DNAbarcodingandphylogenetic analysisofSearsia

MakhadoTshililo,MichellevanderBankandSaneleN.S.ShibaTheAfricanCentreforDNABarcoding(ACDB),DepartmentofBotany&PlantBiotechnology,UniversityofJohannesburg,SouthAfrica

Anacardiaceae also known as the “cashew family” consists of 82genera represented by around 800 species. The largest genus in thisfamily is Rhus L. (senso lato) for which issues around its delimitationhas caused a lot of controversy among taxonomists. Searsia, a newlyaccepted genus within Anacardiaceae, was originally part of the Rhuscomplex but was recently removed along with five other genera. Thegenus Searsia has approximately 120 species and is mainlycharacterised by trifoliate leaves (Yang et al., 2016). It is widelydistributed across Africa with 111 species occurring in southern Africaand 3 species occurring in Asia. The African species all belong to thesection Gerontogeae. Recent phylogenetic studies have definedSearsia as a monophyletic group but these findings need to beconfirmed by conducting a broader sampling of the genus (Yang et al.,2016).

The aim of this study is to investigate the phylogenetic relationshipsamong the seven genera within the Rhus complex as well as resolvethe systematics within Searsia.

MATERIAL&METHODS

DNA extraction, amplification and sequencingDNA was extracted using the 10X CTAB method of Doyle & Doyle(1987). Standard protocols were followed for PCR and sequencingusing the core barcoding regions (rbcLa & matK).

Data analysisA phylogenetic tree was generated using Maximum Parsimony (MP) inPAUP* (Swofford, 2002).

INTRODUCTION

RESULTS&DISCUSSION

The results obtained in this study support the monophyly of African representatives ofSearsia and illustrates the inability of core barcoding regions to provide speciesdelimitation within this genus. Further investigation is required with additionalmarkers (ITS, ETS and trnL-F) to provide better delimitation and resolution within thegenus.

Figure 2 (Clade A) shows high support (99%) for the monophyly ofsouthern African Searsia species which supports the findings of Yanget al. (2016).

Clade B shows high support (100%) for the monophyly of the genusSchinus, having two highly supported sister clades. These findings backGuala (2016) who differentiated S. terebinthifolius from S. areira and S.molle by the presence of pubescent leaflets.

Figure 2 (Clade C) shows weak support (53%) for the monophyly ofLannea. There is also moderate support (80%) for the monophyly ofClade D, with the African species Protorhus nestled within it. Thissupports findings by Von Teichman and Van Wyk (1996) who groupedOzoroa and Protorhus due to morphological similarities.

Results also show no support for the monophyly for the Rhus speciesincluded in this study (Figure 2). In 2007, Yi et al. cited themorphological heterogenousity of Rhus sensu Barkley (1937).

CONCLUSION

Doyle, J.J. & Doyle, J.L. 1987. A rapid isolation procedure for small amounts of leaf tissue. Phytochemical Bulletin, 19: 11-15.Guala, M.S., Lapissonde, M.O., Elder, H.V., van Baren, C.M., Bandoni, A.L. & Dellacassa, E. 2006. Essential Oils in FoodPreservation, Flavor and Safety.Swofford, D.L. 2002. PAUP* - Phylogenetic Analysis Using Parsimony (*and Other Methods). Version 4. SinauerAssociates, Sunderland, MA.Yang, Y., Meng, Y., Wen, J., Sun, H. & Nie, Z. 2016. Phylogentic analyses of Searsia (Anacardiaceae) from eastern Asia andits biogeographic disjunction of its African relatives. South African Journal of Botany, 106: 129-136.Yi, T., Miller, A. J. & Wen, J. 2007 . Phylogeny of Rhus (Anacardiaceae) based on sequences of nuclear NIA-i3 intron andchloroplast trnCtrnD. Systematic Botany 32: 379-391.Von Teichman, I. & Van Wyk, A. E. 1996. Taxonomic significance of pericarp and seed structure in Heeria argentea(Thunb.) Meisn. (Anacardiaceae), including reference to pachychalazy and recalcitrance. Botanical Journal of the LinneanSociety, 122: 335-352.

REFERENCES

CotinusMill.

