267
http://journals.tubitak.gov.tr/botany/
Turkish Journal of Botany Turk J Bot
(2015) 39: 267-279
© TÜBİTAK
doi:10.3906/bot-1406-23
Pollen morphology of the genus Alchemilla L. (Rosaceae) in Iran
Marzieh Beygom FAGHIR1,*, Farideh ATTAR2, Robabeh Shahi SHAVVON1, Atefeh MEHRMANESH1
1
Department of Biology, Faculty of Science, University of Guilan, Rasht, Iran
2
Central Herbarium of Tehran University and School of Biology, University College of Science, Tehran, Iran
* Correspondence: marziehbeygomfaghir@gmail.com
1. Introduction
The genus Alchemilla L. (Rosaceae), with ca. 1000 species,
is one of the most species-rich Holarctic plant genera,
widespread mainly in western Eurasia. Some species
of the genus also show a preference for mountains of
South India, Sri Lanka, Java, China, Japan, Africa, and
Madagascar (Izmailow, 1981). The genus belongs to a
critical and taxonomically difficult group. Several authors
divided the genus into different subgenera, sections,
groups, subgroups, series (De Candolle, 1825; Focke,
1888; Buser, 1892; Lagerheim, 1894; Rothmaler, 1944;
Fröhner, 1995; Kalkman, 2004; Notov and Kusnetzova,
2004; Hayirlioglu-Ayaz and Inceer, 2009), and a large
number of micro-species and species complexes. This
is most likely due to confusion resulting from frequent
apomixis, polyploidization, and hybridization observed in
this group (Asker and Jerling, 1992; Sepp, 2000; Hörandl,
2004; Gehrke et al., 2008). The genus was thought to be
related to the tribe Sanguisorbeae (Juzepczuk, 1941) due
to superficial similarity caused by reduction in flower
parts (Gehrke et al., 2008). However, its relation to the
tribe Potentilleae was noted by Schulz-Menz (1964) and
has been supported by molecular analysis (Eriksson et al.,
1998, 2003). Alchemilla, Aphanes L., and Lachemilla Rydb.
are placed in the subtribe Alchemillinae based on anther
structure (one elliptic theca) and style position (subbasal
to basal) (Soják, 2008). Alchemilla L. is represented
by 31 species in Flora Iranica (Fröhner, 1969) and 24
representatives in Flora of Iran (Khatamsaz, 1993). They
are mainly distributed in the N and NW but some species
grow in W and C Iran. Among them, 14 representatives
are endemic to Iran (Fröhner, 1969; Khatamsaz, 1993).
The species of the genus are herbaceous perennial plants,
preferring open meadows, stony slopes, shady places, river
banks, and forest edges of alpine and subalpine regions
from 1700 to 3300 m altitudes (Juzepczuk, 1941; Fröhner,
1969; Khatamsaz, 1993). The palynological data of the
genus are poorly understood and only limited information
focused on pollen morphology of Rosaceae genera was
presented (Hebda et al., 1988; Hebda and Chinnappa,
1990). The aim of the current survey was to describe
the pollen morphological characters of Iranian species
of Alchemilla and to determine how these traits relate to
species relationship.
2. Materials and methods
In this survey, 18 Iranian species of Alchemilla underwent
palynological analysis using a light microscope (LM) and
scanning electron microscope (SEM). Out of them 16 are
included in the numerical analysis. We used pollen grains
Abstract: This paper reports the morphological characters of 18 Iranian species of the genus Alchemilla using light and scanning
electron microscopy. The pollen grains are monad, radially symmetrical, isopolar, or subisopolar; small to medium in size; triand tetracolporate; rectangular to cylindrical (from equatorial view) and triangular to circular (from polar view) in outline;
and prolate-spheroidal to subprolate and prolate in shape. The exine ornamentation is psilate and microechinate. Based on the
exine sculpturing and microechinate distribution pattern, three main types, four subtypes, and two categories of pollen grains
were recognized. We used cluster analysis and principal component analysis to determine the potential contribution of pollen
morphological characters to the species relationships. Our findings revealed the significance of palynological evidence in explaining
the species relationship. The results of two multivariate analyses showed a close affinity among the seven studied species (A. amardica,
A. sericata, A. fluminea, A. kurdica, A. hyrcana, A. sedelmeyeriana, and A. pesudocartalinica).
Key words: Alchemilla, Rosaceae, palynological characters, numerical analysis, Iran
Received: 08.06.2014 Accepted: 05.10.2014 Published Online: 16.03.2015 Printed: 10.04.2015
Research Article
FAGHIR et al. / Turk J Bot
268
of both fresh (collected from 2010 to 2012, during spring
and later summer) and dried herbarium specimens of
Guilan University Herbarium (GUH), Tehran University
Herbarium (TUH), and Herbarium of the Research
Institute of Forests and Rangelands of Iran (TARI). The
voucher specimens of newly collected samples were
deposited in Guilan University Herbarium (GUH). A list
of specimens used in this analysis is presented in Table 1.
