De-extinction

The Pyrenean ibex, or bucardo, is the first animal to have survived de-extinction past birth.

De-extinction, or resurrection biology, or species revivalism is the process of creating an organism, which is either a member of, or resembles an extinct species, or breeding population of such organisms. Cloning is the most widely proposed method, although selective breeding has also been proposed. Similar techniques have been applied to endangered species.

There is significant controversy over de-extinction. Critics assert that efforts would be better spent conserving existing species, that the habitat necessary for formerly extinct species to survive is too limited to warrant de-extinction, and that the evolutionary conservation benefits of these operations are questionable.

Methods

Cloning

Cloning is the method discussed as an option for bringing back extinct species, by extracting the nucleus from a preserved cell from the extinct species and swapping it into the egg of the nearest living relative. This egg can then be inserted into a relative host. It is important to note that this method can only be used when a preserved cell is available. This means that it is most feasible for recently extinct species.

Pictured above is the process used to clone the Pyrenean ibex in 2003. The tissue culture was taken from the last living, female Pyrenean ibex named Celia. The egg was taken from a goat (Capra hircus) and the nuclei removed to ensure the offspring was purely Pyrenean ibex. The egg was implanted into a surrogate goat mother for development.

Although de-extinction efforts have not yet succeeded in producing viable offspring of a previously extinct species, the same process has been applied successfully to endangered species. The banteng is an endangered species that was successfully cloned, and the first to survive for more than a week (the first was a gaur that died two days after being born). Scientists at Advanced Cell Technology in Worcester, Massachusetts, United States extracted DNA from banteng cells kept in the San Diego Zoo's "Frozen Zoo" facility, and transferred it into eggs from domestic cattle, a process called somatic cell nuclear transfer. Thirty hybrid embryos were created and sent to Trans Ova Genetics, which implanted the fertilized eggs in domestic cattle. Two were carried to term and delivered by Caesarian section. The first hybrid was born on April 1, 2003, and the second two days later. The second was euthanized, but the first survived and, as of September 2006, remained in good health at the San Diego Zoo.

Scientists from the University of Newcastle and the University of New South Wales, including Andrew French, Michael Mahony, Simon Clulow and Mike Archer reported in May 2013 the successful cloning of the extinct frog Rheobatrachus silus using the process of somatic cell nuclear transfer. The embryos developed for several days but died. In an important development the scientists from Newcastle reported associated technologies that provide a "proof of concept" for the proposal that frozen zoos (also referred to as genome banks and seed banks) are an effective mechanism to provide an insurance against species extinction and the loss of population genetic diversity. They connected the circle between de-extinction and the prevention of extinction for threatened animal species. The important advances were the capacity to successfully recover live frozen embryonic cells from animals that produce large yolky eggs (anamniotes such as fishes and amphibians) When this development is combined with somatic cell nuclear transfer (SCNT) it enables the genome to be recovered. The scientists point out that many embryonic cells can be frozen and when combined with frozen sperm storage enables the genetic diversity of populations to be stored. With groups of vertebrates such as the amphibians facing an extinction crisis they propose this as an effective means to prevent extinction while the causes of declines can be identified and remedied. The technical difference between frozen tissue samples commonly used for genetic studies (e.g. phylogenetic reconstruction) and those in a frozen zoo is the use of cryoprotectants and special freezing rates at the time of freezing and thawing.

Genome editing

With recent, rapid advances in genome sequencing and assembly, and gene editing with CRISPR-Cas9, it may soon be possible to insert relevant genes from extinct species into the genomes of their closest extant relatives. Germ cells may be edited directly, so that the egg and sperm produced by the extant parent species will produce offspring of the extinct species, or somatic cells may be edited and transferred via somatic cell nuclear transfer. Because it is possible to sequence and assemble the genome of extinct organisms from highly degraded tissues, this technique enables scientists to pursue de-extinction in a wider array of species, including those for which no well-preserved remains exist.

