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The new taxon, Paraeustigmatos columelliferus, was isolated from a mat of the filamentous alga Zygnema on the shore of Lake Monticello in Arkansas, USA. The results of the phylogenetic analysis of 18S rDNA and rbcL sequence data suggest that this alga is likely allied with the Eustigmatales; alternately, it may represent a new third linage of the class. Light and electron microscopy reveal cellular features similar to other members of the Eustigmatophyceae; however, the cell wall bears unusual structures that resemble the columellae of the walls of pollen grains.

An algal strain from an acidic pool near the shore of Lake Macha, Czech Republic, was identified as Tetraedriella subglobosa Pascher by light microscopy. Phylogenetic analysis of nuclear 18S rDNA and plastid rbcL sequence data indicated that this alga is a member of the Eustigmatophyceae, rather than the Xanthophyceae as presently classified. This is the first report of T. subglobosa since Pascher's description of the species in 1930.

Isolates of the genera Monoraphidium Kom.-Legn., Ankistrodesmus Corda and Raphidocelis Hindak emend. Marvan et al. were cultured from two areas in Minnesota and North Dakota, USA. These isolates were identified to species level (when possible), using light microscopy and standard monographs and then characterized by 18S rDNA sequence analysis. Phylogenetic analyses indicated that in some cases, 18S rDNA sequences from these isolates were very similar, but not identical to the sequences of other isolates of the same morphospecies from different parts of the world. However, some isolates that were identified as the same species actually belong to different lineages within the Selenastraceae, whereas other isolates with identical or nearly identical 18S rDNA sequences possessed rather different morphologies. Overall, our data suggest that the application of a broad morphospecies concept to the Selenastraceae has resulted in an underestimation of the species diversity of this family and probably erroneous conclusions about the distribution of species.

We characterized 81 isolates of picoplankton and other small planktonic coccoid green algae from Lake Itasca, Itasca State Park, MN, USA. Sequence analysis and examination by light microscopy revealed three common algae: Pseudodictyosphaerium/Korshpalmella spp. (Chlorophyceae), Choricystis spp. (Trebouxiophyceae) and a previously undescribed autosporic coccoid. The Pseudodictyosphaerium and Korshpalmella species that were evaluated all possessed identical 18S ribosomal DNA (rDNA) and rbcL sequences, but this 18S rDNA sequence differed from the published 18S rDNA sequence for P. jurisii isolated from Europe. These chlorophycean algae were isolated from samples collected from Lake Itasca during all seasons. The 18S rDNA sequence of some of the Itasca isolates of Choricystis was nearly identical to the published sequence for C. minor; however, six different rbcL sequences were detected among the Choricystis isolates and none of these rbcL sequences matched published sequences for isolates from sever...

A broadly halotolerant new isolate of a small asexual coccoid chlorophyte and six new, related freshwater isolates provided the impetus for a phylogenetic analysis of the so-called ‘Nannochloris-like’ algae within the Trebouxiophyceae. Previous taxonomic disagreements concerning this group had not been rigorously tested with molecular phylogenetic analyses. We show with 1 8S ribosomal DNA (rDNA) sequence phylogeny that 19 of 22 isolates previously assigned to either Nannochloris or Nanochlorum fall within a diverse sister clade to a clade including the four ‘true’ Chlorella species sensu lato. In addition, Marvania geminata, Gloeotila contorta, Chlorella sp. Yanaqocha RA1, Koliella spiculiformis, ‘Chlorella minutissima’ C-1. 1.9, and new Koliella, Gloeotila and Marvania isolates were included in the Nannochloris-like clade. Distinct freshwater and marine or saline lineages comprise at least three major subclades, generally corresponding to cell division pattern. Seven of 14 marine or saline isola...

Current hypotheses on the distribution of freshwater microchlorophytes lead to predictions of low diversity and wide environmental tolerances. Thus, the same few species should be found worldwide in many different habitats. However, these hypotheses are based on a morphospecies concept, which precludes the possibility of numerous cryptic species among these organisms. In this study, we examined the diversity of coccoid green microalgae and chlamydomonads (Chlorophyta) isolated from sites in Minnesota and North Dakota (USA) using techniques of 18S rDNA sequence analysis. Of 93 distinct 18S rDNA sequences identified from among 273 isolates examined by molecular techniques, all but four are new to science. The spatial distribution of organisms represented by these 18S rDNA sequences was not uniform, because some lakes and ponds yielded distinct 18S rDNA types not found at other sites. In addition, organisms generally considered to be cosmopolitan, such as Chlamydomonas reinhardtii and Chlorella vulgaris, were not found. These results challenge predictions of low species number and wide environmental tolerances among these eukaryotic microorganisms.

