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Plant Systematics and Evolution ISSN 0378-2697Volume 300Number 4 Plant Syst Evol (2014) 300:689-708DOI 10.1007/s00606-013-0913-8

Comparative pollen morphology of annualTrigonella L. (Fabaceae) in Turkey

N. Munevver Pinar, Hasan Akan, TalipCeter, Zeki Aytac, Murat Ekici, AydanAcar & Selin Akdogan

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ORIGINAL ARTICLE

Comparative pollen morphology of annual Trigonella L.(Fabaceae) in Turkey

N. Munevver Pinar • Hasan Akan • Talip Ceter •

Zeki Aytac • Murat Ekici • Aydan Acar •

Selin Akdogan

Received: 31 July 2013 / Accepted: 30 August 2013 / Published online: 11 September 2013

� Springer-Verlag Wien 2013

Abstract Pollen morphology of 38 Trigonella species

was investigated with light microscopies and scanning

electron microscopies. Pollen slides were prepared using

Wodehouse technique. The pollen grains of Trigonella

members are radially symmetrical and isopolar, their out-

lines are oblong in equatorial view and circular in polar

view. Amb is semicircular. The pollen grains are prolate-

spheroidal, oblate-spheroidal, suboblate, spheroidal, and

prolate with the polar axes 13.5–49.9 lm and the equato-

rial axes 10.4–43.7 lm. The smallest dimensions are

observed in T. spinosa and T. brachycarpa, and the largest

in T. carica and T. rhytidocarpa. The pollen grains of

Trigonella taxa are usually tricolporate or rarely tricolpate,

pantocolporate, tetracolporate, and tricolpodiporate. Vari-

ous ornamentation types were observed: Microreticulate

(section Samaroideae, Pectinatae, Falcatulae, Cylindricae,

Bucerates, Reflexae, Isthmocarpae, Capitatae and Foenum-

graecum), rugulate-scabrate (section Lunatae, Uncinatae),

perforate (T. halophila, T. rigida and T. carica from sec-

tion Bucerates) and retipilatae (section Biebersteinianae) at

equator region and psilate-perforate (section Samaroideae,

Pectinatae, Bucerates, Reflexae, Isthmocarpae, Foenum-

graecum, T. coelesyriaca, T. cilicica), rugulate-scabrate

(section Lunatae, Uncinatae), microreticulate (Falcatulae,

Cylindricae and Foenum-graecum) and retipilatae (section

Biebersteinianae) at polar region. Ornamentations, pollen

shape and the aperture type have been observed as

important morphological characters.

Keywords Fabaceae � Trifoliae � Trigonella �Pollen morphology � SEM � Turkey

Introduction

The tribe Trifolieae in the family Fabaceae includes six

genera: Trigonella L., Medicago Inc., Trifolium L., Meli-

lotus L., Parochetus Buch.-Ham and Ononis L. (Heyn

1981; Lock and Simpson 1991; Mabberley 1997). Trigo-

nella is one of the largest genera in the tribe Trifolieae and

comprises about 135 species widely distributed in dry

regions around the East Mediterranean, West Asia, South

Europe, North and South Africa, with only one species

growing in South Australia (Townsend and Guest 1974;

Polhill and Raven 1981, Kawashty et al. 1998; Ceter et al.

2012; Ranjbar et al. 2012a, b). It has been postulated by

Vavilov that the near Eastern region extends from Palestine

through Syria, southern Turkey into Iran and Iraq, and the

Mediterranean region including Spain, Morocco and Iran,

and that Afghanistan is the center of origin for Trigonella

(Ranjbar et al. 2012a, b). In Turkey, the genus includes 49

species with two subspecies in 13 sections growing in

different phytogeographical regions. There are 21 taxa,

with 42 % endemic to the country (Davis 1970; Akan et al.

2005; Ceter et al. 2012).

N. M. Pinar � A. Acar � S. Akdogan

Department of Biology, Science Faculty, Ankara University,

Ankara, Turkey

H. Akan

Department of Biology, Arts and Science Faculty, Harran

University, Sanlıurfa, Turkey

T. Ceter (&)

Department of Biology, Arts and Science Faculty, Kastamonu

University, Kastamonu, Turkey

e-mail: talipceter@gmail.com

Z. Aytac � M. Ekici

Department of Biology, Science Faculty, Gazi University,

Ankara, Turkey

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Plant Syst Evol (2014) 300:689–708

DOI 10.1007/s00606-013-0913-8

Author's personal copy

Trigonella species are annual or perennial herbs with

pinnately trifoliate leaves, often emitting an odor, and like

other grain legumes, are important in food and medicine

production (Chopra et al. 1956; Girardon et al. 1986; Ba-

lodi and Rao 1991; Bhatti et al. 1996; Dangi et al. 2004).

But all the members in Turkey are annual (Davis 1970;

Akan et al. 2005).

Taxonomic problems of this genus cannot be ignored.

Species of Trigonella are similar to each other in their

morphological characters such as floral architecture and

leaf shape. Several investigators have attempted to employ

the taxonomy of the genus Trigonella. Sirjaev (1934) gave

an elaborate and systematic account of its taxonomy in

Latin. Vasil’chenko (1953a, b) published a synopsis in

Russian discussing the genus within the family Fabaceae.

