Berkeleya Transfretana sp. Nov., a New Marine Diatom from Ceuta (North Africa)

: The diatom Berkeleya transfretana is described as a new species from samples collected at Playa Punta Sauciño, an intertidal coastal area in Ceuta, North Africa. Its main features are the comparatively small size in relation to other Berkeleya species and the very ﬁne and dense striae pattern (more than 40 striae per 10 µ m). The new species is here described under light and scanning electron microscopy and compared to related taxa. The benthos of these rocky beaches are interesting spots of biodiversity still to be explored.


Introduction
Diatoms are one of the most diversified groups of microalgae, with >200,000 estimated species [1], occurring in a wide variety of aquatic ecosystems. Some genera form colonies by means of characteristic structures such as macroscopic mucilage tubes. One genus, Berkeleya Greville, was first escribed as a macroalga due to its macroscopic habit and its resemblance to filamentous brown algae. This genus was described for the first time by Greville [2] for a group of pennate diatoms living within mucilage tubes attached to macroalgae or macrophytes [3,4]. The high variability of tube structures was initially used as a taxonomic character. However Grunow [5] restricted its use to the definition of varieties. Ever since, frustular ultrastructure has been regarded as the most important diagnostic feature for this genus.
In its modern sense, Berkeleya cells are either solitary or form mucilage tubes or films. Valves are linear to linear-lanceolate, with bluntly rounded or slightly capitate poles. Striae are uniseriate, consisting of simple poroids closed by hymens. External raphe endings are straight or slightly deflected towards the secondary side. The mature cingulum consists of at least five open bands, each of which bears two rows of round or oval poroids. The plastid is unique, consisting of two girdle-appressed plates connected by a narrow isthmus [6]. According to Guiry & Guiry [7], there are 45 species names of Berkeleya at present, as well as 58 infraspecific names, but only 13 of them are currently accepted taxonomically. In spite of the ecological importance of these colonial forms, they have been scarcely studied [8]. Among the studies carried out, the taxonomic works of Cox [3,9,10] in England, Mizuno [11,12] in Japan, Lobban [4,[13][14][15] in North Atlantic waters and Houpt [16] in Europe are of particular relevance. More recently, Hamsher and Saunders (2014) found un unexpected genetic diversity and extensive cohabitation among lines of the Berkeleya rutilans (Trentepohl ex Roth) Grunow complex [17].
In a preliminary account of the diatom flora of Ceuta (North Africa) [18], it came to our attention that an unknown Berkeleya population, which is here described, is a new species.

Study Site
The Autonomous City of Ceuta is a Spanish enclave located in North Africa, on the southern coast of the Strait of Gibraltar. The peninsula where it is located has a surface area of 20 km 2 and a perimeter of 28 km. The coast is composed of heterogeneous environments conditioned by its natural configuration, as well as by the intense port activity. At least 70% of Ceuta's coast is a natural hard bottom [19]. The coast of Ceuta is located in the Atlantic-Mediterranean interface, which causes important environmental and biogeographic gradients [20]. The very irregular underwater topography of this coast determines a great diversity in habitat types. The surface waters that enter from the Atlantic have an important influence, especially in the northern part of this coast [21].

Methodology
The material analyzed in this study was collected in November 2016 in Playa Punta Sauciño, a beach in the northern coast of the peninsula (Figure 1). Water parameters were measured in the field with COLEMETER portable digital probes to measure conductivity, total dissolved solids, pH and water temperature. Water chemical variables (ammonium, nitrites, nitrates, carbonate hardness and phosphates) were also determined in situ with a VISOCOLOR ECO reagent kit (Altmann Analytik GmbH & Co. KG). A sample of epilithon was collected from intermareal rocks with a toothbrush, and preserved using formaldehyde (4% v/v). A suspension of clean frustules was obtained in the laboratory by oxidation of organic matter with hydrogen peroxide (30% v/v) and heated to 70-90 • C to accelerate the reaction. Some drops of hydrochloric acid (3 M) were added to remove calcium carbonate inclusions. Light microscopy (LM) slides were mounted using a synthetic resin with a high optical refractive index (Naphrax ® ). The identification and counting of the diatom species were carried out at 1000× magnification using a DIC-light microscope (LM) Olympus BX60, equipped with an OPTIKA camera. A subsample was analyzed by scanning electron microscopy (SEM) at the Electron Microscopy Unit (University of Jaen, Spain), by placing a drop of the cleaned sample on a conductive metal structure and allowing it to dry at room temperature. The samples were subsequently coated with a 10 nm thick gold layer using a modular high vacuum metallization system (QUORUM Q150T ES). SEM images were obtained using a MERLIN (Carl Zeiss) microscope operating at 20 kV. Images were processed with GIMP software [22]. our attention that an unknown Berkeleya population, which is here described, is a new 49 species. 50

