Genomics of the capybara, two emblematic Colombian species María José Gómez-Hughes¹, Santiago Herrera-Álvarez1,2, Andrew J. Crawford¹ ¹Department of Biological Sciences, Universidad de los Andes, Bogotá, 111711, Colombia. ²Department of Ecology and Evolution, University of Chicago, Chicago, IL 60637, USA. Abstract Capybaras, which are native to South America, are not only the largest rodents in the world, but they also have a number of other characteristics that make them unique. They are semi-aquatic, grazing mammals and live in large groups where females engage in communal breeding. Males communally defend the territory through scent-marking with a specialized gland called the morillo and with two anal glands. Here we present the first genome assembly and annotation for the lesser capybara, Hydrochoerus isthmius, as well as the first transcriptome assembly for the capybara, H. hydrochaeris, both of which are comparable in completeness with previously published rodent genomes, and compared them with the previously published genome assembly for the capybara. We found evidence of reduction on the effective population size of both species, as well as big regions of genomic rearrangement with the guinea pig. Our phylogenetic analysis is consistent with previous phylogenies reported for the suborder Hystrichomorpha, but species related there is evidence for the capybara being a paraphyletic species. We hope that this study contributes for conservation efforts on these species, as well as a better understanding of all the characteristics that make them unique. Resumen Los chigüiros, nativos a América del Sur, no solamente son los roedores más grandes del mundo sino también tienen otras características que los hacen únicos. Son especies de mamíferos semiacuaticas que pastean y viven en grandes grupos en los que las hembras crían comunitariamente a sus crías y los machos defienden sus territorios mediante marcajes con el morillo, una glándula especializada, y dos glándulas anales. Aquí presentamos el primer ensamblaje genómico del chigüiro menor, Hydrochoerus isthmius, y el primer transcriptoma del chigüiro, H. hydrochaeris, como también comparaciones con el genoma del chigüiro publicado anteriormente. Encontramos evidencia de reducciones poblacionales de ambas especies, como también rearreglos genómicos en comparación con el conejillo de indias. Nuestro análisis filogenético es consistente con análisis publicados previamente para el suborder Hystricomorpha, pero hay evidencia para la parafilia del chigüiro. Esperamos que este estudio contribuya a esfuerzos de conservación en estas especies, como también a un mejor entendimiento de esas características que los hacen únicos. Keywords: Hydrochoerus sp., chigüiro, populational genomics, conservation genomics, 10X genomics, genome assembly. Ethics Statement: Tissue samples of the lesser capybara and capybara were obtained under research and collecting permit No. 1177 issued to the Universidad de los Andes by the Autoridad Nacional de Licencias Ambientales (ANLA; National Authority of Environmental Permits). Anesthetic and euthanization protocols used were approved by the Universidad de los Andes’ Comité Institucional de Cuidado y Uso de Animales de Laboratorio (CICUAL; approval number C.FUA_14-023). Introduction The Order Rodentia is the most diverse group of mammals in the world in terms of species and ecological diversity as well as morphological variation (Samuels, 2009; Fabre et al., 2012). Rodents comprise almost 40% of all mammal species (Burgin et al., 2018) and inhabit almost all terrestrial biomes (Hafner & Hafner, 1988). There are currently five recognized suborders of rodents: Sciuromorpha (dormices, mountain beavers, marmots, squirrels and squirrel-like rodents), Castorimorpha (beavers, kangaroo rats and pocket gophers), Myomorpha (hamsters, jerboas, mice, rats and mouse-like rodents), Anomaluromorpha (scaly tailed squirrels and springhares), and Hystricomorpha (chinchillas, guinea pigs, gundis, porcupines and others), which are composed of 33 families (Wilson & Reeder, 2005). Among these groups, in the order Hystricomorpha and family Caviidae (Wilson & Reeder, 2005), are the capybaras (genus: Hydrochoerus). Capybaras are known for being the largest extant rodents (Figure 1A; Moreira et al., 2013), their semi-aquatic habits (Macdonald, 1981), and for being social animals with communal breeding and communal defense (Macdonald, 1981). Capybaras are also known for having distinctive feeding and scent marking behaviors. Feeding related, capybaras graze on both aquatic and terrestrial herbaceous vegetation that undergo multiple passes through their digestive tracts, either via regurgitation or cropography (Lord, 1994). Scent marking related, capybaras possess two types