Ancient DNA Analysis and Phylogeography - A CodonCode Aligner User Showcase
CodonCode Aligner user Logan Kistler studies ancient DNA and phylogeography at the Pennsylvania State University in the Molecular Evolution / Ancient DNA Lab of Dr. Beth Shapiro and in the Environmental Archaeology Lab of Dr. Lee Newsom.
Logan uses CodonCode Aligner for two different projects. The goal of his first research project is to determine the domestication of the prehistoric eastern North American Chenopod (a weedy crop) by comparing modern and ancient DNA on a phylogenetic level. In addition to ancient DNA analysis, Logan studies the phylogeography of the bottle gourd (Lagenaria siceraria) and its arrival in the New World in his second project.
Ancient DNA Analysis of the Prehistoric Eastern North American Chenopod
by Logan Kistler
At the time of European contact, eastern North American Indians relied heavily on maize (Zea mays L.), beans (Phaseolus vulgaris L.), and squash (Cucurbita pepo L.), as well as an assortment of wild plants and animals. However, this “three sisters” agricultural complex is strictly a product of late prehistoric times, with beans having been introduced to the Eastern Woodlands from Mexico via the American Southwest sometime after approximately A.D. 1,300.
Prior to the rise of maize agriculture and its eventual pairing with beans for their combined ecological and nutritive benefits, a group of weedy crops was cultivated on a large scale throughout eastern North America, particularly in the Ohio and Mississippi Valley areas. Some of these, including sunflower (Helianthus annuus L.) and sumpweed (Iva annua L.), are known to have been domesticated in the Eastern Woodlands during the fifth millennium before present (B.P.). Others, including gourd/squash (Cucurbita pepo L.) and chenopod (Chenopodium berlandieri Moq.), might have also been brought under domestication in the Eastern Woodlands; but it has been proposed based on morphological analyses that they were introduced from Mesoamerica. The goal of this research is to determine the site of domestication of the chenopod member of the eastern agricultural complex using modern and ancient DNA analysis.
of archaeological chenopods for DNA analysis.
Chenopod was arguably the most important pre-maize cultivar in the Eastern Woodlands. During parts of the Early Woodland Period (2,200-3,000 B.P), it is estimated to have comprised greater than fifty percent of all dietary intake, based on paleofecal analysis. It was stored in caches of several million seeds, and provided a year-round source of nourishment. Its small seeds, similar to quinoa (a close relative), underwent several morphological changes during domestication that reflect the novel artificial selection pressures imposed by human cultivators. The testa (seed coat) became significantly reduced or was lost completely, the starchy endosperm increased in volume, and the shape changed to accommodate additional nutritive tissue. Also, the plant architecture changed, with the seeds becoming concentrated in terminal clusters to facilitate efficient harvesting.
The primary hypothesis is that the eastern chenopod cultivar was brought under domestication from wild varieties in the North American midcontinental riverine region, along with sunflower, sumpweed, and possibly gourd/squash. There is archaeological evidence for use of a wild type chenopod as early as 8,000 B.P. in parts of Illinois, and other ecological evidence supports this model. However, there is no direct evidence of a transitional phase during which chenopod was modified from its wild form but not yet showing a fully domesticated morphology. The domesticated form appears somewhat abruptly during the fourth millennium B.P., and rapidly comes to dominate archaeobotanical assemblages throughout eastern North America.
The alternative hypothesis is that the eastern chenopod was an introduced form of a Mexican cultivar of the same species known as huazontle or chia. The Mexican crop, still grown commercially today, is morphologically indistinguishable from the chenopod remains found archaeologically in the Eastern Woodlands. Although there is no archaeological evidence directly linking Mesoamerica with the Eastern Woodlands at this time, and certainly none to suggest the long-range movement of tropical crop plants, the morphological similarities are compelling evidence for the possibility of a Mesoamerican introduction. However, there is not currently sufficient evidence to definitively support either of these two models for the origin of the eastern chenopod.
A phylogenetic approach is being used to determine the chenopod’s site of domestication. Because the eastern crop is no longer cultivated, and no viable germplasm has been preserved, it is necessary to extract and analyze DNA from well-preserved archaeological specimens. The first phase of the project, now complete, involved sampling of numerous wild and cultivated Chenopodium sp. specimens from throughout the U.S. and Mexico to establish the group’s phylogeny. A total of 5 taxa were sampled, including three wild members of Chenopodium berlandieri, Mexican huazontle, and Chenopodium album as an outgroup. Several thousand base pairs of DNA was screened in two of the taxa to identify the most informative regions. Polymorphic regions were selected for full scale analysis, and were sequenced in all members of the sample. Sequences were aligned and a maximum likelihood tree was constructed to reveal phylogenetic relationships among the modern taxa.