While the methods needed to carry out genome editing for de-extinction are advancing, there are still several limitations. First, the more degraded and old the tissue from the extinct species is, the more fragmented the resulting DNA will be, making genome assembly more challenging. Additionally, a close relative must be living and able to be reared in captivity for insertion of edited cells. Because a near relative is used as a host for the edited cells, the resulting individuals will not be exactly the same as the extinct species, but may also possess traits of the extant relative.

Selective breeding

Selective breeding is the process by which living relatives of the extinct species are identified and specifically mated to reproduce the traits of the extinct species. This method can recreate the traits of an extinct species, but the genome will differ from the original species.

The aurochs, which became extinct in 1627, could possibly be brought back by taking DNA samples from bone and teeth fragments in museums in order to obtain genetic material to recreate its DNA. Researchers would then compare the DNA to that of modern European cattle to determine which breeds still carry the creature's genes, and then undertake a selective breeding program to reverse the evolutionary process. The intention would be that with every passing generation, the cattle would more closely resemble the ancient aurochs.

The quagga, a subspecies of zebra which has been extinct since the 1880s, has been revived using selective breeding of zebras. Since the new animal is not genetically identical to the extinct subspecies, the new animal is called the Rau quagga.

Chelonoidis elephantopus, an extinct tortoise, originally discovered on the Galapagos Islands by Charles Darwin, has hopes of being revived through selective breeding. A group of scientists have collected over 1,700 blood samples from tortoises on Isabela Island during an expedition in 2012 having identified 80 tortoises with traces of the extinct species DNA.

Arguments

Arguments against de-extinction

Opponents of de-extinction have claimed that efforts and resources used to resurrect extinct species could have been better used trying to conserve endangered species that might themselves become extinct. Featuring in the journal Life Sciences, Society and Policy, a 2014 article claimed that the cost of reviving just a single species could amount to millions of dollars. At the same time, more than 20,000 extant species are currently threatened with extinction.

In some cases, the negative impacts on extant species could be highly direct. For instance, it is proposed that the Asian elephant would act as a surrogate mother for the woolly mammoth embryo. The Asian elephant is currently endangered, and it could be detrimental to its existence to use the few individuals left to bring back another species.

It has also been noted that a resurrected species, while being genetically the same as previously living specimens, will not have the same behaviour as its predecessors. The first animal to be brought back will be raised by parents of a different species (the fetus's host), not the one that died out and thus have differing mothering techniques and other behaviors. Another concern is that de-extinction plans have not discussed bringing back parasites or mutualists which co-existed with extinct species, which may have strong effects on the survival and success of resurrected species.

Other scholars have published ethical concerns regarding de-extinction. In Conservation Biology, Robert Sandler argues that introducing extinct species to environments may produce harm to modern species, as invasive species. Issues regarding scientific hubris, human and animal health, and the ecology of sensitive environments have been raised by the scientific community. Further research must be performed regarding de-extinction to investigate advantages and disadvantages to the technology. New technological practices must be examined to prevent environmental hazards.

Arguments for de-extinction

Counter arguments have been made, however, in regards to the benefits of bringing back extinct species. Harvard geneticist, George Church, gives an example of the positive effects of bringing back the extinct woolly mammoth would have on the environment. He explains that if the newly developed mammoth hybrids were to be placed in areas such as Siberia and Alaska, the outcome may reverse the damage that global warming has caused. He and his fellow researchers predict that mammoths would eat the dead grass allowing the sun to reach the spring grass; their weight would allow them to break through dense, insulating snow in order to let cold air reach the soil; and their characteristic of felling trees would increase the absorption of sunlight. If the theories are proven true, global warming could eventually be lessened.

Scientific American, in an editorial condemning de-extinction, pointed out that the technologies involved could have secondary applications, specifically to help species on the verge of extinction regain their genetic diversity, for example the black-footed ferret or the northern white rhinoceros. It noted, however, that such research "should be conducted under the mantle of preserving modern biodiversity rather than conjuring extinct species from the grave."