The genus was divided into three subgenera (Trigonella,

Trifoliastrum and Foenum-graecum) on the ban’s of the

form and shape of calyx and pod by Hutchinson (1964) and

Tutin and Heywood (1964). Numerical taxonomic studies

were done on 62 species of Trigonella and were separated

into 12 sections (Falcatulae, Callicerates, Uncinatae, Cy-

lindricae, Samaroideae, Capitatae, Pectinatae, Erosae,

Verae, Spinosae and Foenum-graecum) of the classical

monographer (Small 1987). According to ‘‘Flora of Tur-

key’’ the genus represented with 52 taxa, after completed

this study, the number of taxa decreased to 50 (49 species

and one subspecies) in Turkey. In Turkish Flora, the genus

was revised by Davis (1970) and Akan et al. (2005) who

divided it into 13 sections: section Samaroideae, Pectina-

tae, Lunatae, Falcatulae, Verae, Cylindricae, Bucerates,

Reflexae, Isthmocarpae, Uncinatae, Capitatae, Bieberstei-

nianae and Foenum-graecum.

Generic circumscription has long been a problematic

question in the tribe Trifolieae subtribe Trigonellinae. In

particular, many studies have been undertaken on ascer-

taining the prope relationships between the genera Medi-

cago and Trigonella (Small 1988; Small et al. 1981a, b, c;

Small and Jomphe 1989). However, no strict delimitation

could be proposed.

Several cytological investigations have been conducted

on Trigonella (Singh and Roy 1970; Singh and Singh 1976;

Agarwal and Gupta 1983; Ahmad et al. 1999; Dundas et al.

2006; Aykut et al. 2009; Martin et al. 2008, 2010, 2011a,

b). Chromosome numbers of 45 taxa of Turkish Trigonella

belonging to 13 sections were determined as 2x = 14, 16,

30, and 46 by Martin et al. (2011a, b).

Seed coat characters have provided an approach to the

systematic relationships among the species of the Trigo-

nella (Small et al. 1989; Gupta 1991; Taia 2004a). Seeds

morphology of 37 taxa of Trigonella from various regions

in Turkey was examined with a scanning electron micro-

scope (SEM) and light microscope (LM). Based on seed

features such as shape, colour, and surface ornamentation

pattern, seven morphological types were recognized Ceter

et al. (2012).

Studies on the pollen grains of the family Fabaceae have

dealt mainly with the description of the pollen of certain

genera or sometimes tribes (e.g., Clarke and Kupicha 1976;

Ferguson and Skvarla 1979, 1981, 1982; Ferguson 1978,

1980, 1981; Ferguson and Strachan 1982; Diez and Fer-

guson 1994; Perveen and Qaiser 1998; Pinar et al. 2009a;

Ceter et al. 2013a, b). Pollen morphology of Melilotus,

Trifolium, Medicago, Factorovskya, and Trigonella of tribe

Trigonelleae was examined using LM and SEM by Small

et al. (1981a, b, c). Investigation on pollen–ovule patterns

(Small 1986) performed to resolve relationships within and

between these genera. A comparative study on the Egyp-

tian species of genus Trigonella showed the presence of ten

species belonging to five sections (Hassan 1992). Ming-Jou

and Tseng-Chieng (1999) studied the pollen morphology

and seed of Trigonella hamosa Linn. by SEM that repre-

sent as new record to the flora of Taiwan. Gazar (2003)

studied the pollen morphology of 28 species belonging to

the three genera (Melilotus, Trifolium and Trigonella) of

subfamily Papilionoideae in Egypt to establish the taxo-

nomic position of the species with in genera. Lashin (2006)

described the pollen grains of Egypt Trigonella foenum-

graecum L. and T. stellata Forssk. species by LM and

SEM. The pollen morphology of Tunisia T. foenum-grae-

cum, T. maritima Delile ex Poir., T. stellata and T. mons-

peliaca L. was studied by Haouala and Beji (2008). The

effect of maleic hydrazide (MH) has been studied on pollen

fertility of T. foenum-graecum to test its cytotoxicity (Jabee

et al. 2008). The pollen morphological characters of Egypt

T. foenum-graecum were investigated by the aid of LM and

SEM (El-Sayed et al. 2010). T. lasiocarpa Ranjbar and

Hajmoradi, T. binaloudensis Ranjbar and Karamian, T.

stipitata Ranjbar and Joharchi and T. torbatejamensis

Ranjbar, T. heratensis Rech. and T. adscendens Aphan. and

Gontsch from Iran are compared with their closest relatives

using morphology and pollen micromorphology (Ranjbar

et al. 2012b). The pollen morphology of T. yasujensis

Ranjbar, Hajmoradi, Karamian and T. elliptica Boiss. was

studied by Ranjbar et al. (2012a).

There are some other studies focusing on the Trigonella

species out of Turkey; (Sirjeav 1935), Meusel and Jager

(1962), Baum (1968), Small et al. (1981a, b), Classen et al.

(1982), Small (1988), Danin and Small (1989), Small and

Jomphe (1989), Al-Habori et al. (1998), Sheoran et al.

(1999), Ram and Verma (2000), Murakami et al. (2000),

Oncina et al. (2000), Sur et al. (2001), Sabir et al. (2002)

and Petropoulus et al. (Petropoulos 2002).The members of

Trigonella and particularly of the cultivated species T.

foenum–graecum (fenugreek) were known and used for

different purposes in ancient times, especially in Greece

and Egypt (Petropoulos 2002). In N. Africa, it has been

690 N. M. Pinar et al.

123

Author's personal copy

cultivated around the Saharan oases since very early times

(Duke 1986).

