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Radiocarbon dates reveal that Lupinus arcticus plants were grown from modern not Pleistocene seeds

2009; Wiley; Volume: 182; Issue: 4 Linguagem: Inglês

10.1111/j.1469-8137.2009.02818.x

1469-8137

Grant D. Zazula, C. R. Harington, Alice M. Telka, Fiona Brock,

Pleistocene-Era Hominins and Archaeology

In 1967, Porsild et al. published the unprecedented report of Arctic lupine (Lupinus arcticus Wats., Fabaceae) plants grown from seeds of presumed Pleistocene age [>10 000 yr before present (bp)]. These seeds were recovered from an ancient rodent burrow in frozen silt considered to have been deposited during the Pleistocene. At the time of publication, these Arctic lupine seeds (Porsild et al., 1967) were considered to be several thousand years older than any other ancient seeds previously reported to have successfully germinated (Godwin, 1968). As such, Porsild et al.'s publication was remarkable for the understanding of seed longevity and the potential for the preservation of viable ancient organisms in permafrost (Black, 1967; Kjøller & Ødum, 1971; Fabel et al., 2002). However, Porsild et al.'s Arctic lupine germination has been regarded with controversy in the discussion of ancient seed viability (Godwin, 1968; McGraw et al., 1991; Gugerli et al., 2005). Some cite the Pleistocene Arctic lupine as the greatest example of extreme seed longevity (Taylorson & Hendricks, 1976; Leck, 1980; Basu, 1995; Duarte et al., 1996; Gugerli, 2008), whereas others criticize the validity of this report because of the lack of independent dates (Roos et al., 1996; Baskin & Baskin, 2001; Daws et al., 2007; Sallon et al., 2008a). To help resolve this issue, we used accelerator mass spectrometry (AMS) analysis to radiocarbon (14C) date two of the remaining Arctic lupine seeds and an associated lemming (Dicrostonyx torquatus) skull from the burrow to determine their actual age. We report here the first independent radiocarbon dates that reveal that Porsild et al. (1967) did not germinate and grow Pleistocene Arctic lupine seeds as presumed, but instead grew modern seeds that apparently contaminated their ancient sample. In order to put our new radiocarbon data into context, it is crucial to relate the events that led to the discovery and germination of the presumed ancient Arctic lupine seeds. Harold Schmidt, a mining engineer and placer gold miner, discovered a number of rodent burrows exposed c. 3–6 m below the surface within a ‘muck’ deposit (a local term for frozen organic silt of loessic origin) which overlay gold-bearing gravel at his mining site on Miller Creek, Yukon Territory, Canada (64°00′N, 140°49′W) (C. R. Harington field notes, June 14, 1966). Several of the burrows contained rodent nests, fecal pellets, skulls, skeletons and seeds. Schmidt collected the rodent skull and a number of seeds from one of the burrows. The scientific significance of the burrow contents was not recognized until palaeontologist C. R. Harington visited Schmidt 12 years later. Schmidt gave the skull and c. 30 of the seeds to Harington to take them back to the National Museum of Canada in Ottawa for study. The seeds from Schmidt's rodent burrow were then presented to A. E. Porsild, chief of the Botany Division at the National Museum, for identification. The seeds were readily identified as belonging to Arctic lupine (Lupinus arcticus), a common herbaceous plant of the present-day boreal forest across northern Canada. Chromosome counts performed on the seeds showed that they were identical to modern specimens. As Porsild noted that half the seeds were remarkably well preserved, he passed those on to G. A. Mulligan (a research scientist at the Plant Research Institute, Department of Agriculture in Ottawa) for testing. Mulligan placed them on wet filter paper in a Petri dish, where six germinated within 48 h. The young plants were transferred to pots where they grew into normal, healthy plants indistinguishable from Lupinus arcticus, with one plant developing a few flowers after 11 months (Hinds, 1967; Porsild et al., 1967). As the rodent burrow and associated seeds were discovered within frozen silt that was well documented to have been deposited during the Pleistocene, Porsild et al. (1967) assumed that their plants were grown from seeds that were at least 10 000 yr old. Porsild et al. (1967) suggested that these lupine seeds were of similar age to Pleistocene ground squirrel burrows and nests discovered in the mining district near Fairbanks, Alaska that were radiocarbon dated to 14 860 ± 840 yr bp (Péwéet al., 1965). Identification of the skull associated with the Arctic lupine seeds as being that of a collared lemming (Dicrostonyx torquatus) (C. R. Harington field notes, June 14, 1966), a rodent of Arctic and alpine tundra that is no longer found in the vicinity of Miller Creek, Yukon, further suggested that these lupine seeds were indeed ancient. As the discovery was made before the development of AMS radiocarbon dating, there was no way of providing an independent age assessment on the skull or the Arctic lupine seeds to support the presumed Pleistocene age. Since the work by Porsild et al. (1967), many other rodent burrows and nests have