Morphological and Molecular Cladistic Analyses of Heliobrychis Section (Onobrychis Genus)

Abstract

Abstract
According to Flora Iranica, Heliobrychis section has 21 species. In this study, morphological and molecular cladistic analyses were performed for 29 Onobrychis taxa belong to Heliobrychis section. The result of morphological cladistic analysis indicate that the relationship of O. aucheri subsp. aucheri and O. aucheri subsp. psammophila were resolved (BP=52%), and O. aucheri subsp. teheranica was sister to them with 94% bootstrap support. In molecular cladistic analysis O. aucheri subsp. psammophila and O. aucheri subsp. teheranica were closest subspecies with high bootstrap support (BP=79%) but their relationships with O. aucheri subsp. aucheri were not resolved. In morphological and molecular cladistic analyses, there were no close relationships between O. menaotricha var. melanotricha and O. melanotricha var. villosa and each one formed sister group with other species. So contrast to Rechinger classification,O. melanotricha var. melanotricha and O. melanotricha var. villosa was raised to the rank of species as O. melanotricha and O. villosa.
Keywords: Cladistic analyses, O. aucheri, O. psammophila, O. teheranica, O. melanotricha,
O. villosa.

Keywords


Original Research

 


                 Research on Crop Ecophysiology                                   Vol.12/1, Issue 1 (2017), Pages: 25 - 35

 

 

Morphological and Molecular Cladistic Analyses of Heliobrychis Section (Onobrychis Genus)

SanamSafaeiChaeikar1,Farangis Ghanavati2andHosien Amirabadi-zadeh3

1-Tea Research Center, Harticultural Sciences Research Institute, Agricultural Research, Education and Extension Organization, Lahijan, Iran.

2-Seed and Plant Improvement Institute, Karaj, Iran

3- Agricultural and Natural Resources Research Center of KorasanRazavi Province, Mashhad, Iran

 

 

Corresponding author: safaei.sanam@gmail.com

Received:                                   Accepted:

 

Abstract

According to Flora Iranica, Heliobrychis section has 21 species. In this study, morphological and molecular cladistic analyses were performed for 29 Onobrychis taxa belong to Heliobrychis section. The result of morphological cladistic analysis indicate that the relationship of O. aucheri subsp. aucheri and O. aucheri subsp. psammophila were resolved (BP=52%), and O. aucheri subsp. teheranica was sister to them with 94% bootstrap support. In molecular cladistic analysis O. aucheri subsp. psammophila and O. aucheri subsp. teheranica were closest subspecies with high bootstrap support (BP=79%) but their relationships with O. aucheri subsp. aucheri were not resolved. In morphological and molecular cladistic analyses, there were no close relationships between O. menaotricha var. melanotricha and O. melanotricha var. villosa and each one formed sister group with other species. So contrast to Rechinger classification,O. melanotricha var. melanotricha and O. melanotricha var. villosa was raised to the rank of species as O. melanotricha and O. villosa.

Keywords: Cladistic analyses, O. aucheri, O. psammophila, O. teheranica, O. melanotricha,
O. villosa.

Introduction

The genus Onobrychis Miller (Hedysareae, Papilionoideae, Leguminosae) with 130 perennial and annual species wordwide, is distributed mainly in the north temperate regions, but its centers of diversity are in the eastern Mediterranean area and western Asia (Yildiz et al. 1999). The genus was subdivided into two subgenera and nine sections (Rechinger 1984).

The taxonomy of the genus Onobrychis continues to be a subject of much confusion, mainly because of the different approaches to species delimitation, resulting in varying numbers of recognized species (Boissier 1872, Sirjaev 1925, Hedge 1970, Ball 1978,Rechinger 1984, Duman and Vural 1990, Aktoklu 2001). Inconsistency within the taxonomy of Onobrychis genus can be resolved by determining the phylogenetic relationships of Onobrychis species with morphological and molecular phylogenies. Defining of the phylogenetic relationships of these species, beside the introduction of new species and determination of taxonomic position of the species would enhance the efficiency of Onobrychis breeding program.

