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“Subfossil” Koala Lemur Megaladapis Edwardsi

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“Subfossil” Koala Lemur Megaladapis Edwardsi Evolutionary and phylogenetic insights from a nuclear genome sequence of the extinct, giant, “subfossil” koala lemur Megaladapis edwardsi Stephanie Marciniaka, Mehreen R. Mughalb, Laurie R. Godfreyc, Richard J. Bankoffa, Heritiana Randrianatoandroa,d, Brooke E. Crowleye,f, Christina M. Bergeya,g,h, Kathleen M. Muldooni, Jeannot Randrianasyd, Brigitte M. Raharivololonad, Stephan C. Schusterj, Ripan S. Malhik,l, Anne D. Yoderm,n, Edward E. Louis Jro,1, Logan Kistlerp,1, and George H. Perrya,b,g,q,1 aDepartment of Anthropology, Pennsylvania State University, University Park, PA 16802; bBioinformatics and Genomics Intercollege Graduate Program, Pennsylvania State University, University Park, PA 16082; cDepartment of Anthropology, University of Massachusetts, Amherst, MA 01003; dMention Anthropobiologie et Développement Durable, Faculté des Sciences, Université d’Antananarivo, Antananarivo 101, Madagascar; eDepartment of Geology, University of Cincinnati, Cincinnati, OH 45220; fDepartment of Anthropology, University of Cincinnati, Cincinnati, OH 45220; gDepartment of Biology, Pennsylvania State University, University Park, PA 16802; hDepartment of Genetics, Rutgers University, New Brunswick, NJ 08854; iDepartment of Anatomy, Midwestern University, Glendale, AZ 85308; jSingapore Centre for Environmental Life Sciences Engineering, Nanyang Technological University, Singapore 639798; kDepartment of Anthropology, University of Illinois Urbana–Champaign, Urbana, IL 61801; lDepartment of Ecology, Evolution and Behavior, Carl R. Woese Institute for Genomic Biology, University of Illinois Urbana–Champaign, Urbana, IL 61801; mDepartment of Biology, Duke University, Durham, NC 27708; nDepartment of Evolutionary Anthropology, Duke University, Durham, NC 27708; oDepartment of Conservation Genetics, Omaha’s Henry Doorly Zoo and Aquarium, Omaha, NE 68107; pDepartment of Anthropology, National Museum of Natural History, Smithsonian Institution, Washington, DC 20560; and qHuck Institutes of the Life Sciences, Pennsylvania State University, University Park, PA 16802 Edited by Nicolas J. Rawlence, University of Otago, New Zealand, and accepted by Editorial Board Member Richard G. Klein May 13, 2021 (received for review November 2, 2020) No endemic Madagascar animal with body mass >10 kg survived a (primates) species is 6.8 kg (2), well below that of the 17 described relatively recent wave of extinction on the island. From morpho- extinct subfossil lemur taxa, for which estimated adult body masses logical and isotopic analyses of skeletal “subfossil” remains we can ranged from ∼11 kg to an incredible ∼160 kg (3). reconstruct some of the biology and behavioral ecology of giant Despite a tropical and subtropical environment in which nucleotide ANTHROPOLOGY lemurs (primates; up to ∼160 kg) and other extraordinary Malagasy (nt) strands rapidly degrade, in a select subset of Malagasy subfossil megafauna that survived into the past millennium. Yet, much about samples, ancient DNA (aDNA) is sufficiently preserved for pale- the evolutionary biology of these now-extinct species remains un- ogenomic analysis (4–10). In our group’s previous study (6), we known, along with persistent phylogenetic uncertainty in some reconstructed complete or near-complete mitochondrial genomes cases. Thankfully, despite the challenges of DNA preservation in trop- from five subfossil lemur species, with population-level data in two ical and subtropical environments, technical advances have enabled cases. As part of that work, we identified one Megaladapis edwardsi the recovery of ancient DNA from some Malagasy subfossil speci- (body mass ∼85 kg) (3, 11) sample with an especially high proportion ∼ × mens. Here, we present a nuclear genome sequence ( 2 coverage) of endogenous aDNA. We have subsequently performed additional for one of the largest extinct lemurs, the koala lemur Megaladapis ∼ edwardsi ( 85 kg). To support the testing of key phylogenetic and Significance evolutionary hypotheses, we also generated high-coverage nuclear genomes for two extant lemurs, Eulemur rufifrons and Lepilemur Based on “subfossil” skeletal remains it is known that multiple mustelinus, and we aligned these sequences with previously pub- now-extinct giant lemur (primate) species with estimated body lished genomes for three other extant lemurs and 47 nonlemur ver- masses of up to ∼160 kg survived on Madagascar into the past tebrates. Our phylogenetic results confirm that Megaladapis is most millennium. In this study, we used ancient DNA methods to closely related to the extant Lemuridae (typified in our analysis by sequence the nuclear genome of one of these megafaunal le- E. rufifrons) to the exclusion of L. mustelinus, which