1

RESEARCH ARTICLE

Nucleotide diversity and phylogenetic relationships among Gladiolus cultivars and

related taxa of family Iridaceae

NIRAJ SINGH1, BALESHWAR MEENA1, ASHISH KUMAR PAL1, ROOP KUMAR

ROY1, SRI KRISHNA TEWARI, SUSHMA TAMTA2 AND TIKAM SINGH RANA1*

1CSIR-National Botanical Research Institute, Rana Pratap Marg, Lucknow -226001, India

2Department of Botany, D. S. B. Campus, Kumaun University, Nainital-263001, Uttarakhand,

India

Running Title: Nucleotide diversity in Gladiolus cultivars

E-mail: ranatikam@gmail.com; ranats@nbri.res.in

Keywords Gladiolus, Iridaceae, Plastid genome, Phylogenetic analyses

Abstract

The plastid genome regions of two intergenic spacers, psbA-trnH and trnL-trnF, were

sequenced to study the nucleotide diversity and phylogenetic relationships among Gladiolus

cultivars. Nucleotide diversity of psbA-trnH region was higher than trnL-trnF region of

chloroplast. We employed Bayesian, maximum parsimony and Neighbor-Joining approaches

for phylogenetic analysis of Gladiolus and related taxa using combined datasets from

chloroplast genome. The psbA-trnH and trnL-trnF intergenic spacers of Gladiolus and related

taxa like Babiana, Chasmanthe, Crocus, Iris, Moraea, Sisyrinchium, Sparaxis and two out

group species (Hymenocallis littoralis, Asphodeline lutea) were used in the present

investigation. Results showed that Sub-family Iridoideae have sister lineage with sub-family

Ixioideae and Crocoideae. Hymenocallis littoralis and Asphodeline lutea were separately

2

attached at the base of tree as the diverging Iridaceae relative’s lineage. Present study

revealed that psbA-trnH region are useful in addressing questions of phylogenetic

relationships among the Gladiolus cultivars, as these intergenic spacers are more variable and

have more phylogenetically informative sites than the trnL-trnF spacer, and therefore, are

suitable for phylogenetic comparison on a lower taxonomic level. Gladiolus cultivars are

extensively used as an ornamental crop and showed high potential in floriculture trade.

Gladiolus cultivation still needs to generate new cultivars with stable phenotypes. Moreover,

one of the most popular methods for generating new cultivars is hybridization. Hence,

information on phylogenetic relationships among cultivars could be useful for hybridization

programmes for further improvement of the crop.

Introduction

The genus Gladilous L. is comprised of about 265 species in the world, and is one of the

largest genera of family Iridaceae. The Cape of Good Hope (South Africa) is considered to be

the centre of diversity for the genus Gladiolus. It is distributed throughout tropical Africa,

Madagascar, Arabian Peninsula, the Mediterranean basin, Europe and Asia including Iran and

Afghanistan (Goldblatt and Manning 1998; Goldblatt et al. 2001). The basic chromosome

number is x=15, ranging from diploid (2n=30) to hypododecaploid (2n=180), (Bamford

1935; Ohri and Khoshoo 1983). Hybridization and polyploidy have been greatly responsible

for the evolution of Gladiolus (Ohri and Khoshoo 1983). Gladiolus is out-breeding in nature,

and exhibited diverse pollination mechanism (Ohri and Khoshoo 1983; Goldblatt and

Manning 2002). Gladiolus cultivars possess enormous diversity in colours, sizes, textures,

numbers and types of flowers and forms of inflorescence (Buch 1978; Ohri and Khoshoo

1985b; Anderson and Park 1989).

In India, Gladiolus is primarily grown in the states of Uttar Pradesh, Uttarakhand, Himachal

3

Pradesh, Haryana, Delhi, Karnataka, Punjab, West Bengal, Assam, Sikkim and Meghalaya,

having sub tropical climates. Artificial hybridization and selection for desired phenotypes

have already produced large number of cultivars. Phenotypic characters have been widely

used to characterize and identify the cultivars; however, it is often difficult and even

misleading in case of specific cultivars. Therefore, molecular characterization of different

cultivars of Gladiolus is very significant in understanding the genetic relationships among the

cultivars and its close relatives in the family Iridaceae. Characterization of different

cultivars/genotypes using molecular markers will not only help in establishing the

relationship and affinities among various taxa, but will also pave the ways for selection of

elite germplasm from large sets of parental genotypes, and could further be used in

broadening the genetic base of Gladiolus through breeding (Pragya et al. 2010). Earlier,

Gladiolus cultivars have been investigated using RAPD (Takatsu et al. 2001; Jingang et al.

