Behind bars and waiting for science: the power of genetic testingfor the Addis Fifteen

Jenny R. Isaacs

Mongabay.com

They languished behind bars in squalid conditions, their very survival in jeopardy. Outside, an international team of advocatesstrove to bring worldwide attention to their plight. With modern genetics, the experts sought to prove what they had long believed: that these individuals were special. Like other cases of individuals waiting for rescue from a life of deprivation behind bars, the fate of those held captive might be dramaticallyaltered with the application of genetic science to answer questions of debated identity. Now recent DNA analysis has made it official: this group is special and because of their scientifically confirmed distinctiveness they will soon enjoy greater freedom.

Many long suspected that the fifteen languishing lions kept within the archaic Addis Ababa Lion Zoo in Ethiopia were not youraverage lions. The lions are descended from the private lion collection of the last Ethiopian ruler, Emperor Haile Selassie (crowned in 1930), who captured their ancestors from the wild andbrought them to his palaces in the 1940s. These modern day survivors persist in the same antiquated holding pens as their descendants, lacking proper nutrition or socialization (see below). All lions (Panther leo) are endangered and rare, but these lions seemed special; as scientists would say, they exhibited certain observable and distinguishing (i.e. phenotypic) differences from other, more common subspecies of lion. Visiting biologists took note of several remarkable features of appearancewhich hinted that the group was distinctive.

"The male lions at Addis Ababa Zoo are characterized by a large, dark brown mane extending down the chest through the front legs, down the back below the shoulders and the length of the belly through the groin…Compared to male lions from eastern and southern Africa [they] are also characterized by lower body mass…

and smaller body size," wrote the authors of a new study in the European Journal of Wildlife Research.

Could these be the relatives of an extinct lion species? It had been suggested that the Addis Ababa lions might be the last true relatives of North African Barbary lions or South African Cape lions, both extinct. Or even more special, might they be an entirely genetically distinctive population of lion? If so, proving the lions’ genetic identities was urgent due to breeding biological time constraints and poor zoo conditions.

"The conditions at the Addis zoo are dreadful," biologist ClaudioSillero of the University of Oxford’s Wildlife Conservation Research Unit told Africa Geographic, describing it as an old Victorian menagerie, with paint peeling from thinning walls and no soft ground.

"The lions are housed in a 4 x 5 x 2.5-metre den with eight roomsthat can accommodate eight pairs of adult lions. The capacity of the cubs’ lair is two. There is no maternity cage. The male and female lions are kept in isolation. They are fed about 10 kilograms each day of non-deboned beef and given water. No other food or supplements are provided," local veterinarian, Melaku Tefera of Haramaya University, Ethiopia wrote in a 2003 Biodiversity and Conservation article.

Addis Lion zookeepers and city administrators were so overwhelmedfrom caring for the animals that they reached out to their German‘sister city’, Leipzig, for help in 2006.

"The mayor of Addis asked the mayor of Leipzig for help as Addis Lion Zoo was under pressure because of the poor keeping conditions. Vets of Leipzig zoo went to Addis and checked the health status of most of the lions in Addis zoo," Frank Oberwemmer, of the Leipzig Zoo, told mongabay.com.

Granted access to the lions, the team of vets took the opportunity to research the lion identities and so, Oberwemmer adds, "blood samples were taken for genetic investigations as groundwork for an international breeding

program in the case of confirmation of an own subspecies or at least an own genetic construction."

An international effort was mobilized. Hofreiter and other scientists, including Susann Bruche of the Imperial College London, Markus Gusset and Carlos Driscoll of the University of Oxford, of the University of York, and experts from the Max Planck Institute for Evolutionary Anthropology (which funded the study) and UK’s Durham University, compared Addis zoo lion DNA samples with samples from other African and Asian lions.

"Without genetic studies," the scientists wrote in their paper published in the European Journal of Wildlife Research (October 2012), "cryptic species such as the Addis lions could remain unknown or go extinct without ever being recognized."

While the lions spent day after day in the dilapidated zoo, the team was hard at work. Like much of today’s scientific work, proving the identities of the lions would be an intensely collaborative effort, drawing on the specialization of many academic disciplines.

