Identification of non-model mammal species using the MinION DNA sequencer from Oxford Nanopore.

Background: The Neotropics harbors the largest species richness of the planet; however, even in well-studied groups, there are potentially hundreds of species that lack a formal description, and likewise, many already described taxa are difficult to identify using morphology. Specifically in small mammals, complex morphological diagnoses have been facilitated by the use of molecular data, particularly from mitochondrial sequences, to obtain accurate species identifications. Obtaining mitochondrial markers implies the use of PCR and specific primers, which are largely absent for non-model organisms. Oxford Nanopore Technologies (ONT) is a new alternative for sequencing the entire mitochondrial genome without the need for specific primers. Only a limited number of studies have employed exclusively ONT long-reads to assemble mitochondrial genomes, and few studies have yet evaluated the usefulness of such reads in multiple non-model organisms. Methods: We implemented fieldwork to collect small mammals, including rodents, bats, and marsupials, in five localities in the northern extreme of the Cordillera Central of Colombia. DNA samples were sequenced using the MinION device and Flongle flow cells. Shotgun-sequenced data was used to reconstruct the mitochondrial genome of all the samples. In parallel, using a customized computational pipeline, species-level identifications were obtained based on sequencing raw reads (Whole Genome Sequencing). ONT-based identifications were corroborated using traditional morphological characters and phylogenetic analyses. Results: A total of 24 individuals from 18 species were collected, morphologically identified, and deposited in the biological collection of Universidad EAFIT. Our different computational pipelines were able to reconstruct mitochondrial genomes from exclusively ONT reads. We obtained three new mitochondrial genomes and eight new molecular mitochondrial sequences for six species. Our species identification pipeline was able to obtain accurate species identifications for up to 75% of the individuals in as little as 5 s. Finally, our phylogenetic analyses corroborated the identifications from our automated species identification pipeline and revealed important contributions to the knowledge of the diversity of Neotropical small mammals. Discussion: This study was able to evaluate different pipelines to reconstruct mitochondrial genomes from non-model organisms, using exclusively ONT reads, benchmarking these protocols on a multi-species dataset. The proposed methodology can be applied by non-expert taxonomists and has the potential to be implemented in real-time, without the need to euthanize the organisms and under field conditions. Therefore, it stands as a relevant tool to help increase the available data for non-model organisms, and the rate at which researchers can characterize life specially in highly biodiverse places as the Neotropics.

DNA barcodes of birds from northern Colombia.

DNA barcode datasets are a useful tool for conservation and aid in taxonomic identification, particularly in megadiverse tropical countries seeking to document and describe its biota, which is dropping at an alarming rate during recent decades. Here we report the barcodes for several low elevation bird species from northern Colombia with the goal to provide tools for species identification in this region of South America. We blood-sampled birds in a lowland tropical forest with various degrees of intervention using standard 3 × 12 m mist-nets. We extracted DNA and sequenced the COI barcode gene using standard primers and laboratory methods. We obtained 26 COI sequences from 18 species, 10 families and three orders and found that barcodes largely matched (but not always) phenotypic identification (> 90%) and they also facilitated the identification of several challenging passerine species. Despite our reduced sampling, our study represents the first attempt to document COI barcodes for birds (from blood samples) in this part of Colombia, which fills a considerable gap of sampling in this part of South America.

Toxoplasma gondii infection in Colombia with a review of hosts and their ecogeographic distribution.