2006; Pragya et al. 2010), ISSR (Jingang et al. 2008; Raycheva et al. 2011) and AFLP

(Ranjan et al. 2010) to study the variability. It is well known that most of the angiosperm

species, the nuclear genome is bi-parentally inherited and can spread by pollens and seeds,

whereas chloroplast and mitochondrial genome is maternally inherited and spread only by

seeds (Greiner et al. 2015). However, there is no comprehensive study available on the

characterization of varied Gladiolus cultivars using chloroplast DNA regions like psbA-trnH

and trnL-trnF intergenic spacers. However, plastid DNA regions, rbcL, rps4, trnL intron,

trnL-F, matK, and rps16 have been used to infer phylogenetic analysis and molecular

systematics of family Iridaceae (Souza-Chies et al. 1997; Reeves et al. 2001; Goldblatt

2011). The chloroplast genome has been extensively used for phylogenetic reconstructions in

plant systematics, because of its relatively small size and conservative mode of evolution. In

the present study, we therefore, aimed to estimate the nucleotide diversity and establish

phylogenetic relationships among Gladiolus cultivars and related taxa of family Iridaceae

4

using two plastid genome markers.

Material and methods

Plant materials and isolation of genomic DNA

In the present study 66 Gladiolus cultivars were procured from the Botanic Garden of CSIR-

National Botanical Research Institute (CSIR-NBRI), Lucknow (India). Total genomic DNA

was extracted from young fresh leaves of all the collected genotypes following CTAB method

(Doyle and Doyle 1990). Quantitation of purified DNAs was carried out by using NanoDrop

ND-1000 Spectrophotometer (NanoDrop Technologies Inc., USA).

PCR amplification and sequencing

The primer sequences of psbA (5′-GTTATGCATGAACGTAATGCTC-3′), trnH (5′-

CGCGCATGGTGGATTCACAAATC-3′), trnL (5’-AAAATCGTGAGGGTTCAAGTC-3’),

trnF (5’-GATTTGAACTGGTGACACGAG-3’) available in the public domain (Shinozaki et

al. 1986; Taberlet et al. 1991) were custom synthesized from Sigma Aldrich Chemicals Pvt.

Ltd. India. The amplification of chloroplast region psbA-trnH and trnL-trnF intergenic

spacers was performed in 20 μl reaction volume with the following components: 10 μl 2X

phusion master mix, 1 μl (10 picomole) forward primer, 1 μl (10 picomole) reverse primer, 2

μl genomic DNA (40ng) and water 6 μl (protease, DNase, RNase free) using Proflex PCR

System (Applied Biosystems, Life Technologies, USA). After initial denaturation at 98 °C for

30 s, each cycle consisted of 10 sec denaturation at 98 °C, 20 s of annealing at 54 °C, 30 s of

extension at 72 °C and a final 5 min extension at 72 °C at the end of 35 cycles.

The amplified products obtained were electrophoresed on 0.8% agarose gels in 1x TBE

buffer at a constant voltage 5 V/cm. After electrophoresis gel was stained in ethidium

5

bromide, visualized and archived using gel documentation system (UV Technology, UK). A

known DNA ladder of 100 base pair differences was also loaded on the first well of the gels.

Since the size of psbA-trnH and trnL-trnF regions of Gladiolus cultivars were known

(approximately 600 bp and 700 bp respectively), the bands of interest were identified with

reference to DNA ladder loaded in the gels, were excised for purification of DNA. PCR

products eluted from the agarose gel were purified using QIAquick Gel Extraction Kit

(QIAGEN). Quantitation of purified PCR products was carried out by UV

spectrophotometery using NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies

Inc., USA).

Sequencing was conducted using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied

Biosystems, Foster City, CA, USA) and the ABI 3730 (Applied Biosystems). After the

completion of capillary gel electrophoresis the fluorescence data were displayed as an

electropherogram with the help of data collection software (ABI PRISM® DNA Sequencing

Analysis Software v.5.0). Only, sequence data with reliable read lengths were considered in

the present study. The sequences determined for the amplified psbA-trnH and trnL-trnF

regions were uploaded to the EMBL/GenBank nucleotide database for storage and archiving.

Phylogenetic analysis

In the present phylogenetic analyses, a total of 86 accessions of psbA-trnH and trnL-trnF

intergenic spacer sequences, comprising 66 Gladiolus cultivars, 18 accessions of other taxa

representing family Iridaceae (Babiana, Chasmanthe, Crocus, Iris, Moraea, Sisyrinchium,

Sparaxis) and two out group species Hymenocallis littoralis and Asphodeline lutea were

considered. The sequences of interest available in the NCBI database were downloaded to use

as a supplement in the phylogenetic study of Gladiolus cultivars. The names of the taxa

6

downloaded from GenBank have been presented in table 1, along with their accession

numbers.