"A study as the one published by us requires input from differentsides. There is the administration part to allow sample taking and transport which was coordinated by the Zoo Leipzig (Dr Jörg Junhold, Prof. Klaus Eulenberger) as well as overall project and funding coordination (Prof. Micheal Hofreiter), the veterinary task of taking the blood samples (Prof. Klaus Eulenberger) and the work in the laboratory to process the samples and the analysis of the data (Susann Bruche, Dr. Sebastian Lippold). Afterwards, the results have to be brought in context with other knowledge in the research area in order to draw conclusions and we were lucky to have input from experts in the field of feline research (Dr. Markus Gusset, Dr. Ross Barnett, Dr. Carlos Driscoll)," Bruche explained to mongabay.com.

Importantly, the case of the Addis lions is also a testament to the power of genetic testing to alter lives. To prove that the Addis lions were special enough to deserve better treatment, Susann Bruche told mongabay.com that it was essential to use

genetics "as a method to confirm the distinctiveness of populations that had been recognized to be different in some way"adding that "genetic analysis helps to identify the truly distinct populations that warrant conservation."

Geneticists provide such answers which may result in a change of conservation strategy, such as in the case of the Addis lions, but geneticists’ power is limited. Professor Michael Hofreiter tells mongabay.com, that the scientists have no control over whathappens to the actual lions: "the day-to-day well-being of the lions is the domain of the zoo keepers and vets, and genetics researchers have nothing to do with these issues." Yet there is, he explains, a behind-the-scenes role for scientists to play in affecting change for genetically-contested individuals, such as the lions behind bars in Addis Ababa.

"Genetic research certainly contributes data on how important theconservation of a certain population is."

Dr. Carlos Driscoll, a geneticist on the project, who also works with the NIH in Maryland and the Wildlife Institute of India, described to mongabay.com the essential role of genetics within conservation.

"Before you can conserve anything, or even know what to conserve,you have to identify it. And, in practical terms, that also usually means that it has to have a name attached to it so that the lawyers (and journalists) have a handle to hold on to when talking about it."

Today "geneticists are hired guns" says Driscoll, employing widely accepted techniques of genetic testing that have been standardized just over the last twenty years. He tells mongabay.com that these days there is a "fair" amount of certainty to the science itself and that "the genetic techniques and questions you would ask are the same for a lion as for a squirrel." Driscoll says genetic analysis is now common and cheapenough that most research these days is all application to particular cases, in this and similar instances applying genetics

to prove that isolated and suspected-to-be-unique source populations-- like the Addis lions-- are indeed distinctive.

But confirming genetic distinctiveness hasn’t always been so easyor accurate.

"Before the development of genetic analysis, biologist relied on observations" such as superficial or behavioral differences "for the identification of conservational significant units," Bruche says.

Dr. Driscoll described in a paper on tiger classification how in the era before molecular taxonomy (genetics), subspecies definitions were based on visual criteria; such as geographical origin, size, and superficial variations such as hair length, color, etc. Because the identifications were sometimes based on asingle (possibly mutant) individual and not confirmed at the genetic level, classifications of species suffered from a lack ofconsensus, repeated revision, and endless argument.

Even today, "there is continued debate about the genetic distinctiveness of different lion populations, a discussion delaying the initiation of conservation actions for endangered populations" wrote the Addis Abba lion study authors.

With the science established and results widely accepted by society, genetics has become the essential conservation tool which can trigger real, actual improvement for endangered and rare species. In a 2009 PLoS One paper, associating rarity with "value and need," Driscoll described how accurate and systematic genetic assessment would "properly allow conservationists to identify those populations of the greatest value and need in order to formulate policy, to disseminate conservation funds, andto manage endangered populations."

As an example of how genetics can inform wildlife management and land use decisions that affect endangered species, Dr. Driscoll employed genetic science to show that the extinct Caspian tiger (Panthera tigris virgata) and the Critically Endangered Amur tiger (P. t.

altaica) were closely related enough to warrant new possibilities in conservation strategy. Using biogeography and genetic analysis(phylogeography), Driscoll established that Amur tiger reintroductions into former Caspian tiger habitat in Central Asiawere scientifically justified. Here, genetic proof of historic close relation created new possibilities for conservation biologists trying to identify areas of appropriate and safe habitat for endangered species to stage a comeback.

