Unraveling the genomes of ancient Iberian Canis

Unraveling the genomes of ancient Iberian Canis Ludmilla Blaschikoff | Octávio Serra | João Luís Cardoso | Carlos Fernández-Rodríguez | Ana Catarina Sousa | Marta Moreno-Garcia | Silvia Guimarães | 2 3 9 10 10,11 Fernanda Simões | Cleia Detry | Anders Götherström | Catarina Ginja | Ana Elisabete Pires 1 2 3,4,5 6 3 7 8 Departamento de Biologia, Universidade de Aveiro, Portugal, 2Unidade de Biotecnologia e Recursos Genéticos, Instituto Nacional de Investigação Agrária e Veterinária, I.P., Portugal, 3UNIARQ - Centro de Arqueologia da Universidade de Lisboa. Faculdade de Letras de Lisboa. Universidade de Lisboa, Portugal, 4ICArEHB - Interdisciplinary Center forArchaeology and the Evolution of Human Behavior; FCHS; Universidade do Algarve; Campus de Gambelas, Portugal, 5Departamento de Ciências Sociais e de Gestão, Universidade Aberta, Portugal, 6Departamento de Historia. Facultad de Filosofía y Letras. Universidad de León, España, 7Instituto de História, GI Paleoeconomía y subsistencia de las sociedades preindustriales, Instituto de Historia, CSIC, Madrid, Spain, 8Consejo Superior de Investigaciones Científicas (CISC), Institució Milà i Fontanals, Archaeology of Social Dynamics, Barcelona, Spain, 9Archaeological Research Laboratory, Stockholm University, Stockholm, Sweden, 10CIBIO-InBIO, Centro de Investigação em Biodiversidade e Recursos Genéticos, Universidade do Porto, Portugal, 11Faculdade de Medicina Veterinária, Universidade Lusófona de Humanidades e Tecnologias, Portugal. 1 INTRODUCTION MATERIALS Ÿ Ancient DNA is in general of low quantity and highly degraded [8] and its reconstruction and study is Ÿ Ÿ Ÿ Ÿ B A very demanding. Bioinformatic tools have revealed new insights into genomics of ancient specimens [15]. Domestic dogs exist in the Iberian Peninsula at least since ca. 16,000 BP [17]. Genetic studies of Iberian extant dogs shown four mtDNA Haplogroups (Hg): A, B, C and D haplogroup A and B are dominant, whereas clade D is the least represented [11,12]. A recent study [13] focusing on the genetic composition of 6 Mesolithic Iberian dogs reported a high frequency (85%) of dog HgA in the pre-Neolithic period, contrasting with a previous study of other parts of Europe [3] that indicates that HgA is rare or absent in European Canis older than 3,000 years (cal BP). C Fig. 2. A. sample LYEP51; B. sample LYEP11; C. sample LYEP9. Note: no picture is available for sample LYEP53, nor LYEP27 (wolf). Photos by Carlos Fernandez-Rodrigues (remains from Spain) and José Paulo Ruas (remains from Portugal). Fig. 1. Location of the Iberian Chalcolithic archaeological sites that provided the samples. AIMS Table 1. Samples information analysed in this study. Generate whole-genome data for 5 Chalcolithic Iberian Canis (Table 1) - dogs (n=4) and wolf (n=1); Ÿ Assign mtDNA sequences to the major dog and wolf Hgs; Ÿ Describe the genetic basis of some of their phenotypic traits; Ÿ Identify their sex; Ÿ Provide data to understand the origin and diversity of the Chalcolithic dogs. Ÿ METHODS Bioinformatics XV Encontro Nacional de Biologia Evolutiva - Matosinhos, Portugal Graphical report (Fastqc) raw reads (.fastq) Trimming of adapters and low-quality bases (cutadapt) mapping mapdamage unmapped reads (.sam) Filter unmaped reads (samtools) alignment to Camfam 3.1 (BWA) mapped reads mapped reads (.sam) (.sam) variant calling rescaled reads (.bam) GATK Quantifying pos-mortem damage/rescaling of quality scores (mapdamage) raw genotype calls (.vcf) locally realigned and reassembled reads (.bam) trimmed reads (.fastq) Merge reads (AdapterRemoval) merged reads (.collapsed) Sex determination mapped reads (.sam) alignment to composite genome (BWA) remove duplicates (picartools) reads w/o duplicates (.bam) reads filtered (.bam) filter by flag and quality (samtools) only variants bases (.vcf) GATK filtered and recalibrated variants (.vcf) GATK SNP effects Read depth based method - as in [3] variants effects (.vcf) ra o of reads/Mbp of autosomes and sex chromosomes 1:1 1:1/2 X_ x_ X_ y_ RESULTS AND DISCUSSION Ÿ The mitogenomes recovered are the only available worldwide for this chronology. A small fragment of the Control Region (CR) was used (Fig. 4) since there are not other contemporaneous mitogenomes available for comparison. Ÿ Chalcolithic dogs carried mtDNA variants that segregate within dog mtDNA Hg A and C (Fig. 4) . Ÿ The Chalcolithic wolf sequence segregates together with extant wolf sequence within the wolf mtDNA Hg H1. Although few ancient samples (wolf=1; dog=18) were analysed we suspect that, by the Chalcolithic time period, Iberian