Billie A. Gould 2,5 , Blanca Le Ó N 3 , Aron M. Buffen 4,6 , and Lonnie G
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American Journal of Botany 97(9): 1579–1584. 2010. BRIEF COMMUNICATION E VIDENCE OF A HIGH-ANDEAN, MID-HOLOCENE PLANT COMMUNITY: AN ANCIENT DNA ANALYSIS OF GLACIALLY PRESERVED REMAINS 1 Billie A. Gould 2,5 , Blanca Le ó n 3 , Aron M. Buffen 4,6 , and Lonnie G. Thompson 4 2 Department of Ecology and Evolutionary Biology, Cornell University, Ithaca, New York 14850 USA; 3 Herbarium, Plant Resources Center, University of Texas, Austin, Texas 78712 USA; and 4 Byrd Polar Research Center and School of Earth Sciences, The Ohio State University, Columbus, Ohio 43210 USA • Premise of the study : Around the world, tropical glaciers and ice caps are retreating at unprecedented rates because of climate change. In at least one location, along the margin of the Quelccaya Ice Cap in southeastern Peru, ancient plant remains have been continually uncovered since 2002. We used genetic analysis to identify plants that existed at these sites during the mid- Holocene. • Methods: We examined remains between 4576 and 5222 yr old, using PCR amplifi cation, cloning, and sequencing of a frag- ment of the chloroplast trnL intron. We then matched these sequences to sequences in GenBank. • Key results : We found evidence of at least fi ve taxa characteristic of wetlands, which occur primarily at lower elevations in the region today. • Conclusions : A diverse community most likely existed at these locations the last time they were ice-free and thus has the po- tential to reestablish with time. This is the fi rst genetic analysis of vegetation uncovered by receding glacial ice, and it may become one of many as ancient plant materials are newly uncovered in a changing climate. Key words: ancient DNA; Andes; bofedal; Holocene; puna. Historically, much of what we know about ancient plant creasingly fragmented over time as a result of hydrolysis and communities comes from morphological examination of plant oxidation. DNA naturally preserved in cold and/or dry environ- microfossils and macrofossils. Over the past 2 decades, how- ments, however, now has been successfully extracted from ever, we have gained the exciting ability to directly analyze ge- specimens upward of 500 000 yr old, with the theoretical limit netic material of ancient plants, and this can add greatly to what of preservation estimated at about 1 million years ( Willerslev we know about plant community change over long time scales. et al., 2004 ; Willerslev et al., 2007 ). Ancient DNA fragments Ancient DNA (aDNA) analysis of plants, although not common can be amplifi ed with PCR or can be directly sequenced with in the literature, has now successfully been used to characterize sequencing-by-synthesis technology (e.g., Shapiro et al., 2004 ; Holocene and Pleistocene vegetation ( Kuch et al., 2002 ; Willerslev Noonan et al., 2006 ). Modern, intact DNA is heavily favored et al., 2003 ), to examine the history of domestication in crops over fragmented aDNA during amplifi cation, so great care must ( Allaby et al., 1999 ; Jaenicke-Despres et al., 2003 ), and to track be taken to avoid and identify potential modern contaminants of genetic change in wild populations ( Raniello and Procaccini, ancient genetic material. 2002 ; Tani et al., 2003 ). Ancient plant DNA is sometimes pre- In this study, we used a PCR-based approach to isolate se- served in wood or other monotypic archaeological remains, but quences from ancient plant remains recently uncovered by the it also can exist in bulk samples that contain a mix of taxa, such retreating Quelccaya Ice Cap in southeastern Peru. The rapid as lake sediments, rodent middens, coprolites, and permafrost, retreat of this Andean ice cap — recently as fast as ~60 m per yr peat, or ice cores ( Willerslev et al., 1999 ; Willerslev et al., for its largest outlet glacier, Qori Kalis— has been well docu- 2004 ; Gugerli, et al., 2005 ; Willerslev et al., 2007; Gilbert et al., mented over the past 4 decades and is closely linked to modern 2008 ). Ancient DNA is of low quality because it becomes in- climate warming (Thompson et al., 2006). As it retreats, rooted plant remains have been uncovered at sites along the central- western margin, near what are now meltwater-fi lled lakes lo- 1 Manuscript received 12 February 2010; revision accepted 6 July 2010. cated at elevations near 5200 m a.s.l. ( Table 1 ). Buffen et al. The authors thank A. Talaba, L. Stenzler, I. Lovette, M. Rasmussen, (2009) radiocarbon-dated the remains at two sites to roughly M. Arakaki, J. Walters, M. Carling, and A. Chang for all their generous 4700 and 5100 calibrated yr before present (cal. y.b.p.) and re- help and advice on this project. This research was funded by the Andrew mains embedded in sediments at a third site to between roughly W. Mellon Foundation and the Cornell Department of Ecology and 