gills of oysters cultivated in marine farms (Takano 1956 as T. decipiens).Losses were estimated in 58 millions of yens at that time.
None of these species were reported in previous investigations (v.review of Moreira-Filho et al. 1990;Procopiak et al. 2006;Tremarin et al., 2008) in Paranaguá Bay, a large estuarine environment near the coastal shelf where our study was carried out.It might be that the lower salinity, ranging from 18 to 32, precluded the growth of the three species in the bay.Salinity in the adjacent coastal waters where our samples were collected ranged from 34 to 37.
Material examined: Paraná, Santa Catarina and Rio Grande do Sul.Abundant in November and December in Paraná coast; temperature 21-26 o C, salinity 34.0-36.5.

Table 1 .
Comparative valve measurements for T. diporocyclus recorded from the literature and the present study.
*: Described as radial or forming sectors, but author's illustrations show fasciculate pattern sensu

Table 2 .
Comparative valve measurements for T. minuscula recorded from the literature and the present study.

Table 3 .
Comparative valve measurements for T. mala recorded from the literature and the present study.