been discovered from Pleistocene permafrost deposits of Eurasia, Alaska and Yukon (Harington, 1977; Guthrie, 1990; Fraser, 1995; Gubin et al., 2003; Zazula et al., 2007). Indeed, Harington (1997) noted several ground squirrel burrows with nests at Sixtymile Loc. 3, a fossil locality c. 1.6 km northeast of the site at which the Arctic lupine seeds were found. Research by Zazula documented over 100 such Pleistocene fossil nests from the Klondike mining district of central Yukon (Zazula et al., 2005, 2007; Zazula, 2006). It was determined that most of the burrows and nests were constructed by Arctic ground squirrels (Spermophilus parryii), although the recovery of bones from lemming (Dicrostonyx and Lemmus) and vole (Microtus) indicated that microtine rodents also used these subterranean tunnels (Harington, 2003; Zazula et al., 2007). The recovery of these nests in close stratigraphical association with Pleistocene volcanic ash beds of known age, and many radiocarbon dates, indicated that the nests spanned the duration of the Late Pleistocene ∼100 000–10 000 yr bp (Zazula et al., 2005, 2007; Zazula, 2006). Palaeobotanical analysis of the nest contents has resulted in an exceptional record of fossil seeds, fruits and leaves from over 60 plant taxa, although Arctic lupine was conspicuously lacking (Zazula, 2006; Zazula et al., 2007). Considering the controversy surrounding the age of the Arctic lupine seeds studied by Porsild et al. (1967), and the fact that seeds of this species were absent from the extensive collection of nests analysed by Zazula (Zazula, 2006; Zazula et al., 2007), we undertook the present study to date the remaining seeds and skull (Fig. 1) using the AMS radiocarbon method. Photograph of lemming (Dicrostonyx torquatus) skull (occlusal view) and Arctic lupine (Lupinus arcticus) seeds taken before display in Life Through the Ages Hall, Canadian Museum of Nature, Ottawa, ON, Canada (C. R. Harington). Two of the remaining Arctic lupine seeds recovered in 1954 by Harold Schmidt were selected for AMS radiocarbon dating. As they were received in 1966 by Harington, these seeds were curated in the Quaternary Zoology Collection at the National Museums of Canada in Ottawa (now the Canadian Museum of Nature). However, the seeds had been embedded in paraffin wax in a Petri dish for a museum display between the years 1971 and 2004. Because of the potential contamination of residual carbon from the paraffin wax into the Arctic lupine seeds, we needed to use a refined extraction technique before AMS radiocarbon dating. The seeds were decontaminated and dated at the Oxford Radiocarbon Accelerator Unit. As a test to determine the success of the paraffin wax decontamination, we also coated an Indian paintbrush (Castilleja sp.) seed capsule with paraffin wax that was subsampled from a Pleistocene fossil nest (GZ.05.44; YG 343.42) that had previously been radiocarbon dated to 25 800 ± 310 yr bp (Beta-210522) (Zazula et al., 2007). Initially, the seeds were individually subjected to a thorough Soxhlet-type solvent extraction, consisting of sequential extractions with tetrahydrofuran, chloroform, petroleum ether, acetone and methanol for a minimum of 8 h each to remove any paraffin wax (Bruhn et al., 2001). The samples were left to air-dry before undergoing an acid–base–acid (ABA) pre-treatment consisting of sequential washes with 1 M hydrochloric acid (80°C, 20 min), 0.2 M sodium hydroxide solution (80°C, 20 min), 1 M hydrochloric acid (80°C, 1 h) and 2.5% (w/v) sodium chlorite solution at pH 3 (80°C, 5 min), with thorough rinsing with ultra-pure water after each wash. The samples were freeze-dried before being combusted at 1000°C and measured for their elemental and stable isotopic composition using a CF-IRMS system consisting of a PDZ-Europa Robo-Prep biological sample converter (combustion elemental analyser) coupled to a PDZ-Europa 20/20 mass spectrometer (PDZ-Europa, Northwich, Cheshire, UK). Carbon dioxide from each sample was cryogenically distilled and reduced to graphite over an iron catalyst in the presence of excess hydrogen (Dee & Bronk Ramsey, 2000) before AMS radiocarbon measurement. To test whether the Arctic lupine seeds and the collared lemming skull from Miller Creek were contemporaneous, an ∼46-mg portion of the left zygomatic arch from the collared lemming skull (CMN 12062) was removed and submitted to the KECK Carbon Cycle AMS facility for radiocarbon dating. The collared lemming maxilla yielded a radiocarbon age of 23 380 ± 130 yr bp (UCIAMS-48241), confirming its Pleistocene age. However, the two Arctic lupine seeds yielded ages of 1.03492 ± 0.00307 F14C (OxA-18596) and 1.04487 ± 0.00295 F14C (OxA-18999), which calibrate approximately to the years ad 1955–57 using the OxCal 4.05 (Bronk Ramsey, 2001) and the Northern Hemisphere Zone 1 calibration curve dataset (Hua & Barbetti, 2004). The Indian paintbrush seed capsule yielded an age of 23 700 ± 300 yr bp (OxA-18521). The radiocarbon age on the collared lemming skull confirms that Harold Schmidt did indeed collect Pleistocene fossil remains from the frozen silt at Miller Creek, Yukon. This age is comparable with ages on nests recovered from similar burrows in central Yukon by Zazula et