Two new marine species of Cocconeis (Bacillariophyceae) from the west coast of Sweden

This paper is part of a project of studying benthic diatom biodiversity on marine coastal regions of Sweden with focus on rare and less known species. Two new species of Cocconeis Ehrenb. are described from Vrångö, a small island in the west coast of Sweden. Both species were found as epiphytic on the green alga Ulva intestinalis L. Cocconeis magnoareolata Al-Handal, Riaux-Gob., R.Jahn & A.K.Wulff sp. nov. is a small species not exceeding 9 μm in length and characterized by having large subquadrangular areolae on the sternum valve. Cocconeis vrangoensis Al-Handal & RiauxGob. sp. nov. appears similar to some taxa of the ‘Cocconeis scutellum complex’, but differs by its stria density on both valves and variable features of the areola and valvocopula ultrastructure. Detailed descriptions based on light and electron microscopy examination, a comparison with closely related taxa, as well as a description of the habitat of both species are here presented.


Introduction
Cocconeis Ehrenb. is one of the most diversifi ed genera of diatoms in terms of number of species and variability of habitats. It is found in all types of aquatic habitats from freshwater to sea water, from the tropics to the polar regions, with a wide variability in valve ultrastructure (Riaux-Gobin 1991; European Journal of Taxonomy 497: 1-16 (2019) Riaux-Gobin & Compère 1996;Romero & Navarro 1999;De Stefano et al. 2000;Suzuki et al. 2001;Riaux-Gobin et al. 2015). Although some taxa are found in the plankton (Sar et al. 2003), the majority of the species of Cocconeis are epiphytic, attached by its raphe valve to various types of macroalgae (De Stefano et al. 2008). New species of Cocconeis are always being described and the number of species given by VanLandingham (1979) (320 species) has increased remarkably during the last three decades. According to Guiry & Guiry (2018), 812 species and intraspecifi c taxa of Cocconeis have been published, although several have not been validated.
Morphological similarities between numerous closely related taxa of Cocconeis have led to uncertainties in the identifi cation of many species. Because of this, several works attempted to typify such species by examining fi ne structure of type material and compare it with recent specimens examined under electron microscopy (e.g., De Stefano et al. 2008;Jahn et al. 2009). One of such problematic species is C. scutellum Ehrenb., with ca 15 varieties being separated on the basis of the fi ne structure of the areolae and valvocopulae (De Stefano et al. 2008). Such distinction between several varieties is important in the context of biodiversity, but it seems rather unhelpful in ecological surveys where morphological features may not appear different using light microscopy.
The present work describes C. magnoareolata Al-Handal, Riaux-Gob., R.Jahn & A.K.Wulff sp. nov. and C. vrangoensis Al-Handal & Riaux-Gob. sp. nov. as two new marine species, using light microscopy and scanning electron microscopy. Both species were found as epiphytic on the chlorophyte Ulva intestinalis L. These two new species may, under light microscopy, look like members of the 'Cocconeis scutellum complex', but their ultrastructure appear different.

Material and methods
Samples were collected from the eastern side of Vrångö Island (57°34´ N, 11°47´ E), in the strait of Kattegat on the west coast of Sweden (Fig. 1). Vrångö Island is part of the archipelago of Kattegat which, together with Skagerrak strait, connect the Baltic Sea with the North Sea. Thalli of the chlorophyte Ulva intestinalis were collected from the intertidal zone and kept in a plastic container fi lled with water from the same locality to which 10 ml of glutaraldehyde were added for preservation. In the laboratory, samples were vigorously shaken to remove diatom frustules. The suspension containing diatoms was fi rst washed with deionized water by fi ltration and then boiled in 30% hydrogen peroxide for 20 min. Few milliliters of 50% hydrochloric acid were added to remove the carbonates. After several rinses with deionized water, one milliliter of the cleaned diatom suspension was left to dry on a cover slip and then mounted in Naphrax ® . Light microscopy images were taken using a Zeiss Axioimager 2 microscope with differential interference contrast objectives at the Botanisches Museum, Berlin. Scanning electron microscopy images were taken using Hitachi S-4500 SEM operated at 5 kv in Perpignan University, France, and a Hitachi S-4500 SEM in Botanisches Museum, Berlin, Germany.

Results
Ulva intestinalis is a widely distributed macrophyte on all the coasts of Sweden, from the brackish water of the Baltic to the marine water on the west coast (Leskinen et al. 2004). The exam of the epiphytic diatoms on this macrophyte revealed a number of species in varying occurrence and densities. The most abundant species, which covered most of the thallus, was Striatella unipunctata (Lyngb.) C.Agardh. Several taxa of Cocconeis were observed in moderate densities such as C. scutellum Ehrenb., C. stauroneiformis (W.Sm.) Okuno, C. magnoareolata sp. nov., and C. vrangoensis sp. nov. Some other rare taxa, including species of the genera Mastogloia Thwaites ex W.Sm. and Fragilaria Lyngbye, as well as several other unidentifi ed species were present. Salinity of the surface water in the Kattegat varies between 25 and 30 psu, being affected by water discharge from rivers and by water masses coming from the Baltic Sea (Kristiansen & Eyvind 2015). Both new species were found as epiphytic on Ulva intestinalis which were common in marine water pools between rocks at the high tide level. These species were not found on other macroalgae, including brown and red algae, collected from the same site, nor from the sediment in the Vrångö Island location.