The chloroplast sequences were aligned with Clustal W (Thompson et al. 1994) using Bio

Edit Sequence Alignment Editor (Hall 1999), MEGA6 software (Tamura et al. 2013) and

MUSCLE (Edgar 2004). The alignment of sequences was further examined and edited

manually as well. The boundaries of the psbA-trnH regions (psbA gene, psbA-trnH intergenic

Spacer, trnH gene) and trnL-trnF region (trnL gene, trnL-trnF intergenic spacer, trnF gene

were determined by aligning with the existing sequences reported in the database as a

reference (Valente et al. 2011). To infer relationships between the Gladiolus cultivars, a

phylogenetic tree was constructed using the neighbor-joining (NJ) and maximum parsimony

(MP) method as implemented in the MEGA 6 (Tamura et al. 2013), and robustness of each

internal branch in NJ tree was evaluated with 1000 bootstrap replicates.

The MRBAYES 3.1.2 program (Huelsenbeck and Ronquist 2001) was employed to sample

trees using a Bayesian Markov Chain Monte Carlo (B/MCMC) approach. The combined data

set was partitioned into the two parts (psbA-trnH and trnL-trnF), and each partition was

allowed to have its own parameters (Nylander et al. 2004). A nucleotide substitution model

was selected using the Bayesian information criterion (BIC) as implemented in the program

jModelTest (Posada 2008). The analyses were performed assuming the general time-

reversible model of nucleotide substitution (Rodriguez et al. 1990) including estimation of

invariant sites and assumption of gamma distribution with six rate categories and allowing

site-specific rates (GTR+I+G) for the combined analyses. Two parallel runs of 2 million

generations were made, starting with a random tree and employing 12 simultaneous chains

each. Every 100th tree was saved into a file. The first 200000 generations (i.e., 2000 trees)

were deleted as the “burn in” of the chains.

We used AWTY (are we there yet?) program (Nylander et al. 2007) to compare split

7

frequencies in different runs and to plot cumulative split frequencies to ensure that stationary

was reached. Of the remaining 36000 trees (18000 from each parallel run), a majority-rule

consensus tree with average branch lengths was calculated using the sumt option of

MRBAYES.

The basic sequence statistics, including conserved sites, variable sites, parsimony informative

sites, singleton sites were also analyzed with MEGA6 software. Average pairwise differences

between sequences for each base pair, nucleotide diversity (π) (Nei and Li 1979) and

haplotype diversity (Hd) were calculated with DnaSP version 5.10 (Librado and Rozas 2009).

This analysis was carried out for each DNA fragment alignment and for a concatenated

alignment of all fragments.

Results

Nucleotide diversity of psbA-trnH Intergenic spacer region of Chloroplast

The chloroplast sequences of all the taxa were aligned using Clustal W program in MEGA6

software. The aligned cpDNA sequences of psbA-trnH region of Gladiolus cultivars varied

from 547 bp to 557 bp with an average length of 555 bp. The average frequencies of Adenine

(A) and Thymine (T) contents were 30.3% and 33.6%, respectively, showing 63.9% mean

A+T contents in 555 bases average length of psbA-trnH sequence. Similarly, the average

frequencies of Guanine (G) and Cytosine (C) were 18.3% and 17.8%, respectively; showing

36.1% mean G+C contents throughout the entire psbA-trnH sequences. The nucleotide pair

frequencies like identical pairs (ii), transitional pairs (si), transversional pairs (sv) of psbA-

trnH sequences were also calculated (table 2). The average base substitutions recorded in

psbA-trnH region was 3 (i.e., si = 1, sv = 2). The basic statistics such as conserved sites,

variable sites, parsimony informative sites and singleton sites were calculated after the

complete deletion of the missing/gap sites from all the sequences (table 2). The aligned psbA-

trnH sequences (including psbA gene and psbA-trnH intergenic spacer) formed in a matrix of

8

531 nucleotide sites, of which 510 sites were conserved, 21 sites were variables and 8 sites

were parsimony informative and 13 sites were singleton.

Nucleotide diversity of trnL-trnF Intergenic spacer region of chloroplast

The aligned sequences of trnL-trnF region of Gladiolus cultivars varied from 671 to 678 bp

with an average length of 672 bp. The average frequencies of Adenine (A) and Thymine (T)

contents were 36.2% and 29.1% respectively; showing 65.3% mean A+T contents in 672

bases average length of trnL-trnF sequences. Similarly, the average frequencies of Guanine

(G) and Cytosine (C) were 18.5% and 16.2% respectively; showing 34.7% mean G+C

contents throughout the trnL-trnF sequences. The average base substitutions recorded in trnL-

trnF region was 1 (i.e., si = 1, sv = 0). The aligned trnL-trnF sequences (including trnL gene

and trnL-trnF intergenic spacer) formed in a matrix of 678 nucleotide sites, of which 669 sites

were conserved, 5 sites were variables, 4 sites were parsimony informative and 1 singleton

site in Gladiolus cultivars.

Phylogenetic relationships among Gladiolus cultivars

The cumulative phylogeny of chloroplast genome regions (psbA-trnH and trnL-trnF) was

inferred using the Neighbor-Joining method, which resulted in an optimal tree separating all

the Gladiolus cultivars into eight groups with very weak bootstrap support (figure 1) Group I

consisted the cultivars like Roshni, Picardy, Snow Princess, Her-Majesty, Neelima, Tambri,

Sydney Percy-Lancaster, Classic Pink, Thamboliana, Video, Acharya JC Bose and Victor.

