Faecal DNA to the rescue: Shotgun sequencing of non-invasive samples reveals two subspecies of Southeast Asian primates to be Critically Endangered species

Abstract

16A significant number of Southeast Asian mammal species described in the 19 th and 20 th 17 century were subsequently synonymized and are now considered subspecies. Many are 18 affected by rapid habitat loss and there is thus an urgent need to re-assess the conservation 19 status based on species boundaries established with molecular data. However, such data are 20 lacking for many populations and subspecies. We document via a literature survey and 21 empirical study how shotgun sequencing of faecal DNA is a still underutilized but powerful 22 tool for accelerating such evaluations. We obtain 11 mitochondrial genomes for three 23 subspecies in the langur genus Presbytis through shotgun sequencing of faecal DNA (P. 24 femoralis femoralis, P. f. percura, P. siamensis cf. cana). The genomes support the 25 resurrection of all three subspecies to species based on multiple species delimitation 26 algorithms (PTP, ABGD, Objective Clustering) applied to a dataset covering 40 species and 27 43 subspecies of Asian colobines. For two of the newly recognized species (P. femoralis, P. 28 percura), the results lead to an immediate change in the IUCN status to Critically Endangered 29 due to small population estimates and fragmented habitat. We conclude that faecal DNA 30 should be more widely used for clarifying species boundaries in endangered mammals. 32 Human impacts on the environment have rapidly accelerated species extinction via habitat 33 degradation and climate change. Recent report by Intergovernmental Science-Policy Platform 34 on Biodiversity and Ecosystem Services (IPBES) predicts that climate change has already 35 affected the distribution of nearly half (47%) of land-mammals 1 . Conservation efforts are 36 urgently needed but are hampered by the lack of data for a large number of mammal species, 37 subspecies, and populations which face imminent extinction 2,3,4 . A typical example is Asian 38 primates for which 70% of the species are threatened with extinction 5 . Effective conservation 39 programs are needed but they require a robust understanding of species numbers and 40 boundaries based on up-to-date taxonomic information 6,7 . Unfortunately, this information is 41 lacking for many rare, globally threatened, and elusive mammalian species. Many lack 42 molecular data and collecting these data is difficult because invasive sampling that would 43 yield fresh tissues is often not feasible. 44This leaves only three alternative sources of DNA. The first is museum specimens, but the 45 number of samples in museums tends to be small and many were collected in the 19 th or early 46 20 th century thus reflecting (historic) genetic diversity prior to extensive habitat loss. The 47 second is tissue samples obtained from specimens that died of "natural causes" such as road 48 accidents. The third source of genetic material is non-invasive samples such as hair and 49 faeces. Arguably, faecal samples are still an underappreciated source of information although 50 they could be collected in good numbers during ro...

Dietary habits of free-ranging banded langur (Presbytis femoralis) in a secondary-human modified forest in Johor, Malaysia

“…Nonetheless, there is a potential for building upon our findings with more detailed investigations, such as ones exploring more extensive ecological factors influencing dietary adaptations. This information is necessary to support the conservation efforts and management decisions of this species proposed by the immediate change in IUCN status to Critically Endangered by Ang et al (2020b).…”

“…They chose instead to differentiate P. f. femoralis in Malaysia with the previously listed junior synonym P. neglectus . A recent shotgun sequencing of faecal DNA from Asian colobines, however, concludes that those three subspecies of P. femoralis are indeed unique species and as such are elevated from their subspecific status (Ang et al 2020b). As part of this assessment, the conservation status of the three subspecies was also evaluated.…”

“…Johor and its borders (Pahang state), including Singapore, has been assessed as Critically Endangered (Ang et al 2020a) as a result. Indeed, it has been estimated that there are less than 250 mature individuals of P. femoralis in Malaysia and Singapore (Abdul-Latiff et al 2019a, Ang et al 2020b).…”

“…The banded langur, Presbytis femoralis , is a member of the subfamily Colobinae found on the Malay Peninsula and Sumatra and was originally classified as three subspecies (Ang et al 2020b, Nijman 2020, Roos et al 2014): Presbytis f. femoralis (listed as Vulnerable on the IUCN Red List), Presbytis f. percura (Data Deficient) and Presbytis f. robinsoni (Near Threatened). Abdul-Latiff et al (2019a) hypothesised using a molecular phylogeny approach analysing Asian colobine DNA sequences that these three subspecies classifications for P. femoralis are not suitable because each taxonomic group exhibited separation at the species level in the D- loop region of mitochondrial DNA.…”