In this study, the pollen grains of 38 Trigonella taxa

showing morphological similarities were investigated with

LM and SEM, so that palynological comparisons could be

made and inter- and intra-generic relationships based on

pollen morphological data could be discussed. This study

attempts to clarify the systematics of this genus.

Materials and methods

Plant materials

Materials used for this study were collected from wild

populations. Collectors and localities are shown in the

specimens investigated list (Table 1). The specimens of the

plants are deposited in the Gazi (GAZI) and Harran (HRU)

Universities Herbaria. The order of the species was adapted

from Davis (1970) and Akan et al. (2005). All the speci-

mens are deposited in GAZI and HRU.

Palynological analysis

Pollen slides were prepared using the Wodehouse (1935)

technique. Pollen grains were stained with glycerin gel

with safranin, and slightly heating the slide and then a

coverslip was placed on it. The prepared slides were

studied under the light microscope. Their photographs were

taken with the Leica DM3000 digital photomicrograph

system (Germany). Measurements were based on 20 or

more pollen grains per specimen. For scanning electron

microscopy (SEM) studies, dried pollen grains were

transferred on aluminum stubs and coated with gold for

4 min in a sputter coater (Pinar et al. 2009b; Altuner et al.

2012; Ceter et al. 2013c). Morphological observations were

made in a Jeol JSM 6490LV Scanning electron microscope

at TPAO Research Center SEM Laboratory, Ankara.

Terminology was adopted from Faegri and Iversen

(1992), Punt et al. (1994, 2007), Hesse et al. (2009), Pınar

et al. (2009a), and shape classification follows that of Er-

dtman (1969) based on P/E ratio in Table 2.

Numerical analysis

The Simpson and Roe graphical test (van der Pluym and

Hideux 1997) was used for statistical calculations (Fig. 1).

Pollen characters of the taxa, coefficient of correlation

were determined, and they were grouped using the clus-

tering analysis method (UPGMA, dissimilarity, standard-

ized variable).

Table 1 The collections data of investigated Trigonella

Taxon Locality Voucher

T. cretica (L.) Boiss. C2 Burdur Akan & Ekici 3480

T. plicata Boiss & Bal. C4 Konya Akan, Aytac &

Ekici 3789

T. brachycarpa (Fisch.) Moris. C3 Antalya Akan & Ekici 3486

Trigonella lunata Boiss. C6 Adana Akan & Ekici 4687

T. corniculata L. C2 Mugla Akan & Ekici 2827

T. spinosa L. C2 Mugla Akan & Ekici 5655

T. coelesyriaca Boiss. C6

Gaziantep

Akan & Ekici 2610

T. spruneriana var. spruneriana

Boiss.

C3 Antalya Akan 3330

T. spruneriana var. sibthorpii

Fenzl.

C6 Antalya Akan & Ekici 3976

T. mesopotamica Desv. C8 Mardin Akan & Ekici 5578

T. cilicica C6 Adana Akan & Ekici 4682

T. filipes Boiss. C7

Sanlıurfa

Akan & Ekici 3200

T. velutina Boiss. A9 Kars Akan, Aytac &

Ekici 3743

T. strangulata Boiss. C8 Mardin Akan & Ekici 5577

T. smyrnea Boiss. C3 Antalya Akan & Ekici 4630

T. arenicola Hub.-Mor. C3 Antalya Akan & Ekici 3311

T. fischeriana Ser. B5 Kayseri Akan 3654

T. tenuis Fisch. A9 Kars Akan, Aytac &

Ekici 3742

T. astroites Fisch & Mey. B5 Aksaray Akan & Ekici 4750

T. halophila Boiss. C5 Mersin Akan & Ekici 4555

T. crassipes Boiss. B2 Denizli Akan & Ekici 3452

T. rigida Boiss.& Bal. C6 Adana Akan & Ekici 4737

T. pamphylica C3 Antalya Akan & Ekici 3331

T. carica Hub. Mor. C2 Mugla Akan & Ekici 3366

T. monantha C.A. Meyer subsp.

monantha

C2 Burdur Akan & Ekici 3466

T. orthoceras A9 Kars Akan & Ekici 3738

T. monspeliaca L. A7

Trabzon

Akan & Ekici 3690

T. isthmocarpa Boiss. & Bal. B5 Aksaray Akan & Ekici 4751

T. rhytidocarpa Boiss. & Bal. C5 Nigde Akan & Ekici 3590

T. spicata Sibth & Sm. A5 Samsun Akan & Ekici 4715

T. cephalotes C2 Mugla Akan & Ekici 2830

T. procumbens B3 Konya Akan & Ekici 4650

T. capitata Boiss. C2 Denizli Akan & Ekici 4767

T. coerulescens (Bieb.) Hal. C1 Aydın Akan, Aytac &

Ekici 3439

T. gladiata Stey Fischer B1 Izmir, Akan & Ekici 4622

T. foenum-graecum Boiss. C6

Gaziantep

Akan & Ekici 3227

T. macrorrhyncha Boiss. C5 Mersin Akan & Ekici 3293

T. raphanina Boiss. C5 Icel Akan & Ekici 2848

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Five palynological (consisting of nominal and qualita-

tive) characters were selected to distinguish the 38 taxa

(OTUs) of the genus Trigonella. Five morphological

characters state and their values or scales are given at

Table 2. A primary mixed data matrix was created for 38

taxa (OTUs) and five palynological characters (Table 2)

for the analysis. Since Gower’s formula (Jabee et al. 1971)

modified by Podani (1999) allows the inclusion of ordinal

variables and missing scores in the data matrix, it was used

to calculate the primary mixed data for dissimilarities.

