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Diversity Analysis in AmorphophallusUsing Isozyme MarkersShirly Raichal Anila, E. A. Sirila & S. Suhara Beevya

a Department of Botany, University of Kerala, Kariavattom,Thiruvananthapuram, IndiaAccepted author version posted online: 22 Jul 2013.Publishedonline: 27 Aug 2014.

To cite this article: Shirly Raichal Anil, E. A. Siril & S. Suhara Beevy (2014) Diversity Analysis inAmorphophallus Using Isozyme Markers, International Journal of Vegetable Science, 20:4, 305-321,DOI: 10.1080/19315260.2013.803509

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International Journal of Vegetable Science, 20:305–321, 2014Copyright © Taylor & Francis Group, LLCISSN: 1931-5260 print / 1931-5279 onlineDOI: 10.1080/19315260.2013.803509

Diversity Analysis inAmorphophallus UsingIsozyme Markers

Shirly Raichal Anil, E. A. Siril, and S. Suhara Beevy

Department of Botany, University of Kerala, Kariavattom, Thiruvananthapuram,India

A wide range of morphological variability exists within accessions of Amorphophallus.A study to describe variability and genetic structure in the genus was undertakento illustrate genetic diversity. Eight species of Amorphophallus, including seven wildspecies, and 18 morphotypes of A. paeoniifolius (Dennst.) Nicolson including two cul-tivated Elephant foot yam [A. paeoniifolius (Dennst.) Nicolson var. campanulatus(Decne.) Sivad.], cv. Karunaikizhangu (T10), and cv. Gajendra (GJ), and the wild form[A. paeoniifolius Nicolson var. paeoniifolius (Decne.) Sivad.], were collected from fourbiogeographic zones in India. The materials were studied using isozyme variation at12 loci for the enzyme systems esterase, polyphenol oxidase or laccase, malate dehy-drogenase, catalase, and diaphorase. There was little variation in number of allelesper locus (range 1.00 to 1.67). Mean percentage of polymorphic loci of the eight specieswas 58.33% polymorphism. Mean expected heterozygosity of populations ranged from0.11 to 0.375. There was high genetic divergence across populations. A dendrogramof populations was developed using genetic distances among taxa. Grouping of popu-lations consistent with taxonomic species, determined in cluster analysis, confirmedthat the eight species studied were natural, with clear boundaries between them,except for population P19 (which belongs to A. paeoniifolius var. paeoniifolius, fromPuttur, Karnataka), which formed a separate cluster with A. bulbifer (Roxb.) Bl. andA. hohenackeri (Schott.) Engl. et Gehrm. The T10 population was well apart in a smallcluster within the larger group of the A. paeoniifolius group and well apart from GJ.Based on Nei’s genetic distance and the dendrogram, the most diverged population wasof A. smithsonianus Sivad. Morphotypes of A. paeoniifolius clustered together, indicat-ing their species identity in spite of morphological variability. The excess heterozygosityobserved may be the result of out-crossing along with asexual means of reproduc-tion prevalent in the genus. A small amount of inbreeding exists in the genus due tobiparental inbreeding. Thus, a mixed mating system along with vegetative propagationcontributes to variability and wide adaptation.

Address correspondence to Shirly Raichal Anil, Central Tuber CropsResearch Institute, Sreekariyam, Thiruvananthapuram 695 017, India. E-mail:shirlyanil@yahoo.comColor versions of one or more of the figures in this article can be found online atwww.tandfonline.com/wijv.

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306 S. R. Anil et al.

Keywords Amorphophallus paeoniifolius complex, F-statistic, Inbreeding coefficient,Morphotypes, Wild species complex.

Several species in the genus Amorphophallus Blume ex Decne. have ediblerhizomes (Grob et al., 2002; Jaleel et al., 2011; Sedayu et al., 2010; Van derHam et al., 2005). Many species are rare or endangered. The corm tubersof Elephant foot yam [A. paeoniifolius (Dennst.) Nicolson var. campanulatus(Decne.) Sivad.] and A. bulbifer (Roxb.) (Bl.), are rich in starch and used as astaple food and have emerged as a cash crop. These species require minimalinputs and exhibit a high response to fertilizers. Increased production and useof Amorphophallus can improve smallholders’ income (Misra et al., 2001). Thewild species possess many medicinal properties (Anil et al., 2011).

