Rogers, Helen

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Digital Kenyon: Research, Scholarship, and Creative ExchangeKenyon Summer Science Scholars Program Summer Student Research ScholarshipSummer 2013Arboral Diversity in Sub-ecotypes of the PeruvianAmazon Blackwater Rainforest: Barcoding fromCambial TissueHelen RogersFollow this and additional works at: https://digital.kenyon.edu/summerscienceprogramPart of the Biology CommonsThis Poster is brought to you for free and open access by the Summer Student Research Scholarship at Digital Kenyon: Research, Scholarship, andCreative Exchange. It has been accepted for inclusion in Kenyon Summer Science Scholars Program by an authorized administrator of Digital Kenyon:Research, Scholarship, and Creative Exchange. For more information, please contact noltj@kenyon.edu.Recommended CitationRogers, Helen, "Arboral Diversity in Sub-ecotypes of the Peruvian Amazon Blackwater Rainforest: Barcoding from Cambial Tissue"(2013). Kenyon Summer Science Scholars Program. Paper 221.https://digital.kenyon.edu/summerscienceprogram/221    •  Previous studies have  put forth estimates of  >11,000 tree species in the Amazon basin, with density estimates of 500-700 trees per square hectare 1,3.          Study Site: Tamshiyacu Nature Preserve in the lowland Amazonian forest floodplain near the banks of the Tahuayo River (Fig 2), a tributary of the Amazon River upstream and south of Iquitos, Peru. Acknowledgements:  Funded through Howard Sacks’ McGregor Grant for Rural By Design--local and global sustainability, a Kenyon Academic Partnership Grant, Kenyon College Office of the Provost, and Kenyon Summer Science Fellowships.  Thanks to William Hudobenko, Ally Schmidt, and Elizabeth Katoa for assistance with data collection.  Figure 5. The distribution of tree DBH in the sub-ecotypes of the Tahuayo River Igapo Forest Floodplain. DBH was grouped in 2 cm increments for all sub-ecotypes and followed a logarithmic curve (n=1200 for LR, n=748 for HR, and n=654 for B). Chi-Square, X2=47.490,df=30,p=0.022     References: 1.  Pennington, Reynel and Daza, Pennington. 2004. Trees of Peru. David, Hunt, Sherborn, England, 473 pp. 2. Junk, Wolfgang. 1997. The Central Amazon Floodplain. Spring, Germany, 521 pp. 3. Mittelbach, G. G., Schemske, D. W., Cornell, H. V., Allen, A. P., Brown, J. M., Bush, M. B., Harrison, S. P., Hurlbert, A. H., Knowlton, N., Lessios, H. A., McCain, C. M., McCune, A. R., McDade, L. A., McPeek, M. A., Near, T. J., Price, T. D., Ricklefs, R. E., Roy, K., Sax, D. F., Schluter, D., Sobel, J. M. and Turelli, M. 2007.  Evolution and the latitudinal diversity gradient: speciation, extinction and biogeography. Ecology Letters, 10: 315–331.  4. Mailyn Adriana Gonzalez, Christopher Baraloto, Julien Engel, Scott A. Mori, Pascal Pétronelli, Bernard Riéra, Aurélien Roger, et al. (2009). Identification of Amazonian Trees with DNA Barcodes. PLoS ONE. doi:10.1371/journal.pone.0007483 5. Dick C. and Kress J., 2009. Dissecting Tropical Plant Diversity with Forest Plots and   a Molecular Toolkit. BioScience , Vol. 59: 745-755  6. Kress, W. John, et al. "Plant DNA barcodes and a community phylogeny of a tropical forest dynamics plot in Panama." Proceedings of the National Academy of Sciences 106.44 (2009): 18621-18626. 7. Costion, Craig, et al. "Plant DNA barcodes can accurately estimate species richness in poorly known floras." PloS one 6.11 (2011): e26841. 8. Xin, Zhanguo, and Junping Chen. "A high throughput DNA extraction method with high yield and quality." Plant methods 8.1 (2012): 26. 9. Tibbits, Josquin FG, et al. "A rapid method for tissue collection and high-throughput isolation of genomic DNA from mature trees." Plant Molecular Biology Reporter 24.1 (2006): 81-91. 10. Colpaert, N., Cavers, S., Bandou, E., Caron, H., Gheysen, G., & Lowe, A. J. (2005). Sampling tissue for DNA analysis of trees: trunk cambium as an alternative to canopy leaves. Silvae Genetica, 54(6), 265-269.         • Bajial, the sub-ecotype with the highest flood level, had the highest density of trees (1200/ha) and the lowest avg. tree diameter (20.1 cm.) (Fig 6) . •  The rarely flooded HR had the second highest density of trees (748/ha) and contained the second highest average tree diameter (20.6), behind the LR (Fig 6).   •  The Bajial yielded the lowest average DNA concentration and nucleic acid purity.  It also had the lowest proportion of amplification success (Table 1).  •  The HR yielded the highest average DNA concentration and nucleic acid purity.  It had the highest proportion of amplification success.  •  The 5 most effective primers were 1f+r724 (rbcLa), KIM3f+KIM1r (matK), 1329f + 320r (matK), 17f + 25r (ITS), and psbA3f + trnH (psbA3-trnH) (Table 2). Figure 6. A) Density of trees per ha at the TRARC in the various sub-ecotypes of the Tahuayo River Amazon floodplain. LR (654), HR (748) , and B (1200).  B) The average tree diameter of each sub-ecotype studied. Error bars=SEM, ANOVA, F=0.42, p=0.655.      