dogs and wolves were already structured populations based on their mtDNA haplotypes, as in extant populations. Ÿ The two Paleolithic Iberian wolves (*) segregate within extant Iberian dogs Hg A and C together with Mesolithic dogs. The latter kept the genetic signature of ancient Iberian wolves, transmiting it up to present-days dogs. genes annotation (snpEff) only passed variants (.vcf) consensus sequence consensus sequence w/ Ns and SNPs (.fa) Aplying VCF variants (bcftools) consensus sequence w/ Ns (.fa) reporting and masking genome coverage (bedtools) Fig. 3. Schematic representation of the pipeline. preprocessing Ÿ Due to the endogenous DNA poor preservation (Table 1), variants within specific nuclear genes related to coat color, coat texture or body size could not be recovered (very low coverage). A comparison between the nuclear genome - recovered from NGS analysis - and the reference dog genome revealed SNPs of possible high impact, which are being analysed. Ÿ Regarding sex determination, comparing the proportion of reads/Mbp (Fig. 5) for each chr, we observed that: 1) for LYEP9, LYEP11, LYEP53and LYEP27 only half of the reads/Mbp aligned with the X chr, in contrast to the proportion observed for the autosomes. Fewer reads aligned against Y chr because the reference used here is only a part of the dog Y chr (its real size is not know, but is expected to be ~20Mbp long); 2) LYEP51 has the same proportion of reads/Mbp aligning along all the autosomal and X chr. LYEP9 LYEP9 LYEP51 LYEP51 LYEP11 LYEP11 LYEP53 LYEP27 LYEP27 Fig. 5. Histograms representing the proportions of reads mapping each chromosome and the subsequent sex determination.‘Y (Blast)’ bar represents specific reads that matched only to dog MSY. CONCLUSIONS Ÿ Ÿ Fig 4. Median-Joining network (based on a partial fragment of 66 bp of the CR) depicting the relationship between Chalcolithic data and reference data sequences: 61 extant dogs and 23 extant wolves [11, 14, 4, 5, 2]; 25 ancient dogs and 3 ancient wolves [13] - Iberian samples only. Due to a low coverage of the region of interest, the sample LYEP27 (wolf) was merged with data obtained by a 2nd generation NGS method – 454 from a previous study [13].* These haplotypes belong to Paleolithic Iberian wolves [13] that segregate within wolf Hg 2 (wolf Hgs as defifined in [9] [data not shown]). REFERENCES [1] Arana, M.C. & Rodríguez, C.F. (2013). 277–282. [2] Bjornerfeldt, S. et al (2006). Genome Res., 16, 990–994. [3] Frantz, L.A.F. et al (2016). Science, 352, 1228–1231. [4] Koblmüller, S. et al. (2016). J. Biogeogr., 43, 1728–1738. [5] Koepfli, K.-P. et al. (2015). Curr. Biol., 25, 2158–2165. [6] Leigh, J.W. & Bryant, D. (2015). Methods Ecol. Evol., 6, 1110–1116. [7] Librado, P. & Rozas, J. (2009). Bioinformatics, 25, 14511452. [8] Lindahl, T. (1993). Nat. Publ. Gr., 362, 709–715. [19] Pilot, M. et al (2010). BMC Evol. Biol., 10, 104 View publication stats Ÿ Ÿ Ÿ We successfully applied NGS methods to recover ancient DNA - mitogenomes from Iberian dogs and wolves remains. Chalcolithic Iberian dogs harboured haplotypes segregating within haplogroups A and C Their European counterparts exibited more diversity (Hg A, B, C and D were presented). The distinct genetic composition of Iberian dogs is under investigation. The coverage of nuclear genome needs to be improved in order to study some genes related with phenotype. NGS Illumina applied on ancient DNA has potential to provide data for further investigations concerning domestication and evolutionary trajectories of dogs in Iberian. ACKNOWLEDGMENT [10] Pires, F., Cardoso, J.L. & Petrucci-Fonseca, F. (2001). Câmara Municipal de Oeiras, 2, 183-247.. [11] Pires, A.E. (2006). J. Hered., 97, 318–330. [12] Pires, A.E. et al. (2017). Quat. Int., 471, 132–146. [13] Pires, A.E. et al. (2019). J. Archaeol. Sci., 105, 116–129. [14] Randi, E. et al. (2000). Conserv. Biol., 14, 464–473 [15] Shendure, J. & Ji, H. (2008). Nat. Biotechnol., 26, 1135–1145. [16] Sousa, A.C. (2010). Universidade de Lisboa, Faculdade de Letras. [17] Vigne, J.-D. (2005). MUNIBE, 57, 279–287. This study is being developed within the Woof project - PTDC/HAR-ARQ/29545/2017 (CIBIO-InBIO), supported by portuguese funds by FCT/MCTES and co-supported by Fundo Europeu de Desenvolvimento Regional (FEDER) throughout COMPETE - POCI – Programa Operacional Competividade e Internacionalização (POCI-01-0145FEDER-029545), as well as by the post-doctoral grant SFRH/BPD/112653/2015 (AEPires), SFRH/BPD/108236/2015 (CDetry) and IF/00866/2014 (CGinja). CONTACTS Ludmilla Blaschikoff: lud_0911@hotmail.com Ana Elisabete Pires: aepires@cibio.up.pt