One of the contradictions in taxonomy of Onobrychis genus is about Heliobrychis Bunge ex Boiss. section. This section was described by Rechinger (1984) which comprises 21 species. On the basis of Amirabadi-zadeh et al. (2007) results, general characteristics of the section are described as perennial or annual plants; crestless pods covered with pinnate bristles, orbicular, stipitate, along with a curved suture bearing seeds.

The aim of this research was to present result of phylogenetic studies on Heliobrychis section and to determine accurate taxonomic positions of species belong to this section.

 

Material and Methods

 

Taxon sampling

 

Twenty-nine taxa representing Heliobrychis section of Onobrychis genus were included as in-group taxa in the analyses. Based on molecular phylogenies using nrDNA ITS (SafaeiChaeiKar et al. 2012), two species of the other genus of tribe Hedysareae were selected as out-groups. Details of these taxa, including accession identities, geographical origins and gene bank sequence accession numbers, are given in Table 1.

 

Characters and characters states

 

Characters used in the cladistic analyses were obtained through examination of fresh materials in the field and herbarium specimens deposited at Herbarium of National Plant Gene Bank of Iran (NPGBI), Herbarium Research Center of Khorasan-e-Razavi Agricultural and Natural Resources center (MRCH), Mashhad, Iran and Herbarium of the Research Institute of Forests and Rangelands (TARI). Sixty-one characters which were used in the present analysis included: longevity, vegetation form, presence of prickle; stem (presence of stem, presence of hair, state of stem); stipule (tissue, position of stipule, presence of hair, length of stipule, shape of stipule); leaf (number of basal leaflets, number of cauline leaflet, leaflet form, size of leaflet width, length of leaflet, state of tip of the leaflet, presence of mucronateor apiculate, presence of hair at upper surface of leaflet, presence of hair at inferior surface of leaflet, size of petiole); peduncle (size of peduncle rather than leaves, state of peduncle at the end); inflorescence (shape of raceme, number of flowers per raceme); bract (figure of bract, cover of bract, length of bract); calyx (teeth figure of calyx, length of calyx, teeth rather than tube, presence of hair); corolla (color of flower, appearance of corolla); standard (figure of standard, length of standard, state of tip, presence of claw, presence of hair); wing (length of wing, cover of wing, state of tip, wing rather than calyx, figure of wing); keel (cover of keel); ovary (cover of ovary, presence of stipe, number of ovule); pod (size of pod, figure of pod, appearance of pod, presence of hair, presence of stipe, presence of crest, state of margin, number of seed, state of dorsal suture of pod, state of surface of disc, number of loculus, shape of loculusat surface of disc, curvature).

 

DNA extraction, PCR and sequencing

 

Sequence data from nuclear ribosomal DNA internal transcribed spacers (nrDNA ITS) were obtained in order to analyze phylogenetic relationships of species. Total genomic DNAS were isolated from dried leaf material using the modified CTAB (hexadecyltrimethyl ammonium bromide) method (Doyle & Doyle 1987). The complete nrDNA ITS region was amplified using ITS4 and ITS5 primers (White et al. 1990). PCR amplification were carried out on a thermal cycler using the following parameters: initial denaturation at 94˚c for 3 min followed by 30 cycles of denaturation at 94˚c for 30s, annealing at 52˚cfor 45s and extension at 72˚cfor 1 min and a final extension at 70˚c for 10 min.

Successfully amplified samples were purified using a gel purification kit (USA, Bioneer, Inc.). Nucleotide sequences of purified PCR products were determined using cycle sequencing and an automated DNA sequencer through Bioneer Co. The same nrDNA ITS primers ITS4 and ITS5 were utilized for cycle sequencing reactions. The sequences from the forward and reverse primers in each sample were aligned to generate a consensus sequence. As the sequences were of high quality, the forward and reverse sequences were identical, except for a few cases. These few discrepancies were resolved by repeated PCR and sequencing. Finally, each sequence related to each species was registered at the NCBI and a sequence accession number was obtained.

