Morphometrics and Phylogenomics of Coca (Erythroxylum spp.) Illuminate Its Reticulate Evolution, With Implications for Taxonomy.

South American coca (Erythroxylum coca and E. novogranatense) has been a keystone crop for many Andean and Amazonian communities for at least 8,000 years. However, over the last half-century, global demand for its alkaloid cocaine has driven intensive agriculture of this plant and placed it in the center of armed conflict and deforestation. To monitor the changing landscape of coca plantations, the United Nations Office on Drugs and Crime collects annual data on their areas of cultivation. However, attempts to delineate areas in which different varieties are grown have failed due to limitations around identification. In the absence of flowers, identification relies on leaf morphology, yet the extent to which this is reflected in taxonomy is uncertain. Here, we analyze the consistency of the current naming system of coca and its four closest wild relatives (the "coca clade"), using morphometrics, phylogenomics, molecular clocks, and population genomics. We include name-bearing type specimens of coca's closest wild relatives E. gracilipes and E. cataractarum. Morphometrics of 342 digitized herbarium specimens show that leaf shape and size fail to reliably discriminate between species and varieties. However, the statistical analyses illuminate that rounder and more obovate leaves of certain varieties could be associated with the subtle domestication syndrome of coca. Our phylogenomic data indicate extensive gene flow involving E. gracilipes which, combined with morphometrics, supports E. gracilipes being retained as a single species. Establishing a robust evolutionary-taxonomic framework for the coca clade will facilitate the development of cost-effective genotyping methods to support reliable identification.

Archaeobotanical evidence supports indigenous cucurbit long-term use in the Mesoamerican Neotropics.

The squash family (Cucurbitaceae) contains some of the most important crops cultivated worldwide and has played an important ecological, economic, and cultural role for millennia. In the American tropics, squashes were among the first cultivated crop species, but little is known about how their domestication unfolded. Here, we employ direct radiocarbon dating and morphological analyses of desiccated cucurbit seeds, rinds, and stems from El Gigante Rockshelter in Honduras to reconstruct human practices of selection and cultivation of Lagenaria siceraria, Cucurbita pepo, and Cucurbita moschata. Direct radiocarbon dating indicates that humans started using Lagenaria and wild Cucurbita starting ~ 10,950 calendar years before present (cal B.P.), primarily as watertight vessels and possibly as cooking and drinking containers. A rind directly dated to 11,150-10,765 cal B.P. represents the oldest known bottle gourd in the Americas. Domesticated C. moschata subsequently appeared ~ 4035 cal B.P., followed by domesticated C. pepo ~ 2190 cal B.P. associated with increasing evidence for their use as food crops. Multivariate statistical analysis of seed size and shape show that the archaeological C. pepo assemblage exhibits significant variability, representing at least three varieties: one similar to present-day zucchini, another like present-day vegetable marrow, and a native cultivar without modern analogs. Our archaeobotanical data supports the hypothesis that Indigenous cucurbit use started in the Early Holocene, and that agricultural complexity during the Late Holocene involved selective breeding that encouraged crop diversification.

The history of Coast Salish "woolly dogs" revealed by ancient genomics and Indigenous Knowledge.

Ancestral Coast Salish societies in the Pacific Northwest kept long-haired "woolly dogs" that were bred and cared for over millennia. However, the dog wool-weaving tradition declined during the 19th century, and the population was lost. In this study, we analyzed genomic and isotopic data from a preserved woolly dog pelt from "Mutton," collected in 1859. Mutton is the only known example of an Indigenous North American dog with dominant precolonial ancestry postdating the onset of settler colonialism. We identified candidate genetic variants potentially linked with their distinct woolly phenotype. We integrated these data with interviews from Coast Salish Elders, Knowledge Keepers, and weavers about shared traditional knowledge and memories surrounding woolly dogs, their importance within Coast Salish societies, and how colonial policies led directly to their disappearance.

Trans-Holocene Bayesian chronology for tree and field crop use from El Gigante rockshelter, Honduras.