Preservation and availability of a high level of genetic diversity of theseresources is required (Given, 1987). Information on genetic patterns needs tobe gathered. Despite the economic importance of Amorphophallus, knowledgeof its genetic diversity and differentiation is scarce. To improve the informationbase it is crucial to collect available genetic resources and characterize geneticdiversity of accessions.

Molecular polymorphism can be used to characterize genetic diversity ofendangered species (Bouza et al., 2002). Diversity analysis is often impor-tant when considering in situ and ex situ conservation strategies (Sosa, 2001).Genetic variation represents the evolutionary potential of a species (Wright,1978). Species-specific traits such as life cycle, breeding system, seed dispersalmechanism, pollinators, and geographical range should affect the level and dis-tribution of genetic diversity within plant species (Hamrick and Godt, 1996),with breeding system appearing to have the greatest influence on geneticdiversity and its distribution (Hamrick and Godt, 1989). Genetic variabilityin cultivars and wild relatives of cultivated crops can irrecoverably disappearif left unnoticed, as reported by Dansi et al. (2000) in yam (Dioscorea alataL.). Survey, extensive germplasm exploration, collection, characterization, andconservation are needed to overcome genetic erosion.

Isozyme variability analysis is useful in genetic evaluation and is used toestimate the genetic diversity of many crops (Bhandari et al., 2006; Freitaset al., 2000; Jain et al., 2006). Isozyme variations can assist in tracing the evo-lutionary line of a particular species and its relatives. Enzyme electrophoresishas been used to describe the genetic structure and diversity of many taxa(Massey and Hamrick, 1998). However, no information is available on conser-vation genetics, population genetics, and systematic evolution using isozymeanalysis in Amorphophallus.

Banding patterns can be used to assess genetic variation betweenAmorphophallus spp. Morphological variability analysis of various accessionsof A. paeoniifolius was indicated, especially in the petiole pattern (Anil et al.,2011). The current project was undertaken to measure genetic diversity ofAmorphophallus on the basis of isozyme pattern.

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Diversity Analysis in Amorphophallus 307

MATERIALS AND METHODS

Plant Material and Isozyme ExtractionA random stratified sampling was made in selected biogeographic zones of

India where Amorphophallus is distributed. The area includes southwesternGhats (Kerala and Karnataka states, India) where many endemic species andmorphotypes (morphological variants) of A. paeoniifolius are available. Thesemi-arid zones are represented by Gujarat State, India, the Gangetic Plainsis represented by Bihar State, and the Deccan Plateau is represented by TamilNadu and Andhra Pradesh. A total of 250 individuals from 25 populationsof eight species, including seven wild species of Amorphophallus and 18 pop-ulations of A. paeoniifolius, which include its morphotypes, were selected.Wild species of A. bonaccordensis Sivad. Mohanan & Rajkumar, A. smithso-nianus Sivad., and A. hohenackeri (Schott.) Engl. et Gehrm., unlike other wildspecies, are endemic to Western Ghats (Table 1). Amorphophallus paeoniifoliusis widespread compared to all other species. Individual plants were grown inearthen pots (25 cm diameter) filled with a mix of sand : soil : cow manure(1:1:1) during March–April. Plants were irrigated by hand daily in the morn-ing. No fertilizer or pesticides were applied and potted plants were maintainedin an open space in the Botanical Garden of University of Kerala. When plantsdefoliated in October they were harvested.

Approximately 200–300 mg of young leaf tissue (unopened leaf) wasground using a precooled mortar and pestle to which 1 mL of extractionbuffer (0.10 M Tris-HCl, pH 7.4) and 4% polyvinyl polypyrrolidone was added.The extract was centrifuged at 17,700 × g and the supernatant (sample)was stored at −80◦C. Samples were mixed with equal volumes of loadingdye containing 0.5 M Tris HCl (pH 6.8), sucrose (16% w/w), distilled water(30% v/v), bromophenol blue (0.1% w/v), and β-mercaptoethanol (20% v/v)prior to electrophoresis. The mixture was loaded (50 µL) in wells for nativepolyacrylamide gel (7.5%) electrophoresis.

