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Waking the Dead: Bringing Extinct Species Back to Life

TIME Video: Conservationists and scientists have a saying, “extinction is forever.” But soon biologists will clone long-gone animals in the hope to redefine that axiom to “DE-extinction is forever.” Stewart Brand and Ben Novak share their thoughts on de-extinction and Revive & Restore’s flagship project, The Great Passenger Pigeon Comeback.

The Great
Passenger Pigeon

This is the first project to revive an extinct animal using its museum-specimen DNA. Once it succeeds, the techniques will be applicable to hundreds of other extinct species.

› Project goals
› Why passenger pigeons?
› Progress to date
› UC Santa Cruz Paleogenomics Lab partnership
› History of the passenger pigeon
› Population ecology

Project goals

The goal of The Great Passenger Pigeon Comeback is to bring the passenger pigeon all the way back using the genome of the band-tailed pigeon and state-of-the-art genomic technology.

Female passenger pigeon. Painting byTim Hough.

Female passenger pigeon. Painting by Tim Hough.

The genomes of the two birds will be compared in close detail, to determine which differences are most crucial. The data and analysis will begin with the process of converting viable band-tailed DNA into viable passenger pigeon DNA. Later stages in the project will involve newly developed and advancing techniques for CRISPR genome editing and germ line transfer to generate live passenger pigeons from the DNA.

Research for the Great Passenger Pigeon Comeback will encompass not only the stages of comparative genomics, bio-engineering, captive breeding, and then wild release, but also the topics of population genetics, paleo-ecology, dietary investigation, habitat assessment, experimental ecology, and finding a pathway for altering band-tailed pigeons to produce the optimal surrogate parent. These areas of research are proving to be important considerations for all candidates for de-extinction as well as endangered candidate species for genetic assistance.

Why passenger pigeons?
The passenger pigeon is a compelling choice for de-extinction. Humans hunted them to extinction from a population of billions until 1914 when none remained. The return of this iconic species by human hands would be a suitable and extraordinary twist in the story of the passenger pigeon. In developing how science approaches de-extinction, a series of criteria for a de-extinction candidate has emerged. According to this criteria, the passenger pigeon offers relative technical practicality for the scientific work of de-extinction. No single species offers an easily achievable, prime candidate for de-extinction, however the passenger pigeon is a model species poised at the optimal middle ground of ease and difficulty. It is a species that is not only feasible to successfully bring back, but also presents enough challenges to push the science forward and open up the possibility of de-extinction to many more species. An extinct mouse would be an easy win for de-extinction, but it does not challenge us to produce the methods necessary to revive birds or reptiles. The passenger pigeon is also a model species for thoroughly testing the process of de-extinction. There are sufficient specimens and enough recorded history to establish a great understanding of the species’ past. This allows us to plan its future in a knowledgeable and responsible manner and also offers an ideal opportunity to learn about extinction. This knowledge will be essential to understanding and helping other species that are critically endangered.

Progress to date
To fully understand the passenger pigeon genome we need to sequence the genome of a single individual to a high degree of quality and then analyze the genetic diversity of more passenger pigeons to understand which mutations are variable and which mutations are fixed between the passenger pigeon and its relative the band-tailed pigeon. In the past year, DNA has been sequenced from 12 passenger pigeons to analyze if the species’ DNA can be used to assemble the full genomic code. The passenger pigeon samples came from the collections of the Royal Ontario Museum and the Field Museum of Natural History. Their DNA has already proven to be quite good in quality and has moved our project closer to obtaining our goal of a full passenger pigeon genome. From these first 12 specimens it was deduced that testing the DNA of more passenger pigeons could produce the most viable full genome sequencing candidate, one that may be better in quality than the this first test set of 12 specimens. A high quality specimen, ROM, aka “Passenger Pigeon 1871”, was selected for full genome sequencing, and 20 specimens were selected for population genetics sequencing, which commenced October, 2013.

Initial data for the full genome of an individual band-tailed pigeon, “Sally”, and the individual passenger pigeon “Passenger pigeon 1871”,  have been generated and are in various stages of data processing. The project currently has DNA sequence data sets for a second individual band-tailed pigeon, obtained from the American Museum of Natural History Ornithology Collections, courtesy of collections manager Dr. Paul Sweet. This band-tailed pigeon was collected in Whatcom County, Washington, 2003. The DNA from this individual is not of high quality as that obtained from “Sally”, the captive bred pigeon living at Exotic Wings International Aviary, under the care of expert aviculturist Sal Alvarez. But with the reference genome of “Sally”, the DNA sequences from this second specimen can be mapped to generate a second full genome. A second passenger pigeon specimen has been selected from among our population genomics data set to sequence further for an additional full genome. This specimen is ROM 40360, aka BN1-9, a pigeon shot in Montreal, Quebec, 1880.  With multiple genomes of each species we’ll be able to discern significant mutations that differentiate the two species versus mutations unique to single individuals. Evolutionary mutations are the backbone of the de-extinction of the first new living passenger pigeon, while unique mutations between passenger pigeons are necessary for the conservation genetics of restoring a breeding population.