For LM, flower buds were acetolyzed following the method
described by Harley (1992). Prepared slides were studied
with an Olympus BH-2 microscope and photographed by
a Nikon Coolpix S10 camera. Measurements were taken
from at least 25 grains per species. The pollen characters
were measured under a ×40 eyepiece, and are summarized
in Tables 2 and 3. For SEM observation, grains were fixed
on aluminum stubs 12.5 mm in diameter covered with
double-sided cellophane tape and then sputter coated
(Emitech k450) with gold. The micrographs were taken
using an SEM model VEGA/TESCAN in Razi Metallurgical
Research Center (RMRC), Tehran. The pollen terminology
in general follows Erdtman (1952), Eide (1981), Ueda and
Tomita (1989), and Punt et al. (2007).
Table 1. The species used in the current analysis.
Species IRAN: Province, Collector, Date Accession No.
1. A. amardica Rothm Guilan: Deylaman; Shahe shahidan; Chaichi, Faghir and Shahi; 6. 2012. 4872 (GUH)
2. A. caucasica Buser
Mazandaran: Kojur; Firozabad village; 1700 m; Ghahreman and Attar; 19.6. 1997.
Mazandaran: Karaj - Chalus, Zangule Bridge; 3000 m; Nazarian; 10.1997
20598 (TUH)
33155 (TUH)
3. A. citrina Fröhner Guilan: Deylaman; Shahe Shahidan; Chaichi, Faghir and Shahi; 6.2012. 4876 (GUH)
4. A. condensa Fröhner Guilan: Masal; Chaichi; 2012.
Guilan: Deylaman, Larikhani, 1500 m; Saeidi; 20.5.1993.
4871 (GUH)
18845 (TUH)
5. A. erythropoda Juz..
Mazandaran: Kojour, Firoozabad village; 1700 m; Ghahreman and Attar; 8.1999
Mazandaran: Kojur; Kikuh Mont, Zinoosht Rangeland. 2000–2300 m, Khatamsaz and Gholoizadeh.
Gilan: mountain above Damash-east of Rudbar: 1900 m; P.Wendelbo & Ann Ala.
Azarbayejan: Shahbil, Kohe Sabalan, 3450 m; Foroughi and Assadi.
20595 (TUH)
751847(TARI)
18232(TARI)
24248(TARI)
6. A. fluminea Fröhner Guilan: Deylama, Larikhani, 1530 m; Ghahreman and Attar. 18844 (TUH)
7. A. hessii Rothm. Mazandaran: Kojur, Firozabad Village; 1700 m; Ghahreman and Attar; 19.6. 1997.
Mazandaran: Kandovan; Ghahreman, Augustine and Sheikholeslami; 6.1974.
20600/1 (TUH)
19418 (TUH)
8. A. hyrcana (Buser) Juz. Guilan: Deylaman; Shahe Shahidan; Chaichi, Faghir and Shahi; 6.2012.
Mazandaran: Kojur; Firozabad village; 1700 m; Ghahreman and Attar; 19.6. 1997.
4873 (GUH)
20597 (TUH)
9. A. kurdica Rothm. Bornm. Guilan: Masal; Khashkhami; Chaichi, Faghir, and Shahi; 6.2012. 4875 (GUH)
10. A. pectinoloba Fröhner Guilan: Deylama; Larikhani; 1530 m; Saeidi; 5.1993 18837 (TUH)
11. A. persica Rothm.
Mazandaran: Tonokabon, Jannat Rudbar, 1600 m; Ghahreman, Attar, and Khatamsaz; 20.6.1997.
Azarbijan: Arasbaran, Veighan; Makidan; 1400 m; Ghahreman, Attar, and Hamzehei; 2006.
Mazandaran: Karaj to Chalus road, Polezangule bridge, 2600 m; Nazarian; 24.6. 1999.
Tehran: Darake; Mobayen; 1969.
Mazandaran: Chalus road; Mobayen; 20.4.1965
20603 (TUH)
35575 (TUH)
33440 (TUH)
19419 (TUH)
8603 (TUH)
12. A. plicatissima Fröhner Guilan: Almas pass; Chaichi, Faghir, and Shahi; 8.2012. 4869 (GUH)
13. A. pseudocartalinica Juz. Mazandaran: Kojur; Firozabad village; 1700 m; Ghahreman and Attar; 19.6. 1997. 20602 (TUH)
14. A. rechingeri Rothm. Mazandaran: Kojur; Firozabad village; 1700 m; Ghahreman and Attar; 19.6. 1997. 20601 (TUH)
15. A. retinervis Buser.
Mazandaran: Kojur: Firozabad village, 1700 m; Ghahreman and Attar; 19.6. 1997.
Azarbaijan: Maku, Southwest Mountain Church Kennedy, 2400 m to 2650 m; Mozaffarian Asadi 11.9.2009.
20599 (TUH)
30 336(TARI)
16. A. rigida Buser, Guilan: Espili; Larikhani; 1510 m; Saeidi; 5.1993.
Mazandaran: Kojur; Firozabad Village; 1700 m; Ghahreman and Attar; 19.6. 1996.
18842 (TUH)
20596 (TUH)
17. A. sedelmeyeriana Juz. Mazandaran: Kojur: Firozabad village; 1700 m; Ghahreman and Attar; 19.6. 1997.
Mazandaran: Firozabad Mont, 2200m, Khatamsaz and gholizade.
