Abstract
We report the first chromosome-length genome assemblies for three species in the mammalian order Pholidota: the white-bellied, Chinese, and Sunda pangolins. Surprisingly, we observe extraordinary karyotypic plasticity within this order and, in female white-bellied pangolins, the largest number of chromosomes reported in a Laurasiatherian mammal: 2n = 114. We perform the first karyotype analysis of an African pangolin and report a Y-autosome fusion in white-bellied pangolins, resulting in 2n = 113 for males. We employ a novel strategy to confirm the fusion and identify the autosome involved by finding the pseudoautosomal region (PAR) in the female genome assembly and analyzing the 3D contact frequency between PAR sequences and the rest of the genome in male and female white-bellied pangolins. Analyses of genetic variability show that white-bellied pangolins have intermediate levels of genome-wide heterozygosity relative to Chinese and Sunda pangolins, consistent with two moderate declines of historical effective population size. Our results reveal a remarkable feature of pangolin genome biology and highlight the need for further studies of these unique and endangered mammals.
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Data availability
The Whole Genome Shotgun project for P. tricuspis has been deposited at DDBJ/ENA/GenBank under the accession JAQQAC000000000 within the BioProject PRJNA905096. The version described in this paper is version JAQQAC010000000. The short-read sequencing data generated for the Hi-C scaffolding of the three pangolin species have been deposited into the NCBI Sequence Read Archive under the accessions SRX8933295 (Phataginus tricuspis), SRX9606524 (Manis javanica), and SRX9606525 (Manis pentadactyla), part of BioProject PRJNA512907. The P. tricuspis assembly and the upgraded M. javanica and M. pendactyla assemblies are available at https://www.dnazoo.org/assemblies?search=pangolins. Custom scripts used for this study have been deposited on GitHub at https://github.com/thainsCEB/PangolinCytogenomics.
Abbreviations
- PAR :
-
Pseudo-autosomal region
References
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215(3):403–410. https://doi.org/10.1016/S0022-2836(05)80360-2
Bao W, Kojima KK, Kohany O (2015) Repbase Update, a database of repetitive elements in eukaryotic genomes. Mob DNA 6:11. https://doi.org/10.1186/s13100-015-0041-9
Beklemisheva VR, Perelman PL, Lemskaya NA, Proskuryakova AA, Serdyukova NA, Burkanov VN, Gorshunov MB, Ryder O, Thompson M, Lento G, O’Brien SJ, Graphodatsky AS (2020) Karyotype evolution in 10 pinniped species: variability of heterochromatin versus high conservatism of euchromatin as revealed by comparative molecular cytogenetics. Genes 11(12):1485. https://doi.org/10.3390/genes11121485
Bernardi G (2015) Chromosome architecture and genome organization. PLoS One 10(11):e0143739. https://doi.org/10.1371/journal.pone.0143739
Brashear WA, Bredemeyer KR, Murphy WJ (2021) Genomic architecture constrained placental mammal X chromosome evolution. Genome Res 31(8):1353–1365. https://doi.org/10.1101/gr.275274.121
Burgin CJ, Collela JP, Kahn PL, Upham NS (2018) How many species of mammals are there? J Mammal 99(1):1–14. https://doi.org/10.1093/jmammal/gyz052
