Why do we study phylogeny? Mobile! A tree is like a mobile! “Tree of Life” (Klimt, Austria) “Tree of Life” project http://tolweb.org/tree/ PHYLOGENETICS

In biology, phylogenetics is the study of evolutionary relatedness among various groups of organisms (for example, species or populations), which is discovered through molecular sequencing data and morphological data matrices. The term phylogenetics is of Greek origin from the terms phyle/phylon (φυλή/φῦλον), meaning "tribe, race," and genetikos (γενετικός), meaning "relative to birth" from genesis (γένεσις, "birth"). , the classification, identification, and naming of organisms, has been richly informed by phylogenetics but remains methodologically and logically distinct.[1] The fields overlap however in the science of phylogenetic systematics –often called "cladism" or "cladistics" –, where only phylogenetic trees are used to delimit taxa, which represent groups of lineage-connected individuals.[2] In biological systematics as a whole, phylogenetic analyses have become essential in researching the evolutionary tree of life. • It is a genome world!

- The first genome data: Haemophilus influenzae (1995)

about 1.8M bp.

- Homo sapiens (begun in 1990 and completed in 2001)

about 3.0G bp. <$400,000,000

- in 2011, more than 30 genomes are determined.

 In 2013, 40 angiosperm genomes have been sequenced. • Two examples for the effect of phylogeny in the text book:

1) the phylogeny transformed to systematics (classifications)  16S rDNA phylogeny: suggested three domains in the life. (c.f.: Svedverg value)

2) the phylogeny showed evolutionary information  HIV gene (env) phylogeny: gave evidence of infection pathway.  HIV genome stored evolutionary information  recount the very recent history of its spread.

► Not only just pure application to the systematics but also the tracing evolutionary history.

Other examples of interesting phylogenetic studies giving great impacts to the classification

• Soltis et al. (1999): - Dicots are NOT monophyletic group - Amborella is the basal-most angiosperm • Manatee: a sister to elephant • Whale: a sister to hippo • white stork (황새): a sister to condor

And minor impact  my studies on Magnoliaceae ;-) Summarized angiosperm phylogeny

Asterales 국화, 초롱꽃 Dipsacales 인동, 산토끼꽃 Apiales 당귀, 인삼 Aquifoliales 감탕나무 Campanulids Garryales 두충 Gentianales 용담, 꼭두서니 Laminales 꿀풀, 금어초

Solanales 토마토, 메꽃 Lamiids Ericales 진달래, 앵초 Cornales 층층나무, 수국

Sapindales 귤, 단풍나무 Malvales 무궁화 Core-

Brassicales 애기장대, 무우 Malvids Fagales 자작나무, 참나무 Cucurbitales 박, 베고니아 Rosales 장미, 느릅나무 Rosids Traditional 콩, 자귀나무 Fabales Dicots part I Zygophyllales 남가새 Celestrales 노박덩굴 Fabids Oxalidales 굉이밥 Malpighiales 버드나무, 제비꽃 Geraniales 쥐손이풀 Myrtales 바늘꽃 Saxifragales 범의귀, 돌나물 Caryphyllales 카네이션, 선인장 Santalales 단향, 겨우살이 Beberidopsidales 매발톱나무 Gunnerales Buxaceae 회양목 Trochodendraceae Proteales 연꽃, 버즘나무 Sabiaceae 나도밤나무 Basal EUDICOTS eudicots 미나리아재비 양귀비 Ranunculales Euptelea Ceratophyllales 붕어마름

벼, 백합 옥수수 MONOCOTS

Acorus 창포 Chloranthales 홀아비꽃대 Basal Canellales Angiosperms Piperales 후추, 족도리풀 Magnoliales 목련, 튜립나무 magnoliids Laurales 녹나무, 아보카도 Traditional Austrobailales 붓순나무, 오미자 Dicots part II Nymphaeaceae 수련 Hydatellaceae Amborella

EXTENT GYMNOSPERMS Amborella is a sister to all other angiosperms! http://www.amborella.org/

Amborella trichopoda KIM, et al., (2004) Phylogeny and B-function of diversification genes in MADS-box angiosperms: evolutionary and functional implications a 260-million-year-old of duplication. Botany American Journal of 21: 2102-2118. Journal cover Manatee (and Dugong) is a sister to elephant!