El Gigante rockshelter in western Honduras provides a deeply stratified archaeological record of human-environment interaction spanning the entirety of the Holocene. Botanical materials are remarkably well preserved and include important tree (e.g., ciruela (Spondias), avocado (Persea americana)) and field (maize (Zea mays), beans (Phaseolus), and squash (Cucurbita)) crops. Here we provide a major update to the chronology of tree and field crop use evident in the sequence. We report 375 radiocarbon dates, a majority of which are for short-lived botanical macrofossils (e.g., maize cobs, avocado seeds, or rinds). Radiocarbon dates were used in combination with stratigraphic details to establish a Bayesian chronology for ~9,800 identified botanical samples spanning the last 11,000 years. We estimate that at least 16 discrete intervals of use occurred during this time, separated by gaps of ~100-2,000 years. The longest hiatus in rockshelter occupation was between ~6,400 and 4,400 years ago and the deposition of botanical remains peaked at ~2,000 calendar years before present (cal BP). Tree fruits and squash appeared early in the occupational sequence (~11,000 cal BP) with most other field crops appearing later in time (e.g., maize at ~4,400 cal BP; beans at ~2,200 cal BP). The early focus on tree fruits and squash is consistent with early coevolutionary partnering with humans as seed dispersers in the wake of megafaunal extinction in Mesoamerica. Tree crops predominated through much of the Holocene, and there was an overall shift to field crops after 4,000 cal BP that was largely driven by increased reliance on maize farming.

The taming of the weed: Developmental plasticity facilitated plant domestication.

Our experiments with crop progenitors have demonstrated that these species exhibit dramatic plasticity in key traits that are affected by domestication, including seed and fruit morphology. These traits can be altered by cultivating crop progenitors for a single season, in the absence of any selection for domesticated phenotypes. We hypothesize that cultivation caused environmental shifts that led to immediate phenotypic changes in crop progenitors via developmental plasticity, similar to tameness in animals. Here we focus on the loss or reduction of germination inhibitors in an annual seed crop because seeds with high dormancy are undesirable in crops, and also present a serious barrier to selective pressures that arise from seed-saving and planting by humans. Data from four seasons of observation of the crop progenitor Polygonum erectum L. suggest that the low plant density conditions of an agroecosystem trigger a phenotypic response that reduces germination inhibitors, eliminating a key barrier to further selection. The timing of the harvest can also be used to manipulate the germinability of seed stock. These observations suggest that genetic assimilation may have played a role in the domestication of this plant. More experimental work with crop progenitors is needed to understand whether or not this phenomenon played a part in the domestication of other plants, and to accurately interpret the significance of ancient plant phenotypes in the archaeological record.

A plastome phylogeny of Rumex (Polygonaceae) illuminates the divergent evolutionary histories of docks and sorrels.

The genus Rumex L. (Polygonaceae) provides a unique system for investigating the evolutionary development of sex determination and molecular rate evolution. Historically, Rumex has been divided, both taxonomically and colloquially into two groups: 'docks' and 'sorrels'. A well-resolved phylogeny can help evaluate a genetic basis for this division. Here we present a plastome phylogeny for 34 species of Rumex, inferred using maximum likelihood criteria. The historical 'docks' (Rumex subgenus Rumex) were resolved as monophyletic. The historical 'sorrels' (Rumex subgenera Acetosa and Acetosella) were resolved together, though not monophyletic due to the inclusion of R. bucephalophorus (Rumex subgenus Platypodium). Emex is supported as its own subgenus within Rumex, instead of resolved as sister taxa. We found remarkably low nucleotide diversity among the docks, consistent with recent diversification in that group, especially as compared to the sorrels. Fossil calibration of the phylogeny suggested that the common ancestor for Rumex (including Emex) has origins in the lower Miocene (22.13 MYA). The sorrels appear to have subsequently diversified at a relatively constant rate. The origin of the docks, however, was placed in the upper Miocene, but with most speciation occurring in the Plio-Pleistocene.

Emerging evidence of plant domestication as a landscape-level process.

The evidence from ancient crops over the past decade challenges some of our most basic assumptions about the process of domestication. The emergence of crops has been viewed as a technologically progressive process in which single or multiple localized populations adapt to human environments in response to cultivation. By contrast, new genetic and archaeological evidence reveals a slow process that involved large populations over wide areas with unexpectedly sustained cultural connections in deep time. We review evidence that calls for a new landscape framework of crop origins. Evolutionary processes operate across vast distances of landscape and time, and the origins of domesticates are complex. The domestication bottleneck is a redundant concept and the progressive nature of domestication is in doubt.

Phylogenomic analysis points to a South American origin of Manihot and illuminates the primary gene pool of cassava.