Group II clustered together cultivars like Suvarna, Oscar, Hallmark, Friendship Pink,

Urvashi, Nichole, Peter Pear’s, American Beauty Black, American Beauty Pink and Chanson.

Group III comprised Amethyst and Rose Supreme, whereas Group IV grouped Eurovision,

Topaz, Priscilla, Zeus, Friendship White, Invertation and Tropic Sea. Group V grouped

9

together Tiger Flame, Legeand, Orange Zinger, Interpet, Lalima, Parade, White Prosperity,

Garden Glory, Grock, Shagun, Green Wood Pecker and Snow Flower. Group VI clustered

cultivars like Wine & Rose, Red Beauty, Candyman, Pacifica, My Love and Red Beauty

Spot, while group VII clustered together Yellow Stone, Aldebaran, Arka Keshar, Jester,

Vink’s Glory, Inter Pearl, Regency, Praha and Lavender Puff. Cultivars like Sylvia, Heerak,

Rashmi, Agni, Usha, Acidanthera, American Beauty Blue and Fedelio clustered together in

group VIII. One close relative and morphologically distinct species Iris rossii used as out

group was separately attached at the base of the tree as the diverging Gladiolus relative’s

lineage (figure 1).

Phylogenetic relationship within family Iridaceae

Phylogenetic trees were generated using NJ method on the basis of the evolutionary distances

computed using MEGA6 program. The relationships and affinities of Gladiolus to other

members of family Iridaceae were studied in NJ trees (figure 2). In phylogenetic tree of the

family Iridaceae all the representative members were separated into two clades with strong

bootstrap support (BS). Clade-I consisted of all the species of Iris belonging to subfamily

Iridoideae, (I. setosa, I. koreana, I. minutoaurea, I. odaesanensis, I. pseudacorus and I. rossii)

with 96% BS, nested with Moraea species (Moraea simplex and Moraea pallida) with 84%

BS and Sisyrinchium angustifolium with 99% BS. Clade-II comprised members of

Crocoideae and Ixioideae subfamilies and was further divided into IIa and IIb sub-clade. Sub-

clade IIa consisted members of Ixioideae, Babiana species nested with Sparaxis variegata

(61% BS) and Chasmanthe aethiopica (96% BS). Sub-clade IIb divided into IIb1 and IIb2.

Sub-clade IIb1 clustered together Crocus species of Crocoideae with 76% BS, while sub-

clade IIb2 grouped all Gladiolus cultivars together with G. palustris and G. illyricus of sub-

10

family Ixidoideae at 97% BS.

Maximum Parsimony (MP) showed similar topologies with NJ analysis. However, Crocus

species of subfamily Crocoideae grouped together with Babiana and Sparaxis species of

subfamily Ixioideae with moderate bootstrap support (65%) (figure S1).

In B/MCMC phylogenetic tree analysis members of family Iridaceae formed a strongly

supported monophyletic group, which further divided into two sister groups with significant

support (Figure 3). Clade I consisted Iris species nested with Moraea and Sisyrinchium

species and clade II consisted members of Ixioideae and Crocoideae sub-families, and was

further divided into IIa and IIb sub-clade. Sub-clade IIa consisted of the member of sub-

families, Crocoideae (Crocus species) and Ixioideae (Babiana species) nested with Sparaxis

and Chasmanthe species, whereas sub-clade IIb comprised member of sub-family Ixioideae.

Discussion

The present study contributes to our understanding of nucleotide diversity and phylogenetic

relationships within the Gladiolus cultivars and other related taxa of the family Iridaceae.

Three methods namely, Neighbour-Joining, Maximum Parsimony and Bayesian analysis

were used for phylogeny reconstruction. Phylogenetic relationships were studied using

combined dataset of 26 variable sites (21 of psbA-trnH and 5 of trnL-trnF spacer,

respectively), which formed eight groups of the Gladiolus cultivars with weak bootstrap

support. Group I consisted of 12 cultivars, out of which Roshni (Friendship Pink x Red

Beauty), JC Bose (Friendship Pink x Red Beauty) and SP Lancaster (Friendship Pink x

Priscilla) clustered together revealing affinities towards their parentages. Neelima (Snow

Princess x Tropic Sea) showed close affinity with female parentage Snow Princess, while

Suvarna (Fidelio x Hallmark) clustered with male parent (Hallmark) in Group II. Heerak

(Yellow Stone x My Love), Usha (Friendship white x My Love) and Rashmi (Friendship

11

White x Nichole) clustered together in Group VIII, whereas Amethyst, Lalima, Urvashi did

not show any affinity to their parentage. Parentage of other Gladiolus cultivars used in the

present study was not known (figure 1). Although Acidanthera is morphologically distinct

from Gladiolus, it revealed close affinity with Gladiolus cultivars in the present investigation.