Whales are sister to hippos! 9 66 d1 Mich. cavaleriei Phylogeny of Magnoliaceae 1 8 M. pealiana 68 d1 Mi 2 3 Mich. baillonii 72 d1 5 Michelia Terminal 2 Mich. champaca 11 Elmerrillia 4 3 sect. Maingola 2 Mich. figo 98 d4 91 d3 Mich. odora sect. Alcimandra A 12 9 sect. Aromadendron 86 d4 6 M. cathcartiiE. ovalis 3 Al 64 d1 16 Ar 5 8 M. elegans Bu 3 100 d8 Bu 5 77 d2 M. biondii Yu 3 8 14 3 M. kobus 100 d8 100 d13 Yu subgen. Yulania 2 B 11 Base changes 7 Yu 95 d5 2 100 d6 M. dawsonianaCy 29 M. denudataM. campbelliiTu 80 d1 5 M. cylindrica Bootstrap /Decay value 2 Mt sect. Manglietiastrum 20 d0 3 1 C 18 Pachylarnax 1 M. sinica 100 d15 Gy sect. Gynopodium 3 P. praecalva M. acuminata D 28 K. duperreanaM. nitida 100 d26 2 K. septentrionalis Kmeria 12 1 66 d2 M. panamensis Th 2 9 M. virginiana 20 d0 M. tamaulipana Ma 2 E 15 M. grandiflora Th sect. Theorhodon s.s. 100 d14 3 sect. Magnolia 1 Th 6 M.guatemalensis Axillary flower 48 d1 Th 31 d1 70 d1 5 Gw 3 2 7 Bl sect. Gwillimia F 17 4 1 Gw sect. Lirianthe 100 d16 6 M. coco 64 d1 Li sect. Blumiana 3 6 M. gigantifolia M. henryi Bl 45 d1 7 G 9 Ta 4 100 d8 17 M. pterocarpaTa sect. Talauma 82 d3 13 M. liliifera sect. Splendentes M. mexicanaSp 33 d1 1 1 86 d2 2 4 H M. splendens Manglietia 17 M. dodecapetala 100 d13 6 Mang. grandis

7 4 Mang.Ry aroma 9 Mang. conifera 143 I 5 98 d4 Ry sect. Rytidospermum s.s. 100 d137 21 M. officinalis 92 d3 6 Mang.Oy glaucasect. Oyama 14 98 d6 M. fraseri M. tripetalaOy 1 J 17 var. fraseri Ry 1 100 d17 M. macrophyllavar. pyramidata Ry 5 M. fraseri K 19 M. dealbata Ry M. macrophylla 4 M. fraseriM. wilsoniiM. sieboldii 100 d17 Ry M. dealbata

52 27 L. tulipifera Phylogeny of Magnoliaceae changes the classification of the family.

L. chinense • Two examples for the effect of phylogeny in the text book:

1) the phylogeny transformed to systematics (classifications)  16S rDNA phylogeny: suggested three domains in the life.

2) the phylogeny showed evolutionary information  HIV gene (env) phylogeny: gave evidence of infection pathway.  HIV genome stored evolutionary information  recount the very recent history of its spread.

► Not only just pure application to the systematics but also the tracing evolutionary history. Phylogeny of HIV: provide an evidence of infection pathway Another example:

3) Phylogenetic tree based on DNA sequences provide the identification of unknown biological samples!

- DNA barcoding: Molecular identification method of species name based on the sequences of standard gene(s).

- An example of my study: “DNA barcoding study of Korean ” based on three chloroplast DNA regions Two-dimensional barcode QR code barcode

DNA-barcoding Lamiaceae (꿀풀과) Immature Molecular identifications of biological samples are possible EVEN with chopped or grounded samples.

