Generic circumscription and relationships of southern African representatives of Hypoxis and allies (Hypoxidaceae, Asparagales)

Sanele Shiba, Michelle van der BankTheAfricanCentreforDNABarcoding(ACDB),DepartmentofBotany&PlantBiotechnology,UniversityofJohannesburg,SouthAfrica

ABSTRACTBackground. The popularity of Hypoxis L. as a medicinal plant has resulted in unsustainable harvesting practices of rhizomes from the wild. This exploitation has accelerated theneed for correct species names and circumscriptions. However, species delimitation in Hypoxis is problematic and despite several attempts, the systematics of the genus remainslargely unresolved. This is mainly due to the lack of distinct morphological boundaries separating species. Here, we explore the generic circumscriptions of Hypoxis and alliedgenera within Hypoxidaceae using five plastid DNA regions (rbcLa, matK, trnL-F, ycf1 and trnS-G). Results. Findings from our study indicate that Hypoxis is not monophyletic andis represented by at least three distinct lineages. Using the phylogeny produced from the study Hypoxis samples sold at traditional medicinal markets in South Africa could besuccessfully identify. Significance. We proposed to transfer Rhodohypoxis Nel back into Hypoxis as well as the newly described genus Sinocurculigo from China to CurculigoGaertn.

Hypoxis L. represents the largest genus in theHypoxidaceae R. Br. with about 90 species (Kocyan et al.,2011).

The popularity of Hypoxis as a medicinal plant has resultedin unsustainable harvesting practices of rhizomes from thewild. This exploitation has accelerated the need for correctspecies names and circumscriptions (Zimudzi & Kativu,2006; Singh, 2009).

However, species delimitation in Hypoxis is problematicand despite several attempts, the systematics of the genusremains largely unresolved. This is mainly due to the lackof distinct morphological boundaries separating species(Wiland-Szymanska, 2009).

Aim: To resolve the phylogenetic relationships withinHypoxidaceae as well as producing a species levelphylogeny for the approximately 90 species of the genusHypoxis.

Doyle, J.J. & Doyle, J.L., 1987. A rapid isolation procedure for small amounts of leaf tissue. PhytochemicalBulletin 19: 11-15.

Swofford, D. L. (2002) PAUP*. Phylogenetic Analysis Using Parsimony (*and Other Methods). Version 4. SinauerAssociates, Sunderland, MA.

Kocyan, A., Snijman, D.A., Forest, F., Devey, D.S., Freudenstein, J. V., Wiland-Szymańska, J., Chase, M.W. &Rudall, P.J. 2011. Molecular phylogenetics of Hypoxidaceae - Evidence from plastid DNA data andinferences on morphology and biogeography. Molecular Phylogenetics and Evolution. 60(1):122-136. DOI:10.1016/j.ympev.2011.02.021.

Kocyan, A. & Wiland-Szymanska, J. 2016. Friedmannia: a new genus from the Seychelles and the beginning of ageneric realignment of Curculigo (Hypoxidaceae). Phytotaxa. 283(1): 54-64.

Snijman, D.A. & Kocyan, A. (2013) The genus Pauridia (Hypoxidaceae) amplified to include Hypoxis sect. Ianthe,Saniella and Spiloxene, with revised nomenclature and typification. Phytotaxa 116: 19-33.

Wiland-Szymanska, J. 2009. The Genus Hypoxis L. (Hypoxidaceae) in the East Tropical Africa: variability,distribution and conservation status. Biodiv. Res. Conserv. 14: 1-129.

Zimudzi, C. & Kativu, S. 2006. The African potato (Hypoxis hemerocallidea): identity and status in Zimbabwe.Kirkia. 18(pt. 2): 175-176.

MATERIAL & METHODSSamplingA total of 93 taxa were included in the data set. Sampleswere either collected in the field, sourced from herbariumvouchers or downloaded from GenBank.

DNA extraction, amplification and sequencingDNA was extracted using the 10X CTAB method of Doyle& Doyle (1987). Standard protocols were followed for PCRand sequencing, using three plastid regions (rbcLa, trnL-Fand trnS-G).

Data analysisA phylogenetic tree was generated using MaximumParsimony (MP) in PAUP* (Swofford, 2002).

INTRODUCTION

FIGURE 1: Representatives of the southern African genera of Hypoxidaceae

RESULTS & DISCUSSION

FIGURE 2: Phylogenetic tree for the family Hypoxidaceae based on the combined data set showing four well supported clades.

REFERENCES

ACKNOWLEDGEMENTS

CONCLUSIONAlthough the backbone of the phylogeny was not resolved, four well supportedclades could be identified (Clades A – D). Based on the results we propose totransfer Rhodohypoxis back into Hypoxis as well as the newly described genusSinocurculigo to Curculigo.

In Figure 2, there is a 100% support for the monophyly of the familyHypoxidaceae, with four major clades being identified.

There is 100% support for Clade A. The genus Rhodohypoxis was established in1914 to accommodate the Hypoxis plants with white, pink or red flowers. The onlyother morphological difference between Rodohypoxis and Hypoxis is theconcealed stamens and style in Rhodohypoxis.

Within Clade B (99%), Molineria is nested within Curculigo. Since Curculigo hasnomenclatural priority, Molineria species should be transferred to Curculigo withCurculigo seychellensis being the exception. Clade B also suggests that thenewly described genus Sinocurculigo from China should be transferred to thegenus Curculigo. Curculigo seychellensis forms a monophyletic Seychelles cladewith Hypoxidia maheensis and Hypoxidia rhizophylla. A new monotypic genus(Friedmannia), endemic to the Seychelles was proposed to accommodate thesespecies (Kocyan & Wiland-Szymanska, 2016).