The genus Manihot, with around 120 known species, is native to a wide range of habitats and regions in the tropical and subtropical Americas. Its high species richness and recent diversification only c. 6 million years ago have significantly complicated previous phylogenetic analyses. Several basic elements of Manihot evolutionary history therefore remain unresolved. Here, we conduct a comprehensive phylogenomic analysis of Manihot, focusing on exhaustive sampling of South American taxa. We find that two recently described species from northeast Brazil's Atlantic Forest were the earliest to diverge, strongly suggesting a South American common ancestor of Manihot. Ancestral state reconstruction indicates early Manihot diversification in dry forests, with numerous independent episodes of new habitat colonization, including into savannas and rainforests within South America. We identify the closest wild relatives to Manihot esculenta, including the crop cassava, and we quantify extensive wild introgression into the cassava gene pool from at least five wild species, including Manihot glaziovii, a species used widely in breeding programs. Finally, we show that this wild-to-crop introgression substantially shapes the mutation load in cassava. Our findings provide a detailed case study for neotropical evolutionary history in a diverse and widespread group, and a robust phylogenomic framework for future Manihot and cassava research.

Extraction and high-throughput sequencing of oak heartwood DNA: Assessing the feasibility of genome-wide DNA methylation profiling.

Tree ring features are affected by environmental factors and therefore are the basis for dendrochronological studies to reconstruct past environmental conditions. Oak wood often provides the data for these studies because of the durability of oak heartwood and hence the availability of samples spanning long time periods of the distant past. Wood formation is regulated in part by epigenetic mechanisms such as DNA methylation. Studies of the methylation state of DNA preserved in oak heartwood thus could identify epigenetic tree ring features informing on past environmental conditions. In this study, we aimed to establish protocols for the extraction of DNA, the high-throughput sequencing of whole-genome DNA libraries (WGS) and the profiling of DNA methylation by whole-genome bisulfite sequencing (WGBS) for oak (Quercus robur) heartwood drill cores taken from the trunks of living standing trees spanning the AD 1776-2014 time period. Heartwood contains little DNA, and large amounts of phenolic compounds known to hinder the preparation of high-throughput sequencing libraries. Whole-genome and DNA methylome library preparation and sequencing consistently failed for oak heartwood samples more than 100 and 50 years of age, respectively. DNA fragmentation increased with sample age and was exacerbated by the additional bisulfite treatment step during methylome library preparation. Relative coverage of the non-repetitive portion of the oak genome was sparse. These results suggest that quantitative methylome studies of oak hardwood will likely be limited to relatively recent samples and will require a high sequencing depth to achieve sufficient genome coverage.

Pleistocene origins, western ghost lineages, and the emerging phylogeographic history of the red wolf and coyote.

The red wolf (Canis rufus) of the eastern US was driven to near-extinction by colonial-era persecution and habitat conversion, which facilitated coyote (C. latrans) range expansion and widespread hybridization with red wolves. The observation of some grey wolf (C. lupus) ancestry within red wolves sparked controversy over whether it was historically a subspecies of grey wolf with its predominant "coyote-like" ancestry obtained from post-colonial coyote hybridization (2-species hypothesis) versus a distinct species closely related to the coyote that hybridized with grey wolf (3-species hypothesis). We analysed mitogenomes sourced from before the 20th century bottleneck and coyote invasion, along with hundreds of modern amplicons, which led us to reject the 2-species model and to investigate a broader phylogeographic 3-species model suggested by the fossil record. Our findings broadly support this model, in which red wolves ranged the width of the American continent prior to arrival of the grey wolf to the mid-continent 60-80 ka; red wolves subsequently disappeared from the mid-continent, relegated to California and the eastern forests, which ushered in emergence of the coyote in their place (50-30 ka); by the early Holocene (12-10 ka), coyotes had expanded into California, where they admixed with and phenotypically replaced western red wolves in a process analogous to the 20th century coyote invasion of the eastern forests. Findings indicate that the red wolf pre-dated not only European colonization but human, and possibly coyote, presence in North America. These findings highlight the urgency of expanding conservation efforts for the red wolf.

Evolutionary and phylogenetic insights from a nuclear genome sequence of the extinct, giant, "subfossil" koala lemur Megaladapis edwardsi.