Similar results were also found in phylogenetic relationships of Opuntia using ISSR and

trnL-trnF intergenic spacer regions (Realini et al. 2015). Present study revealed that psbA-

trnH region is more suitable than the trnL-trnF region in addressing the questions of

phylogenetic relationships among Gladiolus cultivars as these intergenic spacers are more

variable and have more phylogenetically informative sites than the later, and therefore, are

informative for phylogenetic comparison at a lower taxonomic level. The present findings

also corroborated with the earlier phylogenetic investigations of sub-tribe Sonchinae

(Asteraceae) using chloroplast sequence data of psbA-trnH region (Kim et al. 1999), while

Chandler et al. (2001) found it more informative at higher taxonomic levels, particularly at

the intergeneric level. Evolutionary changes in chloroplast DNA indicated limited variability

within species resulting in a lack of intra-specific sampling in studies examining relationships

at higher taxonomic levels (Terry et al. 2000). The evolutionary rate of the nuclear genome is

the fastest among chloroplast and mitochondrial genomes, and the plant mitochondrial

genome is typically characterized by low point mutation rates (Sloan et al. 2012). It has been

established that overall relative rate of substitution in mitochondrial, chloroplast and nuclear

genes in 27 species of angiosperm is 1:3:16, respectively (Drouin et al. 2008). Analysis of the

total cpDNA sequences indicated that psbA-trnH sequences had higher nucleotide diversity

(4.2 × 10−3) than trnL-trnF sequences (1.78 × 10−3). Similarly, psbA-trnH region has shown

higher nucleotide diversity than atpB-rbcL intergenic region in Japanese Aucuba (Ohi et al.

2003b).

All the three approaches viz., Neighbor-Joining (NJ), Maximum Parsimony (MP) and

12

Bayesian analyses used to analyse the combined dataset produced virtually identical

topologies with high bootstrap support (BS > 80 and PP > 0.95) at majority of nodes.

Gladiolus was retrieved as monophyletic in NJ, MP and Bayesian reconstruction methods

(BS = 97, BS = 94, PP = 0.99), and these results are in congruence with MP and Bayesian

analysis of 150 species of Gladiolus based on five regions of plastid genome (Valente et al.

2011). Phylogenetic trees generated using NJ analysis revealed that Iridoideae have sister

lineage with Ixioideae and Crocoideae. Outgroup species like Hymenocallis littoralis and

Asphodeline lutea were separately attached at base of the tree as diverging relative of the

family Iridaceae. Bayesian and MP analysis also generated similar topologies with strong

bootstrap support at the majority of nodes in different genera of Iridaceae. All three

reconstruction methods indicated monophyletic origin of Iridaceae, which are in agreement

with other phylogenetic analysis of Iridaceae based on chloroplast genes. The sub-family

Crocoideae showed close affinity with sub-family Ixioideae. The results of the present

investigation are supporting the earlier studies on Iridaceae (Souza-Chies et al. 1997;

Goldblatt 2001). Gladiolus cultivars are extensively cultivated as an ornamental crop having

high potential in floriculture trade. There is still lot of demand and need to generate new

Gladiolus cultivars with stable phenotypes, and one of the most popular methods is

hybridization. Therefore, the information generated in the present investigation about

phylogenetic relationships among different Gladiolus cultivars and its relationships with

other taxa of the family Iridaceae will provide new insights for hybridization studies.

Acknowledgments

The study was financially supported by Council of Scientific and Industrial Research, New

Delhi under AGTEC (BSC0110) project.

13

References

Anderson W., Park R. 1989 Growing Gladioli. Portland: Timber Press. 166.

Bamford R. 1935 The chromosome number in Gladiolus. J. Agric. Res. 51, 945–950.

Buch P. O. 1978 The species. In: Kaeing N & Crowley K eds. The world of the Gladiolus.

Edgewood: Edgewood Press. 2–7.

Chandler G. T., Bayer R. J., Crisp M. D. 2001 Amolecular phylogeny of the endemic

Australiangenus Gastrolobium (Fabaceae: Mirbelieae) and allied genera using

chloroplast and nuclear markers. Am. J. Bot. 88, 1675–1687.

Doyle J. J., Doyle J. L. 1990 Isolation of plant DNA from fresh tissue. Focus. 12, 13–15.

Drouin G., Daoud H., Xia J. 2008 Relative rates of synonymous substitutions in the mito-

chondrial chloroplast and nuclear genomes of seed plants. Mol. Phylogenet. Evol. 49,

827–831.

Edgar R. C. 2004 MUSCLE: Multiple sequence alignment with high accuracy and high

throughput. Nucleic. Acids. Res. 32, 1792–1797.

Goldblatt P., Manning J. C., Bernhardt P. 2001 Radiation of pollination systems in Gladiolus

(Iridaceae: Crocoideae) in southern Africa. Ann. Missouri. Bot. Gard. 88, 713-734.