Cotinuscoggygria

ToxicodendronMill.

Toxicodendronradicans

Lannea Engl.

Lannea schweinfurthii

Schinus L.

Schinus terebinthifolia

Rhus L.(S.str.)

RhuschinensisSearsia dentata

Searsia F.A.Barkley

SamplingA total of 205 taxa were included in the study. These includerepresentatives of the genera: Searsia, Rhus, Lannea, Schinus, Cotinusand Toxicodendron (Figure 2).

FIGURE 2: Bootstrap consensus tree generated using PAUP of the combined plastid datasethighlighting Clades A – D, representatives of genus Rhus as well as some representatives of othergenera included in the study.

FIGURE1: DistributionmapoffieldcollectedSearsiasamples.

ACKNOWLEDGEMENTS

PosterID:828

Background. The family Anacardiaceae includes approximately 800 species in 82 genera. Rhus L. is the largest and most widely distributed genus in Anacardiaceae. However, controversy hassurrounded the delimitation of the genus. Searsia F.A. Barkley was originally included into the Rhus complex but recent molecular studies have separated it along with six other genera from the Rhuscomplex and recognised them as separate genera. The genus Searsia, which is the focus of the current study, is represented by approximately 120 species and is widely distributed in Africa with onlythree species currently known from Asia. Here we include an extensive sampling and molecular analyses of Searsia species across its distribution range in Africa to access the relationships within thegenus. Results. In total 205 taxa, representing 35 species of Searsia, were sequenced for the core barcoding regions (matK and rbcLa) along with additional markers, i.e. ITS, ETS, trnL-F and ndhF.Significance. This study contributes a first large sampling of Searsia. An infrageneric classification for the genus will be presented.

ABSTRACT

Searsia chirendensis OM1987

Searsia ciliata Hahn2981

Searsia pyroides OM713 1Searsia tumulicola OM3907Searsia tumulicola var meeseana OM3818

Searsia zeyheri OM2256Searsia leptodictya RBN205Searsia leptodictya RL1645

Searsia leptodictya RL1655

Searsia tenuinervis Hahn2999

Searsia nebulosa Abbott9106

Searsia pyroides var integrifolia OM2477

Searsia discolor OM3911

Searsia fastigiata Abbott9135

Searsia lancea OM1942

Searsia lancea PG0001

Searsia pendulina OM1984

Searsia pondoensis Burrows10242

Searsia pyroides OM1236

Searsia transvaalensis OM0943

Searsia transvaalensis RL1427

Searsia tumulicola var tumulicola OM2028

Searsia tumulicola var tum

ulicola OM3813

Searsia wilmsii OM

3910

Searsia gueinzii OM

0248

Searsia gueinzii OM

0265

Searsia gueinzii OM

0336

Searsia gueinzii OM

3371

Searsia natalensis OM

2655

Searsia pentheri OM

0942

Searsia pentheri OM

0945

Searsia longispina AM0243

Searsia pallens OM

1976

Searsia undulata O

M2940

Searsia lucida M

WC

05809

Searsia crenata O

M1986

Searsia crenata O

M3209

Sea

rsia

neb

ulos

a O

M22

85

Sea

rsia

aco

cksi

i Abb

ott9

154

Sea

rsia

chi

rinde

nsis

OM

2284

Sear

sia

angu

stifo

lia O

M28

47

Sear

sia

diss

ecta

JL

802

4

Sear

sia

inci

sa v

ar in

cisa

OM

3059

Sear

sia

luci

da A

bbot

t923

0

Sear

sia

tom

ento

sa S

M 7

31 3

Sorin

deia

jugl

andi

folia

PM

4827

Sorin

deia

jugl

andi

folia

PM

5106

Sorin

deia

jugl

andi

folia

PM

4949

Loxo

styli

s al

ata

OM

1827

Ozoro

a ob

ovat

a su

bsp

ellipt

ica O

M24

25

Ozoro

a ob

ovat

a su

bsp

ellipt

ica O

M25

11

Ozoro

a ob

ovat

a su

bsp

ellipt

ica O

M2733

Protor

hus l

ongif

olia M

WC28

088

Protorh

us lo

ngifo

lia O

M1764

Ozoroa

barab

etoren

s Burr

ows0

8069

Ozoroa

engle

rii OM11

54

Ozoroa englerii

OM1169

Ozoroa englerii RL1277

Ozoroa insignis s

ubsp reticu

lata OM3580

Ozoroa paniculosa OM1948

Ozoroa sphaerocarpa OM0940

Ozoroa sphaerocarpa OM1106

Heeria argentea PG0016

Rhus thouarsii AY594484Schinus areira AY594488Schinus molle DMP145Schinus molle DMP200Schinus molle DMP224Schinus molle DMP231