It's been argued that revived species can be utilised as a tool to support other conservation initiatives by acting as a "flagship species" - a charismatic organism that generates popular support and funds for conserving entire ecosystems. Along this vein, it is thought that resurrecting the aurochs would boost the European "rewilding" movement, in turn, transforming abandoned farmland into wildlife corridors. De-extinction would act as a kind of "flagship technology' where the excitement stirred from the possibility of seeing an extinct species in the wild strengthens the focus on preserving ecosystems. Similarly, the conservationist Josh Donlan claims that if the passenger pigeon were resurrected, there would inevitably be a legal impetus for the protection of its habitat under the Endangered Species Act.

Current candidates for de-extinction

The woolly mammoth (Mammthus primigenius) is a candidate for de-extinction using either cloning or genome editing.

Woolly mammoth

The existence of preserved soft tissue remains and DNA of woolly mammoths has led to the idea that the species could be recreated by scientific means. Two methods have been proposed to achieve this. The first is cloning, which would involve removal of the DNA-containing nucleus of the egg cell of a female elephant, and replacement with a nucleus from woolly mammoth tissue. The cell would then be stimulated into dividing, and inserted back into a female elephant. The resulting calf would have the genes of the woolly mammoth, although its fetal environment would be different. To date, even the most intact mammoths have had little usable DNA because of their conditions of preservation. There is not enough to guide the production of an embryo. The second method involves artificially inseminating an elephant egg cell with sperm cells from a frozen woolly mammoth carcass. The resulting offspring would be an elephant–mammoth hybrid, and the process would have to be repeated so more hybrids could be used in breeding. After several generations of cross-breeding these hybrids, an almost pure woolly mammoth would be produced. The fact that sperm cells of modern mammals are potent for 15 years at most after deep-freezing is a hindrance to this method. In one case, an Asian elephant and an African elephant produced a live calf named Motty, but it died of defects at less than two weeks old. In 2008, a Japanese team found usable DNA in the brains of mice that had been frozen for 16 years. They hope to use similar methods to find usable mammoth DNA. In 2011, Japanese scientists announced plans to clone mammoths within six years. As the woolly mammoth genome has been mapped, a complete strand of DNA may be synthesised in the future.

Mammoth expert Adrian Lister questions the ethics of such recreation attempts. In addition to the technical problems, he notes that there is not much habitat left that would be suitable for woolly mammoths. Because the species was gregarious, creating a few specimens would not be ideal. He also notes that the time and resources required would be enormous, and that the scientific benefits would be unclear; these resources should instead be used to preserve extant elephant species which are endangered. However, it was reported in March 2014 that blood recovered from a frozen mammoth carcass in 2013 now provides a "High chance" of cloning the woolly mammoth, despite previous hindrances. Another way to revive the woolly mammoth would be to migrate genes from the mammoth genome into the genes of its closest living relative, the Asian elephant, to create hybridized animals with the notable adaptations that it had for living in a much colder environment than modern day elephants. This is currently being done by Harvard geneticist George Church, and they have already successfully made changes in the elephant genome with the genes that gave the woolly mammoth its cold-resistant blood, longer hair, and extra layer of fat. A revived woolly mammoth or mammoth-elephant hybrid may find suitable habitat in the tundra and taiga forest ecozones, and may also find refuge in Pleistocene Park, a Pleistocene rewilding experiment by Russian scientist Sergey Zimov to recreate the mammoth steppe, the former habitat of the woolly mammoth. While mammoths are not required for the recreation of the steppe, they would be highly effective in doing so by quickly clearing brush and forest and allowing grasses to colonize the area, a capability that modern arctic megafauna do not have.