No endemic Madagascar animal with body mass >10 kg survived a relatively recent wave of extinction on the island. From morphological and isotopic analyses of skeletal "subfossil" remains we can reconstruct some of the biology and behavioral ecology of giant lemurs (primates; up to ∼160 kg) and other extraordinary Malagasy megafauna that survived into the past millennium. Yet, much about the evolutionary biology of these now-extinct species remains unknown, along with persistent phylogenetic uncertainty in some cases. Thankfully, despite the challenges of DNA preservation in tropical and subtropical environments, technical advances have enabled the recovery of ancient DNA from some Malagasy subfossil specimens. Here, we present a nuclear genome sequence (∼2× coverage) for one of the largest extinct lemurs, the koala lemur Megaladapis edwardsi (∼85 kg). To support the testing of key phylogenetic and evolutionary hypotheses, we also generated high-coverage nuclear genomes for two extant lemurs, Eulemur rufifrons and Lepilemur mustelinus, and we aligned these sequences with previously published genomes for three other extant lemurs and 47 nonlemur vertebrates. Our phylogenetic results confirm that Megaladapis is most closely related to the extant Lemuridae (typified in our analysis by E. rufifrons) to the exclusion of L. mustelinus, which contradicts morphology-based phylogenies. Our evolutionary analyses identified significant convergent evolution between M. edwardsi and an extant folivore (a colobine monkey) and an herbivore (horse) in genes encoding proteins that function in plant toxin biodegradation and nutrient absorption. These results suggest that koala lemurs were highly adapted to a leaf-based diet, which may also explain their convergent craniodental morphology with the small-bodied folivore Lepilemur.

Exaptation Traits for Megafaunal Mutualisms as a Factor in Plant Domestication.

Megafaunal extinctions are recurring events that cause evolutionary ripples, as cascades of secondary extinctions and shifting selective pressures reshape ecosystems. Megafaunal browsers and grazers are major ecosystem engineers, they: keep woody vegetation suppressed; are nitrogen cyclers; and serve as seed dispersers. Most angiosperms possess sets of physiological traits that allow for the fixation of mutualisms with megafauna; some of these traits appear to serve as exaptation (preadaptation) features for farming. As an easily recognized example, fleshy fruits are, an exaptation to agriculture, as they evolved to recruit a non-human disperser. We hypothesize that the traits of rapid annual growth, self-compatibility, heavy investment in reproduction, high plasticity (wide reaction norms), and rapid evolvability were part of an adaptive syndrome for megafaunal seed dispersal. We review the evolutionary importance that megafauna had for crop and weed progenitors and discuss possible ramifications of their extinction on: (1) seed dispersal; (2) population dynamics; and (3) habitat loss. Humans replaced some of the ecological services that had been lost as a result of late Quaternary extinctions and drove rapid evolutionary change resulting in domestication.

Pre-Columbian transregional captive rearing of Amazonian parrots in the Atacama Desert.

The feathers of tropical birds were one of the most significant symbols of economic, social, and sacred status in the pre-Columbian Americas. In the Andes, finely produced clothing and textiles containing multicolored feathers of tropical parrots materialized power, prestige, and distinction and were particularly prized by political and religious elites. Here we report 27 complete or partial remains of macaws and amazon parrots from five archaeological sites in the Atacama Desert of northern Chile to improve our understanding of their taxonomic identity, chronology, cultural context, and mechanisms of acquisition. We conducted a multiproxy archaeometric study that included zooarchaeological analysis, isotopic dietary reconstruction, accelerated mass spectrometry radiocarbon dating, and paleogenomic analysis. The results reveal that during the Late Intermediate Period (1100 to 1450 CE), Atacama oasis communities acquired scarlet macaws (Ara macao) and at least five additional translocated parrot species through vast exchange networks that extended more than 500 km toward the eastern Amazonian tropics. Carbon and nitrogen stable isotopes indicate that Atacama aviculturalists sustained these birds on diets rich in marine bird guano-fertilized maize-based foods. The captive rearing of these colorful, exotic, and charismatic birds served to unambiguously signal relational wealth in a context of emergent intercommunity competition.

Archaeological Central American maize genomes suggest ancient gene flow from South America.

Maize (Zea mays ssp. mays) domestication began in southwestern Mexico ∼9,000 calendar years before present (cal. BP) and humans dispersed this important grain to South America by at least 7,000 cal. BP as a partial domesticate. South America served as a secondary improvement center where the domestication syndrome became fixed and new lineages emerged in parallel with similar processes in Mesoamerica. Later, Indigenous cultivators carried a second major wave of maize southward from Mesoamerica, but it has been unclear until now whether the deeply divergent maize lineages underwent any subsequent gene flow between these regions. Here we report ancient maize genomes (2,300-1,900 cal. BP) from El Gigante rock shelter, Honduras, that are closely related to ancient and modern maize from South America. Our findings suggest that the second wave of maize brought into South America hybridized with long-established landraces from the first wave, and that some of the resulting newly admixed lineages were then reintroduced to Central America. Direct radiocarbon dates and cob morphological data from the rock shelter suggest that more productive maize varieties developed between 4,300 and 2,500 cal. BP. We hypothesize that the influx of maize from South America into Central America may have been an important source of genetic diversity as maize was becoming a staple grain in Central and Mesoamerica.