Schinus molle JG052Schinus terebinthifolius BS0098

Schinus terebinthifolius DMP237

Schinus terebinthifolius DMP256

Schinus terebinthifolius OM1982

Schinus terebinthifolius PPRI 0027

Mosquitoxylum jamaicens AY594490

Rhus aromatica AY594486

Rhus aromatica AY594494

Rhus copallina AY594485

Rhus hirta AP184

Rhus hypoleuca HQ427342

Astronium graveolens AY594492

Schinopsis brasiliensis AY594477

Thyrsodium puberulum FJ514664

Thyrsodium puberulum FJ514723

Toxicodendron radicans AP422

Toxicodendron rydbergii BAB 0354

Amphipterygium adstring AY594458

Comocladia glabra HM446677

Cotinus coggygria HE966907

Lithrea molleoides AY594470

Loxopterygium huasango AY594471

Pachycormus discolor AY594493

Toxicodendron sylvestre HQ415319

Toxicodendron vernix AY594495

Trichoscypha klainei Tricklai PM5051

Trichoscypha preussii Tricpreu PM5027

Trichoscypha patens Tricpate PM4897

Trichoscypha patens Tricpate PM5076

Trichoscypha acuminata M

PADN

0001

Trichoscypha acuminata M

PADN

0004

Trichoscypha acuminata M

PADN

0277

Trichoscypha arborea MPAD

N0003

Anacardium excelsum

GQ

981932Anacardium

occidentale AY594459A

nacardium occidentale G

H0152

Anacardium

occidentale OM

3645G

luta wallichii A

Y594468

Man

gife

ra in

dica

AY

5944

72

Fegi

man

ra a

frica

na A

Y59

4489

Sem

ecar

pus

aust

ralie

nsi A

Y594

479

Dob

inea

vul

garis

AY

5944

66Pe

gia

nitid

a AY

5944

73Sp

ondi

as m

ombi

n AY

5944

80La

nnea

ant

isco

rbut

ica

OM

2704

Lann

ea d

isco

lor O

M33

22La

nnea

edu

lis O

M19

71La

nnea

edu

lis O

M19

91

Lann

ea s

chwe

infu

rthii O

M34

13

Lann

ea s

chwe

infu

thii O

M13

55

Lann

ea s

chwe

infu

thii O

M24

46

Lann

ea sc

hwein

futh

ii OM

2505

Lann

ea sh

impe

ri OM

2521

Draco

ntom

elon

dao

AY59

4467

Pleiog

ynium

timor

iense

AY59

4474

Pleiog

ynium

timori

ense

AY72

4340

Harpep

hyllu

m caffru

m BS00

58

Harpep

hyllu

m caffru

m DMP20

4

Harpephyllum ca

ffrum O

M1555

Tapirira bethanniana AY594481

Tapirira obtusa AY594482

Tapirira obtusa JQ626383

Choerospondias axillari AY594463

Choerospondias axillari HQ427341

Antrocaryon amazonicum AY594460

Cyrtocarpa procera AY594464

Pseudospondias microcarpa PM5455

Sclerocarya birrea AY594478

Sclerocarya birrea subsp caffra OM0278

Sclerocarya birrea subsp caffra OM0498

Sclerocarya birrea subsp caffra RL1117

Commiphora africana OM0334

Commiphora glandulosa RBN160

Commiphora schimperi OM1361100

71

9589

50

65

88 99

59

77

50

93 62

88

7256

64

62

95

9963

83

8085

90

100

98

85

73

5999

82

75

97

97

9676 89

5175

97

81

53

85

54

76

89

65

CladeC

CladeA

CladeD

CladeB

KEY

Rhus Representatives