Unweighted pair group method using arithmetic averages

(UPGMA) was selected because it is the most commonly

used method (Mohammadi and Prasanna 2003) and it has

advantages to other methods in: accurate reflection of the

similarity matrix as measured by the co-phenetic correla-

tion coefficient of Sokal and Rohlf (1962), symmetrical

hierarchical structure [the ‘‘structural value’’ concept of

McNeill (1979)], and congruence with classification

derived by traditional methods (Ward 1993). All compu-

tations were made by the MVSP 3.1 software.

Table 2 Five palynological characters to distinguish the 38 taxa of the genera Trigonella

Taxa/character states T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 T13 T14 T15 T16 T17 T18 T19

Equatorial orn. 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Polar orn. 0 0 1 1 2 2 0 2 2 2 0 2 2 2 2 0 0 0 0

Aperture orn. 0 1 2 2 0 0 1 0 0 0 0 0 0 0 0 1 1 1 1

Pollen shape 0 0 1 2 2 2 2 3 2 3 4 2 3 0 2 2 2 2 2

Clg 4.2 3.1 1.6 2.7 4.3 2.1 2.3 2.8 2.4 2.6 4.2 3.1 1.8 3.1 2.3 5.2 2.4 4.2 4.2

Taxa/character states T20 T21 T22 T23 T24 T25 T26 T27 T28 T29 T30 T31 T32 T33 T34 T35 T36 T37 T38

Equatorial orn. 2 0 2 0 2 2 0 0 0 0 3 3 0 0 3 0 0 0 0

Polar orn. 3 0 3 0 3 3 0 0 0 0 1 1 0 0 3 0 0 0 0

Aperture orn. 1 1 1 1 1 1 1 1 1 1 2 2 1 1 3 0 0 0 0

Pollen shape 2 0 0 2 0 4 0 0 2 0 2 2 2 5 3 3 3 3 3

Clg 2.1 4.2 4.7 7.8 3.2 4.2 5.2 5.2 4.2 5.2 3.1 4.2 5.3 1.6 2.1 2.1 2.6 2.8 3.6

Fig. 1 Simpson and Roe test

for Trigonella. a Polar axes (P),

b equatorial axes (E)

692 N. M. Pinar et al.

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Table 3 Qualitative pollen features of different taxa of Trigonella (value in lm)