Goldblatt P., Manning J. C. 1998 Gladiolus in Southern Africa. Vlaeberg: Fernwood Press.

Goldblatt P. 2001 Phylogeny and classification of the Iridaceae and the relationships of Iris.

Irises and Iridaceae: biodiversity and systematics. Annali. di. Botanica. 1, 13–28.

Goldblatt P., Manning J. C., Bernhardt P. 2001 Radiation of pollination systems in Gladiolus

(Iridaceae: Crocoideae) in southern Africa. Ann. Missouri. Bot. Gard. 88, 713–734.

Greiner S., Sobanski J., Bock R. 2015 Why are most organelle genomes transmitted

maternally? BioEssays. 37, 80–94.

14

Hall T. A. 1999 BioEdit: a user-friendly biological sequence alignment

editor and analysis program for Windows 95/98/NT. Nucleic. Acids. Sym. Series. 41, 95

–98.

Huelsenbeck J. P., Ronquist F. 2001 MrBAYES: Bayesian inference of phylogenetic trees.

Bioinformatics. 17, 754–755.

Jingang W., Hongkun J., Shufang G., Daidi C. 2006 RAPD Analysis of 12 general species of

Gladiolus hybridus Hort. J. Northeast. Agric. Univ. 13, 112–115.

Jingang W., Ying G., Daidi C., Shenkui L., Chuanpin Y. 2008 ISSR analysis of 26 general

species of Gladiolus hybridus Hort. J. Northeast. Agric. Univ. 15(4), 6–10.

Kim S. C., Crawford D. J., Jansen R. K., Santos- Guerra A. 1999 The use of a non-coding

region of chloroplast DNA in phylogenetic studies of the subtribe Sonchinae

(Asteraceae: Lactuaceae). Plant. Syst. Evol. 215, 85–99.

Librado P., Rozas J. 2009 DnaSP v5: asoftware for comprehensive analysis of DNA

polymorphism data. Bioinformatics. 25, 1451–1452.

Nei M., Li W. H. 1979 Mathematical model for studying genetic variation in terms of

restriction endonucleases. Proc. Natl. Acad. Sci. 76, 5269–5273.

Nylander J. A. A., Ronquist F., Huelsenbeck J. P., Nieves-Aldrey J. L. 2004 Bayesian

phylogenetic analysis of combined data. Syst. Biol. 53, 47–67.

Nylander J. A. A., Wilgenbusch J. C., Warren D. L., Swofford D. L. 2007 AWTY (Are we

there yet?): A system for graphical exploration of MCMC convergence in Bayesian

phylogenetics. Bioinformatics. 24, 581–583.

Ohi T., Kajita T., Murata J. 2003b Distinct geographic structure as evidenced by chloroplast

DNA haplotypes and ploidy level in Japanese Aucuba (Aucubaceae). Am. J. Bot.

90(11), 1645–1652.

Ohri D., Khoshoo T. N. 1983 Cytogenetics of Garden Gladiolus. IV. Origin and evolution of

15

Ornamental Taxa. Proc. Nat. Acad. Sci. 49, 279–294.

Ohri D., Koshoo T. N. 1985b Cytogenetical evolution of garden Gladiolus. The Nucleus. V.

28, 216–221.

Posada D. 2008 jModelTest: Phylogenetic model averaging. Mol. Biol. Evol. 25, 1253–1256.

Pragya, Bhat K. V., Misra R. L., Ranjan J. K. 2010 Analysis of diversity and relationships

among Gladiolus cultivars using morphological and RAPD markers. Indian. J. Agric.

Sci. 80(9), 766 –772.

Ranjan P., Bhat K. V., Misra R. L., Singh S. K., Ranjan J. K. 2010 Genetic relationships of

gladiolus cultivars inferred from fluorescence based AFLP markers. Sci. Hort. 123,

562–567.

Raycheva T., Stoyanov K., Denev I. 2011 Genetic diversity and molecular taxonomy study of

three genera from Iridaceae family in the Bulgarian flora based on ISSR markers.

Biotechnol. Biotechnol. Equip. 25, 2484–2488.

Realini M. F., Gonzalez G. E., Font F., Picca P. I., Poggio L., Gottlieb A. M. 2015

Phylogenetic relationships in Opuntia(Cactaceae, Opuntioideae) from southern

South America Plant. Syst. Evol. 301 (4), 1123–1134.

Reeves G., Chase M. W., Goldblatt P., Rudall P. J., Fay M. F., Cox A. V., Lejeune B., Souza-

Chies T. 2001 Molecular Systematics of Iridaceae: evidence from four plastid DNA

regions. Am. J. Botany. 88, 2074–2087.

Rodriguez F., Oliver J. L., Marin A., Medina J. R. 1990 The general stochastic model of

nucleotide substitution. J. Theor. Biol. 142, 485–501.

Shinozaki K., Ohme M., Tanaka M., et al.1986 The complete nucleotide sequence of the

tobacco chloroplast genome. Plant. Mol. Biol. Report. 4, 110–147.

