Whole-genome phylogenetics, gene regulation and the origin of evolutionary novelty Feather photos: J. Trimble, MCZ “Beast Legends”: A six part adventure in science and myth Fijian shark god Griffin Kraken Wild man Dragon Terror bird Graphics by Invisible Pictures, Inc. Beast Legends – Griffin episode Evolutionary change: genes or gene regulation? Taste receptors in mammals X X Taste receptors on the tongue Birds inherited only the umami (meat) receptor from their dinosaur ancestors el receptor del X gusto dulce se perdió incluso en colibríes ! Hummingbirds can taste sugar due to changes in the gene other birds use to taste meat (or insects) Can taste sugar? Taste receptor T1R1 T1R3 hummingbird ✗ swift ✗ chicken Baldwin et al. 2014. Science 345: 929-933 Non-codingDark matter of the genome: a regulatory network? Karyotype of an Emu CNEEs: evolutionarily conserved non-coding enhancer regions CNEEs = conserved non-exonic elements View of a segment of human chromosome 10 using UCSC Genome Browser Janes et al. (2011) Genome Biol. Evol. 3:102–113 Noncoding enhancers: long-range control of gene expression Levine et al. 2014 Cell 157: 13-25 Phylogenetic hidden Markov model detects CNEEs using Phastcons* *Siepel et al. 2005. Genome Res. 15:1034-1050 A role for gene regulation in the origin of feathers Quanguo et al. (2010) Science Anchiornis Chen et al. (1998) Nature Sinosauroptyerx Archaeopteryx Feather photos: J. Trimble, MCZ Conserved non-exonic elements (CNEEs) act as enhancers for feather genes • 400 abstracts • 193 feather genes • 13,307 feather gene CNEEs (2.2% of total) Bird, amniote- and tetrapod-specific CNEEs near SHox Lowe et al. 2015 Mol. Biol. Evol. 32:23–28 High origination rates of feather CNEEs, but not feather genes, when feathers evolved feathers evolve 1.5 220 mya genes Relative CNEEs number of 1.0 origins 0.5Origin of most feather genes Feather gene pre-dates enhancers feathers evolved 0.0 at almost the same time as feathers Birds Turtles r MammalsReptiles Amphibians Chickens/ducks Crocodilians Bony vertebrates ancient time recent Lowe et al. 2015 Mol. Biol. Evol. 32:23–28 Bird-specific regulatory evolution: what makes a bird a bird? Bird-specific CNEEs associated with genes for limb and body size evolution Chromosome position (chicken) Lowe et al. 2015 Mol. Biol. Evol. 32:23–28 Bird-specific CNEEs associated with genes for limb and body size evolution Lowe et al. 2015 Mol. Biol. Evol. 32:23–28 Bird-specific CNEEs associated with genes for limb and body size evolution Lee et al. 2014. Science 345:562 Correlates of GC content in ~1000 mammalian coding regions Romiguier et al. 2010. Genome Res. 20: 1001-1009 Mechanistic hypotheses for high GC content in a lineage GC content Isochores/Chromo Rate of biased somal location and gene conversion Population size and length (BGC) and life history effects recombination Hughes & Hughes 1995 Nature Lynch & Connery 2003 Science Sessions & Larson 1987 Evolution Consequences of repair of double strand breaks induced by BGC or recombination Frequency of GC alleles among gametes from a heterozygous individual: (b=BGC bias; Ne = effective population size) Covariation of GC content and body mass in birds large small large small populations populations populations populations Nabholz et al./Avian Phylogenomics Consortium Effects of GC variation on phylogenomic analysis Including only genes