Burton JN, Adey A, Patwardhan RP, Qiu R, Kitzman JO, Shendure J (2013) Chromosome-scale scaffolding of de novo genome assemblies based on chromatin interactions. Nat Biotechnol 31(12):1119–1125. https://doi.org/10.1038/nbt.2727
Choo SW, Rayko M, Tan TK, Hari R, Komissarov A, Wee WY, Yurchenko AA, Kliver S, Tamazian G, Antunes A, Wilson RK, Warren WC, Koepfli KP, Minx P, Krasheninnikova K, Kotze A, Dalton DL, Vermaak E, Paterson IC, Dobrynin P, … Wong GJ (2016) Pangolin genomes and the evolution of mammalian scales and immunity. Genome Res 26(10), 1312–1322 https://doi.org/10.1101/gr.203521.115
Choo SW, Chong JL, Gaubert P, Hughes AC, O’Brien S, Chaber AL, Antunes A, Platto S, Sun NC, Yu L, Koepfli KP, Suwal TL, Thakur M, Ntie S, Panjang E, Kumaran JV, Mahmood T, Heighton SP, Dorji D, Gonedelé BS, … Aziz MA (2022) A collective statement in support of saving pangolins. Sci Total Environ 824:153666. https://doi.org/10.1016/j.scitotenv.2022.153666
Contreras LC, Torres-Mura JC, Spotorno AE (1990) The largest known chromosome number for a mammal, in a South American desert rodent. Experientia 46(5):506–508. https://doi.org/10.1007/BF01954248
Damas J, Corbo M, Kim J, Turner-Maier J, Farré M, Larkin DM, Ryder OA, Steiner C, Houck ML, Hall S, Shiue L, Thomas S, Swale T, Daly M, Korlach, J, Uliano-Silva M, Mazzoni CJ, Birren BW, Genereux DP, Johnson J, … Lewin HA (2022) Evolution of the ancestral mammalian karyotype and syntenic regions. Proc Natl Acad Sci US America 119(40):e2209139119. https://doi.org/10.1073/pnas.2209139119
Deakin JE, Ezaz T (2014) Tracing the evolution of amniote chromosomes. Chromosoma 123(3):201–216. https://doi.org/10.1007/s00412-014-0456-y
Dudchenko O, Batra SS, Omer AD, Nyquist SK, Hoeger M, Durand NC, Shamim MS, Machol I, Lander ES, Aiden AP, Aiden EL (2017) De novo assembly of the Aedes aegypti genome using Hi-C yields chromosome-length scaffolds. Science 356(6333):92–95. https://doi.org/10.1126/science.aal3327
Dudchenko O, Shamim MS, Batra S, Durand NC, Musial NT, Mostofa R, Pham M, Hilaire BGS, Yao W, Stamenova E, Hoeger M, Nyquist SK, Korchina V, Pletch K, Flanagan JP, Tomaszewicz A, McAloose D, Estrada CP, Novak BJ, Omer AD, Aiden EL (2018) The Juicebox Assembly Tools module facilitates de novo assembly of mammalian genomes with chromosome-length scaffolds for under $1000. bioRxiv.https://doi.org/10.1101/254797
Dunnum JL, Salazar-Bravo J, Yates TL (2001) The Bolivian bamboo rat Dactylomys boliviensis (Rodentia: Echimyidae), a new record for chromosome number in a mammal. Mamm Biol 66(2):121–126
Durand NC, Shamim MS, Machol I, Rao SS, Huntley MH, Lander ES, Aiden EL (2016a) Juicer provides a one-click system for analyzing loop-resolution Hi-C experiments. Cell Syst 3(1):95–98. https://doi.org/10.1016/j.cels.2016.07.002
Durand NC, Robinson JT, Shamim MS, Machol I, Mesirov JP, Lander ES, Aiden EL (2016b) Juicebox provides a visualization system for Hi-C contact maps with unlimited zoom. Cell Syst 3(1):99–101. https://doi.org/10.1016/j.cels.2015.07.012
Dutoit L, Vijay N, Mugal CF, Bossu CM, Burri R, Wolf J, Ellegren H (2017) Covariation in levels of nucleotide diversity in homologous regions of the avian genome long after completion of lineage sorting. Proc R Soc B: Biol Sci 284(1849):20162756. https://doi.org/10.1098/rspb.2016.2756
