Toward the ultimate phylogeny of Magnoliaceae: phylogenomic approach
Sangtae Kim*1, Suhyeon Park1, and Jongsun Park2
1 Sungshin University, Korea 2 InfoBoss Co., Korea
Mr. Carl Ferris Miller Founder of Chollipo Arboretum in Korea Chollipo Arboretum Famous for its magnolia collection 2020. Annual Meeting of Magnolia Society International Cholliop Arboretum in Korea. April 13th~22th, 2020 http://WWW.Chollipo.org Sungshin University, Seoul, Korea
Dr. Hans Nooteboom Dr. Liu Yu-Hu Twenty-one years ago... in 1998 The 1st International Symposium on the Family Magnoliaceae, Gwangzhow
Dr. Hiroshi Azuma Mr. Richard Figlar Dr. Hans Nooteboom Dr. Qing-wen Zeng
Dr. Weibang Sun Handsome young boy Dr. Yong-kang Sima Dr. Yu-wu Law
Presented ITS study on Magnoliaceae - never published Ten years ago... in 2009
Presented nine cp genome region study (9.2 kbp) on Magnoliaceae – published in 2013 2015
1st International Sympodium on Neotropical Magnoliaceae
Gadalajara, 2019
3rd International Sympodium and Workshop on Neotropical Magnoliaceae Asterales Dipsacales Apiales Why magnolia study is Aquifoliales Campanulids (Euasterids II) Garryales Gentianales Laminales
Solanales Lamiids
important in botany? Ericales Asterids (Euasterids I) Cornales Sapindales Malvales
Brassicales Malvids
Fagales (Eurosids II) • As a member of early-diverging Cucurbitales Rosales Fabales Zygophyllales Celestrales Fabids (Eurosid I) angiosperms, reconstruction of the Oxalidales Malpighiales Vitales Geraniales Myrtales Rosids phylogeny of Magnoliaceae will Saxifragales Caryphyllales Core- Santalales Beberidopsidales eudicots play a key role in understanding Gunnerales Buxaceae Trochodendraceae Proteales Sabiaceae Basal the diversification of angiosperms. eudicots EUDICOTS Ranunculales Euptelea Ceratophyllales
MONOCOTS
Acorus Canellales Basal Piperales Magnoliales Magnoliids Angiosperms Laurales Chloranthus Austrobailales Nymphaeaceae Hydatellaceae Amborella
EXTENT GYMNOSPERMS Why Phylogeny?
Reconstruction of phylogenetic tree is the first step for understanding EVOLUTION and CLASSIFICATION of a group of taxa How can we make phylogenetic tree?
• (Traditionally,) Morphological character analysis
• (After the recognition that DNA is basic genetic material of life,) DNA analysis Toward the ultimate phylogeny… How can we make GOOD phylogenetic tree? 1) Complete sampling representing ALL subgroups of a target plant group
overall resolution
saturation point taxon number 2) Complete data collection: include DNA regions as many as possible (at lease over the saturation point) - have to compare orthologous genes: exclude multiple copy genes - exclude too divergent regions (theoretically, > 25%) overall resolution
saturation point DNA regions History of Molecular Phylogenetic Studies on Magnoliaceae
Kim (in prep.) Complete cp genomes (160 kbp); 130 taxa representing all major sublineages of Magnoliaceae (7 genera, 18 sections) DNA regions
Kim and Suh (2013). 10 cp regions; ndhF, rbcL, matK, trnK 5’ intron, trnK 3’ intron, rbcL-atpB, trnH-psbA, trnL intron, trnL-trnF, ORF350; 7 genera, 18 sections
Kim et al. (2001). ndhF; 7 genera, 18 sections (all genera and sections)
Azuma et al. (2001) analysis I. matK; 6 genera,17 sections
Azuma et al. (1999). Azuma et al. (2001) 5 cp regions: analysis II. matK, trnK 3’intron, trnK 5’ intron, 5 cp regions rbcL-atpB, trnH-psbA; matK, trnK 3’intron, trnK 5’ intron, 3 genera, 10 sections rbcL-atpB, trnH-psbA; 3 genera, 10 sections
Subgroups in Magnoliaceae (subgenera and sections) Chloroplast (160 Kbp) nuclear DNA regions History of Molecular Phylogenetic Studies on Magnoliaceae Studies History ofMolecularPhylogenetic i et al.(2008); Nie ugop nMgoica (subgenera and sections) Magnoliaceae Subgroups in Genome size of Genome PHYA, LFY, GAI1 Magnolia kobus : 1.4~2.0 Gbp : Estimation of genome size of M. kobus based on flow cytometry
1C = 1.42pg (~1.39Gbp)
Standard Solanum lycopersicum 2C=1.96
Magnolia kobus 2C=2.94±0.04
Standard Solanum lycopersicum 4C=3.92 Estimation of genome size of M. kobus by k‐mer analyses
Estimated size is ca. 2 Gbp k=73.
