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Introduction
Diatoms are usually referred to as goldenbrown microalgae, due to the color of their plastids and pigment composition, mainly carotenoids (fucoxanthin, diadinoxanthin, diatoxanthin), which mask chlorophylls a and c.
The pennate marine diatom, Haslea ostrearia (Gaillon) Simonsen, is a tychopelagic or benthic organism, remarkable for its ability to produce a water-soluble blue pigment, commonly called marennine that accumulates mainly in the apical regions of the cells. In oyster ponds of western France, where H. ostrearia regularly dominates, marennine is released into the seawater and is responsible for the “greening” of oysters. This phenomenon, having a locally significant economic impact, has long been known (Sprat 1667). The involvement of a microscopic alga was recognized later but still a long time ago, and the organism was first described as Vibrio ostrearius (Gaillon 1820; Dyer 1877), then as Navicula ostrearia, and finally transferred to the genus Haslea (Simonsen 1974).
At present, the genus Haslea comprises more than 20 species that may show significant differences in their morphology, ecology or physiology. For example, Haslea wawrikae is a planktonic diatom with very long and acute apices, and Haslea gigantea is a mesoplanktonic species with a cell length of up to 500 μm (Simonsen 1974). Most other Haslea species are known for being benthic organisms, and Haslea crucigera is a tube-dwelling species.
Diatoms with blue tips, recorded as H. ostrearia, have also been reported from almost all seas and oceans, in both the Northern and Southern Hemispheres, either from direct observations or through the greening effect on bivalves. In Northwestern Europe, records come from the coasts of Great Britain (Sprat 1667; Hustedt & Aleem 1951), Belgium (M’harzi et al. 1998), Norway (Hendey 1964), Sweden (Wulff et al. 2000), and the Baltic Sea (Snoeijs & Kasperoviciene 1996). In the northern Atlantic Ocean, “blue diatoms” have been found in the Canary Islands (SCCAP, Gert Hansen), different places along the North American coast (Kennett & Hargraves 1991) and possibly in Honduras (Grunow 1877). In the northern Pacific Ocean, H. ostrearia has been reported from the San Juan Islands (Hardy 1973) and from Japan (Ranson 1937). In the Southern Hemisphere, diatoms identified as H. ostrearia have been observed in the Indian Ocean (Simonsen 1974)and reported from eastern Tasmania (Volkman et al. 1994).
A recent approach combining SEM, molecular barcoding and reproductive biology has resulted in the discovery of H. karadagensis Davidovich, Gastineau & Mouget, a new species of bluish diatoms in the Black Sea (Gastineau et al. 2012). In July 2009, a strain of diatoms with blue apices was isolated from the French Mediterranean shores at Boulouris, and kept in the Scandinavian Culture Collection of Algae and Protozoa (SCCAP) under the name of Haslea provincialis (Gastineau et al. 2016). These records suggest that the microalgae producing a blue pigment have a worldwide distribution.
In this paper, we report on a possibly new species from the genus Haslea from the Croatian shore of the Adriatic Sea and its morphological as well as molecular identification.
Materials and methods
Study area and sampling site
In August 2012, diatoms with dark bluish apices were found in Saharun Bay on the island Dugi Otok, near the settlements of Božava, located in the northeastern Adriatic Sea (Croatia) (Fig. 1).
Figure 1
Sampling site in Croatia, Dugi Otok island at Saharun Bay, the Adriatic Sea (Google maps)
Samples were collected from stagnant pools approximately 2–7 m from the seashore harboring ca. 0.3-cm-thick microbial mat, using a sterile scalpel. Haslea sp. densely covered the upper part of Padina sp. at a depth of 5–10 cm (Fig. 2), usually growing in solitary cells.
Figure 2
Field population of Haslea sp. Typical bluish color of diatoms attached to Padina sp. is clearly visible
Subsamples for DNA analysis were preserved in guanidine thiocyanate solution (GES) in 2 ml Eppendorf tubes. GES-solution included 5 M guanidine thiocyanate, 100 mM EDTA with pH 8.0 and 3.4 mM N-lauroyl sarcosine (Abed et al. 2006). Subsamples for further microscopic analysis and morphometry were preserved in 3% formaldehyde solution in environmental water.
