Community analysis of aquatic periphyton using the rbcL gene

Catharina Grubaugh


The rbcL gene, which codes for the large subunit of ribulose-1,5-bisphosphate carboxylase oxygenase (RuBisCO), has been used to barcode algae. Because RuBisCO is a key enzyme in photosynthesis, the rbcL gene is a molecular marker linked to the ecosystem function of algae (Paul et al. 2000). Most algae have one of three subtypes of RuBisCO Form I (Badger and Bek 2008). Sets of primers have been developed to amplify genes coding for a region of the IA and IB Forms, which are found in green algae and cyanobacteria, and for a region of the ID Form, which is found in non-green algae (Paul et al. 2000).

The rbcL gene has been used for whole community analysis of marine phytoplankton (Paul et al 2000, Wawrik and Paul 2004) and phytoplankton found in the effluent from wastewater treatment plants (Ghosh and Love 2011). However, the rbcL gene has not been used for whole community analysis of algae attached to surfaces, known as periphyton.

The purpose of this study was to determine if primers for a 615bp region of the IA and IB Forms of the rbcL gene can be used to assess the biodiversity of green algae and cyanobacteria in periphyton environmental samples. DNA was extracted from two aliquots of the same periphyton sample, and an approximately 600bp region of the rbcL Form IA and IB genes was sequenced. The sequences were compared to known sequences to assess the biodiversity of cyanobacteria and green algae present in the stream, and the community composition of the two aliquots were compared to one another to assess the reproducibility of this method.


Periphyton was sampled on 1 April 2012 from Peters Kill, a rural stream located northwest of New Platz, NY. DNA was extracted from each aliquot on different days. Primers from Paul et al. (2000) were used to amplify a 615 bp region of the Form IA and IB of the rbcL gene.

Following PCR purification, the PCR product was ligated into pGEM-t plasmids and transformed into JM109 cells. The closest match to each DNA sequence was determined using a Basic Local Alignment Search Tool (BLAST) nucleotide search (NCBI). A total of 87 sequences (38 from Aliquot A and 49 from Aliquot B) were matched to sequences in GenBank. Sequences were aligned, and similar sequences were grouped together into the same genotype.


--PCR with the Form IA and IB primers produced a product approximately 600bp in size (Fig. 1). The amount of PCR product decreased with increasing DNA concentration, indicating the presence of a PCR-inhibiting substance in the DNA extract.

--Sequences with 92% or higher nucleotide similarity were grouped together as the same genotype, producing nine genotypes (Table 1). Three of the genotypes matched rbcL barcodes of specific species: Ulothrix zonata, Stigeoclonium helveticum, and Pseudendoclonium akinetum, all of which are green algae.

--Both aliquots had a genotype richness of seven, and the genotype with the highest frequency in both aliquots was U. zonata.

--The community compositions of the two aliquots were significantly different from the community composition that would be expected if the two aliquots were the same (p < 0.05, chi-squared = 20.4, df = 8).


--The most common genotype in both aliquots was U. zonata, a dominant periphyton species in aquatic systems during the winter and early spring, when this sample was taken, at latitudes similar to that of the site where the sample was taken (McMillan and Verduin 1953, Graham et al. 1985).

--The community compositions of the two aliquots in this study were significantly different. This difference could be due to slight differences in aliquot storage.

--Future studies should increase the number of algal rbcL barcodes identified to species to improve the resolution and precision of whole-community periphyton analyses.

Literature Cited

Badger, M. R. and E. J. Bek. 2008. Multiple Rubisco forms in proteobacteria: their functional significance in relation to CO2 acquisition by the CBB cycle. Journal of Experimental Biology 58: 1525-1541.

Ghosh, S. and N. G. Love. 2011. Application of rbcL based molecular diversity analysis to algae in wastewater treatment plants. Bioresource Technology 102: 3619-3622.

Graham, J. M., J. A. Kranzfelder, and M. T. Auer. 1985. Light and temperature as factors regulating seasonal growth and distribution of Ulothrix zonata (Ulvophyceae). Journal of Phycology 21: 228-234.

McMillan, G. L. and J. Verdiun. 1953. Photosynthesis of natural communities dominated by Cladophora glomerata and Ulothrix zonata. The Ohio Journal of Science 53: 373-377.

Paul, J. H., A. Alfreider, and B. Wawrik. 2000. Micro- and macrodiversity in rbcL sequences in ambient phytoplankton populations from the southeastern Gulf of Mexico. Marine Ecology Progress Series 198: 9-18.

Wawrik, B. and J. H. Paul. 2004. Phytoplankton community structure and productivity along the axis of the Mississippi River plume in oligotrophic Gulf of Mexico waters. Aquatic Microbial Ecology 35: 185-196.


Figure 1-PCR results (run on a 1.2% agarose gel) from amplification of a region of the rbcL gene in Aliquot B using primers for rbcL Forms IA and IB. DNA amounts were 0 ng (negative control, NC), 1 ng, 5 ng, 10 ng, and 40 ng. Reaction volume was 25Ál.

Figure 2-Table 1: Community composition of each aliquot, expressed as a percentage of the total sequences in each aliquot matching a particular genotype. % NT indicates the range of percent similarity of the sequences in each genotype to the closest match for that genotype in GenBank.


Previous studies have designed primers for regions of the rbcL gene for use in whole-community analyses of phytoplankton. The purpose of this study was to determine if the rbcL gene can be used to assess the biodiversity of green algae and cyanobacteria in two aliquots of an environmental sample of periphyton. Each of the aliquots had a genotype richness of seven, and the most common genotype in both was Ulothrix zonata. The community compositions of the aliquots were significantly different. Future studies may identify more rbcL sequences from periphyton species, increasing the resolution and precision of whole-community analysis.

Full Paper


I would like to thank Dr. Rubin, Bo Liu, and Xie Xie for their help with the techniques associated with this project. I thank Dr. Rubin, Dr. Wehr, Bo Liu, and Xie Xie for their help in designing this project; Sarah Whorley and Kam Truhn for their help in algae collection and processing, and Rosalind Becker for providing materials and advice. I would like to thank Dr. Plunkett, Dr. Karol, and especially Robin Sleith of the New York Botanical Gardens for use of their machine and expertise in lysing the cells. I also thank Dr. Wehr and Daniel Grubaugh for their help in analyzing the data. This work was supported by the Biological Sciences Department at Fordham University.

This document was last modified 06/19/2012.
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