The enzyme systems alcohol dehydrogenase, superoxide dismutase,polyphenol oxidase or laccase, acid phosphatase, aspartate amino transferase,peroxidase, malate dehydrogenase, esterase (EST), NADH diaphorase, andcatalase (CAT) were studied. The enzyme systems polyphenol oxidase or lac-case (E.C. 1.10.3.1), EST (E.C. 3.1.1.72), NADH diaphorase (E.C. 1.8.1.4), CAT(E.C. 1.11.1.6), and malate dehydrogenase (E.C. 1.1.1.37), which showed con-sistent banding patterns, were considered for further analysis. Isozymes wereseparated into discrete bands by vertical slab (10 × 10 cm, 1 mm thick) gelelectrophoresis (Model TV-100, Scie-Plas, Cambridge, UK) with the resolv-ing gel containing 7.5% (w/v) acrylamide using a Tris-glycine electrophoresisbuffer (pH 8.3) system. Gels were run at constant voltage (100 V) at 4◦Cuntil the tracking dye migrated to the bottom of the gel. Electrophoresis gels

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Tab

le1:

De

tails

of

diff

ere

nt

ac

ce

ssio

ns

eva

lua

ted

usin

giso

zym

em

ark

ers

.

Ac

ce

ssio

nc

od

e

Pla

ce

of

co

llec

tion/

orig

inG

enu

s/sp

ec

ies

Sta

tus:

wild

or

cul

tiva

ted

Latit

ude/

long

itud

eA

ltitu

de

(m)

Ha

bita

t

V2

Vitt

al,

DK

,KA

RA

.ho

he

na

cke

riW

ild12

◦ 47′ N

/75

◦ 6′ E

84O

pe

nfo

rest

un

de

rtr

ee

sV

3V

itta

l,D

K,K

AR

A.b

ulb

ifer

Wild

12◦ 4

7′ N/75

◦ 6′ E

84O

pe

nfo

rest

un

de

rtr

ee

sT2

Vith

ura

,TV

MA

.bo

na

cc

ord

en

sisW

ild8◦ 6

0′ N/77

◦ 15′ E

102.

9O

pe

nu

nd

istu

rbe

dfo

rest

T3V

ithu

ra,T

VM

A.s

mith

son

ian

us

Wild

8◦ 60′ N

/77

◦ 15′ E

91.2

Inte

riorf

ore

sto

nro

cky

cre

vic

es

A.c

(TC

R-1

35)

NBP

GR

A.c

om

mu

tatu

sW

ild—

Inte

riorf

ore

stA

.syl

(TC

R-1

18)

NBP

GR

A.s

ylva

ticu

sW

ild—

Inte

riorf

ore

stA

.d.(

TCR

-105

)N

BPG

RA

.du

biu

sW

ild—

Dist

urb

ed

op

en

pla

ce

V1

Vitt

al,

DK

A.p

ae

on

iifo

lius

var.

pa

eo

niif

oliu

sW

ild12

◦ 47′ N

/75

◦ 6′ E

84D

istu

rbe

do

ute

rfo

rest

are

aV

4-P

Vitt

al,

DK

A.p

ae

on

iifo

lius

var.

pa

eo

niif

oliu

sW

ild12

◦ 47′ N

/75

◦ 6′ E

84D

istu

rbe

do

ute

rfo

rest

are

aT1

Vith

ura

,TV

MA

.pa

eo

niif

oliu

sva

r.p

ae

on

iifo

lius

Wild

8◦ 60′ N

/77

◦ 15′ E

102.

9O

pe

nu

nd

istu

rbe

dfo

rest

M1

Mo

ozh

iya

r,P

TAA

.pa

eo

niif

oliu

sva

r.p

ae

on

iifo

lius

Wild

9◦ 27′ N

/77

◦E

67.8

Un

dist

urb

ed

fore

stn

ea

rro

ad

side

P1

Pu

ttu

r,D

KA

.pa

eo

niif

oliu

sva

r.p

ae

on

iifo

lius

Wild

12◦ 4

4′ N/75

◦ 13′ E

81D

istu

rbe

do

pe

nfo

rest

K3-

1Th

rissu

rA

.pa

eo

niif

oliu

sva

r.p

ae

on

iifo

lius

Wild

10◦ 3

1′ N/76

◦ 20′ E

47.1

Ro

ad

side

ne

arf

ore

st

K3-

2Th

rissu

rA

.pa

eo

niif

oliu

sva

r.p

ae

on

iifo

lius

Wild

10◦ 3

1′ N/76

◦ 20′ E

47.1

Ro

ad

side

ne

arf

ore

st

K3-

3Th

rissu

rA

.pa

eo

niif

oliu

sva

r.p

ae

on

iifo

lius

Wild

10◦ 3

1′ N/76

◦ 20′ E

47.1

Ro

ad

side

ne

arf

ore

st

P17

Pu

ttu

r,D

K,K

AR

A.p

ae

on

iifo

lius

var.

