NCM 2021: Genomic skimming on the MinION uncovers cryptic hybridisation in the buffy-tufted marmoset, one of world’s most threatened primates

Joanna Malukiewicz (German Primate Center, Germany) began her plenary talk by describing the focus of her research: the buffy-tufted marmoset (Callithrix aurita). C. aurita is a small primate, weighing ~400 g, endemic to southeastern Brazil, where it is restricted to montane regions of the tropical Atlantic Forest. With only 10,000–11,000 adults estimated to remain in the wild, C. aurita is listed as endangered on the International Union for Conservation of Nature (IUCN) Red List. The species gives birth to fraternal twins around every six months and subsist partially on a diet of tree gums. Joanna showed a map of Brazil illustrating the distribution of each of the six species within the Callithrix genus, demonstrating how each has a geographically separate domain; C. aurita is the furthest south of these and is found across three states.

Joanna explained that there are multiple ecological and anthropogenic threats to C. aurita. Their natural habitat is disappearing, due to factors such as farming and urban development. The species faces ecological competition from exotic marmoset taxa also present within its range; interspecific hybridisation with these species can also occur. These factors, together with infectious diseases such as Yellow Fever, result in decreasing opportunities for intraspecific mating. Joanna described the phenomenon of ‘anthropogenic hybridisation’: interspecific hybridisation within the Callithrix genus is largely driven by illegal trafficking of the common marmoset (C. jacchus) and black-tufted marmoset (C. penicillata). These are trafficked to be sold as pets; when released into the wild, their relatively young evolutionary age means that the species are able to interbreed. Most anthropogenic hybridisation is concentrated in southeastern Brazil. Joanna noted that the offspring resulting from interspecific hybridisation tend to have a ‘koala bear’ appearance.

Joanna described how C. aurita is a relatively little-studied species, for which limited genetic information is available and no reference genome. Mitogenomics, she explained, has therefore become an important starting point towards understanding Callithrix phylogeny and genetic diversity. She and her team set up a goal to ‘develop a rapid, cost-effective protocol to quickly obtain genomic and genetic data for Callithrix aurita’, in order to provide data critical to conservation of the species. Whilst a conservation programme is in place, this data would provide important information for species identification, breeding decisions, and population management. To this end, they sampled DNA from an individual with a C. aurita phenotype and assessed whether they could use nanopore sequencing on a MinION device to reliably reconstruct its mitogenome for use in phylogenetic and genetic diversity analyses.

After preparing samples for sequencing via the Ligation Sequencing Kit (SQK-LSK108), Joanna and her team performed low-coverage sequencing of the gDNA sample across two MinION Flow Cells, then computationally ‘skimmed’ the data for higher-copy mtDNA sequences. This enabled the construction of a full mitogenome from a ~9x average depth of genome coverage. Joanna displayed a comparison of the nanopore-generated mitogenome sequence with that generated via Sanger sequencing, which showed ‘very high concordance’. A consensus of these two genomes was taken forward for downstream analysis. They then constructed a robust maximum-likelihood phylogeny. The team were expecting, based on the individual’s phenotype, for the sample to group within the C. aurita clade; however, its haplotype in fact grouped within a C. penicillata clade. They found two further instances in which the mitogenome sequences of individuals with C. aurita phenotypes grouped outside of their expected clade — uncovering ‘three unexpected cases of cryptic Callithrix hybridisation’. These results, Joanna highlighted, represented the ‘first published accounts of introgression of genetic material from exotic C. penicillata marmosets into C. aurita’. Joanna and her colleagues also conducted population mitogenomic analysis across four species, using only samples for which phenotype and genotype matched, finding C. aurita to fall in the middle regarding genetic diversity.

Discussing the nanopore methodology they used, Joanna highlighted how ‘this approach really holds great potential for enhancing mitogenomics and genetic diversity studies of data-deficient and/or non-model organisms’. She noted that sample prep took less than a day, and they sequenced their samples for 48 hours, producing data showing high concordance with Sanger sequencing. Joanna highlighted that the data was generated a couple of years ago and, whilst it required a relatively high amount of input material, other protocols are now available that require far less. They also applied computational polishing, using the tool Flye for both de novo assembly and the polishing step. Finally, Joanna described how technological advances in nanopore sequencing are also vastly improving raw nanopore sequence quality.

Joanna described how, with these results and those from a companion paper published earlier in 2021, they have seen very widespread anthropogenic hybridisation of Callithrix species in southeastern Brazil — including C. aurita and some other marmoset species native to the area. She stressed that the fact that the individual selected for sequencing was a cryptic hybrid was ‘unexpected and a surprising result’, and represented the first known instance of one-way genetic introgression from C. penicillata into C. aurita.

Joanna explained that most currently available genomic information for C. aurita is comprised of short mtDNA markers. Due to the relatively young evolutionary age of the species, these provide too low a resolution for phylogenetic analysis. The recent availability of the C. aurita mitogenome therefore ‘opens up a lot of new doors’. She further stressed that larger sampling efforts of Callithrix across its native range is needed to understand the genetic variation present in wild populations and captive populations in the conservation program. Joanna described how the application of their method, combining long nanopore sequencing reads generated on the portable MinION and genome skimming, could ‘address several of these knowledge gaps for C. aurita’. Finally, she encouraged other researchers to combine this molecular data with phenotypic data, as phenotype alone is insufficient for species identification, stressing that integration of this data will lead to a better understanding of hybridisation processes.