Sloan D. B., Alverson A. J., Chuckalovcak J. P., Wu M., McCauley D. E., Palmer J. D. 2012

Rapid evolution of enormous multichromosomal genomes in flowering plant

16

mitochondria with exceptionally high mutation rates. Plos Biol. 10, 1–17.

Souza-Chies T. T, Britar G., Nadot S., Carter L., Besin E., Lejeune B. 1997 Phylogenetic

analysis of Iridaceae with parsimony and distance methods using the plastid gene

rps4. Plant. Syst. Evol. 204, 109–123.

Taberlet P., Gielly L., Pautou G., Bouvet J. 1991 Universal primers for amplification of three

non-coding regions of chloroplast DNA. Plant. Mol. Biol. 17, 1105–1109.

Takatsu Y., Miyamoto M., Inoue E., Yamada Y., Manabe T., Kasumi M., Hayashi M., Sakuma

F., Marubashi W., Niwa M. 2001 Interspecific hybridization among wild Gladiolus

Species of South Africa based on randomly amplified polymorphic DNA markers. Sci.

Hort. 91, 339–348.

Tamura K., Stecher G., Peterson D., Filipski A., Kumar S. 2013 Mega 6: Molecular

Evolutionary Genetics Analysis version 6.0. Mol. Biol. Evol. 30, 2725–2729.

Terry R. G., Nowak R. S., Tausch R. J. 2000 Genetic variation in chloroplast and

nuclearribosomal DNA in Utah Juniper (Juniperus osteosperma, Cupressaceae):

evidence for interspecific gene flow. Am. J. Bot. 87, 250–258.

Thompson J. D., Higgins D. G., Gibson T. J. 1994 CLUSTAL W: improving the sensitivity of

progressive multiple sequence alignment through sequence weighting, position-

specific gap penalties and weight matrix choice. Nucleic. Acids. Res. 22, 4673–4680.

Valente L., Savolainen V., Manning J., Goldblatt P., Vargas P. 2011 Explaining disparities in

species richness between Mediterranean floristic regions: a case study in Gladiolus

(Iridaceae). Glob. Ecol. Biogeogr. 20 (6), 881–892.

Received 11 July 2016, in revised form 1 August 2016; accepted 4 August 2016

Unedited version published online: 8 August 2016

17

Table 1. Details of the Gladiolus cultivars and other taxa of family Iridaceae included in the

present study.

S.No. Vouchar No/Source

Cultivar Name EMBL Acc.No psbA-trnH

EMBL Acc.No trnL-trnF

1. 253201 Roshni KU879113 KU879179 2. 253202 Suvarna KU879114 KU879180 3. 253203 Neelima KU879115 KU879181 4. 253204 Urvashi KU879116 KU879182 5. 253234 Amethyst KU879117 KU879183 6. 253238 Usha KU879118 KU879184 7. 253242 Sydney Percy-Lancaster KU879119 KU879185 8. 253243 Acharya JC Bose KU879120 KU879186 9. 253250 Rashmi KU879121 KU879187 10. 253205 Friendship Pink KU879122 KU879188 11. 253206 Friendship White KU879123 KU879189 12. 253207 Snow Princess KU879124 KU879190 13. 253208 Vink's Glory KU879125 KU879191 14. 253209 Nichole KU879126 KU879192 15. 253210 Green Wood Pecker KU879127 KU879193 16. 253211 Eurovision KU879128 KU879194 17. 253212 Priscilla KU879129 KU879195 18. 253213 Topaz KU879130 KU879196 19. 253214 Thamboliana KU879131 KU879197 20. 253215 Parade KU879132 KU879198 21. 253216 Classic Pink KU879133 KU879199 22. 253217 Shagun KU879134 KU879200 23. 253218 Inter Pearl KU879135 KU879201 24. 253219 Grock KU879136 KU879202 25. 253220 White Prosperity KU879137 KU879203 26. 253221 American Beauty Pink KU879138 KU879204 27. 253222 Tropic Sea KU879139 KU879205 28. 253223 Her-Majesty KU879140 KU879206 29. 253224 Regency KU879141 KU879207 30. 253225 Oscar KU879142 KU879208 31. 253226 Peter Pear's KU879143 KU879209 32. 253227 Yellow stone KU879144 KU879210 33. 253228 Tiger Flame KU879145 KU879211 34. 253229 Aldebaran KU879146 KU879212 35. 253230 Picardy KU879147 KU879213 36. 253231 Sylvia KU879148 KU879214 37. 253232 Praha KU879149 KU879215 38. 253233 Rose Supreme KU879150 KU879216 39. 253235 My Love KU879151 KU879217 40. 253236 Zeus KU879152 KU879218