Taxa P/E Pollen shape Aperture type Ornamentation

Equatorial Polar

Sect: Samaroideae

T. cretica (T1) 1.07 Prolate-spheroidal Tricolporate Microreticulate Psilate-perforate

Sect: Pectinatae

T. plicata (T2) 1.06 Prolate-spheroidal Tricolporate Microreticulate Psilate-perforate

Sect: Lunatae

T. brachycarpa (T3) 0.87 Suboblate Tricolporate Rugulate-scabrate Rugulate-scabrate

T. sirjaevii (T4) 1.28 Subprolate Tricolporate Rugulate-scabrate Rugulate-scabrate

Sect: Falcatulae

T. corniculata (T5) 1.24 Subprolate Tricolporate Microreticulate Microreticulate

T. spinosa (T6) 1.2 Subprolate Tricolporate Microreticulate Microreticulate

T. coelesyriaca (T7) 1.18 Subprolate Tricolporate Microreticulate Psilate-perforate

Sect: Cylindricae

T. spruneriana var. spruneriana (T8) 1.72 Prolate Tricolporate Microreticulate Microreticulate

T. spruneriana var. sibthorpii (T9) 1.34 Prolate Tricolporate Microreticulate Microreticulate

T. mesopotamica (T10) 1.58 Prolate Tricolporate Microreticulate Microreticulate

T. cilicica (T11) 0.96 Oblate- spheroidal Tricolporate Microreticulate Psilate-perforate

T. filipes (T12) 1.3 Subprolate Tricolporate Microreticulate Microreticulate

T. velutina (T13) 1.4 Prolate 60 %Tricolporate

20 %Triporate

20 %Abnormal pollen

Microreticulate Microreticulate

T. strangulata (T14) 1.07 Prolate-spheroidal 10 %Trisyncolporate

90 %Tricolporate

Microreticulate Microreticulate

T. smyrnea (T15) 1.25 Subprolate 10 %Tricolpate

%90 Tricolporate

Microreticulate Microreticulate

Sect: Bucerates

T. arenicola (T16) 1.14 Subprolate 20 %Tricolpate

20 %Pantocolporate

60 %Trizonocolporate

Microreticulate Psilate-perforate

T. fischeriana (T17) 1.13 Subprolate Tricolporate Microreticulate Psilate-perforate

T. tenuis (T18) 1.15 Subprolate %80 Tricolporate

%20 Abnormal pollen

Microreticulate Psilate-perforate

T. astroites (T19) 1.32 Subprolate Tricolporate Microreticulate Psilate-perforate

T. halophila (T20) 1.15 Subprolate Tricolporate Perforate Perforate

T. crassipes (T21) 1.11 Prolate-spheroidal Tricolporate Microreticulate Psilate-perforate

T. rigida (T22) 1.07 Prolate-spheroidal Tricolporate Perforate Perforate

T. pamphylica (T23) 1.25 Subprolate Tricolporate Microreticulate Psilate-perforate

T. carica (T24) 1.14 Prolate-spheroidal Tricolporate Perforate Perforate

T. monantha subsp. monantha (T25) 0.9 Oblate-spheroidal Tricolporate Perforate Perforate

T. orthoceras (T26) 1.02 Prolate-spheroidal %1 Tricolpate

%5 Tetracolporate

%94 Tricolporate

Microreticulate Psilate-perforate

Sect: Reflexae

T. monspeliaca (T27) 1.11 Prolate-spheroidal Tricolporate Microreticulate Psilate-perforate

Sect: Isthmocarpae

T. isthmocarpa (T28) 1.13 Subprolate %80 Tricolporate

%20 Tricolpodiporate

Microreticulate Psilate-perforate

Comparative pollen morphology of annual Trigonella L. (Fabaceae) 693

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Table 3 continued

Taxa P/E Pollen shape Aperture type Ornamentation

Equatorial Polar

T. rhytidocarpa (T29) 1.22 Prolate-spheroidal %80 Tricolporate

%20 Tricolpodiporate

Microreticulate Psilate-perforate

Sect: Uncinatae

T. spicata (T30) 1.3 Subprolate Tricolporate Rugulate-scabrate Rugulate-scabrate

T. cephalotes (T31) 1.15 Subprolate Tricolporate Rugulate-scabrate Rugulate-scabrate

Sect: Capitatae

T. procumbens (T32) 1.2 Subprolate Tricolporate Microreticulate Psilate-perforate

T. capitata (T33) 1 Spheroidal Tricolporate Microreticulate Psilate-perforate

Sect: Biebersteinianae

T. coerulescens (T34) 1.45 Prolate Tricolporate Retipilate Perforate

Sect: Foenum-graecum

T. gladiata (T35) 1.39 Prolate Tricolporate Microreticulate Psilate-perforate

T. foenum-graecum (T36) 1.5 Prolate Tricolporate Microreticulate Psilate-perforate

T. macrorhyncha (T37) 1.67 Prolate Tricolporate Microreticulate Psilate-perforate

T. raphanina (T38) 1.39 Prolate Tricolporate Microreticulate Psilate-perforate

* Martin et al. (2008, 2011a, b)

Table 4 Quantitative pollen features of different taxa of Trigonella (value in lm)

Taxa Chromosome number (2n)/

ploidy level (x)*

Polar axis (P) Equatorial axis (E) Exine Intine Colpus Pore

Min Max Mean Min Max Mean Clt Clg Plt Plg

Sect: Samaroideae

T. cretica (T1) 16 (2x) 24.9 31.2 27.8 22.9 31.2 26 1.8 0.3 4.2 22.4 8.3 7.3

Sect: Pectinatae

T. plicata (T2) 14 (2x) 26 32.2 28.6 22.9 31.2 27.1 1 0.3 3.1 23.9 10.9 8.8

Sect: Lunatae

T. brachycarpa (T3) 16 (2x) 13.5 20.8 17.2 16.6 22.9 19.8 2.4 0.6 1.6 12.5 4.1 2.3

T. sirjaevii (T4) 16 (2x) 15.6 24.8 22.3 15.6 22.9 17.4 2.1 0.5 2.7 18.4 7.4 7.2

Sect: Falcatulae

T. corniculata (T5) 16 (2x) 15.2 29.1 23.1 16.6 21.8 18.6 1.1 0.3 4.3 17.4 5.4 6.7

T. spinosa (T6) 16 (2x) 13.5 18.7 15.6 10.4 16.6 13 0.8 0.3 2.1 12 4.2 2.6

T. coelesyriaca (T7) 16 (2x) 20.8 29.1 24.6 17.7 26 20.8 0.8 0.3 2.3 18.7 9.4 7.3

Sect: Cylindricae

T. spruneriana var.

spruneriana (T8)

16 (2x) 26 32.2 28.6 14.6 20.8 16.6 0.6 0.3 2.8 22.9 5.2 4.2

T. spruneriana var.

sibthorpii (T9)

16 (2x) 15.2 22.9 18.7 11.4 20.8 15.1 0.8 0.5 2.4 15.4 5.4 4.3

T. mesopotamica (T10) 16 (2x) 26 31.2 27.4 14.5 20.8 17.3 0.8 0.3 2.6 22.9 8.3 4

T. cilicica (T11) 16 (2x) 20.8 31.2 23.4 17.6 30.1 24.2 1.4 0.5 4.2 19.7 6.7 4.2

T. filipes (T12) 16 (2x) 25 33.3 29.8 19.8 25.2 22.9 1.3 0.3 3.1 26 6.2 6.2

T. velutina (T13) 16 (2x) 21.4 26 23.9 14.5 20.8 17.7 0.8 0.3 1.8 23.4 7.8 5.2

T. strangulata (T14) 16 (2x) 29.1 34.3 31.2 26 34.3 29.1 1.3 0.3 3.1 26 8.3 6.2

T. smyrnea (T15) 16 (2x) 20.8 29.1 26 16.6 26 20.8 1.1 0.3 2.3 23.9 2.6 1.8

Sect: Bucerates

T. arenicola (T16) 16 (4x) 30.2 41.6 34.8 22.9 35.4 30.4 1.8 0.6 5.2 31.2 6.2 11.4

T. fischeriana (T17) 14 (4x) 31.2 42.6 37.2 29.1 36.4 32.8 1.4 0.5 2.4 28.8 8.8 7.8

T. tenuis (T18) 16 (6x) 21.8 31.2 24.4 18.7 23.9 21.3 0.8 0.3 4.2 19.8 7.3 7.3

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Results

Detailed pollen morphological features of the investigated

taxa are summarized in Tables 3, 4 and representative

pollen grains are illustrated in Figs. 2, 3, 4, 5, 6, 7, 8, 9, 10.

