| Abstract: | No endemic Madagascar animal with body mass >10 kg survived a relatively recent wave of extinction on the island. From morphological and isotopic analyses of skeletal “subfossil” remains we can reconstruct some of the biology and behavioral ecology of giant lemurs (primates; up to ∼160 kg) and other extraordinary Malagasy megafauna that survived into the past millennium. Yet, much about the evolutionary biology of these now-extinct species remains unknown, along with persistent phylogenetic uncertainty in some cases. Thankfully, despite the challenges of DNA preservation in tropical and subtropical environments, technical advances have enabled the recovery of ancient DNA from some Malagasy subfossil specimens. Here, we present a nuclear genome sequence (∼2× coverage) for one of the largest extinct lemurs, the koala lemur Megaladapis edwardsi (∼85 kg). To support the testing of key phylogenetic and evolutionary hypotheses, we also generated high-coverage nuclear genomes for two extant lemurs, Eulemur rufifrons and Lepilemur mustelinus, and we aligned these sequences with previously published genomes for three other extant lemurs and 47 nonlemur vertebrates. Our phylogenetic results confirm that Megaladapis is most closely related to the extant Lemuridae (typified in our analysis by E. rufifrons) to the exclusion of L. mustelinus, which contradicts morphology-based phylogenies. Our evolutionary analyses identified significant convergent evolution between M. edwardsi and an extant folivore (a colobine monkey) and an herbivore (horse) in genes encoding proteins that function in plant toxin biodegradation and nutrient absorption. These results suggest that koala lemurs were highly adapted to a leaf-based diet, which may also explain their convergent craniodental morphology with the small-bodied folivore Lepilemur.Madagascar is exceptionally biodiverse today. Yet, the island’s endemic diversity was even greater in the relatively recent past. Specifically, there is an extensive “subfossil” record of now-extinct Malagasy fauna, with some of these species persisting until at least ∼500 y B.P. (1). The Late Holocene extinction pattern in Madagascar resembles other “megafaunal extinction” patterns in that it is strikingly body-mass structured, with the majority of extinct subfossil taxa substantially larger than their surviving counterparts. For example, the average adult body mass of the largest of the ∼100 extant lemur (primates) species is 6.8 kg (2), well below that of the 17 described extinct subfossil lemur taxa, for which estimated adult body masses ranged from ∼11 kg to an incredible ∼160 kg (3).Despite a tropical and subtropical environment in which nucleotide (nt) strands rapidly degrade, in a select subset of Malagasy subfossil samples, ancient DNA (aDNA) is sufficiently preserved for paleogenomic analysis (4–10). In our group’s previous study (6), we reconstructed complete or near-complete mitochondrial genomes from five subfossil lemur species, with population-level data in two cases. As part of that work, we identified one Megaladapis edwardsi (body mass ∼85 kg) (3, 11) sample with an especially high proportion of endogenous aDNA. We have subsequently performed additional rounds of extraction and sequencing of this sample to amass sufficient data for studying the M. edwardsi nuclear genome.In this study, we analyzed the M. edwardsi nuclear genome to help reconstruct subfossil lemur behavioral ecology and evolutionary biology. Our approach included an unbiased search across the genome for Megaladapis-specific signatures of positive selection at the individual gene level. We also searched for striking patterns of genomic convergence with a set of biologically diverse extant mammals across sets of functionally annotated genes. The results from these analyses may serve to extend current hypotheses or to offer potentially unexpected insights into the evolutionary biology of Megaladapis.Additionally, we aimed to resolve lingering uncertainty over Megaladapis phylogenetic relationships with other lemurs. At one point, a sister taxon relationship between Megaladapis and extant sportive lemurs (genus Lepilemur) was inferred based on craniodental similarities (3, 12). A different phylogeny was estimated, however, following the successful recovery of several hundred base pairs (bp) of the Megaladapis mitochondrial genome in several early aDNA studies (4, 5). Specifically, Megaladapis and the extant Lemuridae (genera Eulemur, Lemur, Varecia, Prolemur, and Hapalemur) formed a clade to the exclusion of Lepilemur. Our more recent aDNA study (6) resolved a similar phylogeny but with greater confidence (e.g., 87% bootstrap support) given the near-complete recovery of the Megaladapis mitochondrial genome (16,714 bp). Still, the mitochondrial genome is a single, nonrecombining locus; in certain cases, true species-level phylogenies are not reconstructed accurately from mitochondrial DNA alone (13). Most recently, Herrera and Dávalos (14) estimated a “total evidence” phylogeny by analyzing the combination of both morphological and genetic characters. Their result was dissimilar to each of the above phylogenies, instead supporting an early divergence of the Megaladapis lineage from all other non-Daubentonia (aye-aye) lemurs.Because the nuclear genome is comprised of thousands of effectively independent markers of ancestry, we expected to achieve a more definitive phylogenetic result with our Megaladapis paleogenome sequence. To distinguish among competing phylogenetic hypotheses, we also needed to generate genome data for representatives of the extant Lemuridae and Lepilemur lineages, which we did for Eulemur rufifrons (red-fronted lemur) and Lepilemur mustelinus (greater sportive lemur), respectively. We aligned the three lemur genome sequences with those previously published for extant lemurs Daubentonia madagascariensis (aye-aye) (15), Microcebus murinus (gray mouse lemur) (16), and Propithecus diadema (diademed sifaka) (17), and with 47 nonlemur outgroup species, for phylogenetic and evolutionary analyses. |