Toxoplasma gondii is one of the most prevalent zoonotic protozoan parasites among warm-blooded animal populations (humans included) around the world, causing multiple clinic manifestations including death in the most severe cases of infection. Due to the versatile life cycle of T. gondii and its diversity of potential hosts, there is a common perception that natural areas and wildlife are highly prevalent reservoirs for the parasite; however, information and reports of the parasite on wildlife populations in Colombia are scarce. Using PRC-based detection analyses of the B1 gene, we evaluated the presence of T. gondii in 49 native small mammal species (10% of the mammal species of Colombia) from 4 different undisturbed natural habitats. Additionally, to understand the ecogeographical distribution of the parasite in Colombia, we developed a literature search of infection reports including information on the host species, density of records and occurrence patterns (using landcover and ecoregions) in natural, rural and urban areas. Our literature review showed a total of 8,103 reports of T. gondii for Colombia of which 86% were related to humans, and 14% to non-human mammals and other categories, with just a single report associated to wildlife; additionally, 82% of all reports were associated to urban areas whereas only 18% to rural sites. Based on the negative results for the presence of T. gondii in our PCR-based analyses and our literature search, we suggest that T. gondii has a synanthropic distribution in Colombia occurring in ecoregions as variable as the xeric scrubs in the northern lowlands and humid montane Andean forests, also we show a lack of information on the parasite relationship with wildlife, a concerning fact given that zoonoses are the leading mechanism for the emergence of infectious diseases.

Rapid mitochondrial genome sequencing based on Oxford Nanopore Sequencing and a proxy for vertebrate species identification.

Molecular information is crucial for species identification when facing challenging morphology-based specimen identifications. The use of DNA barcodes partially solves this problem, but in some cases when PCR is not an option (i.e., primers are not available, problems in reaction standardization), amplification-free approaches could be an optimal alternative. Recent advances in DNA sequencing, like the MinION device from Oxford Nanopore Technologies (ONT), allow to obtain genomic data with low laboratory and technical requirements, and at a relatively low cost. In this study, we explore ONT sequencing for molecular species identification from a total DNA sample obtained from a neotropical rodent and we also test the technology for complete mitochondrial genome reconstruction via genome skimming. We were able to obtain "de novo" the complete mitogenome of a specimen from the genus Melanomys (Cricetidae: Sigmodontinae) with average depth coverage of 78X using ONT-only data and by combining multiple assembly routines. Our pipeline for an automated species identification was able to identify the sample using unassembled sequence data (raw) in a reasonable computing time, which was substantially reduced when a priori information related to the organism identity was known. Our findings suggest ONT sequencing as a suitable candidate to solve species identification problems in metazoan nonmodel organisms and generate complete mtDNA datasets.

Mercury levels in birds and small rodents from Las Orquideas National Natural Park, Colombia.

Mercury (Hg) is a heavy metal known as one of the most toxic elements on the planet. The importance of Hg on living organisms resides on its biomagnification ability. Artisanal gold extraction activities release substantial amounts of this metal, polluting the ecosystems. To assess the impact of gold mining in Las Orquideas National Natural Park (Colombia), total Hg (T-Hg) levels were evaluated from 37 bird and 8 small rodent species collected at two sites within the boundaries of the Natural Park (Abriaqui and Frontino municipalities) that have experienced some gold-extraction history. The mean concentration of T-Hg in bird feathers from both sites was 0.84 ± 0.05 µg/g fw. Differences between species were found according to diet. Total Hg levels were greater on insectivorous (1.00 ± 0.08 µg/g fw), followed by nectarivorous (0.73 ± 0.07 µg/g fw) and frugivorus (0.57 ± 0.09 µg/g fw) species. These Hg levels were greater than those found in feathers from a control sample belonging to the species Penelope perspicax (0.53 ± 0.03 µg/g fw), a frugivorous species living at the Otun Quimbaya Fauna and Flora Sanctuary, a forest without known gold mining. Mercury concentrations in the livers of small rodents were greater in specimens from Frontino (0.15 ± 0.01 µg/g fw) than those from Abriaqui (0.11 ± 0.01 µg/g fw), but levels were not different between species. These results indicate that Hg in birds depends mainly on their diet, but geographical location may affect Hg concentration in rodents. Moreover, Hg sources in natural parks of Colombia may not rely solely on gold mining, atmospheric deposition, among others factors, could be influencing its accumulation in biota.