Size, symmetry and shape

The pollen grains of Trigonella are radially symmetrical

and isopolar. The pollen grains of the Trigonella are pro-

late-spheroidal, oblate-spheroidal, suboblate, spheroidal,

and prolate with the polar axes 13.5–49.9 lm and the

equatorial axes 10.4–43.7 lm (Fig. 1). Their dimensions

are smaller in T. spinosa and T. brachycarpa, and larger in

T. carica and T. rhytidocarpa. Their outlines are oblong in

equatorial view and circular in polar view: amb semicir-

cular. (Tables 3, 4; Figs. 2, 3, 4, 5, 6, 7, 8, 9, 10).

Apertures

The pollen grains of Trigonella are usually tricolporate or

rarely tricolpate, tripantocolporate, tetracolporate, and

colpodiporate. Some species have shown heteromorphic

characteristics. For example, 10 % tricolpate and 90 %

tricolporate in T. smyrnea, 20 % tricolpate, 20 % panto-

colporate and 60 % trizonocolporate in T. arenicola, 1 %

tricolpate, 5 % tetracolporate and 94 % tricolporate in T.

orthoceras, 20 % tricolpodiporate and 80 % tricolporate in

T. isthmocarpa. Colpus is short or long (12–39.5 lm) and

narrow or broad (1.6–7.8 lm) and por is circular or

lalongated. The highest values were observed in T. carica

and T. rhytidocarpa. T. spinosa and T. brachycarpa have

smallest values of colpus. Margins distinct are regular and

ends are acute. Colpus membrane is more or less granulate

(Tables 3, 4; Figs. 2, 3, 4, 5, 6, 7, 8, 9, 10).

Table 4 continued

Taxa Chromosome number (2n)/

ploidy level (x)*

Polar axis (P) Equatorial axis (E) Exine Intine Colpus Pore

Min Max Mean Min Max Mean Clt Clg Plt Plg

T. astroites (T19) 16 (4x) 29.1 34.3 31.7 20.8 28.1 23.9 1.3 0.3 4.2 26 8.3 6.2

T. halophila (T20) 16 (4x) 29.1 36.4 32.2 21.8 33.3 28.1 1.3 0.3 2.1 26 10.9 9.9

T. crassipes (T21) 16 (4x) 25 32.2 28.1 22.9 28.1 25.3 1.7 0.5 4.2 19.8 6.2 6.2

T. rigida (T22) 16 (4x) 30.2 36.4 32.2 25 36.4 30.2 2.1 0.6 4.7 25 11.4 9.4

T. pamphylica (T23) 16 (4x) 36.4 42.6 39.5 29.1 34.3 31.7 1.3 0.8 7.8 31.2 10.4 9.9

T. carica (T24) 16 (4x) 42.6 49.9 46.3 38.4 43.7 40.6 2.3 1 3.2 36.4 17.7 12.5

T. monantha subsp.

monantha(T25)

16,28, 30 (4x) 26 33.3 29.1 29.1 36.4 32.2 1.6 0.3 4.2 23.4 5.7 7.3

T. orthoceras (T26) 46 (4x) 23.9 31.2 27.1 22.9 31.2 26.5 1.1 0.3 5.2 23.9 7.3 6.2

Sect: Reflexae

T. monspeliaca (T27) 16 (2x) 21.8 31.2 26.5 20.8 27.1 23.9 1.4 0.6 5.2 18.7 6.2 5.2

Sect: Isthmocarpae

T. isthmocarpa (T28) 16 (2x) 29.1 36.4 32.3 25 32.2 28.7 1.6 0.5 4.2 29.1 4 4

T. rhytidocarpa (T29) 16 (2x) 39.5 47.8 43.2 29.1 39.5 35.4 3.1 0.6 5.2 39.5 10.4 10.4

Sect: Uncinatae

T. spicata (T30) 16 (2x) 22.9 31.2 26 17.7 23.9 20.8 1.3 0.3 3.1 21.8 9.4 6.2

T. cephalotes (T31) 16 (2x) 20.8 29.1 25 18.7 25 21.8 1.1 0.3 4.2 25 6.8 4.7

Sect: Capitatae

T. procumbens (T32) 18 (2x) 18.7 34.3 27.4 15.6 27.1 22.8 0.5 0.3 5.3 24.3 8.3 7.3

T. capitata (T33) 16 (2x) 16.6 22.9 19.8 16.6 21.8 19.8 1.3 0.6 1.6 15.6 6.2 5.1

Sect: Biebersteinianae

T. coerulescens (T34) 16 (2x) 29.1 40.6 33.3 18.7 26 22.9 2.1 0.3 2.1 26 7.3 7.3

Sect: Foenum-graecum

T. gladiata (T35) 16 (2x) 38.5 31.2 34.3 22.9 28.1 24.6 1.3 0.5 2.1 27.6 8.3 6.2

T. foenum-graecum

(T36)

