Supplementary material

Platypus

Opposum

Wallaby

Armadillo

Sloth

Tenrec

Elephant

Hyrax

Shrew

Hedgehog

Echolocating bat

Fruit bat

Horse

Cat

Dog

Alpaca

Pig

Dolphin

Cow

Rabbit

Pika

Squirrel

Guniea pig

Kangaroo Rat

Mouse

Rat

Tree Shrew

Mouse Lemur

Tarsier

Marmoset

Macaque

Orangutan

Gorilla

Human Chimpanzee Figure S1 Mammals’ species tree from Song et al. 2012[1] The unique incongruence clade between this tree and our final species tree is the Tree Shrew, which labeled in red.

B) phyldog tree

S.cerevisiae C.elegans C.intestinalis C.savignyi Fruitfly Lamprey Spotted gar Zebrafish Cave fish Cod Tetraodon Fugu Stickleback Tilapia Medaka Amazon molly Platyfish Anole lizard Chinese softshell turtle Zebra Finch Flycatcher Duck Turkey Chicken Coelacanth Xenopus Platypus Opossum Wallaby Tasmanian devil Sloth Armadillo Lesser hedgehog tenrec Hyrax Elephant Shrew Hedgehog Cat Dog Ferret Panda Horse Megabat Microbat Alpaca Pig Dolphin Cow Sheep Rabbit Pika Kangaroo rat Squirrel Guinea Pig Mouse Rat Tree Shrew Mouse Lemur Bushbaby Tarsier Marmoset Vervet-AGM Olive baboon Macaque Gibbon Orangutan Gorilla Chimpanzee Human 0.4 Figure S2 Species tree inferred by Phyldog

Gene families

Random selected Random selected Random selected gene families set 1 gene families set 2 gene families set 3 Species tree 1 Species tree 2 Species tree 3 A A A Gene family trees B B B C C C

D D D E E E

Identification the clades with most Inconsistent clades supports from gene family trees

Initial species tree

Final species tree

Figure S3 Alternative species tree inference workflow Here, we only randomly selected three gene families sets to infer the initial species tree and we can select more than three sets in real application.

A nested gene family

Gene 1-Gene 2 read- Gene 1 homologs Gene 2 homologs through homologs

Figure S4 Diagram of nested gene family This diagram displayed the nested gene family caused by read-through genes which read-through two independent genes. And there are more complex nested families, which nested more sub gene families. Petromyzon marinus Lepisosteus oculatus Astyanax mexicanus n23 n 5 Danio rerio n129 n21 Gadus morhua Tetraodon nigroviridis n20 n 8 Takifugu rubripes n18 Gasterosteus aculeatus n11 Oreochromis niloticus n127 n17 Oryzias latipes n16 Poecilia formosa n14 Xiphophorus maculatus Latimeria chalumnae Xenopus tropicalis n126 Anolis carolinensis n36 Pelodiscus sinensis n34 Taeniopygia guttata n124 n26 Ficedula albicollis rate n32 Anas platyrhynchos 0.16 n31 Meleagris gallopavo n29 n122 Gallus gallus Ornithorhynchus anatinus Monodelphis domestica n41 Sarcophilus harrisii n39 Macropus eugenii n121 Dasypus novemcinctus n44 Choloepus hoffmanni n50 Echinops telfairi n49 Loxodonta africana n119 n47 Procavia capensis Erinaceus europaeus n53 Sorex araneus Vicugna pacos n62 n77 Sus scrofa n118 n60 Tursiops truncatus n58 Ovis aries n56 n76 Bos taurus Equus caballus n67 Myotis lucifugus n65 n75 Pteropus vampyrus Felis catus n74 Canis familiaris n117 n72 Ailuropoda melanoleuca n70 Mustela putorius furo Tupaia belangeri n82 Oryctolagus cuniculus n80 0 Ochotona princeps n92 Cavia porcellus n91 Ictidomys tridecemlineatus n89 Dipodomys ordii n87 n116 Rattus norvegicus n85 Mus musculus Otolemur garnettii n95 Microcebus murinus n115 Tarsius syrichta Callithrix jacchus n114 Chlorocebus sabaeus n112 n100 Papio anubis n98 Macaca mulatta n110 Nomascus leucogenys n109 Pongo abelii n107 Gorilla gorilla n105 Pan troglodytes n103 Homo sapiens Figure S5 Average gene duplication retention rates and labeled ancestral nodes The size of cycle on each ancestral branch was determined by the average gene duplication retention rates (detailed in Table S1). There are three ancestral branches showing significantly higher average gene duplication rate among these branches (ks.test in R: p-value = 0.005545). These ancestral branches were reported to undergo genome duplication in previous studies[2-6]. This result verified the correctness of our reconciliation results in some ways. Biological processes bicarbonate transport sodium ion transport Wnt signaling pathway cell-cell signaling by wnt regulation of small GTPase mediated signal transduction monovalent inorganic cation transport cellular potassium ion transport GeneRatio potassium ion transmembrane transport 0.02 regulation of neuron projection development 0.04 sensory organ development 0.06 sodium ion transmembrane transport cell morphogenesis involved in neuron differentiation P extracellular matrix organization extracellular structure organization 0.04 axon development 0.03 axonogenesis 0.02 Ras protein signal transduction 0.01 regulation of transmembrane transport regulation of ion transmembrane transport regulation of membrane potential regulation of metal ion transport cell-cell junction organization cell junction organization multicellular organismal signaling 1R WGD 2R WGD TS WGD ALL