The nrDNA ITS sequence were aligned with Clustal W 1.8 (Thampson et al., 1994) and adjusted manually.

 

Cladistic analyses

 

Phylogenetic analyses were performed on the data matrix (morphological data) and aligned nrDNA ITS sequences using Maximum Parsimony method (MP) as implemented in the version 4.0b 10 of PAUP* (Swofford, 2002). All characters were considered as equally weighted. The heuristic search option was selected using 100 replications of random addition sequence with TBR (tree bisection reconnection) branch-swapping. In the analyses, supports for clades were evaluated by bootstrapping (Felsenstain, 1985) using 100 replications.

 

 

 

Table 1. locality, voucher and sequence accession number of Heliobrychis section of Onobrychis genus

Species

Locality

Voucher number

Sequence accession number

EbenusstellataBioss.

Kerman: 27 Km from Jiroft towards Mahan, near Mohammadabad village, 1680 m.

MRCH 7442

JX 426796

Eversmanniasubspinosa (Fisch.) B. Fedtsch.

Semnan: 28 Km from Shahrood towards Azadshahr, 1500 m.

MRCH 8983

JX 494755

O. aureaRanjbar, Amirabadizadeh&Ghahremani

EasternAzarbayjan: Tabriz, Khajeh, Abkhandari research center, 1450 m.

MRCH 10037

JX 455139

O. sojakiiRech. F.

Kohkiloyeh and Boyerahmad: 45 Km from Khosravi towards Mamsani, near to Babameydan, 1800 m.

MRCH 5996

JX 426798

O. kermanensis (Sirj. &Rech. F.) Rech. F.

Kerman: Sirjan

NPGBI 6236

JX 426797

O. psoraleifolia var. psoraleifoliaBoiss.

Esfahan: Ghomishloo, Baghak, 2000 m.

MRCH 1610

JX 290033

O. andalanicaBornm.

Sanandaj: Abidar park

NPGBI 6143

JX 455124

O. sylvatica

Khorasan: Mashhad

MRCH 5993

JX 290043

O. szovitsiiBoiss.

Azarbayjan: 23 Km southeast of Khoy, SeyedHajin village, 1400-1450 m.

MRCH 6037

JX 455128

O. buhseana Bung ex Boiss.

EasternAzarbayjan: Boostanabad towards Sarab, 1800 M.

NPGBI 3875

JX 412238

O. melanotricha var. villosaBornm.

Zanjan: Zanjan towards Ghorveh, Babarishani village, 1971 m.

NPGBI 6144

JX 455120

O. melanotricha var. melanotrichaBoiss.

Hamedan: Nahavand, 1954 m.

NPGBI 6042

JX 290041

O. oxypteraBoiss.

Fars: 15 Km from Saadatshahr towards Arsanjan, 1800 m.

TAR I87626

JX 412235

O. gypsicolaRech. F.

Khozestan: 16 Km from BagheMalak towards Ramhormoz, 900 m.

MRCH 7757

JX 426799

O. plantagoBornm.

Kerman: Chopar mountain, 2600 m.

NPGBI 6237

JX 455140

O. scrobiculataBoiss.

Gharachaman

NPGBI 2823

JX 290034

O. lunataBoiss.

Hamedan:Malayer, 1994 m.

NPGBI 6041

JX 455127

O. iranshahriiRech. F.

Kerman

MRCH 8265

JX 455125

O. depauperata var. depauperataBoiss.

EasternAzarbayjan: Marand towards Tabriz, Payam village, Mishoodagh hills

MRCH 8184

JX 455136

O. marandensis

EasternAzarbayjan: Marand towards Tabriz, Payam village, Mishoodagh hills, 1900 m.