5200 and 7000 cal. y.b.p., thus providing an estimate of when Evolutionary Biology. these areas were last ice-free. The burial, under ice, of these 5 Author for correspondence (e-mail: [email protected] ) 6 Present address: Department of Geological Sciences, Brown University, plants marks an abrupt mid-Holocene transition of the regional Providence, Rhode Island 02912 USA climate to likely cooler and wetter conditions, which allowed the ice cap to rapidly advance, cover, and preserve existing doi:10.3732/ajb.1000058 plants. Other documented cases exist of retreating ice uncovering American Journal of Botany 97(9): 1579–1584, 2010; http://www.amjbot.org/ © 2010 Botanical Society of America 1579 1580 American Journal of Botany [Vol. 97 Table 1. Location and age of plant remains containing ancient DNA. the Cornell Laboratory of Ornithology (Ithaca, New York, USA). Before this work, no plant material had been processed in that laboratory. PCR amplifi ca- Calibrated 1 σ age tion of the DNA was carried out using three different primer sets (in separate Collection location, elevation, and Sample Collection range reactions): (1) degenerate primers ITS1+2 (5 ′ -GCGGAAGGATCATTGTCR-3 ′ coordinates no. year (cal. y.b.p.) and 5 ′ -GAGCCKAGATATCCGTTGY-3 ′ ), designed to amplify an approxi- mately 250-bp section of the fi rst nuclear ribosomal internal transcribed spacer Base Camp Lake, 5210 m 1 2007 5166 – 5277 (5222) region; (2) trnL ( c / d); and (3) trnL ( g / h). These latter two were developed by (13 ° 55.76 ’ S, 70 ° 51.09 ’ W) 2 2005 5055 – 5188 (5122) Taberlet et al. (2007) to amplify a 254- to 767-bp fragment and a 10- to 143-bp North Lake, 5208 m 3 2005 4953 – 5052 (5003) fragment of the chloroplast leucine transfer RNA ( trnL ) intron, respectively. (13 ° 54.95 ’ S, 70 ° 50.71 ’ W) 4 2005 4524 – 4628 (4576) The short trnL fragment is nested within the large trnL fragment. In simula- 5 2005 4689 – 4762 (4726) tions, the trnL ( g / h ) primers amplify 11 404 sequences across 310 plant families Boulder Lake Sequence, 5143 m 6 2006 5119 – 5185 (5152) listed in GenBank (National Center for Biotechnology Information, Bethesda, (13 ° 56.01 ’ S, 70 ° 51.54 ’ W) Maryland, USA) and identify 80% of these sequences unambiguously to the correct family ( Taberlet et al., 2007 ). Note: Buffen et al., 2009, data reproduced with permission. Cal. y.b.p. = Plant tissue was pulverized with sterile equipment under liquid nitrogen, calibrated yr before present. and DNA was extracted using the DNeasy Plant Mini Kit (Qiagen, Valencia, California, USA). DNA was amplifi ed with Platinum Taq Polymerase (Invitro- vegetation preserved in-place where it once grew ( Bergsma gen, Carlsbad, California, USA) or JumpStart Taq Polymerase (Sigma, St. Louis, Missouri, USA), 1 μ L DNA and 1 μ L bovine serum albumin per et al., 1984 ), but we are aware of no genetic studies of such 20 μL PCR reaction, 250– 375 μ M primer, and 2.5 mM or 4 mM MgCl2 , material. respectively. Amplifi cation was carried out for 55 – 60 cycles. Tissue-free ex- Initial visual inspection of the remains from Peru indicated traction and PCR controls were run in parallel. PCR product was purifi ed using that much of the material might be composed of Distichia mus- the Qiagen PCR cleanup kit and cloned into the TOPO TA vector (Invitrogen). coides (Juncaeae), a cushion-forming rush species common to A total of 578 clones were PCR screened for the presence of an insert, and 316 wetland communities (bofedales) above 3500 m in the region of these were cleaned via ExoI-SAP reaction and sequenced using BigDye v3.1 Terminator Cycle Sequencing (ABI, Foster City, California, USA). ( Young et al., 1997 ; Squeo et al., 2006 ). Bofedales are diverse communities and include many plants from the species-rich Sequence identifi cation— High-quality ( clearly readable), trimmed se- families Poaceae and Asteraceae among others ( Leó n and quences of 15 bp or longer were assembled into contigs by using the built-in Young, 1996 ); however, no other species could be clearly dis- algorithm of CodonCode Aligner (CodonCode Corporation, Dedham, Massa- tinguished by direct examination of the remains. To address the chusetts, USA) with minor editing. Contig consensus sequences and unique question of whether a community of plants existed at these lo- unaligned sequences were compared with sequences in the GenBank environ- cations the last time they were ice-free, we cloned and se- mental (env) and nonredundant (nr/nt) sequence databases in batches using quenced aDNA fragments from fi ve samples of the remains P-BLAST homology search via the Cornell University BioHPC computing cluster. For sequences >20 bp (191 sequences), the search was conducted with found at the soil surface and from one sample of similar age that the BLASTn algorithm with a relaxed E-value cutoff of 10 − 2 . Sequences 15 – 20 bp was preserved in a nearby sedimentary deposit ( Table 1 ).