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Community analysis of aquatic periphyton using the rbcL gene Catharina Grubaugh Department of Biology, Fordham University ABSTRACT Because algae are defined as a functional group rather than by phylogenetic associations, molecular techniques have been challenging. However, primers for regions of the rbcL gene, which codes for RuBisCO, have been used for wholecommunity analysis of phytoplankton. The purpose of this study was to determine if primers for the IA and IB Forms of the rbcL gene can be used to assess the biodiversity of green algae and cyanobacteria in two aliquots of an environmental sample from a periphyton community. Nine algal genotypes were identified in this study, and each of the two aliquots had a genotype richness of seven. The most common genotype in both aliquots was Ulothrix zonata. The community compositions of the two aliquots were significantly different from the composition that would be expected if the two aliquots were equal. Future studies may identify more rbcL sequences from periphyton species increasing the resolution and precision of whole-community analysis in periphyton. Keywords: periphyton, rbcL, whole community analysis INTRODUCTION Because algae form a paraphyletic and polyphyletic functional group, there is no recent common ancestor for the entire algae group. As a result, it is difficult to use molecular techniques for analysis of entire algal communities. However, some genes 1 have been used to barcode algae, including 23S plastid rRNA (Sherwood and Presting 2007), 18S rRNA (Zimmerman et al. 2011), and rbcL (Paul et al. 2000). The rbcL gene is of particular interest because it codes for the large subunit of the protein ribulose-1,5-bisphosphate carboxylase oxygenase (RuBisCO). RuBisCO is a key enzyme in the carbon fixation step of the Calvin Cycle. Therefore, it is a molecular marker that is linked to the ecosystem function of algae (Paul et al. 2000). There are four forms of RuBisCO (forms I, II, III, and IV), and most algae have one of three subtypes of form I. Cyanobacteria have form IA and IB, green algae have form IB, and non-green algae have form ID (Badger and Bek 2008). A set of primers has been developed to amplify genes coding for a region of the IA or IB form, and another set has been developed to amplify genes coding for a region of the ID form (citation in Paul et al. 2000). Previous studies have shown that the rbcL gene can be used for whole community analysis of marine phytoplankton (Paul et al 2000) and phytoplankton found in the effluent from wastewater treatment plants (Ghosh and Love 2011). In addition, mRNA analysis of the rbcL gene can be used to assess the phytoplankton community composition and production (Wawrik and Paul 2004). However, the rbcL gene has not been used for whole community analysis of algae attached to surfaces, known as periphyton. This omission is surprising, as periphyton is a key primary producer in aquatic systems and constitutes an important food source for higher trophic levels (Vadeboncoeur and Steinman 2002, Brito et al. 2006, Torres-Ruiz et al. 2007). The purpose of this study was to determine if primers for the IA and IB Forms of the rbcL gene can be used to assess the biodiversity of green algae and cyanobacteria 2 in environmental samples from a periphyton community. DNA was extracted from two aliquots of the same periphyton sample from a stream in southeastern New York state, and a 615 base pair region of Forms IA and IB of the rbcL genes was sequenced. The resulting rbcL 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. METHODS Periphyton was sampled on 1 April 2012 from Peters Kill, a rural stream located northwest of New Platz, NY. Three rocks were scraped for algae using a toothbrush. The slurry was then split by volume into two aliquots. The both aliquots were stored at 4°C for 24hr. Aliquot A was then stored at -20°C until extraction. Aliquot B was stored at -20°C for three days, thawed, and then stored at -20°C until extraction. DNA was extracted from each aliquot on different days. DNA was extracted from aliquot A on 5 April 2012 and from aliquot B 30 April 2012. Cells were lysed by beadbeating using ceramic beads in a Fast-Prep machine at 4.0m/s for 45s. DNA was extracted using the UltraClean Soil DNA Isolation kit (Mo-Bio, Carlsbad, CA, USA) with extended incubation times. Primers from Paul et al. (2000) were used to amplify a 615 bp region of the Form IA and IB of the rbcL gene, forward: TCIGCITGRAACTAYGGTCG and reverse: GGCATRTGCCAIACRTGRAT. Each PCR reaction contained 12.5µl of 2X GoTaq Green Master Mix (Promega, Madison, WI, USA), 0.5µl of forward primer, 0.5µl of 3 reverse primers, and DNA and dH2O to a final volume of 25µl. In Aliquot A, three different amounts of DNA extract (0ng, 10ng, and 40ng) were used for PCR, and five different amounts (0ng, 1ng, 5ng, 10ng, and 40ng) were used for PCR in Aliquot B. Cycle parameters were: 3 minutes at 95°C; followed by 50 cycles (increased from 35) of 1 minute at 95°C, 1 minute at 52°C, 1.5 minutes at 72°C; and then 20 minutes at 72°C. PCR products were separated on a 1.2% agarose gel, and the PCR product producing the most robust band was used for further analysis. In both aliquots, the PCR product of the reaction with the lowest starting amount of DNA was used. Following PCR purification with QIAQuick PCR Purification kit (Qiagen, Venlo, Netherlands), the PCR product was ligated into pGEM-t plasmids and transformed into JM109 cells. The fifty largest and most independent (i.e. not touching other bacterial colonies) of the resulting bacterial colonies for each aliquot were sent for sequencing using T7 as a primer to Genewiz, Inc. (South Plainfield, NJ, USA). The closest match to each DNA sequence was determined using a Basic Local Alignment Search Tool (BLAST) nucleotide search (NCBI). Some colonies could not be sequenced, and the closest match for one sequence was the cloning vector. These colonies were removed from the data set, resulting in a total of 87 sequences: 38 from Aliquot A and 49 from Aliquot B. Sequences were aligned using Clustal W2 (European Bioinformatics Institute, Cambridge, UK). Similar sequences were grouped together into the same genotype. 