MRCH 10052

JX 455119

O. haussknechtiiBoiss.

Kermanshah

NPGBI 6145

JX 429954

O. gaubaeBornm.

Tehran: the first of Damavand road, 20 Km from Bomhen, 1700 m.

NPGBI 6146

JX 455118

O.mozaffarianiiAmirabadi-zadeh

Esfahan: Semirum, Hanna, between Maurak and Khina to Khafr, 1900 m.

TARI 71263

JX 426794

O. atropatanaBoiss.

Eastern Azarbayjan: Marand towards Zanooz, 1500 m.

NPGBI 3880

JX 412239

O. argyreaBoiss.

Azarbayjan: 2-12 Km west of Zenooz, 1500-1700 m.

NPGBI 10026

JX 412236

O. aucheri subsp. aucheriBoiss.

Ardabil: 15 Km Mianeh towards Ardabil, opposite of Ghazal-Ozan river, 110 m.

NPGBI 6147

JX 455117

O. aucheri subsp. psammophila (Bornm.) Rech. F.

Khorasan: TorbatHeydarieh, Kashmar, Baharieh, 1211 m.

NPGBI 6148

JX 455122

O. aucheri subsp. teheranica (Bornm.) Rech. F.

Khorasan: Neishaboor towards Sabzevar, after Baghjar

NPGBI 6149

JX 455123

O. subacaulisBoiss.

Eastern Azarbayjan: 8 Km Zenooz, Kanglomaraei, 1380 m.

MRCH 8478

JX 455135

O.heliocarpaBioss.

East Azarbayjan: Marand, Zenooz village

NPGBI 6163

JX 429959

O. heterophylla C. A. Mey.

East Azarbayjan: Varzaghan towards Ahar, 20 Km remained to Satarkhan dam, 1721 m.

NPGB 6151

JX 455121

MRCH: Mashhad Research Center Herbarium.

NPGBI: National Plant Gene Bank of Iran.

TARI: Herbarium of the Research Institute of Forests and Rangelands.

 

 

Results

 

Morphological cladistic analysis

 

The phylogenetic analysis based upon equally weighted characters yielded 524 most-parsimonious trees of 256 steps with CI of 0.38 and RI of 0.53. The strict consensus tree of these 524 trees is shown in Figure 1. Species belong to Heliobrychis section formed a strongly supported clade (BP=96%). Species relationships within this section were not properly resolved but O. scrobiculata and O. atropatana formed a weakly supported subclade (subclade a) (BP=60%) and species relationships within clade, b, are well resolved. Within this clade (clade b), O. aucheri subsp. aucheri and O. aucheri subsp. Psammophila were weakly allied taxa (BP=52%) and O. aucheri subsp. teheranica was sister to them (BP=94%). Also O. heliocarpawas sister to clade which include three subspecies belong to O. aucheri (BP= 84%). Some taxonomic distinctions among three subspecies of O. aucheri were mainly associated with state of stem; number of cauline leaflet; figure of wing; presence of stipe; figure of pod; number of ovule and number of seed (Table 2). Furthermore, in Heliobrychis section O. melanotricha var. melanotricha and O. melanotericha var. villosa were not closely related with each other. Some of their morphological differences considered in this study were summarized in Table 3.

 

Molecular cladistic analysis

 

Maximum parsimony (MP) analyze of the nrDNA ITS dataset (characters equally weighted) generated 825 most parsimonious tree with the length of 238 steps, CI=0.82 and RI=0.73. The 50% majority rule consensus tree from the phylogenetic analysis of nrDNA ITS sequences of 29 Onobrychistaxa (taxa belong to Heliobrychis section) and Eversmannia sub spinosa and Ebenusstellata as an out-group species is shown in Figure2.