18

41. 253237 American Beauty Blue KU879153 KU879219 42. 253239 Wine &Rose KU879154 KU879220 43. 253240 Arka Keshar KU879155 KU879221 44. 253241 Interpet KU879156 KU879222 45. 253244 Chanson KU879157 KU879223 46. 253245 Lavender Puff KU879158 KU879224 47. 253246 Jester KU879159 KU879225 48. 253247 Invertation KU879160 KU879226 49. 253248 Garden Glory KU879161 KU879227 50. 253249 American Beauty Black KU879162 KU879228 51. 253251 Red Beauty Spot KU879163 KU879229 52. 253252 Hallmark KU879164 KU879230 53. 253253 Red Beauty KU879165 KU879231 54. 253254 Legeand KU879166 KU879232 55. 253255 Agni KU879167 KU879233 56. 253256 Tambri KU879168 KU879234 57. 253257 Orange Zinger KU879169 KU879235 58. 253258 Candyman KU879170 KU879236 59. 253259 Video KU879171 KU879237 60. 253260 Snow Flower KU879172 KU879238 61. 253261 Parcifica KU879173 KU879239 62. 253262 Victor KU879174 KU879240 63. 253263 Acidanthera KU879175 KU879241 64. 255486 Heerak KU879176 KU879242 65. 255487 Lalima KU879177 KU879243 66. 255488 Fedelio KU879178 KU879244 67. NCBI Gladiolus palustris KM887244 KM887312 68. NCBI Gladiolus illyricus HQ394472 KM887320 69. NCBI Babiana sp. HQ394554 GQ982576 70. NCBI Babiana torta GQ248252 GQ382018 71. NCBI Chasmanthe aethiopica HQ394555 AJ409572 72. NCBI Crocus oreocreticus KF886667 HE864269 73. NCBI Crocus sativus KF886664 HE864251 74. NCBI Crocus niveus EU257491 HE864219 75. NCBI Iris pseudacorus KC584954 KF170871 76. NCBI Iris setosa KC704322 KF170886 77. NCBI Iris odaesanensis KC704309 KF170880 78. NCBI Iris koreana KF170851 KF170878 79. NCBI Iris rossii KC704314 KF170877 80. NCBI Iris minutoaurea KC704306 KF170882 81. NCBI Moraea simplex GQ248344 GQ294108 82. NCBI Moraea pallida GQ248343 GQ294097 83. NCBI Sisyrinchium angustifolium KC704325 KF170891 84. NCBI Sparaxis variegata HQ394559 AJ409582 85. NCBI Hymenocallis littoralis KR857492 JX464401 86. NCBI Asphodeline lutea JQ039289 AB933517

19

Table 2. Results of Basic sequence statistics, Nucleotide diversity, Haplotype diversity,

Nucleotide composition and Nucleotide pair frequencies of psbA-trnH and trnL-trnF

sequences of 66 Gladiolus cultivars.

S. No. Parameters psbA-trnH trnL-trnF 1. Length range (bp) 547 - 557 671-678 2. Total number of sites ((excluding sites with

gaps / missing data) 531 669

3. Conserved sites 510 664 4. Variable (polymorphic) sites 21 5 5. Singleton variable sites 13 1 6. Parsimony informative sites 8 4 7. Total number of InDel sites analysed 26 11 8. Total number of InDels events analysed 18 7 9. Number of InDel Haplotypes 21 9 10. InDel Haplotype diversity 0.853 0.516 11. Number of Haplotypes (h) 12 10 12. Haplotype gene diversity (Hd) 0.559 0.770 13. Nucleotide diversity {π (10−3)} 3.9 1.8 14. Nucleotide frequencies of Adenine (%) 30.3 36.2 15. Nucleotide frequencies of Thymine (%) 33.6 29.1 16. Nucleotide frequencies of Cytosine (%) 17.8 16.2 17. Nucleotide frequencies of Guanine (%) 18.3 18.5 18. Identical pairs (ii) 551 671 19. Transitionsal pairs (si) 1 1 20. Transversional pairs (sv) 2 0

Figure 1. Neighbor-joining clustering based on the sequence variation of the chloroplast

DNA regions (psbA-trnH and trnL-trnF), among the 66 Gladiolus cultivars. Numbers on

nodes are the bootstrap values derived from 1000 replicates.

Figure 2

psbA

from

2. Neighbor-

A-trnH and

m 1000 repli

-joining clus

trnL-trnF r

cates.

stering of Ir

egion. Num

ridaceae bas

mbers on nod

sed on the c

des are the

combined an

bootstrap v

2

nalysis of th

values derive

20

he

ed

221

Figure 3psbAphy

3. The evoluA-trnH and logenetic an

utionary relattrnL-trnF re

nalysis.

tionships of egion. Major

Iridaceae barity-rule con

ased on the nsensus tree

combined aobtained in

2

analysis of ththe MrBaye

22

he es

Figure Sfromvalu

S1. MP boom combinedues derived f

otstrap consed plastid seqfrom 1000 re

ensus tree oquence data eplicates.

f 84 accessanalysis. N

ions of IridNumbers on

aceae and twnodes are

2

wo outgroupthe bootstra

23

p, ap