al. (2007). However, radiocarbon ages on the seeds indicate that the Arctic lupine seeds were not contemporaneous with the burrow and associated collared lemming skull. The radiocarbon age on the Castilleja sp. capsule is comparable with the previous age obtained from the nest sample (GZ.05.44; YG 343.42), thus indicating that the paraffin wax contaminant was effectively removed from this and the Arctic lupine seed samples (Table 1). These radiocarbon ages indicate that Porsild et al. (1967) did not grow Arctic lupine plants from ancient seeds, as presumed, and now effectively negate their Arctic lupine publication from the discussion on ancient seed germination. This revelation is not entirely surprising considering that recent palaeobotanical research on Pleistocene rodent burrows and nests from Yukon failed to recover any Arctic lupine seeds (Zazula, 2006; Zazula et al., 2007). Furthermore, Arctic lupine is a common understorey plant of the present-day boreal forest across northern Canada and would not be expected to have inhabited the cold, arid and, possibly, treeless steppe–tundra ecosystem of Late Pleistocene Beringia (Zazula et al., 2003, 2007). The only other comparable report of Pleistocene seed germination was made by Yashina et al. (2002), in which they germinated seeds of campion (Silene stenophylla Ledeb.) and persicaria (Polygonum spp.) discovered within fossil rodent nests recovered from Siberian permafrost that date between 32 800 ± 400 yr bp (IEMEZH-1178) and 31 800 ± 300 yr bp (Beta-157195). Yashina et al. (2002) placed the sterilized ancient seeds in an in vitro agar nutrient medium containing salts, sucrose and various phytohormones to compensate for the deficiency of endosperm and reserve nutrients within the seed embryo itself. Their experiment resulted in the campion seeds germinating to the initial stage of embryo root growth and the persicaria growing to the stage of cotyledon leaf formation, but neither developing into full plants. If Yashina et al.'s (2002) Siberian seed germination report is authentic, it suggests that permafrost preservation may enable plant cells to remain physiologically viable and able to resume division and growth, even after ∼33 000 yr. The oldest independently dated ancient seeds known to have germinated and grown into full plants include an ∼2000-yr-old date palm (Phoenix dactylifera L.) seed excavated from near the Dead Sea (Sallon et al., 2008b) and an ∼1300-yr-old lotus (Nelumbo nucifera Gaertn.) seed from China (Shen-Miller et al., 1995, 2002). Our new data affirm that no plants have ever been grown to maturity from Pleistocene seeds or from those independently dated to older than ∼2000 yr. Our research highlights the importance of independent radiocarbon dating for any germination experiments involving ancient seeds. Although our data indicate that Porsild et al.'s (1967) claim that Arctic lupine plants were grown from presumably ancient seeds was erroneous, much research has yet to be done on the viability of ancient seeds in permafrost and other archaeological or palaeontological contexts. Most of the research on ancient seed longevity has been conducted in temperate climatic regions, including the Middle East (Sallon et al., 2008b), Asia (Shen-Miller et al., 1995, 2002; Priestly & Posthumus, 1982) and Africa (Daws et al., 2007). We suggest that the permafrost regions of the Holarctic may be an invaluable, but largely unexplored region for similar pursuits. Permafrost deposits in Siberia, Alaska and Yukon are well-known archives of Pleistocene plant and animal genetic material (Willerslev et al., 2003, 2004; Debruyne et al., 2008). Although Yashina et al.'s (2002) report of the successful germination of Pleistocene Silene stenophyllus and Polygonum spp. seeds should be independently replicated to become widely accepted, their experiments represent only the beginning of much needed work on seed longevity in permafrost. Pleistocene rodent nests and burrows from permafrost deposits of northern North America and Eurasia may be the best source of ancient seeds with which to conduct more systematic experiments on ancient seed germination. The burrows and nests are found within well-drained silts that have been permanently frozen for thousands of years. In fact, Arctic ground squirrels are known to collect seeds and store them in winter subterranean hibernation chambers at dry sites, a pattern that extended far back into the Pleistocene (Zazula et al., 2007). Thus, these nests contain a plethora of permafrost-preserved ancient seeds that may be ideal for further germination experiments. With climatically induced permafrost melting and the release of potentially viable Pleistocene seed banks from the cryosphere into the biosphere, there is opportunity for the development of unique plant communities composed of formerly ancient and contemporary organisms. C.R.H. thanks Kieran Shepherd (earth sciences collection manager, Canadian Museum of Nature) for allowing the Miller Creek specimens to be radiocarbon dated. We thank the Schmidt family and other Yukon placer miners for their continued support. This paper greatly benefited from editorial review by Greg Hare and helpful comments from three anonymous reviewers.