The second phase of the project is the extraction and analysis of DNA from archaeological specimens preserved in dry caves and rockshelters throughout the Eastern Woodlands. The regions sequenced in modern accessions were scanned for their most informative short segments, and new primers were designed to target these fragments. This has ensured positive results even with highly fragmented ancient DNA. DNA is extracted from archaeological seeds, and PCR parameters are optimized according to the level of preservation. Sequences are generated for the short amplicons, which are then combined for comparison with the modern phylogenetic tree.
If an eastern origin of the prehistoric chenopod is demonstrated, it will provide the strongest evidence to date for Eastern North America’s status as an independent hearth of agriculture. It will also contribute to the ongoing discussion of multiple domestication events of a single species, which will be revealed in the case of an eastern domestication event. If a Mesoamerican origin is revealed, it will stimulate exciting new lines of inquiry into biogeography and the cultural mechanisms for crop migration, and will greatly alter our ideas about prehistoric interactions between Native American groups.
Phylogeography of the bottle gourd (Lagenaria siceraria [Molina] Standl.) and its New World arrival
by Logan Kistler
Bottle gourd is the earliest domesticated plant present in the Americas, appearing in North American archaeological contexts up to approximately 10,000 years before the present. The large, thick-walled fruits were used as containers by the highly mobile hunters and gatherers of the Paleoindian period (the earliest division of New World prehistory), and continued to be cultivated into historic times. Over sixty landraces and cultivars continue to be grown today, some on a commercial scale. Bottle gourd is native to the southern part of Africa, where it was presumably first domesticated. It dispersed into Asia and the Americas, but it remains unclear whether these migrations were of the wild type or a domestic variety. The timing of these migrations is also currently unknown.
The goal of this research is to better understand how, when, and from where the American bottle gourd originated. Several competing hypotheses have been advanced to account for its arrival, and are being explicitly tested during this research program.
from a freshwater spring in Florida.
The dominant hypothesis for several years was that the domesticated form of bottle gourd had floated on ocean currents from Africa to southeastern North America. This is consistent with the bottle gourd’s ability to travel long distances in seawater and retain viable seeds (and with the gourd family’s tendency at large for long-range water dispersal), and preliminary phylogenetic assessment seemed to cluster modern American bottle gourd landraces with African ones. Additionally, most very early archaeological bottle gourd specimens in the Americas have been recovered from sites in Florida.
Recent ancient DNA work has suggested, however, that prehistoric American bottle gourds were more closely related to Asian varieties than African ones. Two additional hypotheses have been proposed based on this finding. Some researchers argue for a natural transoceanic migration from southeast Asia to the west coast of South America, either of a domestic form or wild type. If a wild type traveled across the Pacific, this would indicate a subsequent independent domestication of bottle gourd in the New World. Others believe it is more likely that the first Paleoindians to migrate through Beringia from Siberia into North America during the late Pleistocene carried bottle gourds and introduced them to the New World, propagating them when possible along the way to maintain viable germplasm.
With the current genetic, ecological, and archaeological evidence, it remains unclear when and from where the bottle gourd came to the New World. To address this topic, germplasm samples have been obtained from landraces and cultivars sampled worldwide. Informative genetic regions are being sequenced in each accession, and sequences will be analyzed using analytical techniques determined based on the nature of the data. Larger-scale phylogenetic analysis than has previously been conducted, combined with datasets from other analyses, will shed light on the geographic origin of the American lineage of bottle gourds. This will help archaeologists and plant systematists better characterize the biogeography of long-distance crop migration. Additionally, this information will aide American archaeologists in our pursuit of a clearer view of the rise of plant cultivation and agriculture throughout the New World.
Using CodonCode Aligner for Ancient DNA Analysis and Phylogeography
of ceramic technology
CodonCode Aligner's most useful features for Logan's research are base calling, contig assembly, manual sequence editing, quality scores, and sequence export for further analysis.
Logan describes his first experience with CodonCode Aligner for his ongoing projects:
"My free trial of CodonCode was used for assembling and aligning the first set of bottle gourd sequences, and was similarly used for curating sequences for the entire modern component of the chenopod project. All PCR products are sequenced in both directions, with forward and reverse runs assembled by CodonCode and checked by eye for discrepancies. CodonCode’s interface is extremely useful for identifying regions of the nucleotide sequences that require additional curation, and for editing them as necessary. Alignments of curated sequences are constructed using CodonCode and exported for further manipulation in other applications. CodonCode facilitates efficient and accurate management of large amounts of sequence data, especially regarding the long intergenic spacers used in the bottle gourd analysis."