16 (2x) 28.1 33.4 31.2 18.7 23.9 20.8 1.8 0.6 2.6 23.9 7.3 5.2

T. macrorhyncha (T37) 16 (2x) 43.7 47.8 52 28.1 33.3 31.2 2.8 0.4 2.8 35.9 13 7.8

T. raphanina (T38) 16 (2x) 39.5 46.8 42.6 27.1 34.3 30.7 1.8 0.3 3.6 31.7 10.4 8.3

* Martin et al. (2008, 2011a, b)

Clg colpus length, Clt colpus width, Plg porus length, Plt porus width

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Exine

The overall thickness of stratified exine ranges from 0.5- to

3.1-lm Ectexine is thicker than endexine. The intine

thickness ranges from 0.3 to 1.3 lm (Tables 3, 4). Various

ornamentation types were observed: microreticulate (sec-

tion Samaroideae, Pectinatae, Falcatulae, Cylindricae,

Bucerates, Reflexae, Isthmocarpae, Capitatae and

Fig. 2 Light microscope microphotograph of Trigonella. 1–4 T. cretica, 5–8 T. plicata, 9–12 T. brachycarpa, 13–16 T. sirjaevii, 17–20 T.

corniculata, 21–24 T. spinosa

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Foenum-graecum), rugulate, scabrate (section Lunatae,

Uncinatae), perforate (T. halophila, T. rigida and T. carica

from section Bucerates) and retipilatae (section Bieber-

steinianae) at equator and psilate-perforate (section

Samaroideae, Pectinatae, Bucerates, Reflexae,

Isthmocarpae, Foenum-graecum, T. coelesyriaca, T. cili-

cica), rugulate-scabrate (section Lunatae, Uncinatae),

microreticulate (Falcatulae, Cylindricae and Foenum-

graecum) and retipilatae (section Biebersteinianae) at polar

region. The lumina in the microreticulate regular and

Fig. 3 Light microscope microphotograph of Trigonella. 1–4 T. coelesyriaca, 5–8 T. spruneriana var. spruneriana, 9–12 T. spruneriana var.

sibthropii, 13–16 T. mesopotamica, 17–20 T. cilicica, 21–24 T. filipes

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0.3–1.5 lm diameter over the equatorial surface and muri

usually deep, concave and 0.075–0.13 lm thickness. The

ornamentation near aperture varies between microreticulate

in section Samaroideae, Falcatulae, Cylindricae, and

Foenum-graecum and psilate in section Pectinatae,

Bucerates, Reflexae, Isthmocarpae, Capitatae,

Fig. 4 SEM microphotograph of Trigonella. 1, 2 T. cretica, 3, 4 T.

plicata, 5, 6 T. brachycarpa, 7, 8 T. sirjaevii, 9, 10 T. corniculata, 11,12 T. spinosa, 13, 14 T. coelesyriaca, 15, 16 T. spruneriana var.

spruneriana, 17, 18 T. spruneriana var. sibthropii, 19, 20 T.

mesopotamica, 21, 22 T. cilicica, 23, 24 T. filipes

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Biebersteinianae and T. coelesyriaca). In Trigonella,

numerical analysis of the differential palynological char-

acters, which became evident during the course of our

investigations, led to the realization of a dendrogram. This

dendrogram shows the similarities or dissimilarities which

exist among the taxa being studied. A dendrogram of

cluster analysis of Turkish Trigonella taxa based on five

character states of 38 taxa (OTUs) has been constructed.

Cluster analysis divided taxa into three main groups,

namely, cluster A, B and C. Cluster A includes:

Fig. 5 Light microscope microphotograph of Trigonella. 1–4 T. velutina, 5–8 T. strangulata, 9–12 T. smyrnea, 13–16 T. arenicola, 17–20T. fischeriana, 21–24 T. tenuis

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T. coerulescens, T. monantha subsp. monantha, T. carica,

T. rigida, T. halophila; Cluster B includes: T. spicata, T.

uncinata, T. brachycarpa, T. sirjaevii; Cluster C1 includes:

T. smyrnea, T. strangulata, T. velutina, T. filipes, T. mes-

opotamica, T. spruneriana var. spruneriana, T. spruneriana

var. sibthropii, T. corniculata, T. spinosa. Cluster C2

includes:T. cilicica, T. capitata, T. gladiata, T. foenum-

graecum, T. macrorhyncha, T. raphanina. Cluster C3

includes two sub-groups, namely, C3a and C3b. C3a

includes: T. coelesyriaca, T. arenicola, T. fischeriana,

Fig. 6 Light microscope microphotograph of Trigonella. 1–4 T. astroites, 5–8 T. halophila, 9–12 T. crassipes, 13–16 T. rigida, 17–20 T.

pamphylica, 21–24 T. carica

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T. tenuis, T. astroites, T. pamphylica, T. isthmocarpa, T.

procumbens. C3b includes: T. cretica, T. pilicata, T.

crassipes, T. orthoceras, T. monspeliaca, T. rhytidocarpa.

Results presented according to palynological data in this

study showed that almost each of taxa separated between

own section (Fig. 11).

Fig. 7 SEM microphotograph of Trigonella. 1, 2 T. velutina, 3, 4 T. strangulata, 5, 6 T. smyrnea, 7, 8 T. arenicola, 9, 10 T. fischeriana, 11, 12T. tenuis, 13, 14 T. astroites, 15, 16 T. halophila, 17, 18 T. crassipes, 19, 20 T. rigida, 21, 22 T. pamphylica, 23, 24 T. carica

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Fig. 8 Light microscope

microphotograph of Trigonella.