황금 곽향 Scutellaria baicalensis 1 (황금) Scutellaria baicalensis 2 (황금) Scutellaria indica var. coccinea (연지골무꽃) Scutellaria indica var. tsusimensis 1 (떡잎골무꽃) Scutellaria indica var. tsusimensis 2 (떡잎골무꽃) Scutellaria insignis 1 (광릉골무꽃) Scutellaria insignis 2 (광릉골무꽃) Anaysis of three DNA Scutellaria moniliorrhiza (구슬골무꽃) Scutellaria regeliana 2 (가는골무꽃) Scutellaria strigillosa (참골무꽃) Scutellaria regeliana 1 (가는골무꽃) regions in chloroplast: Scutellaria pekinensis var. alpina 1 (수골무꽃) Scutellaria pekinensis var. alpina 2 (수골무꽃) Scutellaria Scutellaria pekinensis var. maxima 4 (왕골무꽃) trnH~psbA Scutellaria pekinensis var. ussuriensis 2 (호골무꽃) Scutellaria pekinensis var. ussuriensis 1 (호골무꽃) Scutellaria pekinensis var. maxima 1 (왕골무꽃) Scutellaria pekinensis var. maxima 2 (왕골무꽃) atpF~atpH Scutellaria pekinensis var. maxima 3 (왕골무꽃) Scutellaria pekinensis var. transitra 1 (산골무꽃) Scutellaria pekinensis var. transitra 2 (산골무꽃) Scutellaria pekinensis var. transitra 3 (산골무꽃) psbK~psbI Scutellaria pekinensis var. ussuriensis 3 (호골무꽃) album var. barbatum 1 (광대수염) Lamium album var. barbatum 2 (광대수염) Lamium album var. album 1 (왜광대수염) Lamium takesimense 1 (섬광대수염) Lamium takesimense 2 (섬광대수염) Lamium Lamium album var. album 2 (왜광대수염) Stachys japonica 1 (석잠풀) Stachys japonica 3 (석잠풀) Stachys japonica 2 (석잠풀) Stachys Lamium amplexicaule 1 (광대나물) Lamium amplexicaule 2 (광대나물) Lamium amplexicaule 3 (광대나물) Leonurus japonicus 1 (익모초) Lamium Leonurus japonicus 2 (익모초) Leonurus macranthus 1 (송장풀) Neighbor joining Leonurus macranthus 2 (송장풀) Leonurus Phlomis umbrosa (속단) Dysophylla stellata (물꼬리풀) Phlomis tree Dysophylla yatabeana (전주물꼬리풀) Ajuga decumbens 1 (금창초) Dysophylla Ajuga decumbens 2 (금창초) Ajuga decumbens 3 (금창초) Ajuga spectabilis (자란초) Ajuga Ajuga multiflora 1 (조개나물) Ajuga multiflora 2 (조개나물) Teucrium japonicum (개곽향) Teucrium veronicoides 1 (곽향) Teucrium veronicoides 2 (곽향) Teucrium viscidum (덩굴곽향) Teucrium Clinopodium chinense (층층이꽃) Clinopodium micranthum (두메층층이) Clinopodium gracile (애기탑꽃) Clinopodium Agastache rugosa 1 (배초향) Agastache rugosa 2 (배초향) Dracocephalum rupestreAgastache 1 (벌깨풀) Dracocephalum rupestre 2 (벌깨풀) Meehania urticifolia 1 (벌깨덩굴) Dracocephalum Meehania urticifolia 2 (벌깨덩굴) Lycopus maackianus 1 (애기쉽싸리) Meehania Lycopus maackianus 2 (애기쉽싸리) Lycopus maackianus 3 (애기쉽싸리) Lycopus ramosissimus (개쉽싸리) Lycopus Prunella asiatica (꿀풀) Salvia chanroenica (참배암차즈기) Prunella Salvia plebeia (배암차즈기) Perilla frutescens var. frutescens 1 (들깨) Salvia Perilla frutescens var. frutescens 2 (들깨) Mosla chinensis (가는잎산들깨) Perilla Mosla dianthera (쥐깨풀) Elsholtzia minima (좀향유) Mosla Elsholtzia splendens 1 (꽃향유) Elsholtzia splendens 2 (꽃향유) Isodon inflexus (산박하) Isodon japonicus (방아풀) Isodon 0.005 substitutions/site Elsholtzia Q1 Scutellaria