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\"Could extinct species like mammoths and passenger pigeons be brought back to life? The science says yes. In [this book], Beth Shapiro, evolutionary biologist and pioneer in 'ancient DNA' research, walks readers through the astonishing and controversial process of de-extinction. From deciding which species should be restored, to sequencing their genomes, to anticipating how revived populations might be overseen in the wild, Shapiro vividly explores the extraordinary cutting-edge science that is being used--today--to resurrect the past\"--Amazon.com.

“Conservation genomics” encompasses the idea that genome-scale data will improve the capacity of resource managers to protect species. Although genetic approaches have long been used in conservation research, it has only recently become tractable to generate genome-wide data at a scale that is useful for conservation. In this Review, we discuss how genome-scale data can inform species delineation in the face of admixture, facilitate evolution through the identification of adaptive alleles, and enhance evolutionary rescue based on genomic patterns of inbreeding. As genomic approaches become more widely adopted in conservation, we expect that they will have a positive impact on management and policy decisions.

Environmental DNA (eDNA) metabarcoding is an increasingly popular tool for measuring and cataloguing biodiversity. Because the environments and substrates in which DNA is preserved differ considerably, eDNA research often requires bespoke approaches to generating eDNA data. Here, we explore how two experimental choices in eDNA study design—the number of PCR replicates and the depth of sequencing of PCR replicates—influence the composition and consistency of taxa recovered from eDNA extracts. We perform 24 PCR replicates from each of six soil samples using two of the most common metabarcodes for Fungi and Viridiplantae (ITS1 and ITS2), and sequence each replicate to an average depth of ~84,000 reads. We find that PCR replicates are broadly consistent in composition and relative abundance of dominant taxa, but that low abundance taxa are often unique to one or a few PCR replicates. Taxa observed in one out of 24 PCR replicates make up 21–29% of the total taxa detected. We also observe that sequencing depth or rarefaction influences alpha diversity and beta diversity estimates. Read sampling depth influences local contribution to beta diversity, placement in ordinations, and beta dispersion in ordinations. Our results suggest that, because common taxa drive some alpha diversity estimates, few PCR replicates and low read sampling depths may be sufficient for many biological applications of eDNA metabarcoding. However, because rare taxa are recovered stochastically, eDNA metabarcoding may never fully recover the true amplifiable alpha diversity in an eDNA extract. Rare taxa drive PCR replicate outliers of alpha and beta diversity and lead to dispersion differences at different read sampling depths. We conclude that researchers should consider the complexity and unevenness of a community when choosing analytical approaches, read sampling depths, and filtering thresholds to arrive at stable estimates. Previous studies examining the influence of methodological choices in environmental DNA processing on the resulting observed communities are a great resource in designing methods of a study, and accurately interpreting results. Here, we designed a methodological experiment to address the influences of PCR replication, sequencing depth, and a minimum read threshold on measures of biodiversity. We found stability between PCR replicates in high abundance taxa, and high variation in low abundance taxa, which significantly influence alpha and beta diversity.

The rapid loss of intraspecific variation is a hidden biodiversity crisis. Intraspecific variation, which includes the genomic and phenotypic diversity found within and among populations, is threatened by local extinctions, abundance declines, and anthropogenic selection. However, biodiversity assessments often fail to highlight this loss of diversity within species. We review the literature on how intraspecific variation supports critical ecological functions and nature’s contributions to people (NCP). Results show that the main categories of NCP (material, non-material, and regulating) are supported by intraspecific variation. We highlight new strategies that are needed to further explore these connections and to make explicit the value of intraspecific variation for NCP. These strategies will require collaboration with local and Indigenous groups who possess critical knowledge on the relationships between intraspecific variation and ecosystem function. New genomic methods provide a promising set of tools to uncover hidden variation. Urgent action is needed to document, conserve, and restore the intraspecific variation that supports nature and people. Thus, we propose that the maintenance and restoration of intraspecific variation should be raised to a major global conservation objective. This Perspective highlights how conservation of intraspecific variation is critical for sustaining nature’s contributions to people.