pa

eo

niif

oliu

sW

ild12

◦ 44′ N

/75

◦ 13′ E

81D

istu

rbe

do

pe

nfo

rest

308

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T10

Na

ge

rco

il,TN

A.p

ae

on

iifo

lius

var.

ca

mp

an

ula

tus

Cu

ltiva

ted

8◦ 30′ N

/77

◦ 30′ E

90C

laye

yso

il

G3

Na

vasa

ri,G

UJ

A.p

ae

on

iifo

lius

var.

pa

eo

niif

oliu

sW

ild20

◦ 57′ N

/72

◦ 9′ E

14.1

Dist

urb

ed

fore

stn

ea

rh

um

an

ha

bita

tion

Ga

jen

dra

(GJ)

Kovv

ur,

AP

A.p

ae

on

iifo

lius

var.

ca

mp

an

ula

tus

(te

lea

sed

varie

tyu

nd

ere

xte

nsiv

ec

ulti

vatio

nin

Ind

ia)

Cu

ltiva

ted

17◦ 1

′ N/81

◦ 43′ E

10.8

Cu

ltiva

ted

B1V

aish

ali,

BIH

A.p

ae

on

iifo

lius

var.

pa

eo

niif

oliu

sW

ild24

◦ 52′ N

/85

◦ 15′ E

62.1

Ne

arr

ive

rba

nk

P5

Pu

ttu

r,D

KA

.pa

eo

niif

oliu

sva

r.p

ae

on

iifo

lius

Wild

12◦ 4

4′ N/75

◦ 13′ E

81D

istu

rbe

do

pe

nfo

rest

K4

Thris

sur,

KL

A.p

ae

on

iifo

lius

var.

pa

eo

niif

oliu

sW

ild10

◦ 30′ N

/76

◦ 20′ E

47O

pe

nfo

rest

ne

ar

hu

ma

nh

ab

itatio

nP

18P

utt

ur,

DK

,KA

RA

.pa

eo

niif

oliu

sva

r.p

ae

on

iifo

lius

Wild

12◦ 4

4′ N/75

◦ 13′ E

81D

istu

rbe

do

pe

nfo

rest

P19

Pu

ttu

r,D

K,K

AR

A.p

ae

on

iifo

lius

var.

pa

eo

niif

oliu

sW

ild12

◦ 44′ N

/75

◦ 13′ E

81D

istu

rbe

do

pe

nfo

rest

K5

Thris

sur

A.p

ae

on

iifo

lius

var.

pa

eo

niif

oliu

sW

ild10

◦ 30′ N

/76

◦ 20′ E

47O

pe

nfo

rest

ne

ar

hu

ma

nh

ab

itatio

n

DK

=D

aks

hin

aK

an

na

da

,TV

M=

Thiru

van

an

tha

pu

ram

,N

BPG

R=

Na

tion

alB

ure

au

of

Pla

nt

Ge

ne

ticR

eso

urc

es,

PTA

=Pa

tha

na

mth

itta

,K

AR

=K

arn

ata

ka,T

N=

Tam

ilN

ad

u,G

UJ

=G

uja

rat,

AP

=A

nd

hra

Pra

de

sh,B

IH=

Bih

ar,

KL

=Ke

rala

.

309

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310 S. R. Anil et al.

were rinsed with distilled water and visualized by enzyme activity staining inaccordance with Vallejos (1983). Genetic interpretation of enzyme phenotypeswas made on the basis of differences in zymogram profile (Wendel and Percy,1990) and loci were designated accordingly.

Electrophoretic Patterns and Data AnalysisElectrophoretic bands corresponding to multiple alleles at each locus were

increased numerically starting with 1 for order of mobility from slow to fast,and alleles increased alphabetically beginning with A. Known active sub-unit composition and minimal conserved number in diploid plants for eachenzyme were used to infer numbers of loci and alleles (Huang et al., 1994a,1994b; Wang, 1998; Weeden and Wendel, 1989). Chi-square was used to assessheterogeneity of allele frequencies for each locus among populations.