with low-GC Including only genes with high-GC variation among lineages variation among lineages Avian Phylogenomics Consortium Passerines Parrots Owls Cormorants Penguins Rails Gulls, Auks Pigeons Grebes Ducks, Geese Ratites Jarvis et al. 2014, Science CNEEs and the convergent evolution of flightlessness in Palaeognathae Skeletal modifications for flightlessness Little-spotted kiwi sternum Emu and ostrich keelless sterna De Bakker et al. 2013. Nature 500, 445–448. Convergent losses of flight allow comparative genomics to identify genomic regions for flightlessness Extinct Extant Mitchell et al 2014 11 new palaeognath genomes Little bush moa Rowi Kiwi Great-spotted Kiwi Little Spotted Kiwi Southern Cassowary Emu Lesser Rhea Greater Rhea Elegant-crested Tinamou Thicket tinamou Chilean tinamou Image (all CC): David Cook; Quartl; Jim, the Photographer, Tim Sackton 42-species whole genome alignment for birds using ProgressiveCactus DMRT1 Relative rates of different noncoding markers Edwards et al. 2017. Syst. Biol. 66: 1028 Phylogenomic markers cover c. 3% of total genome length 12,676 CNEEs 5,016 Introns 3,158 Ultraconserved elements (UCEs) Cloutier et al. 2019. Syst. Biol. 10.1093/sysbio/syz019 Relationships of rheas unclear Haddrath & Baker (2012) Mitchell et al. (2014) Smith et al. (2013) 27 nuclear loci mtDNA 60 nuclear loci + mtDNA Moa Moa Tinamou Moa Tinamou Elephant bird 49 Tinamou 1 Kiwi Kiwi Rhea 63 Emu 1 Emu Kiwi 0.67-1 Cassowary Cassowary Emu Rhea Rhea Cassowary Ostrich Ostrich Ostrich Coalescent* analyses resolve the position of rheas and reveal an ancient rapid radiation 0.061 12,676 CNEEs - 4,794,620 bp 0.073 5,016 introns - 27,890,802 bp 3,158 UCEs - 8,498,759 bp Total: 20,850 loci; 41,184,181 bp Branch lengths in coalescent units *MP-EST: Liu et al. 2010. BMC Evol. Biol. Consistent accumulation of phylogenetic signal using MP-EST Cloutier et al. 2019. Syst. Biol. 10.1093/sysbio/syz019 Gene tree distribution suggests a near polytomy at base of ratites Cassowary/ Rheas Emu Kiwis Major topology Cassowary/ Kiwis Emu Rheas Minor topology 1 Cassowary/ Emu Kiwis Rheas Minor topology 2 Cloutier et al. 2019. Syst. Biol. 10.1093/sysbio/syz019 Anomaly zone: most common gene tree does not match the species tree Cloutier et al. 2019. Syst. Biol. 10.1093/sysbio/syz019 Corroboration of coalescent tree from transposable elements • Chicken Repeat1 (CR1) retroelement insertions • (Virtually) homoplasy free • Binary presence/absence • No model misspecification • No GC/rate variation bias • BUT- subject to incomplete lineage sorting TS TS 5' flank D CR1 D 3' flank A TS TS 5' flank D CR1 D 3' flank B 5' flank 3' flank C Example CR1 insertion Most CR1s support the coalescent species tree topology Cloutier et al. 2019. Syst. Biol. 10.1093/sysbio/syz019 Evolutionary change: genes or gene regulation? Searching for evidence of genome-wide amino acid convergence in ratites No evidence for genome-wide convergent amino acid substitutions in ratites R2 = 0.95 ~1-2% of protein-coding genes show evidence of ratite-specific positive selection 271 genes (10% FDR) 104 genes (1% FDR) Sackton et al. 2019. Science 364: 74-78 Non-codingDark matter of the genome: a regulatory network? Karyotype of an Emu CNEEs: evolutionarily conserved non-coding enhancer regions CNEEs = conserved non-exonic elements 284,001 long (* > 50 bp) CNEEs in data set View of a segment of