Many scales, many chromosomes!

Klaus-Peter Koepfli, Marlys Houck, Erez Aiden, and Olga Dudchenko

Pangolins, also known as scaly anteaters, belong to an entirely distinct order of mammals known as the Pholidota (from Greek meaning “horny scale”). DNA evidence has established that the closest living group to the pangolins is the Carnivora, the order containing the cats, civets, mongooses and hyenas as well as the dogs, bears, raccoons, weasels, seals and sea lions. The earliest pangolin fossils date from the Eocene (~34 to 56 million years ago), but molecular dating suggests the ancestors of Pholidota may originated around 80 million years ago in the Cretaceous.

The 8 living species of pangolins were originally classified in the genus Manis, but analyses based on fossils, morphology, and molecular data indicate that the species are divided into three well defined genera: Manis (Asian pangolins, 4 species), Phataginus (African tree pangolins, 2 species) and Smutsia (African ground pangolins, 2 species).

Pangolins are among the most unusual of mammals when it comes to their biology. Along with their bodies being covered in sharp, keratinized scales which provides armor against predators when they roll up into a ball, pangolins lack teeth and instead use their extremely long tongue and sticky saliva to feed on ants and termites. Interestingly, molecular genetic and genome studies have shown that several genes involved in the development of teeth have become pseudogenized in pangolins. They possess long and thick claws at the ends of their powerful limbs which they use to dig burrows, break open ant and termite nests and to climb trees.

Pangolins are considered the most illegally trafficked mammals in the world. They are heavily poached due to the high demand for their scales, which are used in Asian traditional medicines, and for their meat. The eight species are listed as either Threatened or Critically Endangered on the IUCN Red List of Threatened Species. Vigorous international efforts are trying to curtail the illegal trafficking of pangolins in order to prevent their extinction. You can learn more about pangolins and their conservation on the Save Pangolins website.

Pangolins have also been in the news recently because of their possible link to the novel coronavirus that has been infecting people within and outside of China. Several studies have reported evidence that the Malayan pangolin (Manis javanica) to be a possible intermediate host and reservoir of coronaviruses that are related to the novel human coronavirus causing COVID-19 [1, 2, 3]. However, these studies have not been formally peer-reviewed and therefore, the conclusions should be interpreted with caution.

Today, we are proud to share the very first chromosome-length assembly of one of the 8 species of the Pholidota, the white-bellied or tree pangolin (Phataginus tricupis) from Africa. The assembly was generated from a female white-bellied pangolin named “Jaziri” who is housed at the Pittsburgh Zoo & PPG Aquarium, in Pittsburgh, Pennsylvania. The chromosome-length assembly is based on a draft assembly generated using 10x Genomics linked-read sequencing and Supernova version 2.0.1.

Jaziri’s assembly revealed an amazing finding: the presence of as many as 114 diploid chromosomes! This discovery was first observed independently in 2009 in standard giemsa stained and C- and G- banded karyotypes from several individuals of white-bellied pangolin from San Diego Zoo’s Frozen Zoo®. This was unexpected because previous karyotype studies of three Asian pangolin species (Chinese, Indian and Malayan) showed a chromosome complement between 2n=36-42. This would also make white-bellied pangolins the mammal species outside of the Rodentia (mice, rats, squirrels and their allies) with the highest number of chromosomes, among those whose karyotype has been examined. The current record holder for highest chromosome number in mammals is the Bolivian bamboo rat, Dactylomys boliviensis, with a 2n = 118, which in 2001 broke the record of 2n=102, previously held by another rodent, the red vizcacha rat, Tympanoctomys barrerae, which was reported in 1990 [4]. The karyotypes of the San Diego animals show the presence of many small chromosomes and Jaziri’s Hi-C contact map show the presence of many small c-scaffolds, which likely correspond to these very small physical chromosomes. A manuscript describing these results is in preparation.

Jaziri’s assembly was made possible through a collaboration of the following individuals: Tom Smith, Department of Ecology and Evolutionary Biology and Director of the Center for Tropical Research at UCLA; Klaus-Peter Koepfli, Center for Species Survival, Smithsonian Conservation Biology Institute; Kenneth Kaemmerer, Curator of Mammals, and Ginger Sturgeon, Director of Animal Health, Pittsburgh Zoo & PPG Aquarium; Jan Janecka, Department of Biological Sciences, Duquesne University; and Olga Dudchenko, Arina Omer and Erez Aiden, The Center for Genome Architecture, Baylor College of Medicine and Rice University. Karyotypes of the San Diego Zoo pangolins were made possible by Marlys Houck, Julie Fronczek and Ann Misuraca, San Diego Zoo Institute for Conservation Research.