In the case of the fifteen lions descended from Emperor Salassie’s private collection, genetic assessment research has paid off. Results of the study demonstrate that the Addis lions are distinctive, most closely related to lions in Africa, not those in Asia. Dr. Driscoll told Africa Geographic, "Our analysis shows the existence of three African lion clusters: those in Addis Ababa; those in north-eastern Africa, such as Ngorongoro Crater and the Serengeti; and those in south-western Africa in places like Etosha, Kalahari and Kruger," adding, "The Addis zoo lions are a breed apart."

Michael Hofreiter confirmed with mongabay.com that while not everything is known about lion genetics, there is no doubt that the Addis lions are special.

"We do not know how many genetically distinct lion populations exist, as there are not data for all surviving populations," he says, but "the Addis lions should be considered a genetically distinct population, in conservation terms both an ‘evolutionary significant unit (ESU)' and a ‘conservation unit.'"

In their paper, the authors argue forcefully for greater and immediate action to protect these lions.

"These lions are genetically distinct from Asian lions as well asall African lion populations for which comparative data were available. We therefore urge immediate conservation action, including the establishment of a captive breeding programme, to preserve this unique lion population. This is an extremely urgentmatter because population pressure on wild lions was identified as the major threat to this species in Ethiopia and several of

the individuals at Addis Ababa Zoo are reaching the end of their reproductive period."

Already experts are discussing the next steps to take.

"A new conservation breeding facility is required to sustain these lions as a natural heritage of Ethiopia," Yeneneh Teka, director of the Ethiopian Wildlife Conservation Authority, told Africa Geographic. "Hand-in-hand, a detailed study should continue to verify the presence of wild relatives to save these unique lions from extinction."

To that end, the scientists are widening their search for this genetically distinct lion across Africa and in zoos throughout the world.

Since their distinctiveness has been proven through genetic testing, the Addis lions will soon receive much improved care; including translocation to modern facilities where they can thrive, reproduce, and enjoy better lives. In partnership and consultation with the Leipzig Zoo, a new facility was designed; the construction of this facility was started in 2012 and will befinished in 2013.

"This new facility is planned according to the latest knowledge about modern lion keeping and will improve the keeping conditionsof these lions a lot," Frank Oberwemmer of the Leipzig Zoo told mongabay.com,

Construction of the new, state-of-the art holding facility withinan internationally monitored captive breeding program is certainly a victory for the Addis lions and the scientists who work to conserve them. But genetic testing that results in bettertreatment and captive breeding isn’t the end of the lion conservation story. Such expensive and dramatic conservation strategies are only necessary because of the deteriorating conditions for lions in the wild. Lions are today classified as Vulnerable according to the IUCN Red List of Threatened Species.

“The geographical range and population size of lions have both decreased dramatically due toAnthropogenic [human] causes, and this decline has accelerated inmodern times. Once present in most parts of Africa, lions are nowextinct in many regions of their former geographical range and threatened with extinction in others” wrote the authors in their paper. A recent study in Biodiversity Conservation has found thatthe world's lion population dropped from around 100,000 animals fifty years ago to as few as 32,000 today, a 69 percent decline. Other estimates have been even bleaker.

"As for most large species, habitat loss due to increasing human population pressure and direct persecution by humans are the greatest threats" to lions often because "large predators usually…are seen as a threat to livestock and humans directly," Hofreiter said.

Given the worsening conditions of lion habitat, one might ask what are the long-term goals of a proposed captive breeding program for the Addis lions? Will the Addis lions ever be released? If it’s too late for them, what about their descendants? When might such a release happen, under what conditions? How will we know when it’s safe for lions in the wildand who is to decide? It seems for now that many of these of questions are too far down the road.

"The immediate goal of a breeding program would be to conserve the Addis population and its genetic diversity… other questions are not relevant until this is achieved and one will deal with them once (if) they become relevant," Hofreiter notes.