Evans BJ, Upham NS, Golding GB, Ojeda RA, Ojeda AA (2017) Evolution of the largest mammalian genome. Genome Biol Evol 9(6):1711–1724. https://doi.org/10.1093/gbe/evx113
Fong JH, Murphy TD, Pruitt KD (2013) Comparison of RefSeq protein-coding regions in human and vertebrate genomes. BMC Genomics 14:654. https://doi.org/10.1186/1471-2164-14-654
Gaubert P, Antunes A, Meng H, Miao L, Peigné S, Justy F, Njiokou F, Dufour S, Danquah E, Alahakoon J, Verheyen E, Stanley WT, O’Brien SJ, Johnson WE, Luo SJ (2018) The complete phylogeny of pangolins: scaling up resources for the molecular tracing of the most trafficked mammals on Earth. J Hered 109(4):347–359. https://doi.org/10.1093/jhered/esx097
Graphodatsky AS, Sharshov A, Lavryushov S, Sablina OV, Biltueva LS, Perelman PL, Orlov VN, Kozlovsky AI, Nadjafova RS, Bulatova NSH (2000) Chromosomes network. http://www.bionet.nsc.ru/labs/chromosomes/
Graphodatsky AS, Trifonov VA, Stanyon R (2011) The genome diversity and karyotype evolution of mammals. Mol Cytogenet 4:22. https://doi.org/10.1186/1755-8166-4-22
Graphodatsky A, Perelman PL, O’Brien SJ (eds). (2020) Atlas of mammalian chromosomes, Second Edition. Hoboken, NJ: John Wiley & Sons, Inc. 1008
Heinrich S, Wittman TA, Prowse TAA, Ross JV, Delean S, Shepherd CR, Cassey P (2016) Where did all the pangolins go? International CITES trade in pangolin species. Glob Ecol Conserv 8:241–253. https://doi.org/10.1016/j.gecco.2016.09.007
Hoencamp C, Dudchenko O, Elbatsh A, Brahmachari S, Raaijmakers JA, van Schaik T, Sedeño Cacciatore Á, Contessoto VG, van Heesbeen R, van den Broek B, Mhaskar AN, Teunissen H, St Hilaire BG, Weisz D, Omer AD, Pham M, Colaric Z, Yang Z, Rao S, Mitra N, … Rowland BD (2021) 3D genomics across the tree of life reveals condensin II as a determinant of architecture type. Science 372(6545):984–989. https://doi.org/10.1126/science.abe2218
Houck ML, Ryder OA, Váhala J, Kock RA, Oosterhuis JE (1994) Diploid chromosome number and chromosomal variation in the white rhinoceros (Ceratotherium simum). J Hered 85(1):30–34
Hu J-Y, Hao Z-Q, Frantz L, Wu S-F, Chen W, Jiang Y-F, Wu H, Kuang W-M, Li H, Zhang Y-P, Yu L (2020) Genomic consequences of population decline in critically endangered pangolins and their demographic histories. Natl Sci Rev 7(4):798–814. https://doi.org/10.1093/nsr/nwaa031
Keilwagen J, Wenk M, Erickson JL, Schattat MH, Grau J, Hartung F (2016) Using intron position conservation for homology-based gene prediction. Nucleic Acids Res 44(9):e89. https://doi.org/10.1093/nar/gkw092
Keilwagen J, Hartung F, Paulini M, Twardziok SO, Grau J (2018) Combining RNA-seq data and homology-based gene prediction for plants, animals and fungi. BMC Bioinformatics 19(1):189. https://doi.org/10.1186/s12859-018-2203-5
Kiełbasa SM, Wan R, Sato K, Horton P, Frith MC (2011) Adaptive seeds tame genomic sequence comparison. Genome Res 21(3):487–493. https://doi.org/10.1101/gr.113985.110
King M (1993) Species evolution – the role of chromosome change. Cambridge University Press, Cambridge, p 336
Kumamoto AT, Charter SJ, Houck ML, Frahm M (1996) Chromosomes of Damaliscus (Artiodactyla, Bovidae): simple and complex centric fusion rearrangements. Chromosome Res 4(8):614–621. https://doi.org/10.1007/BF02261724
Li H, Durbin R (2009) Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25(14):1754–1760. https://doi.org/10.1093/bioinformatics/btp324