Pyrenean ibex

The Pyrenean ibex was one of four original subspecies of Spanish ibex that roamed on the Iberian peninsula. However, while it was abundant during Medieval times, over-hunting in the 19th and 20th centuries led to its demise. In 1999, only a single female named Celia was left alive in Ordesa National Park. Scientists captured her, took a tissue sample from her ear, collared her, then released her back into the wild, where she lived until she was found dead in 2000, having been crushed by a fallen tree. In 2003, scientists used the tissue sample to attempt to clone Celia and resurrect the extinct subspecies. Despite having successfully transferred nuclei from her cells into domestic goat egg cells and impregnating 208 female goats, only one came to term. The baby ibex that was born had a lung defect, and lived for only 7 minutes before suffocating from being incapable of breathing oxygen. Nevertheless, her birth was seen as a triumph and has been considered to have been the first de-extinction. However, in late 2013, scientists announced that they would again attempt to recreate the Pyrenean ibex. A problem to be faced, in addition to the many challenges of reproduction of a mammal by cloning, is that only females can be produced by cloning the female individual Celia, and no males exist for those females to reproduce with. This could potentially be addressed by breeding female clones with the closely related Southeastern Spanish ibex, and gradually creating a hybrid animal that will eventually bear more resemblance to the Pyrenean ibex than the Southeastern Spanish ibex.

Aurochs

The Aurochs was widespread across Eurasia, North Africa, and the Indian subcontinent during the Pleistocene, but only the European aurochs (Bos primigenius primigenius) survived into historic times. This species is heavily featured in European cave paintings, such as Lascaux and Chauvet cave in France, and was still widespread during the Roman era, in which they were used as fighting animals for entertainment, and were noted by Julius Caesar for their strength and prowess. Following the fall of the Roman empire, however, overhunting of the aurochs by nobility and royalty caused its population to dwindle to a single population in the Jaktorów forest in Poland, where the last wild aurochs, a female, died of natural causes in 1627. However, because the auroch is ancestral to most modern cattle breeds and has close relatives in primitive cattle breeds, it is possible for it (or a superficial ecological replacement) to be brought back through artificial selection. The first attempt at this was by Heinz and Lutz Heck using modern cattle breeds, which resulted in the creation of Heck cattle. This breed has been introduced to nature preserves across Europe; however, it differs strongly from the aurochs in both physical characteristics and behavior, and modern attempts have tried to create an animal that is nearly identical to the aurochs in morphology, behavior, and even genetics. The TaurOs Project aims to recreate the aurochs through selectively breeding primitive cattle breeds over a course of twenty years to create a self-sufficient bovine grazer in herds of at least 150 animals in rewilded nature areas across Europe. This organization is partnered with the organization Rewilding Europe to help restore balance to European nature. A competing project to recreate the aurochs is the Uruz Project by the True Nature Foundation, which aims to recreate the aurochs through a more efficient breeding strategy and through genome editing, in order to decrease the number of generations of breeding needed and the ability to quickly eliminate undesired traits from the new aurochs population. It is hoped that the new aurochs will reinvigorate European nature by restoring its ecological role as a keystone species, and bring back biodiversity that disappeared following the decline of European megafauna, as well as helping to bring new economic opportunities related to European wildlife viewing.

Quagga

The Quagga is a subspecies of the plains zebra that was distinct in that it was striped on its face and upper torso, but its rear abdomen was a solid brown. It was native to South Africa, but was wiped out in the wild due to over-hunting for sport, and the last individual died in 1883 in the Amsterdam zoo. However, since it is technically the same species as the surviving plains zebra, it has been argued that the quagga could be revived through artificial selection. The Quagga Project aims to recreate the quagga through the artificial selection of plains zebras, and aims to release these animals onto the western cape once an animal that fully resembles the quagga is achieved, which could have the benefit of eradicating non-native trees. As of 2014, the project had 110 plains zebras in 10 locations for selective breeding, and individuals began to show a reduction in stripes and a browning of the fur, owing to the success of the project. As of 2016, there were 6 rau quaggas that had been produced through selective mating. These animals appear identical to the quagga but likely have a differing genetic code.

The last known thylacine, named "Benjamin", died from neglect in the Hobart Zoo in 1936.