Data on isozyme expression—that is, allelic data with regard to variouspopulations—were analyzed using genetic data analysis software (POPGENE,ver. 1.0, University of Alberta, Alberta, Canada; Yeh and Boyle, 1997). Forall populations, standard genetic variability measures—that is, percentage ofpolymorphic loci (P), mean observed number of alleles per locus (A), mean genediversity or Shannon’s information index (I), and average observed (Ho) andexpected (He) heterozygosity (genetic diversity)—were computed. Deviationof genotype frequencies from the Hardy-Weinberg equilibrium was estimatedfor each locus by chi-square. Genetic distances among populations and taxawere analyzed as described by Nei (1978). POPGENE 1.0 was used to com-pute Nei’s gene diversity statistics (Nei, 1972, 1978), and NTSYS-pc (Rohlf,1998) to obtain dendrograms using genetic distances of Nei (1978) and theunweighted pair group method of mathematical average (Sneath and Sokal,1973).

Loci were considered polymorphic if more than one allele was detected.Analysis was performed by producing two groups, one with 18 pop-ulations of A. paeoniifolius that included cultivated species and wildrelatives/morphotypes of A. paeoniifolius and a second group with all remain-ing seven wild species. Observed heterozygosity (Ho) and expected heterozy-gosity (He) were calculated for wild species and A. paeoniifolius complexesseparately to assess genetic diversity within the complex. Wright’s fixationindices (F), or inbreeding coefficient, for groups were calculated as F = 1 −Ho/He. The partitioning of genetic diversity (Fis, Fst, and Fit) among all pop-ulations was analyzed using F-statistics (Weir, 1996). Nei’s original measuresof genetic identity (i) and distance (d) (Nei, 1972) were calculated to determinedegree of genetic differentiation between populations. A dendrogram was plot-ted on the basis of Nei’s distance for clustering. The cophenetic correlationcoefficient was calculated to check goodness of fit of the cluster analysis to thematrix.

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Diversity Analysis in Amorphophallus 311

RESULTS AND DISCUSSION

Isozyme VariabilityTwelve loci were detected; seven loci exhibited polymorphism (58.33%).

Two loci were identified for malate dehydrogenase (Mdh; Figure 1), three locifor polyphenol oxidase (Ppo; Figure 2), one each for diaphorase (Dia) and cata-lase (Cat), and five loci for esterase (Est). A total of 23 alleles were detected atthe 12 loci (Table 2), with an average of 1.92 alleles per locus across popula-tions. Genotype frequencies at each locus indicated that with the exception ofPpo-2 and Cat, all loci deviated from Hardy-Weinberg equilibrium (Table 2).

Figure 1: Malate dehydrogenase (Mdh) isozyme pattern in the accessions of Amorphophalus.

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312 S. R. Anil et al.

Figure 2: Polyphenol oxidase (Ppo) isozyme pattern in the accessions of Amorphophallus.

The quaternary structure of the Est enzyme system might be monomericor dimeric, and five loci were detected in each population (Table 2). Only oneallele was found at Est-5 in populations of A. paeoniifolius and was absent inA. bonaccordensis, A. hohenackeri, A. smithsonianus, and A. bulbifer. Est-4 waspresent in three populations, A. bulbifer, A. smithsonianus, and one populationof A. paeoniifolius collected from Karnataka. Two alleles were detected in Est-3 and A. bulbifer was distinguished by a rare allele B and had a genotype BB.The cultivated Elephant foot yam, cv. Gajendra (GJ), had AB genotype and thewild populations of A. paeoniifolius had AA genotype for that locus. Est-2 alsohad three alleles A, B, C, and B was a rare allele found in A. bulbifer and

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Diversity Analysis in Amorphophallus 313

Table 2: Allele frequencies for all the loci analyzed in variousaccessions of Amorphophallus.

Locus Allele Allele frequency (%) χ2

Dia A 0.416 ∗∗∗B 0.583

Ppo-3 A 1.00 ∗∗∗Ppo-2 A 0.80 NS

B 0.20Ppo-1 A 1.00 ∗∗∗

Mdh-2 A 0.02 ∗∗∗B 0.84C 0.10D 0.04

Mdh-1 A 1.00 ∗∗∗

Cat A 0.613 NSB 0.386

Est-5 A 1.00 ∗∗∗Est-4 A 1.00 ∗∗∗Est-3 A 0.785 ∗∗

B 0.214Est-2 A 0.809 ∗∗

B 0.071C 0.119

Est-1 A 0.222 ∗B 0.667C 0.111

NS = nonsignficant. ∗, ∗∗, ∗∗∗Significant deviation between observed andexpected genotype frequency in the polymorphic loci at P ≤ 0.05, P ≤0.01, and P ≤ 0.001, chi-square test.