Etymology
The epithet given to the species refers to the large shape of the areolae on both valves.

Type material
Holotype ( Isotype SWEDEN • Same data as for the holotype; Department of Biological and Environmental Sciences, University of Gothenburg, slide V001.

Sternum valve
Externally, fl at to weakly convex with short mantle . Axial area narrow and straight, reaching valve apices (Fig. 17). Striae uniseriate, reaching valve margin, weakly radiate at the middle and curved on apices. Areolae large, subquandragular with round corners, becoming slightly oblong on the apices (Figs 17-18). Areolae occluded by reticular hymenes with short slits arranged on the margin of the areolae (Fig. 19, arrow). Internally, virgae strongly silicifi ed and more prominent than on the external face, sternum relatively large and straight, ending by a triangular hyaline area on apices (Fig. 16). Areolae more oblong than on the external side but the last areolae near valve margin are round (Fig. 16). SV valvocopula open, fi mbriae short and triangle-like (Figs 20-21).

Cocconeis vrangoensis
Valves broadly elliptic to slightly rhombic, with round apices. Apical axis 13-16 μm (13.9 ± 2.6), transapical axis 12-14 μm (12.9 ± 0.6, n = 72). Raphe straight, ending slightly before apices. Striae parallel in the middle, becoming radiate towards the apices, 24-26 in 10 μm in the RV and 14-16 in 10 μm in the SV. RV with a silica ridge close to the valve margin and following the curvature of the valve, wider near the center and narrower towards the apices.

Etymology
The specifi c epithet of the new species refers to the location where it was collected.

Sternum valve
Convex on the external side, valve face fl at to slightly depressed, with a wide mantle (Fig. 30). Axial area narrow and plain with no central area and terminating before the mantle (Fig. 30). Striae monoseriate becoming tri-to tetraseriate on the mantle. Virgae narrower than the striae and strongly raised on the internal valve face (Fig. 29). Areolae round to sub-quadrangular on both sides (Figs 29-30), sometime the septum between two adjacent areolae become very low or disappear and the areolae appear oblong with round corners (Figs 28-29). In SV internal side, the areolae are complex structure formed of 5-7 chambers (hymenate openings), one in the center and the others surrounding the central one (Fig. 32). The areolae on the mantle are long and composed of several small openings (Fig. 31). Each of these chambers is occluded by hymenes with linear perforations radially arranged (Figs 32-33). Valvocopula closed (Fig. 36), fi mbriae thin, with fi nger-like extensions. On the advalvar side of the valvocopula, basal parts of the fi mbriae are depressed and form grooves below the outer edge of the valvocopula (Fig. 35).

Raphe valve
Externally, RV slightly concave with marginal part of the mantle hyaline and void of areolae (Figs 37-45, arrow). Raphe straight, proximal raphe endings simple and close to each other, terminal raphe endings slightly widening and terminate shortly before apices (Figs 37-45). Axial area very narrow, central area round and small. RV striae uniseriate except on the mantle where they become biseriate. Areolae round and occluded by hymenes with thin perforations radially arranged (Fig. 43). Internally, the RV face is slightly convex with very narrow hyaline area on the inner side of the mantle which is  strongly raised (Figs 40-42). Mantle margin folded inwards with a hyaline area void of areolae (Fig. 41,  arrow). Raphe straight, proximal raphe endings defl ected in opposite directions, terminal raphe endings terminating shortly before the apices and with weakly developed helictoglossae (Figs 40-42). Axial area very narrow, central area small, strongly silicifi ed, round and raised above valve surface (Figs 40-42). Valvocopula with long and thin fi mbriae ending by a hammer-like structure with an oblong papilla (Fig. 45, arrow). Between each pair of well-developed fi mbriae there are short extensions that may be vestigial fi mbriae (Fig. 44, arrow).