 

Table 2. Comparison of morphological characters of three sub-species of O. aucheri

Sub-species

Morphological characters

 

Stem

Leaf

Wing

Ovary

Pod

 

State of stem

Number of cauline leaflet

Figure of wing

Number of ovule

Presence of stipe

Figure of pod

Number of seed

O. aucheri subsp. aucheri

procumbent

1-4 pairs

lanceolate

2 or 3

non stipitate

suborbicular

2 or 3

O. aucher subsp. teheranica

procumbent

> 4 pairs

narrowly oblong

1

stipitate

suborbicular

1

O. aucheri subsp. psammophila

erect

1-4 pairs

deltoid-oblong

2 or 3

stipitate

reniform

2 or 3

 

Table 3. Comparison of morphological characters of two varieties of O. melanotricha

varieties

Morphological characters

 

Stem

Stipule

Leaf

Bract

Calyx

 

Presence of hair

State of stem

Position of stipule

Leaflet form

Presence of mucronate or apiculate

Length of bract

Teeth rather than tube

O. melanotricha var. melanotricha

glabrous

procumbent

sessile

oblong-ovate

without mucronate or apiculate

<5 m.m

equal

O. melanotricha var. villosa

glabrescent

erect

free

oblong-elliptic

mucronate-apiculate

>5 m.m

longer

 

Table 3. Continued

varieties

Morphological characters

 

Standard

Wing

Pod

 

Figure of standard

State of tip

Presence of claw

Length of wing

State of tip

Figure of wing

Figure of pod

O. melanotricha var. melanotricha

ovate

emarginate

with claw

shorter-equal with half the length of the keel

very acute

deltoid-oblong

lunate

O. melanotricha var. villosa

roundish

obtuse

without claw

longer than half the length of the keel

acute

lanceolate

suborbicular

 

 

 

 


 

Figure 1. Maximum- parsimony 50% majority rule consensus tree generated from a phylogenetic analysis of morphological data of twenty-nine Onobrychis taxa (belong to Heliobrychis section) and two genus of tribe Hedysareae as an out-group. Bootstrap values of >50 % are indicated above the branches.

 

Figure 2. Maximum- parsimony 50% majority rule consensus tree generated from a phylogenetic analysis of DNA sequences data from internal transcribed spacers of the nrDNA of twenty-nine Onobrychis taxa (belong to Heliobrychis section) and two genus of tribe Hedysareae as an out-group. Bootstrap values of >50 % are indicated above the branches.

Species of Heliobrychis section made up a well-supported (BP=100%) and partially resolved clade, in which, there were a series of clades and subclades that will be explained about their phylogenetic relationships.

O. aucheri subsp. teheranica and O. aucheri subsp. psammophila formed a well-supported subclade (BP=79%), but these subspecies (O. aucheri subsp. teheranica and O. aucheri subsp. psammophila) were not close relatives to O. aucheri subsp. Aucheri. In other words O. aucheri subsp. teheranica and O. aucheri subsp. psammophilawere closest species with high bootstrap support value of 79%, but they were not allied with O. aucheri subsp. aucheri,and O. aucheri subsp. aucheri was contained in a monoclade. O. melanotricha var. melanotricha and O. melanotricha var. villosa were not closely related with each other, so that O. melanotricha var. melanotricha formed a trichotomy with O. heterophylla and O. sojakii (BP=65%) and O. melanotricha var. villosa and O. oxyptera formed a well-supported subclade (BP=92%) that related to O. lunata (BP= 74%). This result indicates that there was no close association between the two varieties of O. melanotricha.

Also the phylogenetic relationships between O. gaubae and O. mozaffarianii; O. atropatana and O. argyrea; O. buhseana and O. aurea; O. scrobiculata and O. sylvatica was resolved (Figure 2).

 

Discussion

 

Phylogenetic relationships of species belong to Heliobrychis section in molecular cladistic analysis resolved better than in morphological cladistic analysis because of the highest number of parsimony-informative characters in molecular cladistic analysis.

