Mitochondrial Genomes Assembled from Non-Invasive eDNA Metagenomic Scat Samples in Critically Endangered Mammals

Abstract

The abundance of many large-bodied vertebrates, both in marine and terrestrial environments, has declined substantially due to global and regional climate stressors that define the Anthropocene. The development of genetic tools that can serve to monitor population's health non-intrusively and inform strategies for the recovery of these species is crucial. In this study, we formally evaluate whether whole mitochondrial genomes can be assembled from environmental DNA (eDNA) metagenomics scat samples. Mitogenomes of four different large vertebrates, the panda bear (Ailuropoda melanoleuca), the moon bear (Ursus thibetanus), the Java pangolin (Manis javanica), and the the North Atlantic right whale (Eubalaena glacialis) were assembled and circularized using the pipeline GetOrganelle with a coverage ranging from 12x to 480x in 14 out of 18 different eDNA samples. Partial mitochondrial genomes were retrieved from three other eDNA samples. The complete mitochondrial genomes of the studied species were AT-rich and comprised 13 protein coding genes, 22 transfer RNA genes, two ribosomal RNA genes, and a putative D-loop/control region. Synteny observed in all assembled mitogenomes was identical to that reported for specimens of the same and other closely related species. This study demonstrates that it is possible to assemble accurate whole mitochondrial chromosomes from eDNA samples (scats) using forthright bench and bioinformatics workflows. The retrieval of mitochondrial genomes from eDNA samples represents a tool to support bioprospecting, bio-monitoring, and other non-intrusive conservation strategies in species considered 'vulnerable', 'endangered', and/or 'critically endangered' by the IUCN Red List of Threatened Species.

Keywords: eDNA; iDNA; metagenomics; mitochondrial genome.

Figure 1

Circular DNA mitochondrial genome map of the Java pangolin (Manis javanica) assembled from eDNA scats. The annotated map depicts 13 protein-coding genes (PCGs), two ribosomal RNA genes (rrnS: 12 S ribosomal RNA and rrnL: 16 S ribosomal RNA), 22 transfer RNA (tRNA) genes, and the putative control region (not annotated). Mitochondrial genome structure was most similar among the different species analyzed. Photo credit: Firman/INaturalist, CC BY. https://www.inaturalist.org/guide_taxa/178736 (accessed on 28 August 2022).

Figure 2

Heatmap of hierarchically clustered data showing nucleotide use in mitochondrial genomes of the panda bear (Ailuropoda melanoleuca; n = 6 specimens), the moon bear (Ursus thibetanus; n = 2), the Java pangolin (Manis javanica; n = 4), and the the North Atlantic right whale (Eubalaena glacialis; n = 2). Photo credits: Pangolin, Firman/INaturalist, CC BY. https://www.inaturalist.org/guide_taxa/178736 (accessed on 28 August 2022). Moon bear, Guérin Nicolas/CC BY 3.0. https://en.wikipedia.org/wiki/File:Ursus_thibetanus_3_(Wroclaw_zoo).JPG. (accessed on 28 August 2022) Panda bear, Sheila Lau/CC BY 3.0. https://commons.wikimedia.org/wiki/File:Panda_Cub_from_Wolong,_Sichuan,_China.JPG. (accessed on 28 August 2022) North Atlantic right whale, NOAA/CC BY 3.0. https://upload.wikimedia.org/wikipedia/commons/6/6a/Eubalaena_glacialis_with_calf.jpg (accessed on 28 August 2022).

Figure 3

Heatmap of hierarchically clustered data showing relative synonymous codon usage in mitochondrial genomes of the panda bear (Ailuropoda melanoleuca; n = 6 specimens), the moon bear (Ursus thibetanus; n = 2), the Java pangolin (Manis javanica; n = 4), and the the North Atlantic right whale (Eubalaena glacialis; n = 2). Photo credits: Pangolin, Firman/INaturalist, CC BY. https://www.inaturalist.org/guide_taxa/178736 (accessed on 28 August 2022). Moon bear, Guérin Nicolas/CC BY 3.0. https://en.wikipedia.org/wiki/File:Ursus_thibetanus_3_(Wroclaw_zoo).JPG (accessed on 28 August 2022). panda bear, Sheila Lau /CCBY3.0.https://commons.wikimedia.org/wiki/File:panda_Cub_from_Wolong,_Sichuan,_China.JPG (accessed on 28 August 2022). North Atlantic Right Whale, NOAA/CC BY 3.0. https://upload.wikimedia.org/wikipedia/commons/6/6a/Eubalaena_glacialis_with_calf.jpg (accessed on 28 August 2022).

Figure 4

Secondary structure of tRNAs in the mitochondrial genome of the panda bear (Ailuropoda melanoleuca). Out of 22 tRNA genes, 21 exhibit a ‘cloverleaf’ secondary structure. tRNA-Ser1 invariably lacked the entire dihydroxyuridine (DHU) arm (stem + loop). Secondary structures visualized in the Forna web server. The secondary structure of tRNA genes was similar in U. thibetanus and E. glacialis to that found in A. melanoleuca.

Figure 5

Secondary structure of tRNAs in the mitochondrial genome of the Java pangolin (Manis javanica). Out of 22 tRNA genes, 19 exhibit a ‘cloverleaf’ secondary structure. tRNA-Ser1 invariably lacked the entire dihydroxyuridine (DHU) arm (stem + loop), while tRNA-C and tRNA-K lacked the dihydroxyuridine (DHU) loop but not the stem. Secondary structures visualized in the Forna web server.

Figure 6

Visual representation of the D-loop/control region (CR) in the mitochondrial genome of the moon bear (Ursus thibetanus). The CR is divided into the extended terminal association sequence (ETAS), central, and conserved sequence block (CSB) domains. Locations of the ETAS 1 and ETAS 2, CSB1, CSB2, CSB3 blocks, as well as the large highly conserved regions within the central domain are shown. The long repetitive motif is underlined, and its secondary structure is depicted immediately below the visual representation of the CR. The CR of Ailuropoda melanoleuca, Manis javanica, and Eubalaena glacialis were similar. Nonetheless, no tandem repeat located after the CSB1 motif was observed in E. glacialis. Nucleotides are highlighted with different colors to match features in the graphical representation of the CR.