P19 population of A. paeoniifolius. Similarly, allele C was a rare allele found inA. smithsonianus (T3) and the B1 population of A. paeoniifolius, distinguishedby the presence of more cormels and dwarf stature. The corm of T3 and B1 isvery small. A. dubius had a genotype AC for this locus. Est-1 also had threealleles A, B, and C. A. dubius alone had a rare allele C at this locus.

The Ppo enzyme system of laccase or catecol oxidase is composed of amonomeric protein. Three loci were detected, one allele at Ppo-1 and Ppo-3 and two alleles at Ppo-2. The B allele was present in V1, V3, T2, K3-2, P17,and A. sylvaticus was distinguished by presence of this rare allele B in thehomozygous BB.

Catalases are generally tetramers or homodimers. In this study, only onelocus was detected with two alleles A and B. Malate dehydrogenases are usu-ally dimers. Two loci were detected. Mdh-1 had one allele represented bygenotype AA; Mdh-2 had four alleles A, B, C, D. The C allele was rare, presentonly in T3 (CC) and V2 (AC); D, another rare allele, was present only inA. sylvaticus (DD). Diaphorases consist of a hydrogenase dimer representedby single loci with two alleles.

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Genetic DiversityHigh genetic diversity occurred in eight species (Table 2). Mean percentage

of polymorphic loci in the eight species was 58.33%. Mean expected heterozy-gosity (He) was the most appropriate parameter reflecting the level of geneticdiversity. Populations of A. hohenackeri, A. bulbifer, A. bonaccordensis, A. com-mutatus, and A. dubius had higher genetic diversity (He > 0.2500) thanA. paeoniifolius and A. smithsonianus populations (He < 0.200).

Low gene flow (Nm = 0.045) across populations may be due to the lowernumbers of enzyme systems. There was no gene flow between populations ofdifferent species. A high genetic diversity exists among various populationsof Amorphophallus as evident by the high mean genetic diversity (He) value.The mean He within the genus was higher (Table 3) compared to other species(Hamrick and Godt, 1989; Hamrick et al., 1992; Table 4). Analysis betweenwild species and A. paeoniifolius complexes, considered as two groups, indicatehigh genetic diversity values (He = 0.4885, P = 58.33, A = 1.91) in the wildspecies complex (Table 5). The genetic diversity of A. paeoniifolius complex wascomparatively lower. This value is comparable to computed estimates (Table 4)for short-lived perennial species (Hamrick and Godt, 1989, 1996). In the wildspecies complex, the He value was high due to uniqueness of species includedin the complex. Genetic diversity among the 25 populations can be considereda representation of genetic diversity in the genus Amorphophallus in generalbecause it included species ranging from highly endemic A. bonaccordensis andA. hohenackeri to widespread species A. paeoniifolius.

The Amorphophallus diversity can be explained in a way similar to thatin other crops (Ge and Hong, 1999; Hamrick and Godt, 1989, 1996). Overalldiversity of a species may be attributed to three factors. They are biologicalcharacteristics, habitats, and evolutionary history, which are unique to eachspecies. Widespread geographic distribution is a general factor that explains

Table 3: Fixation indices (F) for polymorphic loci in the accessions ofAmorphophallus.

Locus Ho He Fis Fit Fst Nm I

Dia 0.1667 0.5303 −1 0.958∗ 0.9794∗ 0.0053 0.6792Ppo-2 0.32 0.3265 −1 0 0.5∗ 0.2500 0.5004Mdh-2 0.04 0.2882 −1 0.8584∗ 0.9292∗ 0.0191 0.5837Cat 0.5 0.4852 −1 0.2578∗ 0.6289∗ 0.1475 0.6671Est-1 0.222 0.5229 −1 0.9144∗ 0.9572∗ 0.0112 0.8487Est-2 0.0952 0.3333 −1 0.8473∗ 0.9237∗ 0.0207 0.6129Est-3 0.1429 0.3626 −1 0.9578∗ 0.9789∗ 0.0054 0.5196Mean 0.1239 0.2374 −1 0.6917∗ 0.8459∗ 0.0456 0.3676

I = Shannon information index.∗Significant deviation between observed and expected genotype frequency in thepolymorphic loci at P ≤ 0.05, P ≤ 0.01, and P ≤ 0.001, chi-square test.