1–4 T. monantha subsp.

monantha, 5–8 T. orthoceras,

9–12 T. monspeliaca, 13–16 T.

isthmocarpa, 17–20 T.

rhytidocarpa, 21–24 T. spicata,

25–28 T. cephalotes

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Fig. 9 Light microscope

microphotograph of Trigonella.

1–4 T. procumbens, 5–8 T.

capitata, 9–12 T. coerulescens,

13–16 T. gladiata, 17–20 T.

foenum-graecum, 21–24 T.

macrorhyncha, 25–28 T.

raphanina

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Discussion

The grains of the taxa of Trigonella can be ascribed to the

‘‘Astragalus affghanus’’ type by Perveen and Qaiser

(1998), ‘‘1B and 2’’ types by Taia (2004b), on the basis of

pollen shape class (P/E) and exine sculpturing of type 1 by

Lashin (2006) and pollen shape class prolate type and

sculpture microreticulate subtype by El-Sayed et al. (2010).

Pollen grains of the genus Trigonella are heterogenous

and exhibit considerable variation in size (P and E) and

shape (P/E). (Tables 3, 4; Fig. 1). Similarly, Ceter et al.

(2012) found that the seeds of Trigonella are very variable

in size, shape, and colour. According to the values of the

seed length and width, the seeds are classified into four

groups. However, on the basis of pollen shape class (P/E),

there are three distinct pollen types viz., Type 1 (1.33

subprolate, prolate) Trigonella sp., Type 2 (1.22 subpro-

late) Melilotus, Medicago and Trifolium sp., Type 3 (1.11

prolate, spheroidal) Ononis sp. are recognized by Lashin

(2006). The thickness of exine and intine are almost sim-

ilar. (Tables 3, 4).

In general, speciation within Trigonella including

perennial species has occurred on the diploid level

(2n = 2x = 16) whereas with annuals are diploid, tetra-

ploid, and hexaploid with 2n = 2x = 16, 2n = 4x = 32

and 2n = 6x = 48 chromosome numbers (Martin et al.

2008; Aykut et al. 2009; Ranjbar et al. 2011). The annual

taxa of Turkish Trigonella have 2n = 14, 16, 30, and 46

chromosomes numbers (Martin et al. 2011a, b). Some

meiotic irregularities observed in different populations of

T. spruneriana and T. yasujensis included chromosomes

stickiness and formation of micronuclei in tetrad cells and

cytomixis (Ranjbar et al. 2011, 2012a, b). T. spruneriana

var. spruneriana (26–32.2 9 14.6–20.8 lm, prolate) and

T. spruneriana var. sibthorpii (15.2–22.9 9 11.4–20.8,

subprolate) show variation in size (P and E) and shape

(P/E) in Turkey. In addition, Chatuverdi et al. (1990) and

Brochman (1992) reported that pollen grain size strongly

correlates with the level of polyploid. Similarly, maximum

the values of P and E in tetraploid and hexaploid species of

section Bucerates have been observed. (Tables 3, 4;

Figs. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10)

Based on tectum structure, four distinct pollen types

are distinguished in Melilotus sp. (tectate perforate,

reticulate and distinct pore), Medicago and Trigonella sp.

(tectate imperforate, reticulate, verrucate, and pore

indistinct), Trifolium sp. (semi-tectate, reticulate, and

pore indistinct) and Ononis sp. (tectate perforate, rugulate

and pore distinct) by Lashin (2006). Taia (2004b) recog-

nized that Trigonella has reticulate, foveolate, granulate

exine. We found that the pollen of annual Trigonella

members is very variable in exine ornamentation (mic-

roreticulate, rugulate-scabrate, perforate, retipilatae at

Fig. 11 Dendrogram showing dissimilarity distance of the examined taxa

Fig. 10 SEM microphotograph of Trigonella. 1, 2 T. monantha

subsp. monantha, 3, 4 T. orthoceras, 5, 6 T. monspeliaca, 7, 8 T.

isthmocarpa, 9, 10 T. rhytidocarpa, 11, 12 T. spicata, 13, 14 T.

cephalotes, 15, 16 T. procumbens, 17, 18 T. capitata, 19, 20 T.

coerulescens, 21, 22 T. gladiata, 23, 24 T. foenum-graecum, 25, 26 T.

macrorhyncha, 27, 28 T. raphanina

b

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equator region and psilate-perforate, rugulate-scabrate,

microreticulate, and retipilatae at polar region) (Tables 3,

4; Figs. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10).

The general aperture form is tricolporate, but T. smyrnea

(10 % tricolpate and 90 % tricolporate), T. arenicola

(20 % tricolpate, 20 % pantocolporate, and 60 % trizono-

colporate), T. orthoceras (1 % tricolpate, 5 % tetracolpo-

rate, and 94 % tricolporate), and T. isthmocarpa (20 %

tricolpodiporate and 80 % tricolporate) show considerable

aperture type variation (Tables 3, 4; Figs. 1, 2, 3, 4, 5, 6, 7,

8, 9, 10, 11). Variations in pollen size and aperture type

were attributed to heteromorphy in pollen grains by Nair

and Kaul (1965) and Inceoglu (1973)

Acknowledgments The authors thank Scientific and Technological

Research Council of Turkey (TUBITAK, TBAG Project No. 2099)

for financial support.

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