What is clear for now is that genetic testing has facilitated a positive life change for these Addis Ababa Zoo lions and has preserved the hopes of conservationists that perhaps the distant relatives of the Addis Fifteen might someday roam freely across Africa, thriving and safe from harm.

Citation: Susann Bruche & Markus Gusset & Sebastian Lippold & Ross Barnett & Klaus Eulenberger & Jörg Junhold & Carlos A.

Driscoll & Michael Hofreiter. A genetically distinct lion (Panthera leo) population from Ethiopia. European Journal of Wildlife Research. ISSN 1612-4642, DOI 10.1007/s10344-012-0668-5. Springer-Verlag Berlin Heidelberg 2012.

See also:

Driscoll, Carlos A., Yamaguchi N, Bar-Gal GK, Roca AL, Luo S, et al. (2009) Mitochondrial Phylogeography Illuminates the Origin ofthe Extinct Caspian Tiger and Its Relationship to the Amur Tiger.PLoS ONE 4(1): e4125. doi:10.1371/journal.pone.0004125

Dybas, Cheryl Lyn. The Last of the Dark Ones. Africa Geographic. January 2013: p. 53-57.

Lions are the only felids that possess a mane as a secondarysexual character in males. Mane development isdependent on factors such as age, ambient temperature,nutritional status and testosterone level (Patterson 2007).

the purpose of the study was toassess whether specific conservation actions, including fieldsurveys, captive breeding and further genetic analyses, arewarranted for these animals.

allow for comparison with these six populations, see mapAddis Ababa lions' CytB gene sequence has been depositedin GenBank under the accession number JX023542. Thesequence was then compared to other CytB sequences of lionsretrieved from GenBank (see Table 4).

The genotypes of the lions from Addis Ababa Zoo werecompared with the six lion populations genotyped

locus in Hardy–Weinberg equilibrium showed an excessof heterozygosity (FCA006; p00.029). Deviation fromHardy–Weinberg equilibrium and heterozygosity excess

couldbeasignofinbreedingintheAddisAbabalions.However, calculation of FISindices for each of theseven lion populations on the basis of the ten investigatedmicrosatellites revealed that the genetic diversityof the Addis Ababa lions does not seem to be affectedby inbreeding as the Findex calculated as a measureof inbreeding was not significant for them (Table 2).Also, none of the other lion populations showed significantinbreeding.IS

on numbers are in serious decline, with two of only ahandful of evolutionary significant units (Dubach et al.2005; Barnett et al. 2006a; Bertola et al. 2011)alreadyextinct in the wild (North African Barbary lions and SouthAfrican Cape lions) and two others (Asian lions and WestAfrican lions) being close to extinction. Therefore, ex situconservation of genetic diversity is becoming a topic ofincreasing importance as it may preserve the genetic diversityof species until reintroduction programmes can be successfullyimplemented

lion. A substantial loss of genetic diversity is in fact quitelikely for lions as patterns of lion genetic diversity suggestsubstantial population subdivision and reduced gene flow(Antunes et al. 2008), highlighting the importance of preservinglocal lion populations in decline.To our knowledge, the males at Addis Ababa Zoo are thelast extant lions to possess a large, dark mane extendingfrom the head, neck and chest right down to the belly(Fig. 1). However, whether this phenotypic uniqueness isrelated to genetic distinctiveness has been unknown (Tefera

n this study, we compared, based on microsatelliteand mitochondrial DNA data, the lions from Addis Ababa toseveral well-characterized lion populations representative of

their extant range in the wild. Both mitochondrial DNA andmicrosatellite data tend to cluster lions geographically andtherefore allow to some extent for approximation of theirgeographical origin

The results clearly show thatthe lions from Addis Ababa belong to the African lionpopulation rather than to the Asian one

Our population structure analyses show the existenceof three African lion clusters: Addis Ababa, northeasternAfrica (Ngorongoro and Serengeti) and southwesternAfrica (Etosha, Kalahari and Kruger; Figs. 2and 3). As noted previously by other authors (Dubachet al. 2005; Barnett et al. 2006b; Bertola et al. 2011),the genetic distinctiveness between north-eastern andsouth-western African lion populations coincides with aphysical separation of the two groups through theAfrican Rift Valley. Although the principal componentanalysis suggests that the lions from Addis Ababa are—in accordance with their proposed geographical origin—more closely related to north-east African lions, they aresubstantially different from lions in both north-easternand south-western Africa.