O. aucheri was for the first time identified by Boissier (1872). Bornmuller (1905) recognized two species of Onobrychis namely, O. teheranica and O. psammophila, and based on Rechinger (1984) taxonomic work, O. aucheri subsp. aucheri, O. aucheri subsp. psammophila and O. aucheri subsp. teheranica are morphologically similar to each other, and for this reason Rechinger (1984) introduced them as subspecies belong to O. aucheri.Thus, according to Flora Iranica (Rechinger 1984) O. aucheri from sect. Heliobrychis has three subspecies; O. aucheri subsp. aucheri, O. aucheri subsp. teheranica and O. aucheri subsp. psammophila.

Based on morphological and karyotypic studies done by Hatami and Nasirzadeh (2008), O. aucheri subsp. teheranica was diploid (2n=2x=16) but O. auchri subsp. psammophila was tetraploid (2n=4x=32). Karyotypic formula and karyotypic symmetry showed that O. aucheri subsp. teheranica was 7m+1sm, allocated to 1B class and O. aucheri subsp. psammophila was 12m+4m, allocated to 2B class. According to the morphological characteristics, two subspecies have important differences including: stem; number of leaflet; spines on pod, number of seed per pod, size of pod; sepal and vegetation form. So they concluded that due to morphological and karyotypic differences between O. aucheri subsp. psammophila and O. aucheri subsp. teheranica, could not be regarded as subspecies of O. aucheri and the suggestion of Bornmuller (1905) on regarding the two subspecies as two species is preferred.

In this research based on morphological cladistic analysis, relationship between O. aucheri subsp. aucheri and O. aucheri subsp. psammophila were resolved but statistically not well supported (BP=52%), and O. aucheri subsp. teheranica was sister to them with 94% bootstrap support. However, morphological cladistic analysis of this study indicated that these two subspecies (O. aucheri subsp. aucheri and O. aucheri subsp. psammophila) were weakly relative taxa, but they share consecutive morphological characteristics including; number of cauline leaflet, number of ovule, and number of seed. Morphological studies indicated that O. aucheri subsp. aucheri with procumbent stem; lanceolate wing; non-stipitate, suborbicular pod is distinguish from O. aucheri subsp. psammophila. Also, it differs from O.aucheri subsp. teheranica in having less cauline leaflets and 2 or 3 ovules and seeds. In molecular cladistic analysis O. aucheri subsp. psammophila and O. aucheri subsp. teheranica were closest subspecies with high bootstrap support (BP=79%), but their relationships with O. aucheri subsp. aucheri were not resolved. So we conclude that,due to phylogenetic relationships between three subspecies belong to O. aucheri based on morphological and molecular cladistic analyses, they will be considered as subspecies of O. aucheri and the suggestion of Rechinger (1984) is preferred and the Bornmuller (1905) and Hatami and Nasirzadeh (2008) opinions are rejected.

In addition to, Rechinger (1984) recommended O. melanotricha has two varieties namely: O. melanotricha var. melanotricha and O. melanotricha var. villosa. In morphological and molecular cladistic analyses, there were not close relationships between O. menaotricha var. melanotricha and O. melanotricha var. villosa and each one formed sister group with other species. The interspecies relationships of Heliobrychis section displayed by cladistic analyses of morphological and molecular phylogenies differed from those exhibited by traditional taxonomic classification (Rechinger 1984). Due to lake of close relationships between two varietis of O. melanotricha based on morphological and molecular analyses, we propose that this two varieties raised to the rank species as O. melanotricha and O. villosa.

 

Conclusion

 

Based on morphological and molecular cladistic results of this research, it is recommended that the two varieties belong to O. melanotricha (O. melanotricha var. melanotricha and O. melanotricha var. villosa) be introduce as species namely, O. melanotricha and O. villosa respectively.

 

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