Light microscopy and morphometric analyses
Light-microscopic analyses and photomicrographic documentation were carried out using transmitted light, phase contrast, Nomarski differential interference contrast (DIC), illumination and epifluorescence.
Morphometric analysis of cell dimensions was carried out from in-scale camera Lucida projections and scanned photomicrographs using Sigma Scan Image software (Jandel Scientific, Sausalito, CA) with its statistics package. Differences in dimensions were expressed as mean ± standard deviation (n = 30–50).
Molecular analyses
The total DNA was extracted from 1 ml of the Haslea guanidine thiocyanate suspensions using the UltraClean Soil DNA Isolation Kit (MoBio Laboratories, USA) and following the procedure described by Palinska et al. (2006). Five freeze (liquid nitrogen) and thaw (water bath at 60°C) cycles were applied for maximum yields. The 18S ribosomal RNA (rRNA) gene was amplified with the diatom-specific primers described by Zimmermann et al. (2011). The PCR reaction of 50 μl contained 1 × RED Taq PCR Buffer, 200 μM of each deoxynucleotide, 100 μg BSA, 250 ng of each oligonucleotide primer, 2.5 U of RED Taq DNA polymerase (Sigma-Aldrich), and 5µl of DNA extract. The presence of purified PCR products (QIAquick PCR Purification Kit, Qiagen, Germany) was detected by standard agarose gel electrophoresis (1.5%) and ethidium bromide staining.
DNA samples were directly sequenced in both directions by a commercial sequencing laboratory, using the same primers as for amplification. The combination of D512for 18S and D978rev 18S primers (Zimmermann et al. 2011) enabled the amplification of a 466 bp fragment.
The obtained 18S rRNA sequence was aligned with selected GenBank entries using clustalW (http://www.ebi.ac.uk/clustalw/index.html). The percentage identity of the 18S rRNA gene fragment of the studied Haslea population with other sequences from the GeneBank was calculated using the NCBI BLASTN program, version 2.2.10. Sequences were blasted to confirm the identification (http://blast.ncbi.nlm.nih.gov/Blast.cgi).
Results
Morphology
Living cells of Haslea sp. showed two narrow band-like, parietal plastids (Fig. 3) lying appressed on each side of the valve margin and not reaching the apex as observed in other species of Haslea. Cell tips were characteristically intensely blue coloured. This peculiar bluish pigment accumulating at the cell apices does not occur outside the genus Haslea, supporting a close taxonomic relationship within the said genus. Only faint striation was visible in LM (Fig. 3); cells were 41.5 to 90.4 μm long (average 60.1 μm), and 5.8 to 7.8 µm wide (average 7.6 µm) (Table 1).
Morphological features of Haslea sp. (this study) compared with those of H. ostrearia, H. provincialis, H. karadagensis, H. crucigera and H. pseudostrearia obtained from the literature
Haslea sp.
Haslea ostrearia
Haslea provincialis
Haslea karadagensis
Haslea crucigera
Haslea pseudostrearia
Length (μm)
60.1
71.8
65.8
52.5
55.5
95–97
Width (μm)
7.6
7.3
7.4
8.0
8.8
11–121
Color of cell apices
blue
blue
blue
blue-grey
no
no
Figure 3
Light microscopy of Haslea sp., with parietal chloroplasts and apices filled with blue pigment. Scale bar = 30 μm
Diversity of 18S rRNA gene sequences
Our analysis of 18S rRNA gene sequences has revealed that Haslea sp. from Saharun is phylogenetically extremely similar to H. provancialis, strain RG-2015a in 99.7% and H. ostrearia in 97.16%. Further, Haslea sp. showed 94.09% similarity with the sequence from the strain H. spicula BA28, as well as 94.33 and 94.34% sequence similarity with the strain H. crucigera and H. pseudostrearia (Table 2), respectively.
18S rRNA gene fragments sequence similarities (%) among Haslea strains
Haslea sp. (this study)
Haslea ostrearia
Hasle pseudostrearia
Haslea crucigera
Haslea spicula BA28
H. ostrearia
97.16
H. pseudostrearia
94.34
98.06
H. crucigera
94.33
97.99
97.75
H. spicula BA28
94.09
98.35
98.06
98.87
H. sp. RG-2015a
99.70
96.96
93.64
93.62
93.31
Discussion
At present, the genus Haslea includes more than 20 species that show significant differences in their morphology, ecology or physiology.