contradicts murs, Megaladapis edwardsi (∼85 kg). With the power of the morphology-based phylogenies. Our evolutionary analyses identi- nuclear genome, we robustly resolved the phylogenetic rela- fied significant convergent evolution between M. edwardsi and an tionship between Megaladapis and other lemurs, which had extant folivore (a colobine monkey) and an herbivore (horse) in genes been a lingering uncertainty. We also identified multiple sig- encoding proteins that function in plant toxin biodegradation and natures of past positive natural selection across the M. edwardsi nutrient absorption. These results suggest that koala lemurs were genome that support reconstructions of this taxon as a large- highly adapted to a leaf-based diet, which may also explain their bodied, specialized folivore. convergent craniodental morphology with the small-bodied folivore Lepilemur. Author contributions: S.M., E.E.L., L.K., and G.H.P. designed research; S.M., S.C.S., R.S.M., and L.K. performed research; L.R.G., H.R., B.E.C., K.M.M., J.R., B.M.R., A.D.Y., E.E.L., and paleogenomics | megafaunal extinction | phylogenomics | convergent G.H.P. contributed new reagents/analytic tools; S.M., M.R.M., R.J.B., B.E.C., C.M.B., L.K., and G.H.P. analyzed data; and S.M., L.R.G., L.K., and G.H.P. wrote the paper. evolution | dietary reconstruction The authors declare no competing interest. ’ This article is a PNAS Direct Submission. N.J.R. is a guest editor invited by the adagascar is exceptionally biodiverse today. Yet, the island s Editorial Board. endemic diversity was even greater in the relatively recent past. M This open access article is distributed under Creative Commons Attribution-NonCommercial- Specifically, there is an extensive “subfossil” record of now-extinct NoDerivatives License 4.0 (CC BY-NC-ND). Malagasy fauna, with some of these species persisting until at least See online for related content such as Commentaries. ∼ 500 y B.P. (1). The Late Holocene extinction pattern in Madagascar 1To whom correspondence may be addressed. Email: [email protected], [email protected], or resembles other “megafaunal extinction” patternsinthatitisstrik- [email protected]. ingly body-mass structured, with the majority of extinct subfossil taxa This article contains supporting information online at https://www.pnas.org/lookup/suppl/ substantially larger than their surviving counterparts. For example, doi:10.1073/pnas.2022117118/-/DCSupplemental. theaverageadultbodymassofthelargestofthe∼100 extant lemur Published June 23, 2021. PNAS 2021 Vol. 118 No. 26 e2022117118 https://doi.org/10.1073/pnas.2022117118 | 1of12 Downloaded by guest on September 24, 2021 rounds of extraction and sequencing of this sample to amass other paleogenomic studies (23, 24) and overall contributing sufficient data for studying the M. edwardsi nuclear genome. negligibly to the nuclear gene sequence reconstructions we report In this study, we analyzed the M. edwardsi nuclear genome to in this paper. help reconstruct subfossil lemur behavioral ecology and evolution- The set of quality-filtered and damage-masked M. edwardsi se- ary biology. Our approach included an unbiased search across the quence reads were aligned to a version of the human reference genome for Megaladapis-specific signatures of positive selection at genome (hg19) masked to contain only Reference Sequence (RefSeq) the individual gene level. We also searched for striking patterns gene exons ±100 bp. We used a conservative approach to re- of genomic convergence with a set of biologically diverse extant construct orthologous, single-copy M. edwardsi gene coding region mammals across sets of functionally annotated genes. The results sequences with 2× minimum sequence coverage per position (me- from these analyses may serve to extend current hypotheses or to dian proportion of sites reconstructed per gene = 0.29; SI Appendix, offer potentially unexpected insights into the evolutionary biology Fig. S5 and Table S3). Exon sequences reconstructed from shotgun of Megaladapis. sequence reads from the five modern lemur species (including two Additionally, we aimed to resolve lingering uncertainty over Meg- with data newly generated for this study) and a golden snub-nosed aladapis phylogenetic relationships with other lemurs. At one point, a colobine monkey (Rhinopithecus roxellana) were also included in sister taxon relationship between Megaladapis and extant sport- our analysis (25). All of these reconstructed sequences were in- ive lemurs (genus Lepilemur) was inferred based on craniodental tegrated with a canonical gene exon alignment of 46 vertebrate similarities (3, 12). A different phylogeny was estimated, however, species for an overall total alignment with 53 species. following the successful recovery of several hundred base pairs (bp) of the Megaladapis mitochondrial genome
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