20593 (TUH)
57165(TARI)
18. A. sericata Reichen. Azarbaijan: Kaleibar to Makidi; 1510 m; Ghahreman, Mozaffarian, and Sheikholeslami; 5.1993. 17540 (TUH)
FAGHIR et al. / Turk J Bot
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Table 2. Pollen morphology data: Numbers refer to (minimum-) mean ± standard deviation (-maximum), Polar axis (P), Equatorial
axis (E), Polar axis/Equatorial axis (P/E) ratio, Pollen shape (Ps) and Size (Si), Distance between the apices of two ectocolpi/equatorial
diameter (d/D), Mesocolpium (Me), Colpus length/polar axis (Cl/P), Number of Colpi (No. Cl), Colpus length (Cl), Spine height
(Sh), Spine diameter (Sd), Exine thickness (Et), Prolate-spheroidal (PS), Prolate (P), Subprolate (Subp), Small (S), Medium (M).
Species P E=D P/E Ps Si d d/D Me
1. A. amardica 18.92 (19.09 ± 0.18) 19.35 16.18 (16.3 ± 0.17) 16.56 1.17 Subp S 4 (4.07 ± 0.09) 4.2 0.24 7.78 (8.22 ± 0.36) 8.6
2. A. caucasica 19.96 (20.14 ± 0.12) 20.22 18 (18.17 ± 0.15) 18.38 1.10 PS S 7 (7.65 ± 0.55) 8.2 0.42 7.9 (8.15 ± 0.26) 8.5
3. A. citrina 17.5 (17.81 ± 0.34) 18.2 12.9 (13.02 ± 0.12) 13.2 1.36 P S 3.5 (3.9 ± 0.27) 4.1 0.29 7.5 (7.92 ± 0.43) 8.5
4. A. erythropoda 24.24 (24.68 ± 0.37) 25 15.78 (15.86 ± 0.057) 15.91 1.55 P S 4.3 (4.37 ± 0.07) 4.46 0.27 4.6 (4.7 ± 0.09) 4.82
5. A. condensa 22.94 (23.15 ± 0.14) 23.26 14.6 (14.79 ± 0.16) 15 1.56 P S 5.2 (5.36 ± 0.12) 5.5 0.36 4.7 (4.78 ± 0.08) 4.9
6. A. fluminea 26.71 (26.96 ± 0.36) 27.5 19.29 (19.39 ± 0.13) 19.6 1.39 P M 6.74 (7.14 ± 0.37) 7.59 0.36 3.7 (3.86 ± 0.12) 4
7. A. hessii 26.5 (27.42 ± 0.8) 28.2 18.2 (18.34 ± 0.12) 18.51 1.49 P M 4.12 (4.28 ± 0.16) 4.5 0.23 4.26 (4.35 ± 0.09) 4.48
8. A. hyrcana 16.08 (16.4 ± 0.58) 17.28 11.89 (12.19 ± 0.2) 12.32 1.34 P S 3.7 (3.87 ± 0.17) 3.87 0.31 5.15 (5.44 ± 0.38) 6
9. A. kurdica 22.8 (23.4 ± 0.4) 23.66 16.2 (16.35 ± 0.1) 16.45 1.43 P S 4.8 (4.96 ± 0.12) 5.1 0.30 5.6 (5.76 ± 0.16) 5.98
10. A. persica 20 (20.16 ± 0.14) 20.34 16.1 (16.23 ± 0.14) 16.43 1.24 Subp S 5.5 (5.60 ± 0.096) 5.73 0.34 6.52 (6.63 ± 0.10) 6.77
11. A. pectinoloba 19 (19.14 ± 0.16) 19.38 15.8 (16.02 ± 0.26) 16.4 1.19 Subp S 3.87 (4.3 ± 0.39) 4.8 0.26 6.97 (7.47 ± 0.42) 7.97
12. A. plicatissima 16.65 (16.8 ± 0.1) 16.89 10.6 (10.79 ± 0.16) 10.98 1.55 P S 3.17 (3.23 ± 0.08) 3.35 0.29 3.62 (3.73 ± 0.09) 3.83
13. A. pseudocartalinica 21.7 (21.77 ± 0.07) 21.88 13.86 (14.02 ± 0.23) 14.38 1.55 P S 3.51 (4 ± 0.38) 4.39 0.28 3.75 (3.85 ± 0.09) 3.98
14. A. rechingeri 24.8 (25.12 ± 0.27) 25.4 16.7 (16.83 ± 0.125) 17 1.49 P S 3.12 (3.28 ± 0.12) 3.4 0.19 3.6 (3.71 ± 0.08) 3.8
15. A. retinervis 18.09 (18.23 ± 0.14) 18.41 13 (13.13 ± 0.10) 13.25 1.38 P S 5.01 (5.12 ± 0.089) 5.21 0.38 6.24 (6.32 ± 0.089) 6.45
16 .A. sedelmeyeriana 14 (14.21 ± 0.14) 14.34 9.4 (9.57 ± 0.28) 10 1.48 P S 2.8 (2.92 ± 0.09) 3 0.30 2.7 (2.78 ± 0.08) 2.89
17. A. sericata 20.56 (20.79 ± 0.3) 21.22 17.27 (17.44 ± 0.12) 17.55 1.19 Subp S 4.88 (4.97 ± 0.09) 5.1 0.28 9 (9.5 ± 0.4) 9.88
18. A. rigida 14.41 (14.51 ± 0.09) 14.61 9 (9.65 ± 0.43) 9.91.50 P S 3.5 (3.91 ± 0.29) 4.2 0.40 4.28 (4.58 ± 0.21) 4.75
Table 2. (Continued).