A simple method for the purification of PCR- quality DNA from microalgae is presented. This method uses the detergent dodecyltrimethylammonium bromide coupled with cell breakage by agitation in the presence of glass beads and chloroform. A final purification step involves a commercial cartridge system. The procedure requires only about 1–2 mL of algal culture and can be completed in about 20 min. DNA suitable for PCR has been obtained from several algal lineages using this method, including numerous green algae and stramenopiles.

Through the use of molecular techniques, several different types of fusiform and twisted-fusiform microchlorophyte algae (Monoraphidium, Ankistrodesmus, etc.) have been characterized from samples taken from lakes and ponds of Itasca State Park, MN. PCR–RFLP analysis of 18S ribosomal RNA genes was used to categorize 29 different isolates from the Itasca State Park lakes into nine different types. The 18S ribosomal DNA sequences were determined for one isolate from each type for phylogenetic analysis. Light microscopy was used so that morphological characteristics as well as molecular characteristics can be compared and contrasted. The morphological characteristics were consistent for each PCR–RFLP type. Further discussion will include comparisons of the distribution and diversity of these organisms from Itasca State Park to those from Arrowwood National Wildlife Refuge, ND.

Choricystis (Trebouxiphyceae, Chlorophyta) is considered a very common member of the freshwater picoplankton. We have established a culture collection from Itasca State Park (ISP), Minnesota, and Arrowwood National Wildlife Refuge (ANWR), North Dakota, that includes many Choricystis isolates. We examined 109 Choricystis isolates from ISP and 19 isolates from ANWR using PCR-RFLP of the rbcL gene. Twelve types for ISP and 7 types forANWR were distinguished by this technique. Sequence analysis revealed additional diversity within some of the RFLP types. Moreover, none of the Choricystis isolates from ANWR possessed rbcL sequences identical to any isolate from ISP. Phylogenetic analysis of the sequence data revealed at least 2 major lineages. These results indicate that Choricystis is much more diverse than previously thought. This material is based upon work supported by the National Science Foundation under Grant Nos. DEB-0128953, DBI-0070387 and MCB

The Sheyenne River is a third order stream in central and eastern North Dakota. The algae in this river had not been previously studied. For this study, periphytic algal samples were collected Fall 1997, April–November 1998, and May–October 1999. Two hundred sixteen periphytic algal taxa were identified. These taxa included 137 Bacillariophyceae, 33 Cyanophyta, 40 Chlorophyta, 8 Xanthophyceae, and 3 Euglenophyta. Several species of algae found in the river appear to be previously undescribed taxa. Two of these organisms were described as new species, Nitzschia sheyennensis and Nitzschia dakotensis (Bacillariophyceae). The distribution of the periphytic algae was correlated to changes in the selected environmental variables using canonical correspondence analysis (CCA) and mult-dimensional scaling (MDS). The statistically significant environmental variables were nitrite-nitrate, pH, arsenic, potassium, magnesium, manganese, and total dissolved solids. A reservoir, Lake Ashtabula on the river was formed by a dam in 1954. In the initial statistical analysis, the effects of this reservoir on the periphytic algae downstream were so great that an upstream/downstream covariable had to be used. Further statistical analysis was used to determined that this effect could be correlated to changes in the nutrient levels rather than hydrologic changes.

Species of Mychonastes, Pseudodictyosphaerium, and Korshpalmella all have spherical (or nearly so) coccoid cells without pyrenoids. Mychonastes and Pseudodictyosphaerium are apparently frequent components of the picoplankton of lakes, although they had not been reported from North America prior to our studies. We have examined several isolates of Mychonastes, Pseudodictyosphaerium, and Korshpalmella from lakes and ponds in Itasca State Park, Minnesota (USA) and Arrowwood National Wildlife Refuge, North Dakota (USA). These isolates comprise 5 different 18S rDNA sequence types, none of which have a sequence identical to any published sequence for Mychonastes or Pseudodictyosphaerium. Four of these 18S types produce solitary cells, consistent with Mychonastes spp. However, other isolates all have identical 18S rDNA sequences, but are either unicellular, colonial with connecting stalks, or colonial in an amorphous matrix, consistent with Mychonastes spp., Pseudodictyosphaerium spp. or Korshpalmella spp, respectively. For some isolates, the colonial character is plastic, with different morphologies seen with different growth conditions. Ribosomal ITS and rbcL sequences also indicate little or no diversity among the isolates with various morphologies. These results suggest that the colonial habit is not a reliable generic character. Phylogenetic analyses of both rbcL and 18S rDNA sequences indicate that Mychonastes and Pseudodictyosphaerium spp. are a distinct lineage within the Chlorophyceae and should be placed in a new family. This material is based upon work supported by the National Science Foundation under Grant Nos. DEB-0128953, DEB-0129030, DBI-0070387 and MCB-0084188.