Ancient Plant Genomics in Archaeology, Herbaria, and the Environment.

The ancient DNA revolution of the past 35 years has driven an explosion in the breadth, nuance, and diversity of questions that are approachable using ancient biomolecules, and plant research has been a constant, indispensable facet of these developments. Using archaeological, paleontological, and herbarium plant tissues, researchers have probed plant domestication and dispersal, plant evolution and ecology, paleoenvironmental composition and dynamics, and other topics across related disciplines. Here, we review the development of the ancient DNA discipline and the role of plant research in its progress and refinement. We summarize our understanding of long-term plant DNA preservation and the characteristics of degraded DNA. In addition, we discuss challenges in ancient DNA recovery and analysis and the laboratory and bioinformatic strategies used to mitigate them. Finally, we review recent applications of ancient plant genomic research.

A domestication history of dynamic adaptation and genomic deterioration in Sorghum.

The evolution of domesticated cereals was a complex interaction of shifting selection pressures and repeated episodes of introgression. Genomes of archaeological crops have the potential to reveal these dynamics without being obscured by recent breeding or introgression. We report a temporal series of archaeogenomes of the crop sorghum (Sorghum bicolor) from a single locality in Egyptian Nubia. These data indicate no evidence for the effects of a domestication bottleneck, but instead reveal a steady decline in genetic diversity over time coupled with an accumulating mutation load. Dynamic selection pressures acted sequentially to shape architectural and nutritional domestication traits and to facilitate adaptation to the local environment. Later introgression between sorghum races allowed the exchange of adaptive traits and achieved mutual genomic rescue through an ameliorated mutation load. These results reveal a model of domestication in which genomic adaptation and deterioration were not focused on the initial stages of domestication but occurred throughout the history of cultivation.

Extraction of Ancient DNA from Plant Remains.

Ancient plant remains from archaeological sites, paleoenvironmental contexts, and herbaria provide excellent opportunities for interrogating plant genetics over Quaternary timescales using ancient DNA (aDNA)-based analyses. A variety of plant tissues, preserved primarily by desiccation and anaerobic waterlogging, have proven to be viable sources of aDNA. Plant tissues are anatomically and chemically diverse and therefore require optimized DNA extraction approaches. Here, we describe a plant DNA isolation protocol that performs well in most contexts. We include recommendations for optimization to retain the very short DNA fragments that are expected to be preserved in degraded tissues.

A re-evaluation of the domestication bottleneck from archaeogenomic evidence.

Domesticated crops show a reduced level of diversity that is commonly attributed to the "domestication bottleneck"; a drastic reduction in the population size associated with subsampling the wild progenitor species and the imposition of selection pressures associated with the domestication syndrome. A prediction of the domestication bottleneck is a sharp decline in genetic diversity early in the domestication process. Surprisingly, archaeological genomes of three major annual crops do not indicate that such a drop in diversity occurred early in the domestication process. In light of this observation, we revisit the general assumption of the domestication bottleneck concept in our current understanding of the evolutionary process of domestication.

A Guide to Carrying Out a Phylogenomic Target Sequence Capture Project.

High-throughput DNA sequencing techniques enable time- and cost-effective sequencing of large portions of the genome. Instead of sequencing and annotating whole genomes, many phylogenetic studies focus sequencing effort on large sets of pre-selected loci, which further reduces costs and bioinformatic challenges while increasing coverage. One common approach that enriches loci before sequencing is often referred to as target sequence capture. This technique has been shown to be applicable to phylogenetic studies of greatly varying evolutionary depth. Moreover, it has proven to produce powerful, large multi-locus DNA sequence datasets suitable for phylogenetic analyses. However, target capture requires careful considerations, which may greatly affect the success of experiments. Here we provide a simple flowchart for designing phylogenomic target capture experiments. We discuss necessary decisions from the identification of target loci to the final bioinformatic processing of sequence data. We outline challenges and solutions related to the taxonomic scope, sample quality, and available genomic resources of target capture projects. We hope this review will serve as a useful roadmap for designing and carrying out successful phylogenetic target capture studies.