Study Site 52
The Autonomous City of Ceuta is a Spanish enclave located in North Africa, on the 53 southern coast of the Strait of Gibraltar. The peninsula where it is located has a surface 54 area of 20 km² and a perimeter of 28 km. The coast is composed of heterogeneous 55 environments conditioned by its natural configuration, as well as by the intense port 56 activity. At least 70% of Ceuta's coast is a natural hard bottom [19] . The coast of Ceuta is 57 located in the Atlantic-Mediterranean interface, which causes important environmental 58 and biogeographic gradients [20] . The very irregular underwater topography of this coast 59 determines a great diversity in habitat types. The surface waters that enter from the 60 Atlantic have an important influence, especially in the northern part of this coast [21] . 61

Methodology 62
The material analyzed in this study was collected in November 2016 in Playa Punta 63 Sauciño, a beach in the northern coast of the peninsula (Figure 1). Water parameters were 64 measured in the field with COLEMETER portable digital probes to measure conductivity, 65 total dissolved solids, pH and water temperature. Water chemical variables (ammonium, 66 nitrites, nitrates, carbonate hardness and phosphates) were also determined in situ with a 67 VISOCOLOR ECO reagent kit (Altmann Analytik GmbH & Co. KG). A sample of 68 epilithon was collected from intermareal rocks with a toothbrush, and preserved using 69 formaldehyde (4% v/v). A suspension of clean frustules was obtained in the laboratory by 70 oxidation of organic matter with hydrogen peroxide (30% v/v) and heated to 70-90 °C to 71 accelerate the reaction. Some drops of hydrochloric acid (3 M) were added to remove 72 calcium carbonate inclusions. Light microscopy (LM) slides were mounted using a 73 synthetic resin with a high optical refractive index (Naphrax® ). The identification and 74 counting of the diatom species were carried out at 1000× magnification using a DIC-light 75 microscope (LM) Olympus BX60, equipped with an OPTIKA camera. A subsample was 76 analyzed by scanning electron microscopy (SEM) at the Electron Microscopy Unit 77 (University of Jaen, Spain), by placing a drop of the cleaned sample on a conductive metal 78 structure and allowing it to dry at room temperature. The samples were subsequently 79 coated with a 10 nm thick gold layer using a modular high vacuum metallization system 80 (QUORUM Q150T ES). SEM images were obtained using a MERLIN (Carl Zeiss) 81 microscope operating at 20 kV. Images were processed with GIMP software [22] .

Results and Discussion
Phylum Bacillariophyta Class Bacillariophyceae Order Naviculales Family Berkeleyaceae Berkeleya transfretana sp. nov. (Figures 2 and 3 Table 1. Ecology: the type locality was characterized by the presence of very high nutrient concentrations (0.7 mg/L of phosphates, 0.5 mg/L of nitrates and 0.3 mg/L of ammonium) 123 Comments: the new species can be readily ascribed to the genus Berkeleya on the basis 124 of its ultrastructural features, namely the lateral central raphe slits and the uniseriate striae, 125 consisting of rows of simple poroids. Ecologically similar Berkeleya species (e.g. B. fennica 126 and B. micans) have been already recorded in western Mediterranean benthic (epilithic) 127 intermareal communities [23]. This is the first finding of a new diatom taxon from Ceuta 128 (after the description of Cocconeis nigricans var. denudata by Kützing [24] some 177 years 129 ago) and the first phycological study of the region after the review by Blanco et al. [18] 130 who found 268 different diatom taxa in 35 sampling sites (both freshwater and marine). 131 In general, cosmopolite and generalist species were found, with a scarce degree of 132 endemism, but eight new species for the African flora and four for the Spanish flora were 133 found.   Table 1. Ecology: the type locality was characterized by the presence of very high nutrient concentrations (0.7 mg/L of phosphates, 0.5 mg/L of nitrates and 0.3 mg/L of ammonium) and a comparatively low conductivity (7590 µS/cm), with a turbidity of 9390 ppm (total dissolved solids) and a pH of 7.3. Berkeleya transfretana was scarce in the material analyzed, accounting for 5.3% of individuals occurring in the sample. Accompanying species include Navicula perminuta Grunow (14.6%), Nitzschia inconspicua Grunow (13.9%) and Pteroncola inane (Giffen) Round (12.6%), which reveals a community typical of transitional (freshwatermarine) waters. No other Berkeleya taxa were recorded in the type locality.
Comments: the new species can be readily ascribed to the genus Berkeleya on the basis of its ultrastructural features, namely the lateral central raphe slits and the uniseriate striae, consisting of rows of simple poroids. Ecologically similar Berkeleya species (e.g. B. fennica and B. micans) have been already recorded in western Mediterranean benthic (epilithic) intermareal communities [23]. This is the first finding of a new diatom taxon from Ceuta (after the description of Cocconeis nigricans var. denudata by Kützing [24] some 177 years ago) and the first phycological study of the region after the review by Blanco et al. [18] who found 268 different diatom taxa in 35 sampling sites (both freshwater and marine). In general, cosmopolite and generalist species were found, with a scarce degree of endemism, but eight new species for the African flora and four for the Spanish flora were found.
The lack of similar studies in this biogeographically important area prevents any comparative analysis, although the finding of floristic novelties such as B. transfretana make the Northern coast of Africa worthy of further taxonomic investigations.