0 200 400 600 800 1000 1200 1400 Low Restinga  High Restinga Bajial  Tree Density (Ha) Helen Rogers ‘15, Noah Winters ‘15, and Kathryn Edwards  Department of Biology, Kenyon College, Gambier, Ohio   Arboral Diversity in Sub-ecotypes of the Peruvian Amazon Blackwater Rainforest: Barcoding from Cambial Tissue Sub-ecotype Average DNA Yield (ng/ul) Absorb-ance Average (260/280) Amplifica-tion Success (%)  High Restinga  49.73 1.53 36 Low Restinga  43.68 1.40 0.76 Bajial  18.67 1.41 0.8   Table 2.  The genes and primers amplified in each successful           experiment. Conclusions: •   DNA extraction from cambial tissue of bark samples is possible, albeit inconsistent in our preliminary study. •   Differences in sub-ecotype amplification success could be due to flooding that creates anoxic  conditions that lead to death of cambial tissue.   Gene Primer # Samples Attempted Percent Success rbcLa 1f + r724 28 21 % rbcLa SI_For + SI_Rev 15 0 % matK KIM3f + KIM1r 28 21 % matK 1329f + 320r 32 22 % matK KEWXF + MALPR12 19 0 % ITS 17f + 25r 28 29 % trnH-psbA psbA3f + trnH 28 18 %  Objectives:  1)Establish an arboral study of lowland forest floodplain  dynamics across distinct sub-ecotypes in relation to abiotic and biotic parameters. 2) Record size, distribution , and density of the trees within each sub-ecotype  3) Establish a protocol for the extraction and amplification of cambial  tree DNA from bark samples using traditional plant barcoding genes  18.5 19 19.5 20 20.5 21 21.5 Low Restinga High Restinga Bajial Average Tree Diamerer (cm) Sub-ecotype Discussion and Future Work: While DNA extraction from bark was successful, amplification of target genes was less reliable. Potential modifications to protocol include:  1)  Improved on-site extraction methodology and immediately preserving bark samples in silica gel10 2)  Lyophilization of plant tissue prior to DNA extraction8 3)  Addition of NaCl and  Bovine Serum Albumin (BSA) to extraction buffer to remove polyphenolic impurities9 4)  CTAB DNA extraction method to increase DNA yield10     2) DNA extraction and amplification: tissue was homogenized using liquid nitrogen and a mortar & pestle, extracted using a Qiagen DNEasy kit, and amplified using established PCR protocols 7.  DNA Extraction and Amplification:   1)  Isolating cambial tissue: The cambial tissue from 77 samples was isolated by scraping the samples’ underside after removal of wood (Fig 3).  Tree Survey:  Trees ≥ 10 cm, were tagged and measured at breast height (DBH) Figure 3. Bark sample, cambial side up before scraping.  •  The Lowland Amazon Igapo forest  floodplain, identified by the black water that floods it, is home to four distinct sub-ecotypes characterized by differences in annual flood levels: High Restinga (HR), Low Restinga (LR), Bajial (B) and       Palm Swamp (PS) (Fig 4).  •  With up to 2200 mm of precipitation annually, the Igapo floodplain is completely flooded for 2-4 months of the year (Fig. 1).   •  Even without taxonomic species determination, DNA barcoding could provide a reasonable estimate of the species diversity present7.  •  Obtaining DNA from the cambial tissue found in bark elimin-ates the  need to collect leaf tissue from high  up in the canopy 7. Background Information:  Figure 1. Igapo forest floodplain during the wet season. Figure 2.  The TRARC gridded research site southeast of the Tahuayo River divided into 100-hectare regions: Low Restinga (light green), High Restinga (dark green), Bajial (pink), and Palm Swamp (red) . Three, 1 hectare (ha) forest plots (light blue) were surveyed in our preliminary study. Palm swamp was not studied at this time.  •  Simplified species delimitation using DNA barcoding could be useful for baseline estimates of biodiversity and  subsequently for making informed decisions  regarding       conservation management. 0 0.05 0.1 0.15 0.2 0.25 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 >40 Tree DBH Frequency Tree DBH (cm) Bajial Low Restinga High Restinga Table 1.  Average DNA concentration, purity, and amplification success of each sub-ecotype examined.    Results: Methods: Figure 4.  Three sub-ecotypes of the Tahuayo River Igapo forest floodplain organized by increasing annual flooding level: High Restinga (top left), Low Restinga (top right) and Bajial (bottom right). Figure 7.  Amplification of 3 genes known to be valuable tree barcodes.  From left to right: (A) 1f + r724,  (B) KIM3f  + KIM1R, (C) 1329 + 320, (D) SI_For + SI_Rev,  (E) KEW XF + MALPR12, (F) 17F + 25r. Barcoding genes included the conservative coding region rbcL, the protein coding gene matK, the noncoding spacing region trnH-psbA, and the nuclear ribosomal ITS5   

Arboral Diversity in Sub-ecotypes of the Peruvian Amazon Blackwater Rainforest: Barcoding from Cambial Tissue

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Arboral Diversity in Sub-ecotypes of the Peruvian Amazon Blackwater Rainforest: Barcoding from Cambial Tissue

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