k=73, 2,079,028,389bp
k=33, 4,599,062,800bp Michelia cavaleriei 1.00 Mi 79 M. pealiana 1.00 Michelia baillonii Previous 64 Michelia 1.00 Michelia champ 89 Elmerrillia 1.00 1.00 MICHELIA sect. Maingola Chloroplast tree 98 93 MicheliaMichelia figo odora 1.00 sect. Alcimandra 82 E.ovalis sect. Aromadendron 0.98 Michelia cathcartii Al 77 Ar
1.00 M. elegans Bu 100 Bu M. biondii Kim et al. (2013) Yu 0.70 1.00 1.00 M. kobus 70 100 100 Yu YULANIA subgen. Yulania 10 chloroplast regions 1.00 1.00 M. dawsonianaYu 97 = 9.2 Kbp 97 M. campbelliiCy M. denudata Tu M. cylindrica sect. Manglietiastrum 1.00 Mt 1.00 80 GYNOPODIUM Pachylarnax 100 M. sinicaM. acuminataGy sect. Gynopodium Pachylarnax praecalvaTh 1.00 M. nitida Ma 1.00 60 sect. Theorhodon M. panamensisTh THEORHODON sect. Magnolia 100 0.99 M. virginianaTh 63 M. tamaulipana Th 1.00 M. grandiflora 100 M. guatemalensisKMERIA Kmeria
1.00 Kmeria duperreana 1.00 86 MANGLIETIA Manglietia ManglietiaKmeria grand septentrionalis 100 Manglietia aroma 1.00 Manglietia coniferaRy 100 1.00 Ry sect. Rytidospermum s.s. Manglietia glaucaRYTIDOSPERMUM 92 1.00 Oy sect. Oyama 98 M. officinalis M. fraseri Oy M. tripetala Posterior 1.00 var. fraseri Ry M. macrophyllavar. pyramidata FRASERI probability 100 Ry M. fraseri 1.00 M. dealbata Ry M. sieboldii M. macrophylla M. coco M. wilsoniiMACROPHYLLA Bootsrtap 100 M. fraseriRy M. dealbata 1.00 M. gigantifolia Gw 85 M. henryi value Bl sect. Gwillimia 1.00 0.99 M. pterocarpa 1.00 100 Gw GWILLIMIA sect. Lirianthe 100 75 M. liliifera M. splendens Li sect. Blumiana Bl M. mexicana Sp 1.00 sect. Talauma Ta 100 1.00 TALAUMA sect. Splendentes 100 M. dodecapetala Ta
Liriodendron tulipifera Liriodendron chinense 0.001 substitutions/site Yulania
Maingola Alcimandra Aromadendron Manglietiastrum Gynopodium Theorhodon Magnolia Rytidospermum s.s. Oyama Gwillimia Lirianthe Blumiana Talauma Splendentes
Michelia Elmerrillia sect. sect. sect. subgen. sect. Pachylarnax sect. sect. sect. Kmeria Manglietia sect. sect. M. fraseri macrophylla M. M. dealbata sect. sect. sect. sect. sect. MICHELIA YULANIA GYNOPODIUM THEORHODON KMERIA MANGLIETIA RYTIDOSPERMUM FRASERI MACROPHYLLA GWILLIMIA TALAUMA Li Bl Al Bl Ta Ta Mi Ar Mt Sp Tu Th Th Th Th Yu Yu Yu Cy Ry Ry Ry Ry Ry Ry Bu Bu Gy Oy Oy Ma Gw Gw
Liriodendron chinense M. acuminata M. M. dawsoniana fraseri pyramidata M. campbellii M.