Best. Rupert. Ever.

The Indian rhino (Rhinoceros unicornis), also known as the greater one-horned rhino, is a rhinoceros native to the Indian subcontinent. Among terrestrial land mammals native to Asia, the Indian rhinoceros is second in size only to the Asian elephant and can weigh more than one ton! [1]

Indian rhinos are listed as a vulnerable species on the IUCN red list. This is actually good news: the greater one-horned rhino is one of Asia’s biggest success stories, with their status improving from endangered to vulnerable following significant population increases. However, the species still remains under threat from poaching for its horn and from habitat loss and degradation.

Today, we share the chromosome-length assembly for Rupert, an Indian rhino from the Oklahoma City Zoo. That’s him on the photo below and on the blog cover photo (with his mom). Isn’t he the cutie!

One could say Rupert was destined to be sequenced since his parents, Chandra and Niki, met because of their genetic compatibility! Today Rupert is part of the Greater One-Horned Rhinoceros Species Survival Plan (SSP) developed by the Association of Zoos and Aquariums. Today, Rupert lives at Mesker Park Zoo, Indiana. We thank Julia Jones, Liz McCrae, Jennifer D’Agostino and Candice Rennels at the Oklahoma City Zoo for their help with the sample. To learn more about the OKC Zoo visit www.okczoo.org.

This is a $1K genome assembly. For more details about the genome assembly procedure, see Dudchenko et al., 2018.

Rhino Rupert hopping, credit: Lena Kofoed, via Oklahoma City Zoo

Cover photo credits: Nicky and Rupert by Joel Sartore, via Oklahoma City Zoo

Rousette bats: a coronavirus reservoir

Today we release a chromosome-length genome assembly for the Madagascar rousette (Rousettus madagascariensis), a small Malagasy fruit bat, one of ten species in the genus. Rousettus is an old world fruit bat of the family Pteropodidae, like the Madagascar flying fox (Pteropus rufus), whose genome we released at the end of 2019.[i] Members of the family Pteropodidae have been known to be natural hosts for many different types of viruses.

We have worked to release this genome as soon as possible in light of the Wuhan coronavirus nCoV-2019 [ii], which, as of this writing, has infected over 17,000 people, with an early case fatality rate of approximately 2%.[iii] Two weeks ago, an analysis of Wuhan and SARS coronavirus genomes by Xu et al.[iv] came to the conclusion that the human viral sequence is most closely related to a nonhuman viral sequence known as HKU-9-1. HKU-9-1, also known as “Rousettus bat coronavirus”, was first isolated from a bat in genus Rousettus. As such we believe that the reference genome for a species in this genus – even one endemic to Madagascar rather than China – could be relevant to studies of the coronavirus and its reservoir.

Ekipa Fanihy (“Team Fruit Bat” in Malagasy, the native language of Madagascar), led by Dr. Cara Brook (https://carabrook.github.io/team.html) has been studying the dynamics of viral infection in Malagasy fruit bats since 2013. Old World Fruit Bats have been disproportionately linked to the emergence of human viruses in the past two decades, serving as reservoir hosts for rabies and related lyssaviruses, Hendra and Nipah henipaviruses, Ebola and Marburg filoviruses, and the SARS coronavirus[v] [vi]. Several genomic analyses have demonstrated unique adaptations related to the evolution of flight which appear to have elongated bat lifespans and also made them resilient to many of the pathogenic effects of viral hosting.[vii],[viii],[ix] It has also been demonstrated that several zoonotic virus families circulate specifically in the Malagasy bats.[x] DNA Zoo is assembling bat genomes, including all the Madagascar fruit bats, in order to learn more about the mechanisms which underlie these bats’ unique viral tolerance. If you have samples from potential bat vector species, we’d love to be in touch. (Aviva Presser Aiden - Aviva.Aiden@bcm.edu)

Assembly of this genome was financed by the DNA Zoo and NIH grant (R01-AI129822-01) administered by Dr. Jean-Michel Héraud of Institut Pasteur of Madagascar and Dr. Cara Brook of UC Berkeley (link: http://grantome.com/grant/NIH/R01-AI129822-01).