Given observed low inbreeding coefficient in the AddisAbaba lions, their genetic distinctiveness does not seem to be a consequence of repeated inbreeding in captivity.

Thus, ‘both the microsatellite and mitochondrialDNA data suggest the Addis Ababa lions to be geneticallydistinct from all lion populations for which comparative dataexist. Therefore, we suggest that the Addis Ababa lions aretreated as a distinct conservation management unit andpriority should be given to preserving their genetic diversity.’

The genetic uniqueness of the Addis Ababa lions suggeststhat their wild source population is similarly unique. However,given that the wild source population is unknown and considering

the precarious conservation situation of many wild lionpopulations (IUCN/SSC Cat Specialist Group 2006a, b;Gebresenbet et al. 2009), we believe that the Addis Ababalions deserve immediate conservation action. Our results suggestthat the lions at Addis Ababa Zoo are both sufficientlygenetically distinct and harbour sufficient genetic diversity towarrant a captive breeding programme.

‘Establishing a captive breeding programme should beregarded as a first step towards conserving this unique lionpopulation.’

Tefera (2003) mentions that no lions ofthe Addis Ababa Zoo phenotype exist in the wild, mainlydue to hunting for their mane. However, this claim needs to be investigated through field surveys and genetic analysis ofsamples collected during such excursions (if possible noninvasivelyfrom hair and/or faeces).ss release: http://www.sciencedaily.com/releases/2012/10/121011085336.htm (you can take quotes etc.)

The team of researchers, led by the University of York, UK, and the Max Planck Institute for Evolutionary Anthropology, Germany, has shown that captive lions at the Addis Ababa Zoo in Ethiopia are, in fact, genetically distinct from all lion populations for which comparative data exists, both in Africa and Asia.

The results of the study, which also involved researchers from Leipzig Zoo and the Universities of Durham and Oxford, UK, are published in the European Journal of Wildlife Research.

The lion (Panthera leo) is the principal terrestrial predator in Africa and therefore a key species of the savannah ecosystem. Lion numbers are in serious decline and two significant populations of lion -- the North African Barbary lions and the South African Cape lions have already become extinct in the wild.

Principal Investigator Professor Michi Hofreiter, of the Department of Biology at the University of York, said: ‘To our knowledge, the males at Addis Ababa Zoo are the last existing lions to possess this distinctive mane. Both microsatellite and mitochondrial DNA data suggest the zoo lions are genetically distinct from all existing lion populations for which comparativedata exist.

‘We therefore believe the Addis Ababa lions should be treated as a distinct conservation management unit and are urging immediate conservation actions, including a captive breeding programme, to preserve this unique lion population.’

Lead author Susann Bruche, now with Imperial College London, but who conducted the research with the Max Planck Institute for Evolutionary Anthropology, said: ‘A great amount of genetic diversity in lions has most likely already been lost, largely dueto human influences. Every effort should be made to preserve as much of the lion's genetic heritage as possible. We hope field surveys will identify wild relatives of the unique Addis Ababa Zoo lions in the future, but conserving the captive population isa crucial first step. Our results show that these zoo lions harbour sufficient genetic diversity to warrant a captive breeding programme.’

Professor Hofreiter said: ‘A key question is which wild population did the zoo lions originate from and whether this wildpopulation still exists; this would obviously make it a priority for conservation. What is clear is that these lions did not originate in the zoo, but come from somewhere in the wild -- but not from any of the populations for which comparative data is available.’

Article: http://grist.org/list/a-new-species-of-lion-has-been-discovered-in-an-ethiopian-zoo/

Another article: http://www.mnn.com/earth-matters/animals/stories/new-species-of-lion-discovered-with-an-extravagant-dark-mane

Here's your contact:  Bruche, Susann    susann.bruche09@imperial.ac.uk