Species Cl/P No. Cl Cl Sh Sd Et
1. A. amardica 0.76 3–4 14.2 (14.65 ± 0.44) 15.2 0.10 (0.12 ± 0.02) 0.16 0.16 (0.21 ± 0.05) 0.28 0.89 (0.95 ± 0.09) 1.02
2. A. caucasica 0.57 3–4 10.9 (11.52 ± 0.66) 12.19 0.15 (0.18 ± 0.02) 0.19 0.12 (0.16 ± 0.02) 0.18 1.45 (1.90 ± 0.63) 1.90
3. A. citrina 0.64 3 10.7 (11.5 ± 0.7) 12.33 0.10 (0.14 ± 0.02) 0.16 0.18 (0.21 ± 0.03) 0.25 1.05 (1.13 ± 0.11) 1.21
4. A. erythropoda 0.80 3–4 19.68 (19.76 ± 0.09) 19.88 0.29 (0.31 ± 0.01) 0.33 0.18 (0.25 ± 0.05) 0.30 0.80 (1.02 ± 0.31) 1.24
5. A. condensa 0.86 3 19.7 (19.96 ± 0.22) 20.25 0.09 (0.14 ± 0.04) 0.19 0.10 (0.19 ± 0.07) 0.26 1.90 (2.25 ± 0.49) 2.60
6. A. fluminea 0.77 3 20.41 (20.90 ± 0.45) 21.5 0.10 (0.14 ± 0.03) 1.17 0.13 (0.20 ± 0.06) 0.26 1.17 (1.23 ± 0.08) 1.29
7. A. hessii 0.87 3 23.15 (24.04 ± 0.68) 24.8 0.13 (0.14 ± 0.01) 0.15 0.15 (0.23 ± 0.07) 0.33 1.83 (1.90 ± 0.10) 1.98
8. A. hyrcana 0.82 3 12.86 (13.45 ± 0.61) 14.1 0.10 (0.13 ± 0.02) 0.17 0.14 (0.17 ± 0.03) 0.21 1.88 (2.04 ± 0.22) 2.20
9. A. kurdica 0.84 3 19 (19.71 ± 0.51) 20.2 0.12 (0.13 ± 0.02) 0.16 0.12 (0.16 ± 0.04) 0.22 0.94 (1.05 ± 0.16) 1.17
10. A. persica 0.70 3 14 (14.15 ± 0.13) 14.32 - - 1.05 (1.17 ± 0.17) 1.30
11. A. pectinoloba 0.86 3 16.14 (16.59 ± 0.34) 16.96 0.19 (0.20 ± 0.01) 0.21 0.14 (0.21 ± 0.05) 0.28 1.15 (1.24 ± 0.13) 1.34
12. A. plicatissima 0.93 3–4 14.3 (15.71 ± 1.26) 16.85 0.14 (0.17 ± 0.02) 0.21 0.12 (0.17 ± 0.03) 0.21 1.41 (1.53 ± 0.16) 1.65
13. A. pseudocartalinica 0.81 3 17 (17.67 ± 0.89) 19 0.11 (0.12 ± 0.01) 0.10 0.15 (0.19 ± 0.03) 0.23 1.67 (2.23 ± 0.79) 2.80
14. A. rechingeri 0.79 3 19.5 (19.96 ± 0.7) 21 0.11 (0.16 ± 0.03) 0.19 0.18 (0.21 ± 0.03) 0.25 1.05 (1.19 ± 0. 20) 1.34
15. A. retinervis 0.70 3 12.5 (12.78 ± 0.20) 13 - - 0.90 (1.22 ± 0.45) 1.22
16 .A. sedelmeyeriana 0.80 3 11.24 (11.49 ± 0.34) 12 0.09 (0.13 ± 0.01) 0.12 0.13 (0.15 ± 0.01) 0.17 1.82 (1.92 ± 0.14) 2.03
17. A. sericata 0.88 3 18 (18.43 ± 0.33) 18.72 0.13 (0.16 ± 0.03) 0.21 0.17 (0.21 ± 0.04) 0.26 1.73 (1.85 ± 0.17) 1.98
18.A. rigida 0.81 3 11.62 (11.84 ± 0.21) 12.1 0.09 (0.11 ± 0.02) 0.15 0.10 (0.12 ± 0.02) 0.15 1.08 (1.13 ± 0.07) 1.19
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2.1. Data analysis
In the current survey, two numerical analyses including
cluster analysis and principal components analysis
(PCA) were carried out. In these analyses 13 quantitative
and quantitative evidences comprising the mean of
quantitative and coded qualitative characters, as binary/
multistate characters, were involved. The standardized
variables were employed for multivariate statistical
analysis. The palynological characters and character states
used in the numerical analysis of 16 species of Alchemilla
in Iran are presented in Table 4. Cluster analysis
(CA) was undertaken using average taxonomic distance
(Euclidean distance matrix) and unweighted pair-group
method with arithmetic mean (UPGMA) clustering
procedures (SIMINT, SAHN, and TREE). The values of
each character were standardized and the cophenetic
correlation coefficient was determined to find out to what
extent the cluster analysis fits the distance matrix. PCA
was conducted (for analyzing multivariate data) using the
general linear model (GLM) in Minitab statistical software
(Ryan and Joiner, 2001).