M. biondii M.
M. sieboldii M. var. var. M. kobus M. Michelia odora Kmeria duperreana M. elegans M. denudata M. cylindrica M. septentrionalis Kmeria Michelia cavaleriei M. panamensis M. pealiana M. E.ovalis sinica M.
100 wilsonii M. 1.00 praecalva Pachylarnax M. virginiana macrophylla M. M. dealbata M. dodecapetala Michelia champ 97 100
Michelia baillonii 1.00 Michelia figo 1.00 guatemalensis M. Liriodendron tulipifera M. nitida M. gigantifolia M.
79 Michelia cathcartii Manglietia aroma Manglietia glauca 80 M. tamaulipana grandiflora M. tripetala M. M. fraseri M. fraseri M. pterocarpa M. coco M. liliifera M. 1.00 Manglietia grand M. officinalis
93 henryi M. 64 1.00 Manglietia conifera M. mexicana 1.00 60 100 1.00 1.00 89 63 86
1.00 splendens M. 1.00 site 1.00 98 85 100 / 0.99 1.00 1.00 77 1.00 75 100 98 0.99 1.00 0.98 1.00 100 1.00 100 97 1.00 100 1.00 100 82 1.00 1.00 100 1.00 1.00 100 1.00 92 100 70 1.00 1.00 0.70 1.00 100 0.001 substitutions 100 1.00 value Bootsrtap Posterior probability Kim et al. (2013) al. Kim et regions chloroplast 10 = 9.2 Kbp Previous Chloroplast tree Previous
Nuclear tree TALAUMA
Nie et al. (2008) Nuclear PHYA, LFY, GAI1 OYAMA + RYTIDOSPERMUM = 2.3 Kbp + MANGLIETIA
GWILLIMIA
GYNOPODIUM
THEORHODON
FRASERI MACROPHYLLA
YULANIA is NOT monophyletic group
MICHELIA In this study,
• We tried a genome-wide approach on the phylogeny of Magnoliaceae with high-throughput target-enrichment sequencing (Hyb-Seq) using NGS. Overview of NGS based target-enrichment sequencing (Hyb-Seq)
1. Detection of single-copy genes Taxon A genome Taxon B genome
Taxon C genome
2. Probe design for single-copy genes
3. Shred genomes
Taxon A genome Taxon B genome Taxon C genome
4. Add taxon-specific index adaptors 5. Capture target fragments in each taxon and mix them
6. Run NGS 7. Demultiplexing 8. Assemble and alignment A
B
C Target-enrichment sequencing = Hyb-Seq = Targeted sequencing
• Already have been applied in human exome sequencing.