pekinensis var. transitra 2 (산골무꽃) Scutellaria pekinensis var. transitra 3 (산골무꽃) Scutellaria pekinensis var. ussuriensis 3 (호골무꽃) Q2 Q3 Scutellaria pekinensis var. alpina 2 (수골무꽃) Scutellaria pekinensis var. maxima 1 (왕골무꽃) Scutellaria pekinensis var. maxima 2 (왕골무꽃) Scutellaria pekinensis var. maxima 3 (왕골무꽃) Identifying unknown samples (Qs) Scutellaria pekinensis var. maxima 4 (왕골무꽃) Scutellaria pekinensis var. ussuriensis 2 (호골무꽃) Scutellaria pekinensis var. ussuriensis 1 (호골무꽃) Scutellaria pekinensis var. transitra 1 (산골무꽃) Scutellaria pekinensis var. alpina 1 (수골무꽃) Scutellaria moniliorrhiza (구슬골무꽃) Scutellaria regeliana 2 (가는골무꽃) Scutellaria strigillosa (참골무꽃) Scutellaria regeliana 1 (가는골무꽃) Q4 Scutellaria indica var. coccinea (연지골무꽃) Scutellaria indica var. tsusimensis 1 (떡잎골무꽃) Scutellaria indica var. tsusimensis 2 (떡잎골무꽃) Scutellaria insignis 1 (광릉골무꽃) Scutellaria insignis 2 (광릉골무꽃) Scutellaria baicalensis 1 (황금) Scutellaria baicalensis 2 (황금) Q5 Ajuga decumbens 1 (금창초) Ajuga decumbens 2 (금창초) Ajuga decumbens 3 (금창초) Ajuga spectabilis (자란초) Ajuga multiflora 1 (조개나물) Ajuga multiflora 2 (조개나물) Lamium album var. barbatum 1 (광대수염) Lamium album var. barbatum 2 (광대수염) Lamium album var. album 1 (왜광대수염) Lamium takesimense 1 (섬광대수염) Lamium takesimense 2 (섬광대수염) Lamium album var. album 2 (왜광대수염) Stachys japonica 1 (석잠풀) Stachys japonica 3 (석잠풀) Stachys japonica 2 (석잠풀) Lamium amplexicaule 1 (광대나물) Lamium amplexicaule 2 (광대나물) Lamium amplexicaule 3 (광대나물) Q6 Leonurus japonicus 1 (익모초) Leonurus japonicus 2 (익모초) Leonurus macranthus 1 (송장풀) Leonurus macranthus 2 (송장풀) Q17 Phlomis umbrosa (속단) Q7 Dysophylla stellata (물꼬리풀) Dysophylla yatabeana (전주물꼬리풀) Teucrium japonicum (개곽향) Teucrium veronicoides 1 (곽향) Teucrium veronicoides 2 (곽향) Teucrium viscidum (덩굴곽향) Q8 Clinopodium chinense (층층이꽃) Q9 Clinopodium micranthum (두메층층이) Clinopodium gracile (애기탑꽃) Q10 Agastache rugosa 1 (배초향) Agastache rugosa 2 (배초향) Q11 Dracocephalum rupestre 1 (벌깨풀) Dracocephalum rupestre 2 (벌깨풀) Meehania urticifolia 1 (벌깨덩굴) Meehania urticifolia 2 (벌깨덩굴) Q12 Lycopus maackianus 1 (애기쉽싸리) Lycopus maackianus 2 (애기쉽싸리) Lycopus maackianus 3 (애기쉽싸리) Lycopus ramosissimus (개쉽싸리) Prunella asiatica (꿀풀) Salvia chanroenica (참배암차즈기) Salvia plebeia (배암차즈기) Perilla frutescens var. frutescens 1 (들깨) Q16 Perilla frutescens var. frutescens 2 (들깨) Mosla chinensis (가는잎산들깨) Mosla dianthera (쥐깨풀) Elsholtzia minima (좀향유) Elsholtzia splendens 1 (꽃향유) Elsholtzia splendens 2 (꽃향유) Q13 Isodon inflexus (산박하) Q14 Q15 Isodon japonicus (방아풀) 0.005 substitutions/site Consortium for the Barcode of Life (CBOL) is an international collaborative effort which aims to develop a mechanism capable of generating a unique genetic barcode for every species of life on earth.