3. Results
3.1. General pollen morphology
The microphotographs taken by LM (Figure 1) and SEM
(Figures 2–4) revealed interesting pollen morphological
characters of the studied species. The pollen grains are
isopolar to subisopolar radially symmetrical monads,
prolate spheroidal (P/E = 1.11–1.12) (Figure 1A),
subprolate (P/E = 1.19–1.32) (Figure 1B), and prolate
(P/E = 1.38–1.79) (Figures 1C–1F) in shape. The
outline of pollen varies from rectangular to cylindrical
in equatorial view (Figures 1A–1F) and triangular to
subcubic in polar view (Figures 1G–1L). The pollen grains
are small to medium based on Erdtman (1952), with triand tetracolporate apertures (Table 2). The minimum
and maximum polar axis varies from 14.21 µm (in A.
sedelmeyeriana) to 27.42 µm (in A. hessii). The minimum
equatorial axis (9.57 µm) is reported in A. sedelmeyeriana
and the maximum equatorial axis is found (19.39 µm) in A.
fluminea. The minimum P/E ratio (1.1) and maximum P/E
ratio (1.56) are identified in A. caucasica and A. condensa,
Table 3. Species grouping based on sculpturing type.
Species Sculpturing type
T 1 Subtype A Microechinate distribution
1. A. citrina
Circular
Type I
Microechinate only in vicinity of colpi
2. A. pectinoloba
3. A. erythropoda*
4. A. hessii
5. A. pseudo-cartalinica
T 1 Subtype B
6. A. condensa
Striate
7. A. rigida
TII Subtype A
Type II
Microechinate in vicinity of colpi and mid-intercolpium
/ pole psilate
Category 1
8. A. kurdica
9. A. hyrcana Striate
10. A. sericata
Category 2
11. A. sedelmeyeriana
Circular
12. A. fluminea
TII Subtype B
13. A. plicatissima*
14. A. caucasica* Arranged in 2–3 rows
15. A. amardica*
TIII Type III
16. A. rechingeri Microechinate in vicinity of colpi, mid-intercolpium and poles
FAGHIR et al. / Turk J Bot
271
respectively. The mesocolpium length ranges from 2.78
µm (in A. sedelmeyeriana) to 9.5 µm (in A. sericata). The
minimum and maximum colpus length is measured in
A. sedelmeyeriana (11.49 µm) and A. hessii (24.04 µm).
The exine thickness varies from minimum 0.95 µm in
A. amardica to maximum 2.25 µm in A. condensa. The
ratio of colpus length and polar axis varies from 0.57 in
A. caucasica to 0.93 in A. plicatissima. The apocolpium
index (AI) (Punt et al., 2007) or d/D: the ratio of the distance
between the apices of two ectocolpi of a zonocolpate
pollen grain (d) to its equatorial diameter (D) ranges
over an interval of 0.19 µm in A. rechingeri to 0.42 µm in
A. caucasica. The minimum (0.11 µm in A. rigida) and
maximum (0.31 µm in A. erythropoda) spine height were
also measured. A. sedelmeyeriana and A. erythropoda had
the minimum (0.15 µm) and maximum (0.25 µm) spine
diameter, respectively (Table 2).
3.2. Exine sculpture types
We observed three main types of exine sculpturing (Table 3).
Type I: Microechinate only in vicinity of colpi/poles
and mid-intercolpium psilate. Based on the microechinate
distribution pattern the first type is further divided into 2
subtypes:
TI subtype A: consists of the species with circular
microechinate distribution pattern, which includes A.
pseudocartalinica (Figures 2A–2C), A. hessii (Figure 2D),
A. citrina (Figures 2E, 2F), A. pectinoloba (Figures 2G–2I),
and A. erythropoda (Figures 2J–2L).
TI subtype B: comprises the species with striate
microechinate distribution pattern .This subtype is
recorded in A. condensa (Figures 3A, 3B) and A. rigida
(Figure 3C).
Type II: Microechinate in vicinity of colpi, midintercolpium/poles psilate
Table 4. Palynological character and character states used in numerical analysis of 16 species of
Alchemilla in Iran.
Characters Character states No.