Li H, Durbin R (2011) Inference of human population history from individual whole-genome sequences. Nature 475(7357):493–496. https://doi.org/10.1038/nature10231
Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R, 1000 Genome Project Data Processing Subgroup (2009) The Sequence Alignment/Map format and SAMtools. Bioinformatics 25(16):2078–2079. https://doi.org/10.1093/bioinformatics/btp352
Li H (2011) A statistical framework for SNP calling, mutation discovery, association mapping and population genetical parameter estimation from sequencing data. Bioinformatics 27(21):2987–2993. https://doi.org/10.1093/bioinformatics/btr509
Lieberman-Aiden E, van Berkum NL, Williams L, Imakaev M, Ragoczy T, Telling A, Amit I, Lajoie BR, Sabo PJ, Dorschner MO, Sandstrom R, Bernstein B, Bender MA, Groudine M, Gnirke A, Stamatoyannopoulos J, Mirny LA, Lander ES, Dekker J (2009) Comprehensive mapping of long-range interactions reveals folding principles of the human genome. Science 326(5950):289–293. https://doi.org/10.1126/science.1181369
Maden BEH, Dent CL, Farrell TE, Garde J, McCallum FS, Wakeman JA (1987) Clones of human ribosomal DNA containing the complete 18 S-rRNA and 28 S-rRNA genes. Characterization, a detailed map of the human ribosomal transcription unit and diversity among clones. Biochem J 246(2):519–527. https://doi.org/10.1042/bj2460519
Maere S, De Bodt S, Raes J, Casneuf T, Van Montagu M, Kuiper M, & Van de Peer Y (2005) Modeling gene and genome duplications in eukaryotes. Proc Natl Acad Sci U S A 102(15):5454–5459. https://doi.org/10.1073/pnas.0501102102
Makino S, Tateishi S (1951) Notes on the chromosomes of the pangolin, Manis pentadactyla (Edentata). J Fac Sci Hokkaido Univ Series VI Zool 10(3–4):319–323
Manni M, Berkeley MR, Seppey M, Simão FA, Zdobnov EM (2021) BUSCO update: novel and streamlined workflows along with broader and deeper phylogenetic coverage for scoring of eukaryotic, prokaryotic, and viral genomes. Mol Biol Evol 38(10):4647–4654. https://doi.org/10.1093/molbev/msab199
Mayrose I, Lysak MA (2021) The evolution of chromosome numbers: mechanistic models and experimental approaches. Genome Biol Evolution 13(2):evaa220. https://doi.org/10.1093/gbe/evaa220
Nash WG, Menninger JC, Padilla-Nash HM, Stone G, Perelman PL, O’Brien SJ (2008) The ancestral carnivore karyotype (2n = 38) lives today in ringtails. J Hered 99(3):241–253. https://doi.org/10.1093/jhered/esm130
Nie W, Wang J, Su W, Wang Y, Yang F (2009) Chromosomal rearrangements underlying karyotype differences between Chinese pangolin (Manis pentadactyla) and Malayan pangolin (Manis javanica) revealed by chromosome painting. Chromosome Res 17(3):321–329. https://doi.org/10.1007/s10577-009-9027-0
Nie W, Wang J, Su W, Wang D, Tanomtong A, Perelman PL, Graphodatsky AS, Yang F (2012) Chromosomal rearrangements and karyotype evolution in carnivores revealed by chromosome painting. Heredity 108(1):17–27. https://doi.org/10.1038/hdy.2011.107
Pedersen BS, Quinlan AR (2018) Mosdepth: quick coverage calculation for genomes and exomes. Bioinformatics (Oxford, England) 34(5):867–868. https://doi.org/10.1093/bioinformatics/btx699
R Core Team (2018) R: a language and environment for statistical computing. R Foundation for Statistical Computing. https://www.R-project.org/
