Genomic skimming and nanopore sequencing uncover cryptic hybridization in one of world’s most threatened primates

Abstract

The Brazilian buffy-tufted-ear marmoset (Callithrix aurita), one of the world’s most endangered primates, is threatened by anthropogenic hybridization with exotic, invasive marmoset species. As there are few genetic data available for C. aurita, we developed a PCR-free protocol with minimal technical requirements to rapidly generate genomic data with genomic skimming and portable nanopore sequencing. With this direct DNA sequencing approach, we successfully determined the complete mitogenome of a marmoset that we initially identified as C. aurita. The obtained nanopore-assembled sequence was highly concordant with a Sanger sequenced version of the same mitogenome. Phylogenetic analyses unexpectedly revealed that our specimen was a cryptic hybrid, with a C. aurita phenotype and C. penicillata mitogenome lineage. We also used publicly available mitogenome data to determine diversity estimates for C. aurita and three other marmoset species. Mitogenomics holds great potential to address deficiencies in genomic data for endangered, non-model species such as C. aurita. However, we discuss why mitogenomic approaches should be used in conjunction with other data for marmoset species identification. Finally, we discuss the utility and implications of our results and genomic skimming/nanopore approach for conservation and evolutionary studies of C. aurita and other marmosets.

Utilisation of Oxford Nanopore sequencing to generate six complete gastropod mitochondrial genomes as part of a biodiversity curriculum

“…In addition to enriching for mitochondrial DNA experimentally 15 , 54 , the use of adaptive sampling recently developed in ONT allows preselected sequences to be enriched during the sequencing process 55 – 57 . This approach has proven successful in obtaining full mitogenomes of endangered animals by enriching host genomic DNA from fecal samples 56 . Bait sequences can be up to 30% sequence identity divergent, suggesting the feasibility of this approach for sequencing an unknown species using references from distant relatives 55 .…”

Comparison of Mitogenomic and Mitochondrial Markers for the Phylogenetic and Evolutionary Study of Marmosets, with special focus on captive Callithrix jacchus

“…Based on several published mtDNA phylogenies [4, 6, 9], as well as those presented here, one plausible biogeographic hypothesis is that the C. jacchus clade originated in the Caatinga. Although C. jacchus occurs naturally in the Caatinga, Cerrado, and northeastern Brazilian Atlantic Forest biomes (Figure 1), mitochondrial phylogenies show that the species diverged from the C. penicillata Caatinga clade.…”

“…Mitochondrial DNA (mtDNA) has been a valuable and informative genetic marker in resolving Callithrix phylogeny, divergence, biogeography, and hybrid identification [4,6,[8][9][10]. Patterns of interspecific divergence analyzed using the approximately 1200 base pair (bp) rapidly-evolving mtDNA control region, which is much smaller than the approximately 16,500 bp Callithrix mitogenome, are largely concordant with results from full Callithrix mitogenome analyses [4,6,[8][9][10].…”

“…However, there is phenotypic evidence of possible Callithrix jacchus x C. penicillata hybrids present among marmosets kept in US biomedical facilities (personal observation, Malukiewicz). Additionally, previous mtDNA genetic studies show that cryptic hybridization in the wild does seem to occur regularly within the Callithrix genus [4–6, 9].…”

Affordable de novo generation of fish mitogenomes using amplification-free enrichment of mitochondrial DNA and deep sequencing of long fragments

“…To date, the average depth of reads for the mitochondrial genome using PCR-free genome skimming approach with long reads is low [e.g. 9x for the Brazilian buffy-tufted-ear marmoset Callithrix aurita , 78x for fresh liver of a rodent of the genus Melanomys (Franco-Sierra & Diaz-Nieto, 2020; Malukiewicz et al, 2021)].…”