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Black as reservoir of the

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Rat • Three pandemics Wikimedia Commons • The first pandemic (Justinian’s plague): 6th century Ancient and Modern Genomes Reveal • The second pandemic (the ): 14th century Population History of the Black Rat • The third pandemic: 19th century

• Modern outbreaks in Madagascar He Yu Department of Archaeogenetics Max Planck Institute for the Science of Human History

2019.1.13

The black rat ( rattus) Questions

• Ship rat, roof rat … • How did the black rat spread across Europe and the other parts of • One of the most successful commensal species the world?

• Is there any correlation between the black rat population history and outbreaks of the bubonic plague?

Wikimedia Commons Robins et al. 2008, MPE

The spread of black rat Questions

• How did the black rat spread across Europe and the other parts of the world? and

Roman time • Is there any correlation between the black rat population history and outbreaks of the bubonic plague? Europe and North Africa

• De novo assembly of Rattus rattus genome All around the world

Around 16-18th century • Genomic study of ancient black

Replaced by R. norvegicus in most temperate regions

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De novo assembly of Rattus rattus genome Chromosome rearrangements

• Wild-caught male individual from California, USA • Shotgun sequencing: illumina HiSeq4000, ~250Gbp • Dovetail HiRise scaffolding with Chicago and Hi-C data

Total Length 2137.81 Mb Contig number 170944 Contig N50 33.05 kb Scaffold number 5712 Scaffold N50 110.50 Mb Longest scaffold 218.46 Mb Percentage of gaps 0.78% GC content 42.34%

Assembly assessment The missing sex chromosomes

• Repeat masking • RepeatMasker (RepBase 20170127, species rattus) • TandomRepeatFinder Missing • 37.9% ChrX

• BUSCO • Based on Eukaryota odb9 gene set • Comparing with Rnor_6.0 (Rattus norvegicus reference genome)

Complete and Complete and Genome assembly single-copy duplicated Complete Fragmented Missing Total Rattus rattus unmask 264 10 274 10 19 303 mask 259 11 270 14 19 303 Rnor_6.0 unmask 259 18 277 10 16 303 mask 252 18 270 14 19 303

Pairwise alignment with Rnor_6.0 Retrieving sex chromosomes

22 largest scaffolds (>1Mb), 99.4% of the assembly Mapping shotgun reads to Rnor_6.0 reference genome

Selecting reads mapped to chrX/chrY chrX: ~6.5Gbp chrY: ~0.1Gb

De novo assembling with Meraculous chrX: 110Mb, N50=18.9kb chrY: 1.5Mb, N50=7.1kb

Scaffolding with Dovetail HiRise

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Genomic divergence with Rnor_6.0 Population dynamic of Rattus rattus

• Autosomes • 3.85% on average

• ChrX • Meraculous de novo scaffolds • 3.00% on average

Ancient Rat coordinates

AncientRat

All items

Protein-coding gene prediction Ancient black rats • Maker2 pipeline • EST evidence from R. norvegicus • 22 sites • Protein evidence from R. norvegicus and Mus musculus (Swiss-Prot) • SNAP trained by CEGMA default KOG • 1st to 17th century AD • Augustus trained by BUSCO eukaryota odb9 gene set • 105 samples

• OrthoFinder • Comparing with R. norvegicus protein-coding genes (Rnor_6.0, Ensembl release 94)

Total gene Gene with orthologs Percentage R. rattus 23444 15491 66.1% R. norvegicus 29107 19256 66.2%

Genetic relationship with other species DNA quality assessments

• R. rattus, R. tanezumi and R. norvegicus • Shotgun sequencing of 2-10M reads

• Gene flow between R. tanezumi and R. rattus from USA Endogenous Endogenous Site Country Time period N_Sample DNA >1% DNA >30% ed-Dur UAE 1st-4th century AD 1 0 0 Zembra Tunisia 4th-6th century AD 9 9 5 Tróia Portugal 5th century AD 3 3 0 Caricin Grad Serbia 6th century AD 31 30 14 Assos Turkey Byzantine time 2 2 1 Panga Ya saidi Kenya 7th-10th century AD 2 0 0 Chombo Kenya 8th century AD 2 0 0 Aqaba Jordan 13th-15th century AD 1 1 0 Karksi Castle Estonia 13th century AD 2 1 0 Santa maria, Lavezzi Italy 14th century AD 9 9 9 Songo Mnara Tansania 14th-15th century AD 1 0 0 Monte di Tuda France 15th century AD 6 2 1 Nurnberg Germany Middle Age 1 1 0 NJ tree, p-distance castle Sulzbach Germany Middle Age 5 5 5 Kilton Castle UK Post-Medieval 20 7 1 Fort Frederick Mauritius Late 17th century AD 2 2 0 Mbuyuni Kenya unknown 1 0 0 W X Y Z Dstat Z score Paithan India unknown 1 0 0 norvegicus tanezumi rattus_USA rattus_Britain -0.0618 -27.399 Kantharodai Sri Lanka unknown 1 0 0 Manthai Sri Lanka unknown 2 0 0 Shenhour Egpyt unknown 1 0 0 Kalba Oman Multi-period 2 0 0 Total 105 68 36

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Mitogenome analysis Acknowledgements

• 32 samples with high-quality bases > 70% • Johannes Krause, Kay Prüfer, Alexander Stößel, Rodrigo Barquera, Nicole Boivin, William Taylor (MPI SHH, Germany) • 11 haplotypes + 8 modern references • Greger Larson, Alexandra Jamieson, Laurent Frantz (University of • Two distinct lineages in 6th century Oxford, UK) Serbian samples • Ben Krause-Kyora (Kiel University, Germany) • Dovetail Genomics • Chris J. Conroy (University of California, Berkeley, USA) • Steve Donnellan (South Australian Museum, Australia) • Neil Gemmell (University of Otago, New Zealand) • David Orton (University of York, UK)

ML tree, JC model

Whole genome phylogeny

• 33 samples with mean coverage >0.01 • 5 modern genomes • Transversion only after masking

NJ tree, p-distance

Summary

• The first de novo genome assembly of Rattus rattus in high quality

• Ancient rat genomes from a large geographic range, showing population structure among rat populations

• To be continued… • De novo assembly of sex chromosomes • Deep sequencing of high-quality ancient samples • Modern rat genomes as reference panel

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