• Tools for Hyb-Seq - MarkerMiner (Chamala et al., 2015): detecting single copy-genes - HybPiper (Johnson et al., 2016): mapping and alignment
• Recent publications Animals Prum et al. (2015). Birds. Nature Breinholt et al. (2017). Butterflies. Systematic Biology …
Plants Mandel et al. (2016). Asteraceae. Applications in Plant Sciences Schmickl et al. (2016). African Oxalis (Oxalidaceae). Molecular Ecology Resources Stefan et al. (2017). Aristolochiaceae Molecular Phylogenetics and Evolution Tamara et al. (2018). Euphobia (Euphobiaceae). New Phytologist Global Carex Group (submitted). Carex (Cyperaceae). … Materials and Method
Materials • 130 taxa of Magnoliaceae - Representatives of previously reported ALL subgroups of Magnoliaceae (subgenera, sections, major clades) Method • Target regions: chloroplast whole genome (160 Kbp) + 504 nuclear single-copy gene regions (443 Kbp) • Capturing and NGS running: RapidGenomics Co. - 32 Gbp of 100 bp paired-end sequencing using HiSeq3000 • Extraction of othologos gene regions: HybPiper (Johnson et al., 2016) • Matrix combining and sequence statistics: MEGA (ver. 7.0) • Phylogenetic analyses: ML with 500 bootstrap using RAxML (GUI 1.5 beta) Nuclear Target Regions for Hyb-Seq: 504 single-copy genes (443 Kbp)
Preliminary assembly of Magnolia kobus (ver. 0.4): 1.91 Gbp
M. kobus SCGs based on gene clustering (tribe MCL; 1,772 genes)
Shared SCGs with Shared SCGs with expressed M. kobus Amborella genome SCGs from transcriptomes (257 genes) 23 26 230 2 1 2 1,400 7 213 623 75
Shared SCGs (or low copy genes) among Arabidopsis, Populus, Oryza, and Vitis (Duarte et al. , 2010; 959 genes) SCG: single copy gene Results
Chloroplast genome • 130 magnolia cp genomes were COMPLETED (more than 1,000X) - uncovered gaps: only 0.06 % (three regions) later, completed by PCR-based Sanger sequencing • Size of cp genome - subfamily Magnolioideae: 159.1 Kbp (mean of 127 taxa) - subfamily Liriodendroideae: 159.7 Kbp (mean of two taxa)
• Structural variations - trnV-GAC deleted in M.liliiflora - 299 bp deletion in M. acuminate - 658 bp deletion in M. kobus • Aligned matrix: 136.4 Kbp - Phylogenetically informative sites in Magnoliaceae: 6,014 (4.8%) - Phylogenetically informative sites in subfamily Magnolioideae: 4,984 (3.7%) Summary of Chloroplast phylogeny (BS>50%)
99 100 MICHELIA I 99 93 MICHELIA II – Ar. Al. 99 YULANIA I 99 99 100 YULANIA II – M. acu 93 99 GYNOPODIUM 100 KMERIA 75 100 MANGLIETIA 99 100 RYTIDOSPERMUM I 98 100 97 99 RYTIDOSPERMUM II -Oyama 100 THEORHODON 100 100 98 MACROPHYLLA 100 FRASERI 100 GWILLIMIA 92 99 TALAUMA I – M. max 99 99 TALAUMA II – M. bank 100 LIRIODENDRON Results
Nuclear regions • 504 nuclear single-copy genes were determined. (+ introns and adjacent intergenic spacer regions)
Only 20 gene regions were analyzed in this talk
Results
Nuclear regions • 504 nuclear single copy genes were determined. (+ introns and adjacent intergenic spacer regions)
Only 20 gene regions were analyzed in this talk
nuclear 20 gene regons cp genome aligned length (Kbp) 24 136 Magnoliaceae Magnolioideae Magnoliaceae Magnolioideae
# of informative sites (bp) 4,255 3,514 6,014 4,984
% of informative sites (%) 17.8 14.7 4.8 3.7 Summary of nuclear phylogeny (BS>50%)
95 66 MICHELIA I 91 100 YULANIA II – M. acu 87 99 YULANIA I 76 99 MICHELIA II – Ar. Al. 100 KMERIA 88 GYNOPODIUM 94 62 MANGLIETIA 74 60 99 RYTIDOSPERMUM I 70 100 FRASERI 100 RYTIDOSPERMUM II -Oyama 100 100 THEORHODON 100 MACROPHYLLA 99 GWILLIMIA 100 TALAUMA I – M. max 100 TALAUMA II – M. bank
LIRIODENDRON Chloroplast vs. Nuclear (BS>50%)
99 95 100100 MICHELIA I MICHELIA I 66 99 100 91 93 MICHELIA II – Ar. Al. YULANIA II – M. acu 99 99 8787 99 YULANIA I YULANIA I 99 99 100 99 76 YULANIA II – M. acu MICHELIA II – Ar. Al. 93 99 100 GYNOPODIUM KMERIA 100 88 KMERIA GYNOPODIUM 75 100 94 MANGLIETIA MANGLIETIA 62 99 74 100 99 60 100 RYTIDOSPERMUM I RYTIDOSPERMUM I 98 100 70 97 99 RYTIDOSPERMUM II -Oyama FRASERI
100 100 THEORHODON RYTIDOSPERMUM II -Oyama
100 100 100 98 MACROPHYLLA THEORHODON 100 100 100 FRASERI MACROPHYLLA
100 99 GWILLIMIA GWILLIMIA 92 99 100 TALAUMA I – M. max TALAUMA I – M. max 99 99 100 TALAUMA II – M. bank TALAUMA II – M. bank
LIRIODENDRON LIRIODENDRON Strict consensus tree between cp tree and nuclear tree
MICHELIA I Ancient hybridization ? MICHELIA II – Ar. Al.
