Elsevier

Protist

Volume 158, Issue 3, 18 July 2007, Pages 325-336
Protist

ORIGINAL PAPER
Observations on the Diversity and Ecology of Freshwater Nannochloropsis (Eustigmatophyceae), with Descriptions of New Taxa

https://doi.org/10.1016/j.protis.2007.03.003Get rights and content

The genus Nannochloropsis is well known from the marine environment but has only recently been reported from fresh and brackish waters. A single species, N. limnetica, was first documented from shallow lakes in Germany, where it produced spring blooms. A second unnamed isolate from a river in the United States has been characterized by sequence analysis and light microscopy. All of the Nannochloropsis species that have been described, both marine and freshwater, are small spheres with essentially no distinguishing morphological characteristics. Therefore, they must be characterized using molecular techniques. We have cultured numerous isolates of Nannochloropsis from a series of lakes on the James River in the Arrowwood National Wildlife Refuge, North Dakota, USA, and 1 isolate from a pond in Itasca State Park, Minnesota, USA. The diversity among these isolates was determined by light microscopy and DNA sequence analysis. Seven distinct haplotypes of Nannochloropsis were found, one of which possesses 18S rDNA and rbcL sequences identical to those of N. limnetica from Europe. The 6 new haplotypes vary in rbcL sequences and some are morophologically distinct from each other and from N. limnetica. These types are described as the new taxa N. limnetica var. globosa, N. limnetica var. irregularis, N. limnetica var. dystrophica, and N. limnetica var. gutta. All of the Nannochloropsis isolates from Arrowwood and Itasca were cultured only from samples taken during cold-water periods. These results suggest that Nannochloropsis species may be better adapted to cold water conditions, including temperatures near 0 °C and ice cover.

Introduction

Nannochloropsis is a genus comprised of very small (less than 5 μm) coccoid unicells and is known primarily from the marine environment. Some isolates of this genus are important food organisms for aquaculture because they reproduce very rapidly and possess fatty acids that are not found in other types of phytoplankton (Apt and Behrens 1999). The five species that have been described from the marine environment cannot be discriminated by either light or electron microscopy, and therefore the species have been delimited primarily by DNA sequence analysis (Andersen et al. 1998; Karlson et al. 1996; Krienitz et al. 2000; Suda et al. 2002).

Krienitz et al. (2000) were the first to describe a freshwater species of Nannochloropsis, N. limnetica, from a hypertrophic pond and a polytrophic lake in Germany. They isolated this organism from the hypertrophic site and characterized it by sequence analysis of the 18S rDNA, pigments, lipids, light microscopy and transmission electron microscopy. Nannochloropsis limnetica often produced a bloom, with density of up to 5.7×109 cells per liter. Another, unnamed, species of Nannochloropsis (Nannochloropsis sp. 2, Andersen et al. 1998) was isolated from an estuarine site in North Carolina, USA. The 18S rDNA sequence of this isolate differs from that of N. limnetica by only 2 indels out of 1759 nucleotides (Krienitz et al. 2000) and reportedly can grow in freshwater media (R.A. Andersen, personal communication in Krienitz et al. 2000). Nannochloropsis sp. was also reported among the coccoid algae isolated from winter samples from lakes in North Dakota, USA, with identification based on 18S rDNA PCR-RFLP analysis (Phillips and Fawley 2000). Recently, Fietz et al. (2005) identified 3 new strains of Nannochloropsis limnetica from Lake Baikal using 18S rDNA sequence analysis. However, RAPD-PCR analysis showed that the Lake Baikal strains differed from each other, and from the German Nannochloropsis limnetica isolate.

Although both Andersen et al. (1998) and Krienitz et al. (2000) suggested that 18S rDNA is adequate for species characterization in the genus Nannochloropsis, Suda et al. (2002) demonstrated that the rbcL sequences provided over 3 times the variability of 18S rDNA for the molecular characterization of Nannochloropsis species. Suda et al. (2002) concluded that rbcL sequences should be used to distinguish species of Nannochloropsis that cannot be distinguished by morphology.

Our sampling and culture work from the lakes of the Arrowwood National Wildlife Refuge, North Dakota (USA) resulted in a number of isolates tentatively identified as Nannochloropsis sp. by 18S rDNA PCR-RFLP analysis (Phillips and Fawley 2000). In this study, we extend our previous work by examining isolates from samples collected throughout the year from the Arrowwood sites and also isolates from samples obtained from water bodies in Itasca State Park, Minnesota (USA). Isolates were further characterized by sequence analysis, focusing mainly on rbcL, but with some 18S rDNA sequences generated for additional phylogenetic context.

Section snippets

New Taxa Descriptions

Nannochloropsis limnetica Krienitz, Hepperle, Stich and Weiler var. globosa var. nov. (Fig. 1 G).

Cellulae solitariae, globosae vel ovoideae vel guttiformes, pariete laevi, papilla unica prominente praedito. Chromatophorum unicum parietale flavo-viride sine pyrenoide. Cytoplasma granulis plerumque non mobilibus instructa. Dimensiones cellularum 2.5–6.5×3.0–7.0 μm. Propagatio 2 vel 4 vel 6 autosporis. Ordine nucleotidorum geni dicti ‘rbcL’ a taxis ceteris generis differt.

Cells solitary, globose to

Discussion

Twenty-four isolates from the lakes of Arrowwood National Wildlife Refuge, ND, were identified as Nannochloropsis sp. and one additional Nannochloropsis isolate was identified from a dystrophic pond, Tower Pond, in Itasca State Park. We found 8 different rbcL sequences among isolates from the Arrowwood lakes and 1 additional sequence from the ISP isolate. One of these sequences was identical to the rbcL sequence from the type culture of the only previously described freshwater Nannochloropsis,

Methods

Site descriptions: Arrowwood National Wildlife Refuge contains four shallow mesotrophic lakes and marshes on the James River that are stabilized by low weirs. For this study, we sampled four sites, the north end of Arrowwood Lake, the south end of Arrowwood Lake, the south end of Mud Lake and the south end of Jim Lake. For a more comprehensive description of these sites and their algal flora, see Phillips and Fawley, 2002a, Phillips and Fawley, 2002b and Fawley et al. (2004). These four sites

Acknowledgements

We thank Matt Hoffman and Joni Johnson for technical assistance, and Dr. Phillip McClean and Rian Lee for the use of the Beckman sequencer. We thank Mark Vanimann at Arrowwood National Wildlife Refuge for his interest and help in this research. We thank Dr. Lothar Krienitz for the culture of Nannochloropsis limnetica KR1998/3. This material is based on work supported by the National Science Foundation under Grant Nos. DBI-00703387, DEB-0128952, and MCB-0084188, and grants from North Dakota

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