Thylacine

The Thylacine is native to continental Australia, Tasmania and New Guinea. It is believed to have become extinct in the 20th century. The thylacine had become extremely rare or extinct on the Australian mainland before British settlement of the continent, but it survived on the island of Tasmania along with several other endemic species, including the Tasmanian devil. Intensive hunting encouraged by bounties is generally blamed for its extinction, but other contributing factors may have been disease, the introduction of dogs, and human encroachment into its habitat. Despite its official classification as extinct, sightings are still reported, though none has been conclusively proven. The last known thylacine, named Benjamin, died at the Hobart Zoo, on 7 September 1936. It is believed to have died as the result of neglect—locked out of its sheltered sleeping quarters, it was exposed to a rare occurrence of extreme Tasmanian weather: extreme heat during the day and freezing temperatures at night. National Threatened Species Day has been held annually since 1996 on 7 September in Australia, to commemorate the death of the last officially recorded thylacine. Although there had been a conservation movement pressing for the thylacine's protection since 1901, driven in part by the increasing difficulty in obtaining specimens for overseas collections, political difficulties prevented any form of protection coming into force until 1936.

Official protection of the species by the Tasmanian government was introduced on 10 July 1936, 59 days before the last known specimen died in captivity. The thylacine held the status of endangered species until the 1980s. International standards at the time stated that an animal could not be declared extinct until 50 years had passed without a confirmed record. Since no definitive proof of the thylacine's existence in the wild had been obtained for more than 50 years, it met that official criterion and was declared extinct by the International Union for Conservation of Nature in 1982 and by the Tasmanian government in 1986. The species was removed from Appendix I of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) in 2013.

The Australian Museum in Sydney began a cloning project in 1999. The goal was to use genetic material from specimens taken and preserved in the early 20th century to clone new individuals and restore the species from extinction. Several molecular biologists have dismissed the project as a public relations stunt and its chief proponent, Mike Archer, received a 2002 nomination for the Australian Skeptics Bent Spoon Award for "the perpetrator of the most preposterous piece of paranormal or pseudo-scientific piffle." In late 2002, the researchers had some success as they were able to extract replicable DNA from the specimens. On 15 February 2005, the museum announced that it was stopping the project after tests showed the DNA retrieved from the specimens had been too badly degraded to be usable. In May 2005, Archer, the University of New South Wales Dean of Science at the time, former director of the Australian Museum, and evolutionary biologist, announced that the project was being restarted by a group of interested universities and a research institute. The International Thylacine Specimen Database was completed in April 2005, and is the culmination of a four-year research project to catalog and digitally photograph, if possible, all known surviving thylacine specimen material held within museum, university and private collections. The master records are held by the Zoological Society of London. In 2008, researchers Andrew J. Pask and Marilyn B. Renfree from the University of Melbourne and Richard R. Behringer from the University of Texas at Austin reported that they managed to restore functionality of a gene Col2A1 enhancer obtained from a 108-year-old, alcohol-preserved thylacine pouch young specimen. The genetic material was found working in transgenic mice. The research enhanced hopes of eventually restoring the population of thylacines. That same year, another group of researchers successfully sequenced the complete thylacine mitochondrial genome from two museum specimens. Their results were published in the journal Genome Research in 2009.

In December of 2017 it was announced in Nature Ecology and Evolution that the full nuclear genome of the Thylacine had been successfully sequenced, marking the completion of the critical first step toward de-extinction that began in 2008 with the extraction of the DNA samples from the preserved pouch specimen. The Thylacine genome was reconstructed by mapping the thylacine genome to that of one of its closest living relatives, the Tasmanian devil, and generating a reference based assembly of the full nuclear genome. Andrew J. Pask from the University of Melbourne has stated that the next step toward de-extinction will be to create a functional genome, which will require extensive research and development, estimating that a full attempt to resurrect the species may be possible as early as 2027.