Figure S6 GO enrichment results of WGD-affected gene families Class ‘1R WGD’ represents genes from the gene families with ohnologs retention after the first round WGD in vertebrates. Class ‘2R WGD’ represents genes from the gene families with ohnologs retention after the second round WGD. Class ‘TS WGD’ represents genes from the gene families with ohnologs retention after the TS WGD. Class ‘All’ represents genes from gene families with ohnologs retention at least after one of these tree WGDs.

Figure S7 Dated species tree from TIMETREE The concentration of oxygen is displayed under the species tree. The number labeled beside each node represents its age (unit: million years ago, MYA). And the WGD-affected branches are labels in bold and colored in red. 400

300

200 Density

100

0

0.00 0.05 0.10 0.15 Long term duplication preservation rate

Figure S8 Distribution of gene family duplication retention/preservation rates

with X-axis limited to 0.0009 ~ 0.016

A) B) S.cerevisiae S.cerevisiae C.elegans Fruitfly Fruitfly C.elegans C.savignyi C.intestinalis C.intestinalis C.savignyi Lamprey Lamprey Spotted gar Spotted gar Zebrafish Zebrafish Cave fish Cave fish Cod Cod Fugu Tetraodon Tetraodon Fugu Tilapia Tilapia Stickleback Stickleback Medaka Medaka Platyfish Amazon molly Amazon molly Platyfish Xenopus Coelacanth Coelacanth Xenopus Anole lizard Anole lizard Chinese softshell turtle Chinese softshell turtle Flycatcher Zebra Finch Zebra Finch Flycatcher Duck Duck Chicken Turkey Turkey Chicken Platypus Platypus Opossum Opossum Wallaby Wallaby Tasmanian devil Tasmanian devil Sloth Sloth Armadillo Armadillo Lesser hedgehog tenrec Lesser hedgehog tenrec Hyrax Hyrax Elephant Elephant Shrew Shrew Hedgehog Hedgehog Alpaca Pig Pig Alpaca Dolphin Dolphin Cow Cow Sheep Sheep Horse Megabat Megabat Microbat Microbat Horse Cat Cat Dog Dog Ferret Ferret Panda Panda Tree Shrew Rabbit Pika Pika Rabbit Guinea Pig Squirrel Squirrel Guinea Pig Kangaroo rat Kangaroo rat Mouse Mouse Rat Rat Tree Shrew Mouse Lemur Mouse Lemur Bushbaby Bushbaby Tarsier Tarsier Marmoset Marmoset Vervet-AGM Vervet-AGM 1 2 3 Macaque Olive baboon Olive baboon Macaque Gibbon Gibbon 4 5 6 7 Orangutan Orangutan Gorilla Gorilla Human Chimpanzee Chimpanzee Human Figure S9 Inconsistent clades between our initial species tree and Ensembl species tree The clades bearing inconsistent phylogenetic relationships between the initial species tree and the Ensembl species tree are labeled in different colors and the inconsistences are numbered. A) Our initial species tree. B) Ensembl species tree.