4 RESULTS PCR amplification with the Form IA and IB primers resulted in a product slightly larger than 600bp (Figure 1). The amount of PCR product decreased with increasing DNA concentration, indicating the presence of a PCR-inhibiting compound in the DNA extract, which is common in environmental samples (Zhang and Lin 2005). 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 25µl. Using alignment tools, sequences with 92% or greater nucleotide similarity were grouped into genotypes. Nine genotypes were established (Table 1). Three of the genotypes matched rbcL sequences of specific species: Ulothrix zonata, Stigeoclonium helveticum, and Pseudendoclonium akinetum, all of which are species of green algae. The HM538765 sequence had percent nucleotide matches high enough to be grouped into the U. zonata genotype or into the P. akinetum genotype. However, only some of 5 the U. zonata and P. akinetum sequences had high nucleotide match percentages. As a result, the three genotypes could not be grouped together. 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. Closest Match Ulothrix zonata Stigeoclonium helveticum GU132903 GU132900 and GU132909 GU132903 and GU13208 HM538765 GU132904 GU132908 Pseudendoclonium akinetum Aliquot A 55.3 31.6 2.6 2.6 2.6 2.6 2.6 0.0 0.0 Aliquot B 61.2 4.1 20.4 4.1 6.1 0.0 0.0 2.0 2.0 % NT 87 to 89 93 to 98 88 to 89 88 to 90 88 87 86 88 92 Each of the aliquots had sequences from seven of the genotypes, and the genotype with the highest frequency in both aliquots was U. zonata. The community composition 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, χ2 = 20.4, df = 8). DISCUSSION In this study, nine algal genotypes were identified using primers for a region of the Form IA and IB rbcL genes. Each of the two aliquots had a genotype richness of seven. The most common genotype in both aliquots was Ulothrix zonata. This species 6 has been shown to be a dominant periphyton species in the winter and early spring, when this sample was taken, in aquatic systems at similar latitudes to that of Peters Kill (McMillan and Verduin 1953, Graham et al. 1985). Although both aliquots had the same genotype richness and dominant genotype, the community compositions of the two aliquots were significantly different. This difference could be due to slight differences in aliquot storage. Aliquot A was frozen only once before DNA extraction, while Aliquot B was frozen, thawed, and then frozen again before extraction. Hopefully, when more rbcL sequences from periphyton species are added to GenBank, community composition can be determined with greater resolution and greater precision. While whole-community biodiversity analysis of periphyton with species-level resolution may not yet be possible, other applications of these techniques, such as improved gut content analysis in the consumers of algae and mRNA analysis to determine RuBisCO production rates, have the potential to increase understanding of the role of algae in ecosystems. Future studies should increase the number of algal rbcL barcodes to improve later whole-community analyses. ACKNOWLEDGEMENTS 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 7 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. 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. Brito, E. F., T. P. Moulton, M. L. De Souza, and S. E. Bunn. 2006. Stable isotope analysis indicates microalgae as the predominant food source of fauna in a coastal forest stream, south-east Brazil. Austral Ecology 31: 623-633. Ghosh, S. and N. G. Love. 2011. Application of rbcL based molecular diversity analysis to algae in wastewater treatment plants. Bioresource Technology 102: 36193622. 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. Sherwood, A. R. and G. G. Presting. 2007. Universal primers amplify a 23S rDNA plastid marker in eukaryotic algae and cyanobacteria. Journal of Phycology. 43: 605-608. Torres-Ruiz, M., J. D. Wehr, and A. A. Perrone. 2007. Trophic relations in a stream food web: importance of fatty acids for macroinvertebrate consumers. Journal of the North American Benthological Society 26: 509-522. Vadeboncoeur, Y. and A. D. Steinman. 2002. Periphyton function in lake ecosystems. The Scientific World Journal 2: 1449-1468. 8 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. Zhang, H. and S. Lin. 2005. Development of a cob-18S rRNA gene real-time PCR assay for quantifying Pfiesteria shumwayae in the natural environment. Applied and Environmental Microbiology 71: 7053-7063. Zimmerman, J., R. Jahn, and B. Gemeinholzer. 2011. Barcoding diatoms: evaluation of the V4 subregion on the 18S rRNA gene, including new primers and protocols. Organisms Diversity and Evolution 11: 173-192. 9