Polar axis (P) 1
Equatorial axis (E or D) 2
Polar axis/Equatorial axis ratio( P/E) 3
Distance between the apices of two ectocolpi (d) 4
Apocolpium index (d/D) 5
Mesocolpium (Me) 6
Colpus length/Polar axis (Cl/P) 7
Colpus length (Cl) 8
Exine thickness (Et) 9
Number of colpi (No. Cl) 10
0 Prolate
1 Spheroidal Pollen shape (Ps) 11
2 Subprolate
0 Isopolar
Polarity (Pol) 12
1 Subisopolar
0 T1 Subtype A
Sculpturing types (Sculp) 13
1 TI Subtype B
2 TII Subtype A category 1
3 TII Subtype A category 2
4 TII Subtype B
5 TIII
FAGHIR et al. / Turk J Bot
272
This type is further divided into two subtypes:
TII subtype A: Microechinate in vicinity of colpi and
mid-intercolpium. Based on the microechinate distribution
pattern this subtype is divided into 2 categories:
The first category is composed of species with a striate
microechinate distribution pattern and includes three
species: A. kurdica (Figure 3D), A. hyrcana (Figures 3E,
3F), and A. sericata (Figures 3G, 3H). The second category
includes species with a circular microechinate distribution
pattern, which is found in A. sedelmeyeriana (Figures 3I,
3J) and A. fluminea (Figures 3K, 3L).
TII subtype B: Three rows of microechinae in vicinity
of colpi and mid-intercolpium. This type is observed in
three tetracolporate species of A. plicatissima (Figures
4A–4E), A. caucasica (Figures 4F–4H), and A. amardica
(Figures 4I, 4J).
Type III: In this type the mid-intercolpium, colpi edges,
and poles are covered by microechinae. This was recorded
in A. rechingeri (Figures 4K, 4L).
3.3. Cluster analysis
The UPGMA phenogram and all OTUs in this survey are
presented in Figure 5.
The cophenetic correlation was 75.75%,
indicating a reasonable percent of the data similarity
matrix transferred to the phenogram (Rohlf,
1993). Three clusters and branches were formed:
A) a cluster is divided into three subgroups: A1) a branch with
A. amardica, A. pectinoloba, and A. sericata, A2) a branch
Figure 1.A–L. LM micrographs of pollen grains in species ofAlchemilla:A.caucasicaA and
J; A. plicatissima B, K, L; A. condensa C; A. kurdica D; A. erythropoda E; A. rechingeri F, G;
A. retinervis H; A. amardica I (scale bar = 2.5 µm).
FAGHIR et al. / Turk J Bot
273
with A. erythropoda, A. rechingeri, A. fluminea, A. kurdica,
and A. hessii, and A3) a branch with A. condensa; B) a branch
with A. citrina, A. rigida, A. hyrcana, A. pesudocartalinica,
A. plicatissima, and A. sedelmeyeriana; and C) a branch with
A. caucasica at the base of the phenogram.
3.4. Principal component analysis (PCA)
Eigen analysis results from the PCA and loading scores
of three principal components are presented in Tables
5 and 6 and a score plot is shown in Figure 6. The three
components describe 89.9% of the pollen morphological
character variation between 16 individuals.
The projections of the loadings defined by the
first two principal components describe the position
of 16 studied species. The first principal component
(PC1) scoring system is dominated by equatorial axis
(E), polar axis/equatorial axis ratio (P/E), ratio of the
distance between the apices of two ectocolpi (d), and
mesocolpium index (Me). Along the first axis a group
of 10 species: A. amardica, A. pectinoloba, A. sericata,
A. erythropoda, A. rechingeri, A. fluminea, A. kurdica,
A. hessii, A. condensa and A. caucasica with 31.6% of
the total variation was segregated. The second principal
component (PC2) scoring system is dominated by polar
axis (P) and colpus length (CL) (Table 6). In the second
axis, a group of A. citrina, A. rigida, A. hyrcana, A.
pesudocartalinica, A. plicatissima, and A. sedelmeyeriana,
with 29.8% of the total variation was formed. The third
principal component (PC3) scoring system is dominated
by polarity (Polr), number of colpi (ncol), and sculpturing
(Sculp) (Table 6).
Figure 2. A–L. SEM micrographs of pollen grains in species of Alchemilla: A.
pseudocartalinica A–C; A. hessii D; A. citrina E, F; A. pectinoloba G–I; A. erythropoda J,
L (scale bar = 5 µm for A, B, D, E–L; 2 µm for C, F).
FAGHIR et al. / Turk J Bot
274
4. Discussion
The current results revealed the most important
palynological characters within the genus Alchemilla. They
include pollen polarity, outline, shape, and size; colpi length
and number; and exine thickness and sculpturing. Based
on our findings, the studied species have either isopolar
and subisopolar pollen. Hebda et al. (1988) identified the
isopolar pollen of A. occidentalis Nutt.
The pollen shape varies from prolate–spheroidal
(Figure 1A) to subprolate (Figure 1B) and prolate (Figures
1C–1F). The prolate shape is the most dominant type and
present among 14 studied species, while subprolate and
prolate–spheroidal shapes are the least common types.
Murbeck (1901) and Strasburger (1905) reported variation
in shape of some species, especially A. vulgaris, A. alpina,
and A. glabra.
Regarding pollen size, the small grains are the most
dominant (found in 22 species), whereas the medium
pollen is recorded in two species: A. fluminea and A. hessii.
These pollen morphological data are in agreement with
the previous studies by Hebda et al. (1988) and Hebda and
Chinnappa (1990). The size of the pollen grains can be helpful
to distinguish some species, especially A. hessii for the largest
and A. sedelmeyeriana for the smallest pollen grains.