Rao SS, Huntley MH, Durand NC, Stamenova EK, Bochkov ID, Robinson JT, Sanborn AL, Machol I, Omer AD, Lander ES, Aiden EL (2014) A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping. Cell 159(7):1665–1680. https://doi.org/10.1016/j.cell.2014.11.021
Robinson JA, Ortega-Del Vecchyo D, Fan Z, Kim BY, vonHoldt BM, Marsden CD, Lohmueller KE, Wayne RK (2016) Genomic flatlining in the endangered island fox. Curr Biol 26(9):1183–1189. https://doi.org/10.1016/j.cub.2016.02.062
Robinson JA, Räikkönen J, Vucetich LM, Vucetich JA, Peterson RO, Lohmueller KE, Wayne RK (2019) Genomic signatures of extensive inbreeding in Isle Royale wolves, a population on the threshold of extinction. Sci Adv 5(5):eaau0757. https://doi.org/10.1126/sciadv.aau0757
Robinson TJ, Ruiz-Herrera A, Avise JC (2008) Hemiplasy and homoplasy in the karyotypic phylogenies of mammals. Proc Natl Acad Sci U S A 105(38):14477–14481https://doi.org/10.1073/pnas.0807433105
Sexton T, Cavalli G (2015) The role of chromosome domains in shaping the functional genome. Cell 160(6):1049–1059. https://doi.org/10.1016/j.cell.2015.02.040
Smit AFA, Hubley R, Green P (2013–2015) RepeatMasker Open-4.0.2013–2015. http://www.repeatmasker.org
Stanke M, Schöffmann O, Morgenstern B, Waack S (2006) Gene prediction in eukaryotes with a generalized hidden Markov model that uses hints from external sources. BMC Bioinformatics 7:62. https://doi.org/10.1186/1471-2105-7-62
Stanyon R, Galleni L (1991) A rapid fibroblast culture technique for high resolution karyotypes. Bollettino Di Zoologia 58(1):81–83. https://doi.org/10.1080/11250009109355732
Steinegger M, Söding J (2017) MMseqs2 enables sensitive protein sequence searching for the analysis of massive data sets. Nat Biotechnol 35(11):1026–1028. https://doi.org/10.1038/nbt.3988
Sumner AT (1972) A simple technique for demonstrating centromeric heterochromatin. Exp Cell Res 75(1):304–306. https://doi.org/10.1016/0014-4827(72)90558-7
Tang H, Krishnakumar V, Li J (2015) jcvi: JCVI utility libraries. Zenodo. https://doi.org/10.5281/zenodo.31631
Van der Auwera GA, O’Connor BD (2020) Genomics in the cloud: using Docker, GATK, and WDL in Terra. O’Reilly Media Inc., Sebastopol, California
Wang Y, Tang H, Debarry JD, Tan X, Li J, Wang X, Lee TH, Jin H, Marler B, Guo H, Kissinger JC, Paterson AH (2012) MCScanX: a toolkit for detection and evolutionary analysis of gene synteny and collinearity. Nucleic Acids Res 40(7):e49. https://doi.org/10.1093/nar/gkr1293
Wickham H (2016) ggplot2: Elegant graphics for data analysis 2nd ed. Springer-Verlag, New York
Yang F, Graphodatsky AS (2009) Animal probes and ZOO-FISH. In: Liehr T (ed) Fluorescence in situ hybridization (FISH) - application guide. Springer-Verlag, Berlin, pp 323–346
Yang F, Graphodatsky AS, Li T, Fu B, Dobigny G, Wang J, Perelman PL, Serdukova NA, Su W, O’Brien PC, Wang Y, Ferguson-Smith MA, Volobouev V, Nie W (2006) Comparative genome maps of the pangolin, hedgehog, sloth, anteater and human revealed by cross-species chromosome painting: further insight into the ancestral karyotype and genome evolution of eutherian mammals. Chromosome Res 14(3):283–296. https://doi.org/10.1007/s10577-006-1045-6