http://www.boldsystems.org/views/login.php • Early 20C - Sera and antisera studies (George Nuttall): gave most important principle in molecular evolution  degree of similarity between genes reflects the strength of the evolutionary relationship between them

• 1950s - Discovery of the molecular structure of DNA (Watson and Crick in 1953)  evolution can be thought of as changes in the structure of DNA! - The first comparison of amino acid sequences (Sanger et al. in 1955)  cattle, pigs, and sheep  the first study to reveal how species differed at the molecular level.

• 1960s - the first model of molecular evolution - recovering the true number of gene sequence changes which have accumulated over time was one of the central tasks of molecular evolution - Concept of “MOLECULAR CLOCK”  still hotly debated, it is one of the most important properties of gene sequence data as it enables us to place evolutionary history within a timeframe. • Sarich and Wilson (1967) showed relationship between human and great apes using the extent of immunological cross- reaction.  The first example of molecular systematics:the use of macromolecules to reconstruct phylogenetic relationships  Opened “molecule vs. morphology” debate. • From the mid-1980s, Nucleotide (DNA) sequence have replaced proteins as the main source of phylogenetic data, particularly since the invention of the polymerase chain reaction (PCR)

• PCR: A technique for the in vitro amplification of specific DNA sequences by the simultaneous primer extension of complementary strands of DNA (>105).

• PCR method was devised and named by Mullis and his colleagues at the Cetus Corporation (Mullis and Faloona, 1987)

• The principle had been described in detail by Khorana et al. (Kleppe et al., 1971) over a decade earlier WITHOUT Taq. polymerase.

• Kary Mullis is a Novel prize winner (1993). DNA polymerization: DNA polymerase extends a primer by using a complementary strand as a template.

5) Mg++ 3) DNA POLYMERASE 2) PRIMER 5’ 3’ T T T G C A A G G G C T A A A C G T T C C C G A G T T C C T C G A G T G T T A C G T T C T T C T C T A G T G T T A C A A A C G T T C C A C G T T C A A A … 3’ 1) TEMPLETE 5’

T A C A 4) dNTP’s pool A dATP T G C dCTP G C A G C G dGTP C T T dTTP G A T G C AT

Newly synthesized strand

5’ T T T G C A A G G G C T C A A G G A G C T C A C A A T G C A A G A A G A G A T C A C A A T G T T T G C A A G G T G C A A A C G T T C C C G A G T T C C T C G A G T G T T A C G T T C T T C T C T A G T G T T A C A A A C G T T C C A C G T T C A A A … 3’ 5’ http://www.youtube.com/watch?v=2KoLnIwoZKU An example of a PCR method

Pre-denature Denature 100 95 ℃ 95 ℃

Extend Final-extend 80 72 ℃ 72 ℃

60 Temp. ℃ Anneal 40 55 ℃

20 Cycle 1 Cycle 2 ••• Cycle 30 Soak 4 ℃ 0 Hold Cycle Hold program program program Hydrothermal vents Taq. DNA Polymerase:

• Most commonly used DNA polymerase for PCR isolated from Thermus aquaticus  Heat stable polymerase

• PCR technique put to practical use by finding of Taq.

• The use of a heat stable enzyme has two major advantages:

1) replenishment after each heating step is not required, thus simplifying the process

2) the enzyme is active at high temperatures, where annealing of the oligonucleotide primers is more specific and DNA synthesis more rapid. • Such is the sensitivity of PCR that it has even been used to obtain DNA from single human hairs and from tissue samples hundreds, or even thousands, of years old.

CSI, Even from FOSSILS! … 1993 An example of fossil DNA: rbcL sequence from 17MY-old Magnolia

DNAs are successfully extracted from Miocene fossils (17MYBP)

KIM, SANGTAE, Douglas E. Soltis, Pamela S. Soltis, and Youngbae Suh. DNA sequences from Miocene fossils: an ndhF sequence of Magnolia latahensis (Magnoliaceae) and an rbcL sequence of Persea pseudocarolinensis (Lauraceae). American Journal of Botany 91: 615-620. (Berry) Brown

Magnolia latahensis Magnolia Krause et al. 2006. Multiplification of the Krause et al. 2006. Multiplification of the mammoth mitochondial genome and mammoth mitochondial genome and the evolution of Elephantidae. Nature the evolution of Elephantidae. Nature 439: 724-727. 439: 724-727. Mitochondrial genome Whole genome from from mammoth! Neandertal! An example of DNA barcoding: Roots in Jeju Volcanic laval tube system http://amborella.net/Media%20exposure/Media%20exposure.htm