Continued work in the lab has moved to experiments with “enrichment capture” – a method by which the bacterial, fungal, and human contaminant DNA within a DNA extraction is removed by isolating the passenger pigeon DNA using “baits” designed to capture pigeon-like DNA codes. Once the pigeon DNA has been “baited” the non-pigeon DNA is washed away, yielding a DNA extraction with a higher concentration of passenger pigeon DNA. This will allow us to expand our data set of passenger pigeon genes more efficiently.

We are now initiating research designs for the isolation and culturing of band-tailed pigeon primordial germ cells, which are necessary to perform the genome engineering to create passenger pigeons. This is expected to be the major limiting component of the project, both in terms of time and resources; however, experts in the field of avian biotech are currently developing success with similar work for several species beyond the chicken model. Expanding this research to pigeons is the exciting next steps of The Great Comeback.

UCSC Paleogenomics Lab partnership
Revive & Restore is partnering with University of California Santa Cruz’s state-of-the-art Paleogenomics Lab for the Great Passenger Pigeon Comeback.

At the Lab, researchers are incorporating experimental and computational approaches to a wide range of evolutionary and ecological questions— mostly involving the application of genomic techniques— to better understand how species and populations such as the passenger pigeon evolve through time. Evolutionary biologist Dr. Beth Shapiro, an expert in ancient DNA laboratory techniques and paleogenomics, jointly leads the Lab with Dr. Richard “Ed” Green, a bioinformatician and expert in modern and ancient genome assembly and analysis. Dr. Shapiro, whose work focuses on how populations of organisms respond to climate and habitat change over time, has been working in collaboration with Dr. Green for several years to assemble passenger pigeon DNA. Additionally, Dr. Green has already developed tools for the eventual assembly of the full passenger pigeon genome. Revive & Restore’s research consultant Ben Novak has contributed vital input on the research, design and planning of the Great Passenger Pigeon Comeback since he joined the Lab as a visiting researcher in early 2013. Lab researcher, Dr. André Elias Rodrigues Soares is leading the computational analysis of new passenger pigeon data, focusing on the population genetics and demography of the species.

History of the passenger pigeon
The first written record of passenger pigeons occurs in a ship captain’s journal in the year 1534 and recounts “an infinite number of wood pigeons.” Throughout the formation of the early British colonies into the United States, the flocks of passenger pigeons were observed to number in the billions. Several ornithologists have tried to calculate the size of flocks that were recorded to take days to fly over a town. It’s suggested that some flocks were a mile wide and 300 miles long, dense enough to block out the sun. During the 1800s poultry farms and ranches hadn’t established sufficiently to feed the growing population of immigrants to the United States. A trip to the marketplace was filled with the meat of all types of wild game. The pigeon’s giant flocks became an easy source of abundant food. The invention of the telegraph allowed food companies to track the passenger pigeon’s movements and “head them off” with hired trappers and shooters. The completion of the railroad between Chicago and New York City changed the industry from harvesting for local markets to shipping to more profitable city markets, and so the harvest of passenger pigeons boomed. During the 1870s millions upon millions of birds were consumed for food. Their feathers made bed mattresses and pillows. Live birds were caught and shipped by the thousands for trap-shooting tournaments. There were certainly at least one billion passenger pigeons alive in 1878, but by 1890 only tens could be spotted anywhere. The last wild birds were shot between 1900 and 1902. This was a shock to the people of that time who believed their numbers would never be diminished. Unfortunately, no one ever put effort into raising the birds in captivity. At the time, pigeon fanciers were more interested in flashy domestic breeds and exotic species from Southeast Asia. Passenger pigeons were thought to be readily available from the wild, so breeding deemed unnecessary. One captive flock was alive when the last birds in the wild disappeared. The flock was highly inbred and the individuals were already quite old. They died off without producing successful offspring over the next few years, until a female living at the Cincinnati Ohio Zoo, named Martha was the last of the species. On September 1, 1914 Martha died. The passenger pigeon became extinct.

Ben Novak studying Martha, the last passenger pigeon in the world (left). She died on September 1, 1914, at the Cincinnati Zoo. She was preserved in ice and sent to the Smithsonian Institution in Washington DC, where she is occasionally displayed with a male (on right). Note the red eye, iridescent neck feathers, red feet, and (in the male) peach-colored breast and blueish back. Photo credit Ryan Phelan.

Ben Novak studying Martha, the last passenger pigeon in the world (left). She died on September 1, 1914, at the Cincinnati Zoo. She was preserved in ice and sent to the Smithsonian Institution in Washington DC, where she is occasionally displayed with a male (on right). Note the red eye, iridescent neck feathers, red feet, and (in the male) peach-colored breast and blueish back.
Photo credit Ryan Phelan.