Yang Y, Li Y, Chen Q, Sun Y, Lu Z (2019) WGDdetector: a pipeline for detecting whole genome duplication events using the genome or transcriptome annotations. BMC Bioinformatics 20(1):75. https://doi.org/10.1186/s12859-019-2670-3
Acknowledgements
We thank Kang Sa and the laboratory and bioinformatic teams at Psomagen, Inc. for their excellent services and support in generating the linked-read assembly for the white-bellied pangolin, Jaziri. We are grateful to Russ Hansen (National Cancer Institute-Frederick) for assistance with CITES paperwork. We thank Melody Roelke (Leidos Inc), Carlos Driscoll, Christina Barr, Stephen Lindell, Cheryl Marietta, and David Goldman (National Institute on Alcohol Abuse and Alcoholism) for preservation of pangolin cell lines as part of the former NCI-Laboratory of Genomic Diversity collection. We thank Saul Godinez Puido and Zane Colaric for their help with Hi-C sample acquisitions and processing. Hi-C data were created by the DNA Zoo Consortium (www.dnazoo.org). DNA Zoo is supported by Illumina, Inc.; IBM; and the Pawsey Supercomputing Center. Figure 1 photo credits: P. tricuspis: copyright: ehbidault, some rights reserved (CC-BY-NC), via inaturalist.org; M. javanica: Sunda pangolin, photo by budak (CC BY-NC-ND 2.0), via flickr.com; M. pentadactyla: Manis pentadactyla pentadactyla (2010.07.15), photo by Skink Chen (CC BY-NC-ND 2.0), via flickr.com.
Funding
Work was supported by grants from the National Geographic Society (NGS-418C-18) and US Gov. INLEC (S-INLEC-17-GR-1006). E.L.A. was supported by the Welch Foundation (Q-1866), a McNair Medical Institute Scholar Award, an NIH Encyclopedia of DNA Elements Mapping Center Award (UM1HG009375), a US-Israel Binational Science Foundation Award (2019276), the Behavioral Plasticity Research Institute (NSF DBI-2021795), an NSF Physics Frontiers Center Award (NSF PHY-2019745), and an NIH CEGS (RM1HG011016-01A1). A.S. Graphodatsky, P.L. Perelman, and V.R. Beklemisheva were supported by the Russian Science Foundation (Grant No. 19–14-00034p to A.S.G.). G. Tamazian was supported by Peter the Great St. Petersburg Polytechnic University in the framework of the Russian Federation’s Priority 2030 Strategic Academic Leadership Programme (Agreement 075–15-2021–1333).
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M.L.H., K-P.K., T.B.S., E.L.A., and O.D.: conceived the study. A.G., S-L.J., S.J.O., N.T-L.L., J.S.C.C., K.K., G.S., J.G., A.H., M.M., I.G.-T., J.G., R.L.C., J.E.J., R.J.H., J.T.: facilitated and provided experimental materials. M.L.H., S.J.C., J.A.F., A.C.M., P.L.P, V.B.: performed the karyotyping. K-P.K., J.E.J., R.J.H., J.T., T.B.S: led the effort to generate the linked-read genome assembly. R.K., A.O., D.W., E.L.A., O.D.: performed the Hi-C experiments and generated the chromosome-length assemblies. M.L.H., K-P.K., T.H., S.K., A.G., V.G., G.T., E.L.A., O.D.: analyzed the data. M.L.H., K-P.K., P.L.P., E.L.A., O.D.: drafted the manuscript, with subsequent input from all the authors.
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Houck, M.L., Koepfli, KP., Hains, T. et al. Chromosome-length genome assemblies and cytogenomic analyses of pangolins reveal remarkable chromosome counts and plasticity. Chromosome Res 31, 13 (2023). https://doi.org/10.1007/s10577-023-09722-y
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DOI: https://doi.org/10.1007/s10577-023-09722-y