The PacBio ® HiFi sequencing method yields highly accurate long-read sequencing datasets with read lengths averaging 10–25 kb and accuracies greater than 99.5%. These accurate long reads can be used to improve results for complex applications such as single nucleotide and structural variant detection, genome assembly, assembly of difficult polyploid or highly repetitive genomes, and assembly of metagenomes. Currently, there is a need for sample data sets to both evaluate the benefits of these long accurate reads as well as for development of bioinformatic tools including genome assemblers, variant callers, and haplotyping algorithms. We present deep coverage HiFi datasets for five complex samples including the two inbred model genomes Mus musculus and Zea mays , as well as two complex genomes, octoploid Fragaria  ×  ananassa and the diploid anuran Rana muscosa . Additionally, we release sequence data from a mock metagenome community. The datasets reported here can be used without restriction to develop new algorithms and explore complex genome structure and evolution. Data were generated on the PacBio Sequel II System. Measurement(s) DNA • genome • Metagenome Technology Type(s) DNA sequencing • PacBio Sequel System Factor Type(s) organism that had its genome sequenced Sample Characteristic - Organism Mus musculus • Rana muscosa • Fragaria x ananassa • Zea mays Machine-accessible metadata file describing the reported data: https://doi.org/10.6084/m9.figshare.12855527

A low-coverage draft genome sequence from a horse bone recovered from permafrost dated to approximately 560–780 thousand years ago is presented; this represents the oldest full genome sequence to date by almost an order of magnitude. Old horse DNA makes sense of Equus lineage A low-coverage draft genome sequence has been obtained from a horse bone recovered from a permafrost site in the Yukon Territory, Canada, dated to around 560,000–780,000 years before present. This is by far the earliest genome sequence so far determined. The data were compared to draft genome sequences for a Late Pleistocene horse, those of five contemporary domestic horse breeds, a Przewalski's horse and a donkey. Comparative genomics suggest that the Equus lineage that gave rise to all contemporary horses, zebras and donkeys originated about 4.0–4.5 million years ago — much earlier than previously suspected. The data support the contention that Przewalski's horses — an endangered subspecies native to the Mongolian steppes — represent the last surviving wild horse population. The rich fossil record of equids has made them a model for evolutionary processes 1 . Here we present a 1.12-times coverage draft genome from a horse bone recovered from permafrost dated to approximately 560–780 thousand years before present (kyr bp ) 2 , 3 . Our data represent the oldest full genome sequence determined so far by almost an order of magnitude. For comparison, we sequenced the genome of a Late Pleistocene horse (43 kyr bp ), and modern genomes of five domestic horse breeds ( Equus ferus caballus ), a Przewalski’s horse ( E. f. przewalskii ) and a donkey ( E. asinus ). Our analyses suggest that the Equus lineage giving rise to all contemporary horses, zebras and donkeys originated 4.0–4.5 million years before present (Myr bp ), twice the conventionally accepted time to the most recent common ancestor of the genus Equus 4 , 5 . We also find that horse population size fluctuated multiple times over the past 2 Myr, particularly during periods of severe climatic changes. We estimate that the Przewalski’s and domestic horse populations diverged 38–72 kyr bp , and find no evidence of recent admixture between the domestic horse breeds and the Przewalski’s horse investigated. This supports the contention that Przewalski’s horses represent the last surviving wild horse population 6 . We find similar levels of genetic variation among Przewalski’s and domestic populations, indicating that the former are genetically viable and worthy of conservation efforts. We also find evidence for continuous selection on the immune system and olfaction throughout horse evolution. Finally, we identify 29 genomic regions among horse breeds that deviate from neutrality and show low levels of genetic variation compared to the Przewalski’s horse. Such regions could correspond to loci selected early during domestication.