The first molecular phylogeny of some Haslea species constructed using chloroplast 16S rDNA (Pillet et al. 2011) supported the division of this genus into several subgenera. However, with the recent discovery of the new species of blue diatoms: H. karadagensis,
H. provincialis as well as Haslea sp. described here from Croatia, the relative positions of different clusters of species within the genus Haslea and the status of the genus deserve further studies.
Haslea ostrearia Gaillon/Bory (Simonsen) was considered the only species with the worldwide distribution, known for its capacity to synthesize a blue pigment – marennine, which accumulates at cell apices (for historical considerations, see Briée 2010). Recently, new species of diatoms with blue apices producing marennine-like pigments, including the one described here, have been discovered, which questions the alleged uniqueness of H. ostrearia and unravels the blue diatom biodiversity.
Blue pigmented hasleoid diatoms were previously found on Padina sp. in the Gulf of Naples (Funk 1919), on the shores of different Italian islands (Solazzi & Tolomio 1976; Tolomio 1978; Barone 1979), the Tyrrhenian Sea (Tolomio 1984) and the Adriatic (Molisch 1903; Höfler et al. 1956; Solazzi et al. 1972; Tolomio 1976; 1982; 1988; Tolomio & Bullo 2001a,b). In the above cases, however, the identification of diatoms was based only on light microscopy (LM) and the decisive feature was the presence of blue tips. Our study presents the first description of the Adriatic blue Haslea sp. based on light microscopy and 18S rRNA gene fragment sequencing.
Our results demonstrated that the possibly new species of Haslea sp. is morphologically similar to H. ostrearia, H. karadagensis and H.provincialis (Table 1, Fig. 3). Pigmented cells of Haslea sp. are difficult to distinguish from H. ostrearia and H. provincialis, but the pigment color distinguishes it from H. karagadensis. All three species are also genetically separated.
Morphological data show a lower maximum length of Haslea sp. compared to H. ostrearia and H. provincialis and higher compared to H. karadagensis, suggesting that the taxa should be separated. Further, the comparison of molecular data supports the recognition of the new species of diatoms within the same Haslea genus, capable of producing blue pigments.
To date, H. karadagensis and H. provincialis have only been found in their distinct areas of discovery, i.e. H. karadagensis in the Black Sea and H. provincialis on the French shores of the Mediterranean Sea. Interestingly, the molecular analyses reveal that each area is inhabited by a genetically distinct subpopulation of H. ostrearia. One of them occurs in the Atlantic Ocean, the other one in the Kattegat Strait between Sweden and Denmark, and the third one inhabits Coffin Bay in Australia (Gastineau et al. 2013; 2014). Similar distribution patterns have also been documented for Thalassiosira rotula, another cosmopolitan diatom (Whittaker et al. 2012). Our results, describing Haslea sp. only from the Croatian coast as well as showing differences in the overall sequence similarity and morphology compared to other Haslea species inhabiting different geographical areas, support the view that geographically specific distribution may occur among diatoms. Further, we agree with Gastineau et al. (2016) who suggested that H. provincialis, and in our opinion possibly also Croatian Haslea sp., may have been observed in the Mediterranean Sea already before. They were reported by e.g. Sauvageau (1906) and various Italian authors, but confused with H. ostrearia. Unfortunately, due to the lack of preserved specimens or cultures from these studies, no comparison is possible at the moment. It is necessary, however, to reisolate Haslea-like, blue-pigmented diatoms from the entire region of the Mediterranean Sea to verify this statement using modern approaches to species identification.
The discovery of this possibly new species of Haslea sp. suggests that Haslea diversity has been underestimated and needs further detailed studies. This also raises questions about previously published reports and observations of Haslea distribution in the Mediterranean Sea whether these organisms really belong to H. ostrearia.
With the recent discovery of the new species of blue diatoms, the relative positions of different clusters of species within the genus Haslea and the status of the genus deserve further studies. Our study suggests the existence of a new Haslea species, however, this assumption has still to be confirmed by reproductive studies, checking the possibility of interbreeding and sequencing further gene fragments.