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Table 4: Comparison of genetic diversity of Amorphophallus with other plantspecies.

Species character

Geneticdiversity

(He)Polymorphic

loci (%)

Meanalleles per

locus Source

Temperate zonespecies

0.146 48.5 1.91 Hamrick et al. (1992)

Short-livedperennial species

0.116 39.3 1.7 Hamrick and Godt(1989)

Widespreadgeographicalrange

0.205 58.9 2.29

Short-livedperennial withseed dispersal bybirds/animals

0.128 38.3 —

Out-crossingwidespread

0.183 55.3 —

Amorphophallus 0.237 58.3 1.92 Present investigation

Table 5: Parameters to estimate genetic variation in wild species/accessions com-plexes of Amorphophallus.

Group N P A I Ho He F

A. paeoniifolius accessions 4 33.3 1.4 0.1704 0.06481 0.1166 0.44∗Wild species (WS) 7 58.33 1.91 0.2198 0.3622 0.4885 0.26∗

N = number of polymorphic loci; I = Shannon information index.∗Significant deviation between observed and expected frequencies in the two groups atP < 0.05, chi-square test.

diversity in the genus. Amorphophallus has a distribution across tropicalregions of Asia, Africa, and Australia (Mayo et al., 1997) with a wide range ofendemism for a majority of the species. The wide range of adaptation to differ-ent geographic areas may be a reason for diversity of the species. Finally, thebreeding system of the plant is a determinant of genetic diversity (Hamrickand Godt, 1989). Variability in Amorphophallus can be attributed to its out-crossing (insect-pollinated) mechanism. Beetles are the main pollinators ofAmorphophallus (Sivadasan and Sabu, 1989), followed by flies, bees, ants, andcockroaches (Punekar and Kumaran, 2010). Most taxa display features charac-teristic of cantharophily (beetle pollination). This genus reproduces by sexualand asexual means. Whereas sexual reproduction leads to enhanced geneticvariation, asexual methods preserve variation. This combination is generallyassociated with a high level of allozyme variation (Huh et al., 1998). In rootand tuber crops that are clonally propagated, intracrop chromosomal polymor-phism is common (Zohary, 2004), and somatic mutations and ploidy variations(Hahn, 1995), present within the genus, may also be a reason for enrichment

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of diversity. According to Hamrick and Godt (1989), out-crossing plants typi-cally have higher genetic diversity than self-pollinating plants and show lessgenetic differentiation.

Cluster Analysis and Genetic RelationshipsThere was wide genetic distance between populations using POPGENE

based on Nei’s distance (Nei, 1972). The greatest genetic distance was betweenT3 and P5 (2.34), between V2 and P5 (2.34), and between V2 and V1 (2.44).The least genetically distant populations were P1 and K5. The matrix corre-lation coefficient (cophenetic correlation; r = 0.81) implied a good fit of thedendrogram to the matrix. The unweighted pair group method of mathemat-ical average based on Nei’s genetic distance indicated three major clusters,one including only T3; another composed of populations V2, A. sylvaticus,and A. commutatus; and a third cluster, with two subclusters. One subclus-ter included T2, V3, and P19. The second large subcluster was composed ofA. paeoniifolius populations, except the P19 population, which confirmed thespecies status of all the A. paeoniifolius populations despite morphologicalvariations.