human chromosome 10 using UCSC Genome Browser Janes et al. (2011) Genome Biol. Evol. 3:102–113 Convergent loss of function of CNEEs in ratite lineages Tinamous and neognaths Ratites 250 bp 250 Branch-specific Bayesian model of noncoding rate accelerations for noncoding element i Z a = probability of gain of conserved state b = probability of loss of conserved state Hu, Z., et al. 2019. Mol. Biol. Evol. 36: 1086 A convergently accelerated CNEE detected with a novel Bayesian method Anole Turtle2 Turtle1 Gharial Crocodile Alligator2 SNPs Alligator1 Ostrich Moa Tinamou4 Tinamou3 Tinamou2 Tinamou1 Rhea2 Rhea1 accelerated Emu Cassowary Kiwi3 branch Kiwi2 Kiwi1 Mallard Turkey Chicken Mesite Pigeon conserved Cuckoo Swift Hummingbird Killdeer branch Crane Ibis Fulmar Penguin2 Penguin1 Eagle neutral Courol Woodpecker Falcon Budgerigar branch Crow Ground−tit Flycatcher Finch BF1: 72 BF2: 6 r1=0.27, r2=3.08 Branch lengths relative to conserved rate Hu, Z., et al. 2019. Mol. Biol. Evol. 36: 1086 Additional examples of convergently accelerated CNEEs Hu, Z., et al. 2019. Mol. Biol. Evol. 36: 1086 Rapid regulatory evolution near developmental genes Sackton et al. 2019. Science 364: 74-78 Great_Spotted_Kiwi Little_Spotted_Kiwi CNEEs 4,260 CNEE relaxed CNEE on 1 lineage 1 on Rowi_Kiwi Southern_Cassowary Homing in on regulators for for regulators on in Homing Emu Chilean_TInamou Elegent-crested Tinamou Thicket_Tinamou White-throated Tinamou Little_Bush_Moa Lesser_Rhea Greater_Rhea Ostrich 0.7 Same CNEE relaxed Same Great_Spotted_Kiwi through convergence Little_Spotted_Kiwi 1,270 CNEEs 1,270 in 2 lineages Rowi_Kiwi Southern_Cassowary Emu Chilean_TInamou Elegent-crested Tinamou Thicket_Tinamou White-throated Tinamou Little_Bush_Moa Lesser_Rhea Greater_Rhea Ostrich 0.7 Same CNEE relaxed Same Great_Spotted_Kiwi Little_Spotted_Kiwi Rowi_Kiwi CNEEs252 in 3 lineages Southern_Cassowary Emu Chilean_TInamou Elegent-crested Tinamou Thicket_Tinamou White-throated Tinamou Little_Bush_Moa flightlessness Lesser_Rhea Greater_Rhea Ostrich 0.7 Great_Spotted_Kiwi Same CNEE relaxed Same Little_Spotted_Kiwi CNEEs66 Rowi_Kiwi Southern_Cassowary in 4 lineages Emu Chilean_TInamou Elegent-crested Tinamou Thicket_Tinamou White-throated Tinamou Little_Bush_Moa Lesser_Rhea Greater_Rhea Ostrich 0.7 Ratite genomes exhibit higher numbers of convergently accelerating CNEEs than do vocal learners or random trios of taxa 140 ratites random pairs/trios 120 vocal learners Number of 100 convergently accelerated 80 CNEEs 60 40 20 0 2 3 Number of convergent lineages Assay for Transposase-Accessible Chromatin ATAC-Seq identifies DNA with open chromatin, accessible to transcription factors Stage HH24-25 chickens and rheas Buenrostro et al. 2015. Curr Protoc.Biol. 2015; 109: 21.29.1–21.29.9. Differences in ATAC-seq peaks between rhea and chicken suggest changes in limb gene regulation Ratite accelerated element 1317692 is contained under chicken ATAC peaks … 5 kb galGal 11,278,500 11,279,000 11,279,500 11,280,000 11,280,500 11,281,000 11,281,500 11,282,000 11,282,500 11,283,000 11,283,500 11,284,000 11,284,500 11,285,000 11,285,500 11,286,000 11,286,500 11,287,000 11,287,500 CNEECNEE Chick HL 90.2857 _ 0 _ Chicken 82.4286 _ 0 _ hind limb86.6667 _ 0 _ Chick FL 98.5714 _ 0 _ Chicken 93.6667 _ 0 _ 84.8571 _ Forelimb 0 _ Rhea HL 14.8333 _ 0 _ 18.5 _ 0 _ RheaRhea FL 17 _ 0 _ forelimb27.5 _ 0 _ ..