Population ecology
Using the fossil record of the passenger pigeon and ancient pollen grains we now know that passenger pigeons lived and thrived throughout the past 250,000 years – adapting to major climate changes from ice ages to dry hot climates. Gut contents of passenger pigeons that were collected in the 1800s did not survive, but were documented to contain tree mast — the majority of which is comprised of oak acorns. Based on how living pigeon species interact with good food sources, we can hypothesize that the passenger pigeon was dependent on deciduous forests. Its primary food source were the acorns of diverse oak species, and the abundance of acorns may have been a limiting factor in population size. The passenger pigeon’s mega flocks impacted acorn and nut crops each year, each mega flock acting as a “seed-predator” that shaped the diversity of tree types throughout the forest. This relationship caused cycling disturbances of the kind that generate biodiversity turnover, and thus over time the passenger pigeon sustained high biodiversity in forest ecosystems. There are several theories as to how the passenger pigeon reached such large numbers. Some believe that the species was abundant for hundreds of thousands of years, while others have postulated that the species “exploded” in population much more recently, perhaps due to the end of the last ice age or a relationship with Amerindian hunter/gatherers and agricultural communities that developed even more recently. Our population genetics work aims to fill in the details of these many hypotheses that historical data and research cannot fully answer.

Work in Progress

By Beth Shapiro, PhD
Associate Professor, Ecology and Evolutionary Biology
Co-Principal Investigator of the Paleogenomics Lab, University of California Santa Cruz

June 19, 2014

As you may know, a different group (not us — Ouch!) has published a paper in PNAS on June 16 (see abstract and link) in which they use genome sequence data from several preserved passenger pigeons to infer long-term demographic trends in the bird.

It is important to the de-extinction effort because it shows (as our data do) that passenger pigeon populations fluctuated in size through time, as resource availability changed. This means that we probably won’t need to bring back billions of birds in order for their populations to be sustainable, as long as we can keep ourselves from killing them.

The paper maps passenger pigeon genetic data to a published genome from the Rock dove, Columba livia, and uses these data to infer changes in their population size through time. They also do a very nice reconstruction of what niche space would have been available to these birds during the Holocene.

In addition to sequencing a few more passenger pigeon genomes (which will be useful to evaluate how genetically diverse they were), we are assembling and annotating the genome of the band-tailed pigeon. Because the band-tailed pigeon is the most closely related living pigeon to the passenger pigeon, we can use these data to figure out what genetic changes are unique to the passenger pigeon. This will be key to resurrecting passenger pigeon traits in living birds.

Project Update:
The Great Passenger Pigeon Comeback

February 27, 02014

The year 02013 was a productive time for our flagship project: The Great Passsenger Pigeon Comeback. Outreach for 02013 included presentations at TEDxDeExtinction, the Mississippi Flyway Council meeting, the American Ornithologists’ Union annual meeting, the Association of Zoos and Aquariums annual conference, and various education and public presentations engaging diverse audiences from elementary and high school students to researchers, scientists and administrators of academia, zoos, the United States Fish and Wildlife Service, and project enthusiasts of all walks of life. In the lab, under the leadership of Ben Novak (Revive & Restore) and Dr. Beth Shapiro (UC Santa Cruz Paleogenomics Lab) several major milestones for the project have been completed.

Progress continues into 02014 with bioinformatics and ecological research. Upcoming conferences and meetings concerning avian biotech are forming the initiation of project designs for the cellular and genomic editing that will produce living passenger pigeons from the genome studies currently in development.

Next Steps: Bioinformatics

Nearly a century after the last passenger pigeon died in 1914, the team of scientists working on the Great Passenger Pigeon Comeback is on the verge of assembling – arranging to create a representation of the original chromosomes – a “first draft” of the genome of this species.

Dr. André Soares, UCSC Paleogenomics Lab, is leading the computational analysis and assembly of the new passenger pigeon data. The focus of the team’s work is now on finalizing the assembly of the passenger pigeon genome using the rock pigeon and band-tailed pigeon genomes as references. The rock pigeon genome has already been serving as a reference and as soon as the band-tailed pigeon genome is assembled it will fill gaps and produce a higher quality mapping. The team is confident that all essential DNA regions will assemble successfully, paving the way for the de-extinction of the passenger pigeon.

We will be developing ecological studies in order to give insight into the passenger pigeon’s historical populations and how they interacted with the environment. These findings will be used to create a passenger pigeon recovery plan, which includes ultimately identifying and selecting wild release sites, calculating target population size, and predicting ecological impact and management needs.

A 15-year Project in the Making

Our major goal: to bring the Passenger Pigeon all the way back. The work necessary to achieve this goal includes comparing the assembled passenger pigeon genome to the genome of its closest living relative, the band-tailed pigeon, to reveal the DNA regions that make these two species different. This information will allow the team to replace segments of the band-tailed pigeon genome with the essential passenger pigeon sequences. The resulting passenger pigeon genome will be transferred into germ cells of band-tailed pigeons, using techniques still in development, to generate live passenger pigeons. The live birds will be bred in captivity and eventually returned to the wild. Every step of the way, the genetic variability of the passenger pigeon will be prioritized, incorporating diversity from multiple specimens spanning 250 years of ecological history across Northeastern North America, to make sure the end result is a diverse and viable population.