YULANIA II – M. acu
YULANIA I
GYNOPODIUM
KMERIA
MANGLIETIA
RYTIDOSPERMUM I
RYTIDOSPERMUM II -Oyama
FRASERI
THEORHODON
MACROPHYLLA
GWILLIMIA
TALAUMA I – M. max
TALAUMA II – M. bank
LIRIODENDRON Taxonomic structures of possible classification systems proposed in Kim and Suh (2013) based on the analyses of chloroplast 10 regions Strict consensus tree between cp tree and nuclear tree PhyloCode (Cantino and de Queiroz, 2010)
MICHELIA I
CORE EUMAGNOLIA MICHELIA II – Ar. Al.
YULANIA II – M. acu
YULANIA I
GYNOPODIUM
KMERIA EUMAGNOLIA MANGLIETIA
RYTIDOSPERMUM I Basal EUMAGNOLIA RYTIDOSPERMUM II -Oyama
FRASERI MAGNOLIA THEORHODON
MACROPHYLLA
GWILLIMIA Basal TALAUMA I – M. max MAGNOLIA
TALAUMA II – M. bank
LIRIODENDRON LIRIODENDRON SUMMARY
•For 130 taxa representing all subgroups in Magnoliaceae, complete chloroplast genomes and 20 nuclear DNA regions were determined.
• Some conflict relationships were recognized between chloroplast tree and nuclear tree suggesting possible ancient hybridization, especially between ancestors of MICHELIA and YULANIA clades.
• Highly supported clades from both chloroplast tree and nuclear tree were named as EUMAGNOLIA and CORE EUMAGNOLIA based on the concept of PhyloCode.
• Target-enrichment NGS (Hyb-Seq) will be the standard method of future phylogenetic studies. Ultimate Phylogeny Further studies
1) Evolutionary character reconstruction
2) Re-investigation of genome size
3) Suggestion of a minimum set of genes for phylogenetic or barcoding studies Evolutionary character reconstruction Family Magnoliaceae Key characters in the Subfamily Magnolioideae Tribe Magnolieae classification system of Genus Magnolia Nooteboom (1985) Subgenus Magnolia Section Magnolia Section Gwillimia Section Lirianthe Section Rytidospermum Section Oyama Section Theorhodon Section Gynopodium Section Maingola Section Alcimandra Subgenus Yulania Section Yulania Section Buergeria Section Tulipastrum Subgenus Talauma Section Talauma Section Blumiana Section Aromadendron Section Manglietiastrum Genus Kmeria Genus Manglietia Genus Pachylarnax Tribe Michelieae Genus Elmerrillia Genus Michelia Subfamily Liriodendroideae Genus Liriodendron Family Magnoliaceae Terminal flower Subfamily Magnolioideae Tribe Magnolieae Genus Magnolia Subgenus Magnolia Section Magnolia Section Gwillimia Section Lirianthe Section Rytidospermum Section Oyama Section Theorhodon Section Gynopodium Section Maingola Section Alcimandra Subgenus Yulania Section Yulania Section Buergeria Axillary flower Section Tulipastrum Subgenus Talauma Section Talauma Section Blumiana Section Aromadendron Section Manglietiastrum Genus Kmeria Genus Manglietia Genus Pachylarnax Tribe Michelieae Genus Elmerrillia Genus Michelia Subfamily Liriodendroideae Genus Liriodendron Unisexual flower Family Magnoliaceae Subfamily Magnolioideae Tribe Magnolieae Genus Magnolia Subgenus Magnolia Section Magnolia Section Gwillimia Section Lirianthe Section Rytidospermum Section Oyama Section Theorhodon Ovules 4 or more Section Gynopodium Section Maingola Section Alcimandra Subgenus Yulania Section Yulania Section Buergeria Section Tulipastrum Subgenus Talauma