Figure 3. A–L. SEM micrographs of pollen grains in species of Alchemilla: A. condensa A, B;
A. rigida C; A. kurdica D; A. hyrcana E, F; A. sericata G, H; A. sedelmeyeriana I, J; A. fluminea
K, L (scale bar = 5 µm for A, C, D, F, G, I, J, L; 2 µm for B, E, H, K).
FAGHIR et al. / Turk J Bot
275
Previously, 3-colporate pollen was reported in some
species of the genus, e.g., A. occidentalis (Hebda et al.,
1988). We identified 3-colporate pollen grains in the
majority of the studied species (Figures 1G–1I). However,
in some species, especially A. amardica and A. erythropoda
(with 30%), A. caucasica (with 60%–65%) (Figure 1H),
and A. plicatissima (with 60%–70%) (Figures 1K, 1L)
4-colporate pollen grains were also recorded.
The colpus usually spans 80%–90% of the distance
between poles (Hebda et al., 1988; Hebda and Chinnappa,
1990). In this survey, based on the colpus length, three
main groups including 11–14 µm, 15–19 µm, and 20–25
µm were recognized. Each of the first and second groups
includes 8 representatives and the third one consists of two
species.
The colpus membrane and margin are occasionally
covered by microechinae (Figures 4D, 4E). The pore
area is not distinct, because the pore is buried in the
colpus showing microechinae (spinules) (Figures 3E,
3F). The endopore usually is slit-like in the colpus floor,
e.g., in A. rigida and A. kurdica (Figures 3C, 3D) and the
operculum is either fully absent or poorly formed, e.g.,
A. citrina (Figures 2E, 2F) and A. sericata
(Figure 3H). A. sedelmeyeriana (Figure 3J) and
Figure 4. A–L. SEM micrographs of pollen grains in species of Alchemilla: A. plicatissima
A–E; A. caucasica F–H; A. amardica I, J; A. rechingeri K, L (scale bar = 5 µm for A, B, F,
G, I–L; 2 µm for C–E, H).
FAGHIR et al. / Turk J Bot
276
A. rechingeri (Figures 4K, 4L). These pollen grains’
morphological data are consistent with previous studies
by Hebda et al. (1988) and Hebda and Chinnappa (1990).
Thick exine is one of the palynological characteristics
of the genus (Hebda and Chinnappa, 1990). Based on exine
thickness 3 groups were recorded including 9 species with
0.95–1.24 µm, 4 species with 1.53–1.98 µm, and 3 species
with more than 2 µm (2.04–2.25 µm).
The thickness of the exine is increased especially at
the center of intercolpia, which is responsible for giving a
subrectangular outline of the pollen in equatorial view and
shifting the apertures to an interangular position (Reitsma,
1966). However, in A. rechingeri (Figure 1G) the exine is
thickened at the corners and the pores are located at angles.
Ueda and Tomita (1989) reported the importance
of exine sculpture types in the family Rosaceae. Later,
Hebda and Chinnappa (1990) divided the Canadian
rosaceous pollen types into two broad categories and
placed Alchemilla in the second category, containing
psilate with microperforations sculpturing patterns. The
exine sculpturing analysis of the present study revealed
three main types, 4 subtypes, and 2 categories. However,
the exine sculpturing type classes do not support a close
relationship between the species and is of restricted
taxonomic value.
In some species, especially in A. persica, rarely free
functional pollen grains were collected. Occurrence
of nonfunctional pollen was primarily recorded by
Murbeck (1901) and Strasburger (1905) in A. vulgaris, A.
alpina, and A. glabra.
A1
A2
A
A3
B1
B
B2
C
2.71 3.72 4.72 5.72 6.73
Coeicient
A. amardica
A. pectinoloba
A. sericata
A. erythropoda
A. rechingeri
A. fluminea
A. kurdica
A. hessii
A. condensa
A. citrina
A. rigida
A. hyrcana
A. caucasica
A. plicatissima
A. pseudocartalinica
A. sedelmyeriana
Figure 5. Phenogram of the 16 OTUs studied, clustering with UPGMA
method.
Table 5. Eigen analysis of the correlation matrix.
PC1 PC2 PC3
Eigenvalue 3.1661 2.9822 2.2538
Proportion 0.244 0.229 0.173
Cumulative 0.244 0.473 0.646
FAGHIR et al. / Turk J Bot
277
Table. 6. The variable loading scores for each principal component (PC1–PC3) for 16
species of the genus Alchemilla in Iran (the dominant coefficients for PC1, PC2, and PC3
scoring systems are underlined).
Variable PC1 PC2 PC3
P 0.205 -0.496 -0.136
E 0.445 -0.326 -0.099
P.E -0.443 -0.227 -0.068
d 0.444 -0.035 0.103
d.D 0.110 0.267 0.264
Me 0.418 0.163 0.159
cl.p -0.287 -0.305 0.039
Cl 0.049 -0.568 -0.084
Et -0.086 0.003 0.184
PS 0.160 -0.113 0.250
Pol -0.087 0.167 -0.540
NO.Cl 0.205 0.085 -0.506
sculp 0.114 0.182 -0.457
Abbreviations used in Table 6: Polar axis (P), Equatorial axis (E), Polar axis/Equatorial
axis (P/E), Pollen shape (Ps), Distance between the apices of two ectocolpi/equatorial
diameter (d/D), Mesocolpium (Me), Colpus length/polar axis (Cl/P), Colpus length
(Cl), Exine thickness (ET), Number of Colpi (No. Cl), Pollen shape (PS), Polarity (Pol),
Sculpturing types (Sculp).
