Conservation genomic analysis reveals ancient introgression and declining levels of genetic diversity in Madagascar's hibernating dwarf lemurs

Abstract

Madagascar's biodiversity is notoriously threatened by deforestation and climate change. Many of these organisms are rare, cryptic, and severely threatened, making population-level sampling unrealistic. Such is the case with Madagascar's dwarf lemurs (genus Cheirogaleus), the only obligate hibernating primate. We here apply comparative genomic approaches to generate the first genome-wide estimates of genetic diversity within dwarf lemurs. We generate a reference genome for the fat-tailed dwarf lemur, Cheirogaleus medius, and use this resource to facilitate analyses of high-coverage (~30×) genome sequences for wild-caught individuals representing species: C. sp. cf. medius, C. major, C. crossleyi, and C. sibreei. This study represents the largest contribution to date of novel genomic resources for Madagascar's lemurs. We find concordant phylogenetic relationships among the four lineages of Cheirogaleus across most of the genome, and yet detect a number of discordant genomic regions consistent with ancient admixture. We hypothesized that these regions could have resulted from adaptive introgression related to hibernation, indeed finding that genes associated with hibernation are present, though most significantly, that gene ontology categories relating to transcription are over-represented. We estimate levels of heterozygosity and find particularly low levels in an individual sampled from an isolated population of C. medius that we refer to as C. sp. cf. medius. Results are consistent with a recent decline in effective population size, which is evident across species. Our study highlights the power of comparative genomic analysis for identifying species and populations of conservation concern, as well as for illuminating possible mechanisms of adaptive phenotypic evolution.

Fig. 1

Geographic distributions and phylogenetic relationships of Cheirogaleus. a Map of Madagascar showing the sampling locations and IUCN Red List distributions of the four study species. Green star shows sampling location of outgroup Microcebus griseorufus. b Maximum likelihood estimation showing relationships within Cheirogaleus based on 684-bp cytochrome c oxidase subunit II (COII). Individuals used here in genomic analyses are indicated by their species colour. Comparative taxa were taken from the National Center for Biotechnology Information (NCBI) online database. Locations for these comparative samples were taken from NCBI or, if unlisted, their publication. Bootstrap values shown. c Cheirogaleus illustrations, copyright 2013 Stephen D. Nash/IUCN SSC Primate Specialist Group; used with permission

Fig. 2

The relationships assigned to regions of the genome that were identified in the SAGUARO analyses. Discordance to previously published phylogenies indicated. Phylogenies are neighbour joining trees generated from the distance matrices. Phylogenies with the same overall relationships have been concatenated. Colours represent species: Cheirogaleus sp. cf. medius (blue), C. major (red), C. crossleyi (purple), and C. sibreei (yellow)

Fig. 3

F_d test for introgression plotted across the genome. F_d statistic computed in 40-kb windows with a 10-kb sliding window, with the full phylogeny and associated test indicated on the right. Scaffolds are arranged from largest to smallest, alternating in colour. ‘Significant regions' are shown in red. Hibernation-related genes are labelled. a Results from a test of introgression between Cheirogaleus sp. cf. medius and C. major (C. med/C. maj). b Results from a test of introgression between C. sp. cf. medius and C. sibreei (C. med/C. sib). c Isolated scaffolds of interest

Fig. 4

Ancient introgression between C. sp. cf. medius, C. major, C. crossleyi, and C. sibreei. a Overall relationships within the clade and the approximate mean timing of introgression. Thicker dashed line indicates a larger proportion of admixed regions. b The age of introgressed regions between species pairs

Fig. 5

Estimated population sizes and levels of diversity. a Effective population size (N_e; y-axis) estimated for each species over the last 2 million years (x-axis). The bold line represents the MSMC2 estimate for the original data, and the surrounding lines in the corresponding species colours represent 50 bootstrap replicates. Present time is at the origin and time into the past along the x-axis. b Heterozygosity calculated in 100-kb windows across the genome