Differences in Rubisco Large Subunit (rbcL) gene sequences among populations of the freshwater alga Heribaudiella fluviatilis.

John D. Wehr


The Phaeophyceae is a diverse class of multicellular photosynthetic protists, commonly called brown algae. They range in size from microscopic forms to large seaweeds, and occur from arctic to tropical biomes (Bold and Wynne 1978). The Phaeophyceae comprise roughly 265 genera and between 1500 and 2000 species (De Reviers and Rousseau 1999), but less than one percent (seven species) are known to colonize freshwater environments (Starmach 1977, Wehr 2003). Despite their low diversity, species freshwater brown algae have been described and basic ecological studies have been conducted for roughly 150 years. Until recently their classification has been based solely on morphological, reproductive, and ultrastructural characters (Bold and Wynne 1978, Pueschel and Stein 1983, Wehr 2003). Their presumed rarity and apparent disjunct status (Wilce 1966, Wehr & Stein 1985) also raises questions of possible genetic isolation and biogeographic status world-wide.

A number of molecular studies on many marine taxa have emerged within the last ten years (e.g. Tan and Druehl 1994, Draisma et al. 2001, Burrowes et al. 2003). These studies have altered perceptions of the classification of species within the class, as well as their evolutionary and genetic history.

Recently, McCauley (2002) completed the first study using 18S rDNA small subunit and RUBISCO large subunit (rbcL) information to determine the correct placement of freshwater genera within the brown algal class. Her studies demonstrated that (1) all taxa presumed to be members of the Phaeophyceae belong in the class; (2) some of the genera (Heribaudiella, Bodanella, Porterinema) previously placed in the order Ectocarpales do not belong in that order, and (3) the more variable rbcL sequences were effective in discriminating among genera and even among populations of the same species. Sequences from Heribaudiella fluviatilis from western Canada North America were differed from a European population by roughly 31 bp (out of ca. 1100 bp), but by < 10 bp from each other (McCauley 2002). Heribaudiella requires further attention, as it is the mostly widely reported freshwater species (Wehr & Perrone 2004), yet the distribution record is still disjunct, with many populations collected from Europe, a few from Japan, one from China, and about 30 from western North America (Wehr 2003).

This study aims to characterize some of the variation in rbcL gene sequences among populations of the freshwater brown alga Heribaudiella fluviatilis, by comparing nucleotides sequences from two previously described populations (western Canada, Germany) with three newly described populations from Washington state.


Figure 1-Figure 1. Initial visualization of PCR products obtained from first amplification of whole genomic DNA obtained from populations of Heribaudiella fluviatilis (a2 =Ashnola River, BC; G2 = Granite Creek, BC; P1 & P2 = Peshastin Creek, WA; E1, E2 = Entiat River, WA; M1, M2 = Mad River, WA; Lad = ladder; X = negative control).

Figure 2-Figure 2. Visualization of re-amplified PCR products obtained from second amplification of PCR products using nested primers 4 and 5 only, obtained from populations of Heribaudiella fluviatilis (a2 =Ashnola River, BC; G2 = Granite Creek, BC; P1 & P2 = Peshastin Creek, WA; E1, E2 = Entiat River, WA; M1, M2 = Mad River, WA; Lad = ladder; X = negative control).

Figure 3-Figure 3A. Alignments of rbcL sequences obtained from Mad River, WA (5M), Entiat River, WA (5E), Ashnola River, BC (5A), and Peshastin Creek, WA (5A), using reverse primer 5 (rbc-R3). Figure 3B. Alignments of rbcL sequences obtained from Mad River, WA (5M), Entiat River, WA (5E), Ashnola River, BC (5A), and Peshastin Creek, WA (5A), using reverse primer 5 (rbc-R3).

Figure 4-Figure 4. Alignments of rbcL sequences obtained in the present from Peshastin Creek (4P), and from Ashnola River, BC (a2) and Baden Würtemberg, Germany (Heri 13) by McCauley (2002).


Populations. Heribaudiella populations were identified and collected in August 2003 as crusts on rocks from three streams in central Washington state: Peshastin Creek, Entait River, and Mad River (Chelan County, near Leavenworth, WA). Previously sampled populations included Ashnola River (British Columbia; ca. 200 km N of Washington populations) collected December, and Baden Würtemberg (Germany), collected in 1986.