Clustering based on Nei’s distance separated species T3, V2, T2, V3,A. sylvaticus, and A. commutatus from the rest, indicating a separate pat-tern of evolution of isozymes. All of these species had unique alleles for Mdh,Dia, and Est. The cultivated forms had different banding patterns for theenzyme system Ppo. Isozymes can be used as markers for distinguishingaccessions/species (Hamrick et al., 1991). Dia was detected only in five pop-ulations as a rare allele. Based on Nei’s genetic distance and the dendrogram,the most diverged population was A. smithsonianus (Figure 3). Clustering ofA. paeoniifolius populations with minimum intracluster distances agree withNicolson (1987), who noted that variable forms are segregants. These charac-ters may become fixed to give rise to new species or varieties. The populationP19 of A. paeoniifolius from Puttur exists separately in a different cluster alongwith the wild species. Similarly, populations of GJ and T10 were placed indifferent subgroups within the larger subcluster (II subcluster) of A. paeoni-ifolius. Clustering of A. dubius with the A. paeoniifolius complex indicatesthat A. dubius may be the closest relative or the possible ancestor of the lat-ter. GJ and T10 were placed apart (Nei’s genetic distance of 0.44), indicatingthat T10 was closer to A. paeoniifolius var. paeoniifolius than A. paeoniifoliusvar. campanulatus. Variability studies in A. paeoniifolius germplasm, based onmorphological characters, placed GJ and T10 in different subclusters withinthe larger cluster of A. paeoniifolius (Anil et al., 2011), indicating that thoughboth are known as A. paeonifolius var. campanulatus they may be separateentities. Because flowering has not been reported in T10, taxonomic delin-eation based on isozymes and morphological characters suggests a separateentity for this population.

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Figure 3: Unweighted pair group method of mathematical average clustering based on Nei’soriginal distance (Nei, 1972) using isozyme markers.

Genetic StructureThe F-statistic (Table 3) indicated significant Fis (−1) values, indicating an

excess of heterozygosity for all loci examined across the genus. The Fit valuesdiffered from zero in all loci except for Ppo-2. The mean value of Fst acrosspopulations indicated high genetic diversity among populations. The indirectestimate of gene flow (Nm) was very low and found in all loci.

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At all loci except Cat and Ppo-2 there was a deviation from the Hardy-Weinberg equilibrium. The Fis value seems to be lower than zero, indicatinga prevalence of heterozygotes in the germplasm. The Fit and Fst values indi-cated differences implying a deviation from the Hardy-Weinberg equilibrium.The excess heterozygosity observed may be the result of out-crossing, whichis characteristic of this genus along with clonal propagation, which preservesheterozygosity. Vegetatively propagated plants are, as a rule, highly heterozy-gous (Zohary, 2004). Some loci, including Ppo-1, Ppo-3, Mdh-1, Est-4, andEst-5 (monomorphic loci), exhibited an excess of homozygotes, indicating thatinbreeding might have led to a deviation from Hardy-Weinberg equilibrium.This is because clonal propagation by corms, cormels, stolons, or bulbils ismore common in this genus than sexual reproduction. Out-crossing betweenspatially closer clones is possible and equivalent to inbreeding, resulting in adeficiency in heterozygotes in at least some loci on which selection may actto eliminate or maintain homozygosity. Biparental inbreeding, especially insmall populations, can result in small heterozygosity deficits (Sampson et al.,1988). Inbreeding coefficient values for wild species complex and for A. paeoni-ifolius complex (Table 5) indicated that a small amount of inbreeding existsin this genus in addition to its out-crossing character. A mixed mating sys-tem (reproduction by both cross- and self-fertilization), along with vegetativemeans of propagation, is highly dynamic and contributes to wide adaptation(Ronfort and Glemin, 2013). Based on a comparative analysis of estimatedinbreeding coefficients and out-crossing rates, Goodwillie et al. (2005) sug-gested that mixed mating often evolves despite strong inbreeding depression.The overall Fst estimates the interpopulation variation, which in this study iscontributed to by distinct species included in the population.

Unweighted pair group method of mathematical average clustering ofaccessions based on Nei’s distance separated the diverse species A. smith-sonianus (T3), A. hohenackeri (V2), A. bonaccordensis (T2), A. bulbifer (V3),A. sylvaticus, and A. commutatus as one group and all accessions of A. paeoni-ifolius as another group. The average Fst value across accessions indicatedhigh genetic divergence among accessions. The high He value, low Fis, andinbreeding coefficient values for wild species and A. paeoniifolius complexesindicate that a small amount of inbreeding exists in this genus in addition toits out-crossing character, which may be attributed to its propagation throughvegetative means. Based on isozyme pattern (polymorphic loci) and heterozy-gosity values it can be inferred that there was moderate to high geneticdiversity between populations of Amorphophallus.

ACKNOWLEDGEMENTS

The authors thank Dr. Ashalatha S. Nair, Professor and Head, Departmentof Botany, University of Kerala, for providing facilities. S.R.A. is thankful

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to the Director, Central Tuber Crops Research Institute (Indian Councilof Agricultural Research), Thiruvananthapuram, Kerala, for granting studyleave.

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