There is a high support (100%) for the monophyly of the Empodium species(Clade C) included in this study. There is a high support (85%) for the cladecontaining Pauridia, Spiloxene, Saniella and Hypoxis section Ianthe (Clade D).This supports findings by Snijman and Kocyan (2013) who transferred all thespecies of Spiloxene, Saniella and Hypoxis section Ianthe to Pauridia.

Hypoxishirsuta Pauridia aquatica

Saniella verna Spiloxene capensis

Empodium gloriosum Rhodohypoxisbaurii

Astelia alpina KocyanAK981010 1 01 Chase1103Astelia banksii Chase1072Blandfordia nobilis Chase2835Blandfordia punicea AK981013 1 01 Chase519

Curculigo erecta Leiden932789

Curculigo racemosa Leiden970647

Molineria latifolia KocyanAK981023 1 03

Curculigo finlaysoniana CookRixSchneller23

Curculigo orchioides Janarthanam TCMK696

Curculigo pilosa subsp major Ehrich266

Curculigo sinensis KunmingBotanicalGarden

Molineria crassifolia KunmingBotanicalGarden

Molineria capitulata KocyanAK981228 1 01

Sinocurculigo taishanica

Molineria aff latifolia BGM

unich98 3407 Bogner2650

Molineria villosa KocyanAK971031 1 03

Curculigo seychellensis Bonn

Empodium

elongatum Snijm

an1908

Em

podium sp. D

esmet2979

Em

podium flexile S

nijman1706E

mpo

dium

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BG

Mun

ich

Em

podi

um v

erat

rifol

ium

Koc

yanA

K99

0309

1 0

3

Hyp

oxis

acu

min

ata

BNR

H00

0316

1

Hyp

oxis

acu

min

ata

BT13

09

Hyp

oxis

acu

min

ata

BT13

07

Hyp

oxis

ang

ustif

olia

var

. ang

astif

olia

BN

RH

0006

307

Hypo

xis c

osta

ta B

NRH0

0092

68

Hypo

xis h

emer

ocal

lidea

BNR

H000

3965

Hypo

xis in

terje

cta B

NRH0

0091

43

Hypox

is kra

ussia

na B

NRH0006

312

Hypox

is lon

gifoli

a BNRH00

0872

5

Hypox

is lon

gifoli

a BNRH00

0629

7

Hypoxis

obtusa BNRH0003269

Hypoxis parvif

olia BNRH0009285

Hypoxis rigidula var. ri

gidula BNRH0006261

Hypoxis rigidula var. ri

gidula BNRH0006262

Hypoxis colchicifolia BNRH0006375

Hypoxis costata BT1306

Hypoxis galpinii BNRH0006328Hypoxis galpinii BT1305

Hypoxis rigidula var. pilosissima BNRH0006534Hypoxis hemerocallidea BGBerlin635807Hypoxis hemerocallidea BNRH0006241

Hypoxis rigidula cf. rigidula BB14828

Hypoxis parvifolia BNRH0006219

Hypoxis sp. Bonn126

Hypoxis angustifolia KewGardens1973 2957

Hypoxis angustifolia var. buchananii BNRH0009208

Hypoxis argentea BNRH0006257

Hypoxis argentea var. argentea BNRH0002746

Hypoxis aurea KocyanAK426

Hypoxis curtissii Chase108

Hypoxis hirsuta Wipf2209

Hypoxis juncea WhittenK

Hypoxis decumbens KewGardens1976 6038

Hypoxis filiformis Basel KocyanAK438

Hypoxis filiformis BNRH0009185

Hypoxis hygrometrica BGBerlin43336

Hypoxis limicola BNRH0006314

Hypoxis limicola BT1311

Rodohypoxis baurii var. baurii BSP10497

Rodohypoxis baurii var. confecta BN

RH

0023126H

ypoxis parvula NH

556

Rhodohypoxis baurii BG

Basel KocyanAK990522 1 02R

hodohypoxis baurii var. baurii Singh554

Hypoxis setosa ZS

S29 156

Hyp

oxis

vill

osa

Kew

Gar

dens

1972

394

7H

ypox

is g

labe

lla Y

1498

9H

ypox

is o

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enta

lis K

ew22

34B

Sani

ella

occ

iden

tali

Snijm

an20

59Sa

niel

la v

erna

BG

Edin

burg

h199

7 32

02

Spilo

xene

cap

ensi

s Ar

b728

Spilo

xene

alb

a Sn

ijman

2106

Spilo

xene

aqu

atica

Sni

jman

2113

Spilo

xene

mon

ophy

lla P

ater

son

Jone

s913

Spilo

xene

nan

a Sn

ijman

1865

a

Spilox

ene p

usilla

Snij

man18

60

Spilox

ene s

chlec

hteri P

arker4

69

Paurid

ia lon

gitub

a Koc

yanA

K9806

24 1

01

Pauridia longituba Snijm

an1440

Pauridia minuta Snijman1812

Spiloxene flaccida Goldblatt Manning9585

Spiloxene trifurcillata McMaster208084

Spiloxene linearis Snijman1754

Spiloxene minuta Parker452

Spiloxene scullyi Harrower3271

Lanaria lanata Orchard342

Milligania stylosa Kocyan981011 3 01 Chase511

Collospermum hastatum BG875 767

5289100

95

96

100

99

99

85 92

62

61 65

82

95

100100

100

916210

0

100

86

58

79

72

59

94

72

50 87

85

86

55

6886

53

9377

9674

Bootstrap consensus tree

Curculigo scorzonerifolia Gorts et al. 449

Curculigo seychellensis Kuffer 26 11 2002

Hypoxidia m

aheensis Kuffer 26 11 2002

Hypoxidia rhizophylla Kuffer 26 11 2002

Clade A

Clade CClade B

Clade D

HypoxidaceaeKEY

PosterID741