The diversity of Scenedesmus and Scenedesmus-like taxa from Itasca State Park, Minnesota was assessed using light microscopy and molecular techniques. Thirty isolates from various ponds and lakes in Itasca State Park were examined. Light microscopy showed many similarities in morphology among isolates, but PCR-RFLP analysis of the ribosomal ITS region from these isolates revealed twenty different types. A previous study from Itasca State Park using only light microscopy found only six taxa of Scenedesmus; however, our results suggest that there is much greater diversity than previously suspected. DNA sequences of the 5.8S ribosomal subunit and the ITS-2 region from our isolates are presently being determined and will be used to assess this diversity in greater detail.

Several types of fusiform microchlorophyte algae (Monoraphidium, Ankistrodesmus, and related genera) have been characterized from lakes and ponds of Itasca State Park, Minnesota, and Arrowwood National Wildlife Refuge, North Dakota. PCR-RFLP analysis of 18s ribosomal RNA genes was used to categorize 29 different isolates from Itasca State Park into 9 different types, and 15 isolates from the Arrowwood National Wildlife Refuge samples into 8 different types. 18S ribosomal DNA sequences were determined for at least one isolate from each type for phylogenetic analysis. All of these sequences were different than any sequence published in GenBank. None of the types were found in both Itasca State Park and Arrowwood National Wildlife Refuge. These results suggest that fusiform green algae are highly diverse, with little species overlap in different habitats.

The taxonomy of the genus Monoraphidium is unclear due in part to the absence of morphological features to clearly distinguish one species from another. Phytoplankton samples collected from lakes in the Arrowwood National Refuge in eastern North Dakota were found to contain several morphological species of Monoraphidium. Eighteen Monoraphidium isolates were examined with light microscopy and six morphological species were identified. PCR–RFLP of the 18S rDNA was used to type the isolates. Following digestion by Hae III and Taq I, the 18S rDNA PCR–RFLP patterns indicated 10 different types. Presently, the 18S rDNA product is being sequenced for each of the 10 types. By examining morphological characters and 18S rDNA sequences, congruence between morphology and sequence data may be compared. Also, because there is a lack of morphological characters defining Monoraphidium species, diversity within the 18S rDNA sequences may aid in the taxonomy of the genus and its place within the Chlorococcales. Supported by National Science Foundation Grants MCB-0084188 and DBI-0070387.

The coccoid algae (a.k.a ‘little green balls’) are important primary producers in freshwater communities, but very little is known about their diversity. This lack of knowledge results from the extremely small size and simple morphology of these organisms, which makes identification by light microscopy essentially impossible. However, molecular techniques allow differentiation of taxa at various levels and can ultimately lead to species identification. We have isolated several hundred cultures of coccoid algae from lakes and ponds in Itasca State Park in north-western Minnesota and Arrowwood National Wildlife Refuge in central North Dakota. Many of these isolates have been examined by light microscopy, 18S rDNA PCR–RFLP analysis, and 18S rDNA sequence analysis. In addition, some taxa with multiple isolates have been examined by rbcL and ribosomal RNA ITS sequence analysis. Our results indicate a high level of diversity, including possible new species of Chlorella, Muriella, Pseudomuriella and Mychonastes, among others. We will discuss the results of our analyses, including implications for identification of taxa and higher level systematics. Supported by National Science Foundation Grants DBI-00703867, MCB-0084188 and DEB-0128952.

The Sheyenne River is a third order stream that drains a significant portion of central and eastern North Dakota. In 1950, the Baldhill Dam was built and as a result, the reservoir, Lake Ashtabula, was formed. The periphytic algae in this river were extensively studied from 1997 to 1999. During this research, the effects of differences in environmental variables on the algal communities were assessed with Canonical Correspondence Analysis (CCA). In this analysis, the periphytic algal communities in the downstream sites appeared to be quite different from those communities found upriver of Lake Ashtabula. Other research on different rivers has shown profound changes in the algal community after a dam was built along the river. Redundancy Analysis (RDA) was used to determine if the sites downstream are more statistically similar with Lake Ashtabula than with the upstream sites. Finally, CCA was used to examine which environmental variables explain the most variance in Lake Ashtabula and the downstream sites.