Passenger pigeon

The Passenger pigeon numbered in the billions before being wiped out due to commercial hunting and habitat loss. The non-profit Revive & Restore obtained DNA from the passenger pigeon from museum specimens and skins; however, this DNA is degraded because it is so old. For this reason, simple cloning would not be an effective way to perform de-extinction for this species because parts of the genome would be missing. Instead, Revive & Restore focuses on identifying mutations in the DNA that would cause a phenotypic difference between the extinct passenger pigeon and its closest living relative the band-tailed pigeon. In doing this, they can determine how to modify the DNA of the band-tailed pigeon to change the traits to mimic the traits of the passenger pigeon. In this sense, the de-extinct passenger pigeon would not be genetically identical to the extinct passenger pigeon, but it would have the same traits. The de-extinct passenger pigeon hybrid is expected to be ready for captive breeding by 2024 released into the wild by 2030.

Future potential candidates for de-extinction

Birds

• Moa – this group of large (up to 4 m [12 ft] tall and 110 kg [250 lb]), flightless birds became extinct in approximately 1400 AD following the arrival and proliferation of the Maori people on New Zealand; however, intact DNA from both preserved specimens and eggshells makes the moa a candidate for resurrection. New Zealand politician Trevor Mallard has suggested bringing back a medium-sized species.
• Heath hen – this subspecies of the prairie chicken became extinct on Martha's Vineyard in 1932 despite conservation efforts; however, the availability of usable DNA in museum specimens and protected areas in its former range makes this bird a possible candidate for de-extinction and reintroduction to its former habitat.
• Dodo – this large, flightless ground bird endemic to Mauritius became extinct in the 1640s due to exploitation by humans and due to introduced species such as rats and pigs, which ate their eggs. Due to a wealth of bones and some tissues, it is possible that this species may live again as it has a close relative in the surviving Nicobar pigeon. The entire dodo bird genome has been sequenced, but further steps towards de-extinction have not yet been made.
• Elephant bird – of the largest birds to have ever existed, the elephant bird was driven to extinction by the early colonization of Madagascar. Ancient DNA has been obtained from the eggshells but may be too degraded for use in de-extinction.

Mammals

• Cave lion - The discovery of two preserved cubs in the Sakha Republic ignited a project to clone the animal.
• Steppe bison - The discovery of the mummified steppe bison of 9,000 years ago could help people clone the ancient bison species back, even though the steppe bison would not be the first to be "resurrected".

Reptiles

• Floreana Island Tortoise - In 2008, mitochondrial DNA from the Floreana tortoise species was found in museum specimens. In theory, a breeding programme could be established to "resurrect" a pure Floreana race from living hybrids.

Amphibians

• Gastric-brooding frog - In 2013, scientists in Australia successfully created a living embryo from non-living preserved genetic material, and hopes by using somatic-cell nuclear transfer methods, they can produce an embryo that can survive to the tadpole stage.

See also

• Breeding back
• Cryoconservation of animal genetic resources
• Endangered species
• Endling
• Holocene extinction
• List of introduced species
• Pleistocene Park
• Pleistocene rewilding
• Woolly mammoth
• List of resurrected species

Further reading

• O'Connor, M.R. (2015). Resurrection Science: Conservation, De-Extinction and the Precarious Future of Wild Things. New York: St. Martin's Press. ISBN 9781137279293.
• Shapiro, Beth (2015). How to Clone a Mammoth: The Science of De-Extinction. Princeton, NJ: Princeton University Press. ISBN 9780691157054.
• Pilcher, Helen (2016). Bring Back the King: The New Science of De-extinction. Bloomsbury Press ISBN 9781472912251

External links

• TEDx DeExtinction March 15, 2013 conference sponsored by Revive and Restore project of the Long Now Foundation, supported by TEDx and hosted by the National Geographic Society, that helped popularize the public understanding of the science of de-extinction. Video proceedings, meeting report, and links to press coverage freely available.
• De-Extinction: Bringing Extinct Species Back to Life April 2013 article by Carl Zimmer for National Geographic magazine reporting on 2013 conference.