Background Cell-free DNA (cfDNA), present in circulating blood plasma, contains information about prenatal health, organ transplant reception, and cancer presence and progression. Originally developed for the genomic analysis of highly degraded ancient DNA, single-stranded DNA (ssDNA) library preparation methods are gaining popularity in the field of cfDNA analysis due to their efficiency and ability to convert short, fragmented DNA into sequencing libraries without altering DNA ends. However, current ssDNA methods are costly and time-consuming. Results Here we present an efficient ligation-based single-stranded library preparation method that is engineered to produce complex libraries in under 2.5 h from as little as 1 nanogram of input DNA without alteration to the native ends of template molecules. Our method, called Single Reaction Single-stranded LibrarY or SRSLY, ligates uniquely designed Next-Generation Sequencing (NGS) adapters in a one-step combined phosphorylation/ligation reaction that foregoes end-polishing. Using synthetic DNA oligos and cfDNA, we demonstrate the efficiency and utility of this approach and compare with existing double-stranded and single-stranded approaches for library generation. Finally, we demonstrate that cfDNA NGS data generated from SRSLY can be used to analyze DNA fragmentation patterns to deduce nucleosome positioning and transcription factor binding. Conclusions SRSLY is a versatile tool for converting short and fragmented DNA molecules, like cfDNA fragments, into sequencing libraries while retaining native lengths and ends.

Large-scale changes in global climate at the end of the Pleistocene significantly impacted ecosystems across North America. However, the pace and scale of biotic turnover in response to both the Younger Dryas cold period and subsequent Holocene rapid warming have been challenging to assess because of the scarcity of well dated fossil and pollen records that covers this period. Here we present an ancient DNA record from Hall’s Cave, Texas, that documents 100 vertebrate and 45 plant taxa from bulk fossils and sediment. We show that local plant and animal diversity dropped markedly during Younger Dryas cooling, but while plant diversity recovered in the early Holocene, animal diversity did not. Instead, five extant and nine extinct large bodied animals disappeared from the region at the end of the Pleistocene. Our findings suggest that climate change affected the local ecosystem in Texas over the Pleistocene-Holocene boundary, but climate change on its own may not explain the disappearance of the megafauna at the end of the Pleistocene. The impact of late Pleistocene climate change on ecosystems has been hard to assess. Here, the authors sequence ancient DNA from Hall’s Cave, Texas and find that both plant and vertebrate diversity decreased with cooling, and though plant diversity recovered with rewarming, megafauna went extinct.

Significance The domestication of the horse revolutionized warfare, trade, and the exchange of people and ideas. This at least 5,500-y-long process, which ultimately transformed wild horses into the hundreds of breeds living today, is difficult to reconstruct from archeological data and modern genetics alone. We therefore sequenced two complete horse genomes, predating domestication by thousands of years, to characterize the genetic footprint of domestication. These ancient genomes reveal predomestic population structure and a significant fraction of genetic variation shared with the domestic breeds but absent from Przewalski’s horses. We find positive selection on genes involved in various aspects of locomotion, physiology, and cognition. Finally, we show that modern horse genomes contain an excess of deleterious mutations, likely representing the genetic cost of domestication. The domestication of the horse ∼5.5 kya and the emergence of mounted riding, chariotry, and cavalry dramatically transformed human civilization. However, the genetics underlying horse domestication are difficult to reconstruct, given the near extinction of wild horses. We therefore sequenced two ancient horse genomes from Taymyr, Russia (at 7.4- and 24.3-fold coverage), both predating the earliest archeological evidence of domestication. We compared these genomes with genomes of domesticated horses and the wild Przewalski’s horse and found genetic structure within Eurasia in the Late Pleistocene, with the ancient population contributing significantly to the genetic variation of domesticated breeds. We furthermore identified a conservative set of 125 potential domestication targets using four complementary scans for genes that have undergone positive selection. One group of genes is involved in muscular and limb development, articular junctions, and the cardiac system, and may represent physiological adaptations to human utilization. A second group consists of genes with cognitive functions, including social behavior, learning capabilities, fear response, and agreeableness, which may have been key for taming horses. We also found that domestication is associated with inbreeding and an excess of deleterious mutations. This genetic load is in line with the “cost of domestication” hypothesis also reported for rice, tomatoes, and dogs, and it is generally attributed to the relaxation of purifying selection resulting from the strong demographic bottlenecks accompanying domestication. Our work demonstrates the power of ancient genomes to reconstruct the complex genetic changes that transformed wild animals into their domesticated forms, and the population context in which this process took place.