Comparison with Cocconeis arenicola
Cocconeis magnoareolata sp. nov. with its large areolae may be compared to C. arenicola Ryznik, described from tidal fl ats in the Yaquina Estuary, USA, but the later differs by having larger valves (22-27 × 17-20 μm) and by its striae density, almost one third of that in our new species (7 vs 21-22 in 10 μm). Although C. arenicola was abundant in Yaquina Estuary, it has not been observed at any other geographic location. The type slide of this species (ANSP, slide no. 62634) has been examined. R.Z. Riznyk made seven small circles on the cover slip, and only two of these contain valves of C. arenicola. New images have been taken and illustrated here (Figs 8-9) to represent sternum and raphe valves. Sternum valve appears conforming to original description but the raphe valve does not seem to correspond to the description of the type by Riznyk (1973: pl. V, fi g. 6). Striae density of both sternum and raphe valves described by the author is different from that observed in the type specimens. R.Z. Ryznyk gave seven striae in 10 μm on both valves, while the specimens in the type slide show 9-9.8 and 12-16 in 10 μm for sternum and raphe valves, respectively. Because of the very brief original description of this species, as well as of the varying of some features of the type specimens, an emended description is given below based on LM.
The ultrastructure of both C. arenicola and C. nitens has not been studied and therefore comparison with fi ne structure of C. magnoareolata sp. nov. is imposible. However, under LM, C. magnoareolata sp. nov. may have similarity with some taxa of the 'Cocconeis scutellum complex', but the fi ne structure of the areolae on both valves is rather different, e.g., being reticular on the SV (Fig. 18) and with short slit-like perforations located on areolae margin on the RV (Fig. 27). The SV valvocopula is also very different from that of the 'Cocconeis scutellum complex' (Fig. 21).
Morphologically, C. vrangoensis sp. nov. bears similarities to C. sagaraensis Hid.Suzuki and to some members of the 'Cocconeis scutellum complex', such as C. scutellum var. posidoniae M.De Stefano, D.Marino & L.Mazzella. Cocconeis sagaraensis differs by having pointed valve apices, coarser striae on both valves, with 5-7 and 12-14 striae in 10 μm on SV and RV respectively. In the internal face of the RV of C. vrangoensis sp. nov., the region between the hyaline rim and the valve margin has several rows of areolae (Figs 40-42), while in C. sagaraensis only one row of areolae is seen (Suzuki et al. 2005: fi gs 10, 13). The structure of the SV valvocopula is completely different between these two species.
In C. sagaraensis, the SV valvocopula possesses complex thick structures, while the valvocopula in C. vrangoensis sp. nov. is thin with slender fi mbria (Fig. 34).
Cocconeis vrangoensis sp. nov. is similar to C. scutellum var. posidoniae by having almost the same valve outline and striae density, but it can be distinguished by its different structure of areolae and valvocopulae. The SV areolae of C. scutellum var. posidoniae are different in shape and structure where it is composed of two radial bars and with hymenes that are radially arranged (described by De Stefano et al. (2000) as rotate hymenes) on the areolae margins. The SV and RV valvocopulae have fi mbriae that are attached in their outer parts forming large pores while fi mbriae of the SV valvocopulae of C. vrangoensis sp. nov. are plain slender extensions. The RV valvocopulae are hammerhead lobes alternating with short plain lobs in C. vrangoensis sp. nov. (Fig. 44), a feature that is not seen in any member of the 'Cocconeis scutellum complex'.
Under LM and with certain oblong specimens, C. vrangoensis sp. nov. may look slightly similar to C. stauroneiformis, particularly the SV (De Stephano et al. 2008: 237, fi gs 87-92). Stria density and complex areola structure, however, are different between these two species. The RV of C. stauroneiformis can be readily distinguished by the shape of its central area on the RV where it expands narrowly to the sides in a form of fascia reaching the valve margins.
Both C. magnoareolata sp. nov. and C. vrangoensis sp. nov. may appear as members of the 'Cocconeis scutellum complex' due to general morphological similarities. Cocconeis magnoareolata sp. nov., however, is far from this complex, based on its valve size (very small), its areola shape and size (subquadrangular and large), hymenes and SV valvocopulae structure (see above). Cocconeis vrangoensis sp. nov. can be separated from the 'Cocconeis scutellum complex' by having denser striae and different stria density on both valves, whereas most of the members of the 'Cocconeis scutellum complex' possesses almost similar stria densities on both valves except for C. scutellum var. posidoniae, and by some variations in areola and valvocopula structure.
The fourteen varieties of C. scutellum bear enough morphological similarities and led Hustedt (1933) to consider them as synonyms, a concept that also was followed by Hendey (1951). Nevertheless, the examination under SEM revealed structural differences that validate these varieties (e.g., De Stefano et al. 2008). The differences are mostly found in the structure and shape of the valvocopulae, such as the shape of fi mbriae, whether the fi mbriae have free or united ends, the presence and shape of the papillae (see discussion in Riaux-Gobin et al. 2013). According to Holmes et al. (1982), the particular shape or the structure of the valvocopulae is species-specifi c. The fi ve lineages of the 'Cocconeis scutellum complex' proposed by Mizuno (1987), which were based on valve outline and stria density, need to be verifi ed by molecular analysis.

Small valve size range
One of the noticeable characters of both new species is the small valve size range. Although a large number of specimens was measured (60 for C. magnoareolata sp. nov. and 72 for C. vrangoensis sp. nov.), yet valve size distribution was rather small. Valve length variation was only 2.5 μm in C. magnoareolata sp. nov. and 3 μm in C. vrangoensis sp. nov. The distribution of both species is not yet known. They were only found on one macrophyte (Ulva intestinalis) although several other macrophytes and sediment samples from the same site were examined. The very small size of both species may not allow for wide variation in valve dimensions. There are, however, a number of environmental factors that affect cell size in diatoms. It has been demonstrated that small cell-sized diatom taxa with high surface area to volume ratio are more effi cient in nutrient uptake with more ability to harvest light (Reynolds et al. 2002;Litchman et al. 2006).