Ekipa Fanihy is affiliated with UC Berkeley (link: https://ib.berkeley.edu/), the University of Antananarivo (link: http://www.univ-antananarivo.mg/), and Institut Pasteur of Madagascar (link: http://www.pasteur.mg/).

Aviva Presser Aiden, Cara Brook, Olga Dudchenko

[i] E.C. Teeling, M.S. Springer, O. Madsen, P. Bates, S.J. O’brien, W.J. Murphy, A molecular phylogeny for bats illuminates biogeography and the fossil record., Science (80-. ). 307 (2005) 580–4. doi:10.1126/science.1105113.

[ii] Zhou, P. et al. “Discovery of a novel coronavirus associated with the recent pneumonia outbreak in humans and its potential bat origin” Biorxiv, doi: https://doi.org/10.1101/2020.01.22.914952

[iii] https://www.worldometers.info/coronavirus/ accessed 2/3/2020

[iv] Xu, X, et al. Evolution of the novel coronavirus from the ongoing Wuhan outbreak and modeling of its spike protein for risk of human transmission. SCIENCE CHINA Life Sciences; doi: 10.1007/s11427-020-1637-5

[v] C.H. Calisher, J.E. Childs, H.E. Field, K. V Holmes, T. Schountz, Bats: Important reservoir hosts of emerging viruses, Clin. Microbiol. Rev. 19 (2006) 531–45. doi:10.1128/CMR.00017-06.

[vi] C.E. Brook, A.P. Dobson, Bats as “special” reservoirs for emerging zoonotic pathogens, Trends Microbiol. 23 (2015) 172–180. doi:10.1016/j.tim.2014.12.004.

[vii] S.S. Pavlovich, S.P. Lovett, G. Koroleva, J.C. Guito, C.E. Arnold, E.R. Nagle, K. Kulcsar, A. Lee, F. Thibaud-Nissen, A.J. Hume, E. Mühlberger, L.S. Uebelhoer, J.S. Towner, R. Rabadan, M. Sanchez-Lockhart, T.B. Kepler, G. Palacios, The Egyptian Rousette genome reveals unexpected features of bat antiviral immunity, Cell. 173 (2018) 1–13. doi:10.1016/j.cell.2018.03.070

[viii] G. Zhang, C. Cowled, Z. Shi, Z. Huang, K. a Bishop-Lilly, X. Fang, J.W. Wynne, Z. Xiong, M.L. Baker, W. Zhao, M. Tachedjian, Y. Zhu, P. Zhou, X. Jiang, J. Ng, L. Yang, L. Wu, J. Xiao, Y. Feng, Y. Chen, X. Sun, Y. Zhang, G. a Marsh, G. Crameri, C.C. Broder, K.G. Frey, L.-F. Wang, J. Wang, Comparative analysis of bat genomes provides insight into the evolution of flight and immunity, Science. 339 (2013) 456–60. doi:10.1126/science.1230835.

[ix] I. Seim, X. Fang, Z. Xiong, A. V Lobanov, Z. Huang, S. Ma, Y. Feng, A. a Turanov, Y. Zhu, T.L. Lenz, M. V Gerashchenko, D. Fan, S. Hee Yim, X. Yao, D. Jordan, Y. Xiong, Y. Ma, A.N. Lyapunov, G. Chen, O.I. Kulakova, Y. Sun, S.-G. Lee, R.T. Bronson, A. a Moskalev, S.R. Sunyaev, G. Zhang, A. Krogh, J. Wang, V.N. Gladyshev, Genome analysis reveals insights into physiology and longevity of the Brandt’s bat Myotis brandtii., Nat. Commun. 4 (2013) 2212. doi:10.1038/ncomms3212.

[x] C.E. Brook, H.C. Ranaivoson, C.C. Broder, A.A. Cunningham, J.-M. Héraud, A.J. Peel, L. Gibson, J.L.N. Wood, C.J. Metcalf, A.P. Dobson, Disentangling serology to elucidate henipa- and filovirus transmission in Madagascar fruit bats, J. Anim. Ecol. 00 (2019) 1– 16. doi:10.1111/1365-2656.12985.