Section Talauma Section Blumiana Section Aromadendron Capsule like fruit Section Manglietiastrum Genus Kmeria Genus Manglietia Genus Pachylarnax Tribe Michelieae Genus Elmerrillia Genus Michelia Subfamily Liriodendroideae Genus Liriodendron Family Magnoliaceae Subfamily Magnolioideae Tribe Magnolieae Genus Magnolia Subgenus Magnolia Section Magnolia Section Gwillimia Section Lirianthe Section Rytidospermum Section Oyama Section Theorhodon Section Gynopodium Section Maingola Section Alcimandra Anthers introrse Subgenus Yulania Section Yulania Section Buergeria Section Tulipastrum Subgenus Talauma Section Talauma Section Blumiana Section Aromadendron Section Manglietiastrum Genus Kmeria Genus Manglietia Genus Pachylarnax Tribe Michelieae Genus Elmerrillia Anthers latrorse Genus Michelia Subfamily Liriodendroideae Genus Liriodendron Anthers Carpels; fruits Family Magnoliaceae introrse free; ellipsoidal Subfamily Magnolioideae Tribe Magnolieae Genus Magnolia Subgenus Magnolia Section Magnolia Section Gwillimia Section Lirianthe Section Rytidospermum Section Oyama Section Theorhodon latrorse free; cylindrical Section Gynopodium Section Maingola Section Alcimandra Subgenus Yulania Section Yulania Section Buergeria Section Tulipastrum Subgenus Talauma Section Talauma Section Blumiana Section Aromadendron introrse connate; ovoidal Section Manglietiastrum Genus Kmeria Genus Manglietia Genus Pachylarnax Tribe Michelieae Genus Elmerrillia Genus Michelia Subfamily Liriodendroideae Genus Liriodendron
Diverse morphology of magnolia seeds
5 mm
5 mm 5 mm
Well described in Tiffney (1977) Tiffney (1977) MICHELIA I
CORE EUMAGNOLIA MICHELIA II – Ar. Al.
YULANIA II – M. acu
YULANIA I
GYNOPODIUM
KMERIA EUMAGNOLIA MANGLIETIA
RYTIDOSPERMUM I Basal EUMAGNOLIA RYTIDOSPERMUM II -Oyama
FRASERI MAGNOLIA THEORHODON
MACROPHYLLA
GWILLIMIA Basal TALAUMA I – M. max MAGNOLIA
TALAUMA II – M. bank
LIRIODENDRON LIRIODENDRON Further studies
1) Evolutionary character reconstruction
2) Re-investigation of genome size
3) Suggestion of a minimum set of genes for phylogenetic or barcoding studies Genome size estimation • Using a proper standard plant is a key to estimate precise genome size in flow cytometry
• Parris et al. (2010) used just one internal standard (Psium sativum; 2C=0.09)
• Genome size of M. kobus: 1.4 Gbp in this study 2.0 Gbp in Parris et al. (2010)
• Recognition of genome size is very important for the selection of taxon for future intensive genome sequencing Further studies
1) Evolutionary character reconstruction
2) Re-investigation of genome size
3) Suggestion of a minimum set of genes for phylogenetic or barcoding studies Suggestion of a minimum set of genes for phylogenetic or barcoding studies
• In case of Carex Hyb-Seq study, we successfully extracted 10 best nuclear regions showing 80% of clade-resolution compare to total gene (340 genes) analysis. • We may suggest a set of the best regions for the studies of additional taxa.
Bora Lee, Ph.D. student, Sungshin Univ. Thanks to Suhyun Park, Ph.D. student Dr. Jongsun Park, CEO, InfoBoss Co. Many thanks to Dr. Antonia Vazquez Dr. Weerakit Harnpariphan
X 100