Whole genomic DNA was extracted using the Qiagen DNeasy Plant Mini-Kit; PCR products were gel purified before sequencing.
- DNA was amplified using PCR and checked for size (600 to 1100 bp) against a molecular ladder using 0.8% agarose gel.
- Three sets of primers homolgous to different locations along the rbcL gene were based on previous studies:

Primer Direction Sequence (5'-3') Annealing position Reference

JDW 1 & 6
LMrbcL17F F ATG CCT GAA GAT GTG CA rbcL 1-17 McCauley (2002)
LMrbcL1207R R TAC CAC CAC CAA ATT GTA GA rbcL 1187-1207 McCauley (2002)
JDW 3 & 7
LMrbcL130F F ACT GAT ATT CTA GCT CTT T rbcL 111-130 McCauley (2002)
LMrbcL1378R R CCA TAA ATC TAA AGC CGC T rbcL 1397-1378 McCauley (2002)
JDW 4 & 5
rbc-F2.5 F TTC CAA GGC CCA GCA ACA GGT rbcL 454-474 Kawai et al. 2000
rbc-R3 R CCT TTA ACC ATT AAG GGA TC rbcL 1040-1021 Kawai et al. 2000

Sequencing was Nucleotide sequences were determined using the dideoxy chain termination method, with an Amplicycle Sequencing Kit (Applied Biosystems), using single primers from primer set JDW 4 &5, listed above. Nucleotides were read into files using the MacVector program.

Data Analysis
Verification: Sequences were first compared against known sequences using BLASTn on the NCBI website (http://www.ncbi.nlm.nih.gov/BLAST/), to determine if the sequences obtained (1) were from the rbcL gene and (2) likely represent DNA from brown algae, based on their similarity to published algal sequences.
Alignment: The ClustalW program was issued to align the sequences obtained in the present study, and to compare these with Heribaudiella sequences from Ashnola River (British Columbia) and Baden Würtemberg (Germany).

Portions of the rbcL gene were weakly amplified from whole genomic DNA using the three primer sets described above. Bands were seen in lanes corresponding to two of the primer sets for some of the populations (Fig. 1). The most distinct bands were from Peshastin (P2), Entiat (E1, E2) and Mad River M2 populations amplified using primer sets 3 and 7. Next, all putative PCR products were re-amplified using just the nested (internal primer set 4 and 5). Results from this step were more pronounced, with strong bands produced from Ashnola (A2), Peshastin (P2), Entiat (E1, E2,) and Mad (M2) stream populations (Fig. 2).

BLASTn Results

Results of BLAST n tests of rbcL sequences (150 to 180 bp) from Heribaudiella populations demonstrated that all of portions of these sequences were most closely similar (score in bits; and % identity) to rbcL sequences from marine brown algae (Table 1).

Population Primer 4: Nearest taxon Score % Primer 5: Nearest taxon Score %

Ashnola, R. BC (A2) Padina japonia rbcL 139 90 Sphacelaria caespitula rbcL 103 83
Entiat R., WA (E2) Padina japonia rbcL 149 89 Syringoderma phinneyi rbcL 119 87
Mad R., WA (M2) Padina japonia rbcL 133 88 Sphacelaria caespitula rbcL 123 85
Peshastin, WA (P2) Padina japonia rbcL 149 89 Syringoderma phinneyi rbcL 105 85

All of the nearest taxa were small filamentous marine members of the Phaeophyceae.

Alignment Results: North America

Results of alignment tests using Clustal W) from Heribaudiella populations demonstrated very high homology among the Washington and British Columbia rbcL sequences (Fig 3). Using the forward primer sequences, ClustalW found between zero and four bp differences among the Washington populations of Heribaudiella, in the first 130 bases (Fig. 3A). In the same region, six bp differences were observed for the British Columbia population. Using the reverse primer sequences, ClustalW identified between zero and four bp differences among the Washington populations of Heribaudiella, in the first 130 bases (Fig. 3A). In this region, the Ashnola (BC) population differed by no more than two bp from the other three I collected from Washington State. Overall, the Peshastin Creek and Entiat River populations appeared most similar in their rbcL sequences.

Alignment Results: North America versus Germany

A third contrast compared the rbcL gene sequence of Heribaudiella collected in present study from Peshastin Creek (WA; this study), versus sequences obtained by McCauley (2002) from homologous regions from Ashnola River (BC) and Baden Würtemberg (Germany). In this comparison, roughly 160 homologous pieces of about 160 bp were compared (Fig. 4). The ClustalW alignments indicate that sequences from the two North American populations differed by at least two bp (additional nucleotides were undetermined “N”). In contrast, the German population differed from either North American population by 12 to 15 bases.


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Wehr, J.D. 2003. Brown Algae. In Wehr, J.D. & Sheath, R.G. [Eds.], Freshwater algae of North America, pp. 757-773. Academic Press, San Diego, CA.

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I thank Jinsong Qiu and Brian Fox for their expert and cheerful guidance in the laboratory. I also thank Dr. Sylvia Anderson for preparing the DNA primers and offering advice on how to salvage PCR products poorly amplified by the author. Thanks also go to Dr. Berish Rubin for offering space in his laboratory, his excellent instruction and continued patience.

This document was last modified 01/31/2006.
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