Table S1 Average gene duplication retention rate for each branch

Branch Duplication rate Branch Duplication rate

Ornithorhynchus anatinus 0.286 n127 0.1573

Sus scrofa 0.2155 n129 0.0998

Taeniopygia guttata 0.1727 n21 0.0863

Dasypus novemcinctus 0.1326 n5 0.0497

Latimeria chalumnae 0.1299 n23 0.0272

Myotis lucifugus 0.1067 n85 0.0217

Astyanax mexicanus 0.1028 n20 0.0209

Poecilia formosa 0.1016 n14 0.0176

Monodelphis domestica 0.0973 n56 0.0175

Homo sapiens 0.0968 n126 0.0133

Petromyzon marinus 0.0946 n8 0.0123

Callithrix jacchus 0.0869 n41 0.0118

Danio rerio 0.0852 n18 0.0111

Tetraodon nigroviridis 0.0829 n100 0.0087

Anolis carolinensis 0.0725 n121 0.0075

Oryctolagus cuniculus 0.0717 n26 0.0075

Macaca mulatta 0.07 n98 0.0075

Ovis aries 0.0643 n119 0.0074

Otolemur garnettii 0.0605 n11 0.0072

Rattus norvegicus 0.0597 n107 0.007

Xenopus tropicalis 0.058 n124 0.0067

Sarcophilus harrisii 0.057 n17 0.006

Felis catus 0.056 n112 0.0058

Pelodiscus sinensis 0.0523 n122 0.0058

Lepisosteus oculatus 0.0496 n16 0.0054

Ictidomys tridecemlineatus 0.0485 n36 0.0052

Oreochromis niloticus 0.048 n47 0.0048

Gorilla gorilla 0.0479 n103 0.0047

Echinops telfairi 0.0473 n29 0.0046

Cavia porcellus 0.0471 n72 0.0046 Branch Duplication rate Branch Duplication rate

Anas platyrhynchos 0.047 n118 0.0045

Mus musculus 0.0456 n44 0.0045

Oryzias latipes 0.044 n91 0.0045

Loxodonta africana 0.0437 n31 0.0044

Canis familiaris 0.0416 n75 0.0041

Gadus morhua 0.0391 n110 0.004

Gasterosteus aculeatus 0.0386 n34 0.004

Equus caballus 0.0376 n74 0.0038

Meleagris gallopavo 0.0364 n60 0.0036

Takifugu rubripes 0.0343 n32 0.0034

Mustela putorius furo 0.0317 n105 0.0033

Pongo abelii 0.0301 n70 0.0032

Ailuropoda melanoleuca 0.0267 n109 0.0031

Gallus gallus 0.0262 n53 0.0031

Tupaia belangeri 0.0247 n58 0.0031

Nomascus leucogenys 0.0229 n80 0.003

Xiphophorus maculatus 0.0225 n76 0.0025

Erinaceus europaeus 0.0222 n95 0.0025

Bos taurus 0.0214 n89 0.0022

Ochotona princeps 0.0201 n49 0.002

Ficedula albicollis 0.02 n117 0.0018

Sorex araneus 0.0179 n67 0.0014

Choloepus hoffmanni 0.0173 n39 0.0013

Tarsius syrichta 0.0155 n115 0.0012

Microcebus murinus 0.0152 n65 0.001

Macropus eugenii 0.0151 n116 0.0009

Chlorocebus sabaeus 0.015 n87 0.0008

Papio anubis 0.0139 n114 0.0005

Dipodomys ordii 0.0114 n50 0.0005

Tursiops truncatus 0.0098 n77 0.0005

Procavia capensis 0.0091 n62 0.0003 Branch Duplication rate Branch Duplication rate

Pan troglodytes 0.0089 n82 0.0003

Pteropus vampyrus 0.0066 n92 0.0003

Vicugna pacos 0.0064

In this table, ancestral branches are represented by their end-nodes. And the ancestral node represents the ancestral vertebrate species as the Figure S5 displayed. The calculation methods are detailed in section 3 in this file.

2R & TS WGD: Gene families retaining ohnologs both from the TS and 2R WGDs.

1R & 2R & 3R: Gene families retaining ohnologs from all of the three WGDs.

Table S2 PPI enrichment results in the WGD-affected gene family classes

WGDs PPI Gene pairs Enrichment fold P-value

1R WGD 8,340 1,214,080 4.245023 < 2.2e-16

2R WGD 15,690 4,179,953 2.369621 < 2.2e-16

TS WGD 11,838 2,182,655 3.407453 < 2.2e-16

1R & 2R WGD 1,096 340,918 1.880676 < 2.2e-16

1R & TS WGD 2,746 446,232 3.656845 < 2.2e-16

2R & TS WGD 3,144 1,190,374 1.552143 < 2.2e-16

1R & 2R & 3R 428 76,538 3.273032 < 2.2e-16

1R WGD: Gene families that only retaining ohnologs after the first round WGD.

2R WGD: Gene families that only retaining ohnologs after the second round WGD.

TS WGD: Gene families that only retaining ohnologs after the TS (teleost fishes) specific WGD.

1R & 2R WGD: Gene families retaining ohnologs both from the 1R and 2R WGDs.

1R & TS WGD: Gene families retaining ohnologs both from the TS and 1R WGDs

Table S3 Supports of the 7 inconsistent clades from 11,698 gene family trees

Inconsistent cladesa Ensembl species tree Guenomu species tree

Inconsistent clade 1 2066* 1332

Inconsistent clade 2 1592 2172*

Inconsistent clade 3 672 2932*

Inconsistent clade 4 2650* 147

Inconsistent clade 5 2834* 676 Inconsistent cladesa Ensembl species tree Guenomu species tree

Inconsistent clade 6 1731 2752*

Inconsistent clade 7 501 405

*The favored clade a The relative clades can be found in Figure S9

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