Species
A. amardica
A. caucasica
A. citrina
A. condensa
A. erythropoda
A. fluminea
A. hessii
A. hyrcana
A. kurdica
A. pectinoloba
A. plicatissima
A. pseudocartalinica
A. rechingeri
A. rigida
A. sedelmeyeriana
A. sericata
Score Plot
First Component
–3 –2 –1 0 1 2 3 4
Second Component
–4
–3
–2
–1
0
1
2
3
A2
A. sedelmeyeriana
A. citrina
A. caucasica
C
A. rigida
A. plicatissima
A. hyrcana
A. pseudocartalinica
B
A. amardica
A. sericata
A1
A. pectinoloba
A. erythropoda
A. condensa
A. kurdica
A. hessii
A. rechingeri A. fluminea
Figure 6. Principal components analysis score plot expressing the pollen morphological
variation of 16 species of Alchemilla from Iran.
FAGHIR et al. / Turk J Bot
278
Generally, our findings revealed congruence
between the UPGMA clustering and PCA
analyses and the studied species were arranged
into three groups: A) A. amardica, A. pectinoloba,
A. sericata, A. erythropoda, A. rechingeri, A.
fluminea, A. kurdica, A. hessii, and A. condensa;
B) A. citrina, A. rigida, A. hyrcana, A. pesudocartalinica,
A. plicatissima, and A. sedelmeyeriana; and C) A. caucasica
(Figures 5 and 6).
The representatives of each group share some
morphological evidence. For example, in subgroup A1, A.
amardica, A. pectinoloba, and A. sericata are characterized
by having a hairy hypanthium shorter than sepals. Among
them, A. amardica and A. sericata have been considered as
closely related species because their petiole is covered by
appressed hairs (Fröhner, 1969; Khatamsaz, 1993).
There are several morphological affinities
between different species of the large group A,
e.g., all these representatives (except A. hessii)
have a hypanthium shorter than sepals;
A. rechingeri, A. fluminea, A. kurdica, and A. hessii share
a glabrous hypanthium; A. erythropoda and A. condensa
have a hairy hypanthium; and A. erythropoda resembles A.
rechingeri, by having declinate hairs on the petiole of radical
leaves. Among them, A. kurdica and A. fluminea also have
been treated as closely allied species for having hairless
stem, leaves with 7 lobes, and 7 to 9 teeth on each lobe by
Fröhner (1969) and Khatamsaz (1993). These authors also
considered the two endemic of Iran, A. condensa and A.
amardica, as related species for possessing a hypanthium
covered by dense appressed hairs. A. condensa differs from
A. amardica in its broad leaf lobes and triangular sepals.
There are controversies regarding the relationship
between 6 representatives of the second group (B). Based
on Fröhner (1969) A. citrina resembles A. gigantodus (for
its common erecto-patent hairs on the petiole of radical
leaves), while according to Khatamsaz (1993) it has more
common morphological features with A. rigida (for having
hypanthium shorter than sepals and all parts covered by
hairs) and A. caucasica (by erecto-patent hairs on the
petiole of radical leaves). However, earlier, Juzepczuk
(1941) treated A. rigida and A. sericata in the first series
and A. caucasica in the second series of the section
Pubscentes Buser. and subgenus Pes-Leonis
Juz. He also arranged A. hyrcana, A. sedelmeyeriana, and
A. pesudocartalinica in the section Vulgares Buser. (for
sharing pedicels varying in length; inner sepals shorter
than or as long as hypanthia, and glabrous like hypanthia
or more or less hairy). A. hyrcana and A. pesudocartalinica
resemble each other by their hairless pedicel and
hypanthium. According to Flora Iranica and Flora of Iran,
A. hyrcana and A. sedelmeyeriana was treated as related
species because both share a thin stem, hairy leaf (on both
sides), and more or less hairy hypanthium (Fröhner, 1969;
Khatamsaz, 1993).
In addition, Khatamsaz (1993) regarded
three endemics species (A. plicatissima,
A. amardica, and A. condensa) as closely related for
possessing erecto-patent hairs covering all parts.
In conclusion, our findings revealed the palynological
characteristics (e.g., pollen polarity, size, exine
sculpturing) of the genus Alchemilla. These pieces of
evidence are reliable criteria for explaining species
relationships. The current numerical analysis supports
the relationship between some species, especially A.
amardica, A. sericata, A. fluminea, A. kurdica (of group A);
A. hyrcana, A. sedelmeyeriana, and A. pesudocartalinica (of
group B), but its application is restricted for others.
Acknowledgments
This study was supported by grants from research
assistance of Guilan University, Rasht, Iran. We would like
to thank Rahmani from Razi Metallugy Research Institute
(Tehran, Iran) for taking the SEM photographs.
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