of scent marking glands - the morillo, a protuberance that males express in the top of their snouts which size can be predictive of dominance (Rosenfield et al., 2019), and two anal glands - and is the social interaction most seen in them (Emilio & Macdonald, 1994). There are currently two described species of capybaras: the capybara, H. hydrochaeris, and the lesser capybara, H. isthmius (Mones, 1991), inhabiting eastern Colombia, eastern Venezuela, the Guyanas, Ecuador, Peru, northeastern Argentina and Uruguay, and Panama, western Colombia and western Venezuela, respectively (Figure 1B; Reid, 2016; Delgado & Emmons, 2016). However, some dispute exists as to whether there are two or only one species of capybaras, with some still referring to the lesser capybara as a subspecies (see Correa & Jorgenson, 2009 and Carrascal, Linares & Chacón, 2011), but classified as its own species in the database of mammalian taxonomy (Wilson & Reeder, 2005) and by the International Union for Conservation of Nature and Natural Resources (IUCN; Delgado & Emmons, 2016), as well as to where in the Hystricomorpha phylogeny they are localized (see Upham & Patterson, 2015; Álvarez, Arévalo, & Verzi, 2017; Rowe & Honeycutt, 2002). Figure 1. (A) Relative size of the two species of capybara as compared to a 1.75 m tall human. The lesser capybara (Hydrochoerus isthmius) is shown in purple on the left while the capybara (Hydrochoerus hydrochaeris) is shown in blue on the right. (B) Geographic ranges of the capybara (blue) and the lesser capybara (purple) according to the IUCN (Reid, 2016; Delgado & Emmons, 2016). (C) A picture showing a male capybara with its morillo indicated by the red arrow. All images used were labeled for noncommercial use with modifications from Wiki Commons. Currently, the capybara is listed as a species of Least Concern by the IUCN (Reid, 2016), but some concerns have arisen over the years and substantial population declines have been noted in this species (Corriale & Herrera, 2014). The lesser capybara is listed as Data Deficient due to lack of baseline studies on the status of these populations (Delgado & Emmons, 2016). This species has been neglected in studies of conservation despite being harvested for meat, leather and fat (Pinheiro & Moreira, 2013) and despite the threats to its native habitat (Aldana-Domínguez, Vieira-Muñoz & Bejarano, 2013). In this paper we present the first genome assembly for the lesser capybara and the first transcriptome assembly for the capybara, as well as new analyses for the previously published capybara genome assembly by Herrera-Álvarez et al. (2018). We compare demographic changes over time of both species, compare synteny between the two capybara species and between each species and the guinea pig (Cavia porcellus), evaluate distinctiveness of the capybaras as independent monophyletic groups, assess the position of Hydrochoerus in the Hystricomorpha tree, and analyze differentially expressed genes among tissues from the capybara. Materials and Methods Genome assembly of the capybara Tissue from a wild-caught but captive-raised capybara (H. hydrochaeris), reportedly from Bolivia, was donated by the San Diego Zoo’s Frozen Zoo to the 200 Mammalian Genomes Project led by the Broad Institute, which then sequenced and assembled a draft genome using the sequencing and assembly method of DISCOVAR de novo (Weisenfeld et al., 2014). This assembly was then ‘upgraded’ using Chicago libraries provided by Dovetail Genomics (Putnam et al., 2016) and financed by Colciencias. Details on the final assembly can be found in Herrera-Álvarez et al. (2018). Genome assembly of the lesser capybara Tissue samples of the lesser capybara were collected from one juvenile H. isthmius from San Juan del Carare, Santander, Colombia, on 22 June, 2017, that was subsequently accessioned into the mammals collection of the Museo Historia Natural ANDES, Universidad de los Andes, Bogotá, Colombia (field number AJC 7100, voucher number ANDES-M 2300). This sample was sequenced using 10X Genomics linked reads technology in two lanes of Illumina HiSeq X10. The resulting reads were run through longranger v2.2.2 (10X Genomics) to estimate the genome size, heterozygosity, and to process the barcodes. These reads were assembled with Supernova v2.0.1 (Weisenfeld et al., 2017), an assembler created by 10X Genomics that uses a progressively larger contigs approach and its own trimming step to create phased scaffolds from the reads. We included the mkoutput pseudohap option to visualize only one haplotype on the resulting assembly. To enhance this assembly we used the following three scripts. 1) Tigmint v1.1.2 was used to produce an assembly that is both more contiguous and more correct by comparing the alignment of linked reads