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Pollen morphology of the genus Gagea (Liliaceae) in Iran
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Flora 200 (2005) 96–108 www.elsevier.de/flora
Pollen morphology of the genus Gagea (Liliaceae) in Iran Mehdi Zarrei, Shahin Zarre Department of Biology, Faculty of Sciences, University of Tehran, P.O. Box 14155-6455, Tehran, Iran Received 28 July 2003; accepted 26 April 2004
Abstract Pollen grains of 26 species of the genus Gagea distributed in Iran were examined by light and scanning electron microscopy. Detailed pollen morphological characteristics are given for these species. Among the studied species, the newly described G. iranica together with G. olgae and G. graminifolia possess the smallest pollen grains, and the widely distributed G. lutea the largest ones. Our studies show that the sculpturing of exine provides valuable characters for separating the species, sometimes even for closely related ones, and delimitation of natural groups within the genus. The exine of the genus Gagea is in most cases perforated upon tectum or rarely tectate-columellate. The muri are solid or structured, compound, simpli-, dupli- or pluricolumellate. It seems that the structure of muri is very important in recognizing natural groups within the genus. Tectal perforations vary from o0.2 to 2.0 mm in diameter among the studied species. Regarding sculpturing of the exine in proximal face, four basic types of pollen grains can be distinguished: reticulate, microreticulate, foveolate and perforate. Within the reticulate type there is sufficient variation in exine structure at distal face to describe three subtypes: reticulate, microreticulate and perforate. A diagnostic key is given for all studied taxa based on palynomorphological characters. For a limited number of populations of selected Iranian species of Gagea, further aspects of pollen biology were studied. It seems that populations with ploidy levels other than diploidy, show a low percentage of pollen fertility. Moreover, the rate of pollen fertility is correlated with the manner of the reproduction in certain species. r 2005 Elsevier GmbH. All rights reserved. Keywords: Palynology; Liliaceae; Iran
Introduction The genus Gagea Salisb. with about 250 species (Levichev, 1999a) and more than 500 nomenclatural combinations (Levichev, 1999b) is the largest genus among Liliaceae, although earlier estimates considered the number of species between 70 and 127 (Melchior, 1964; Stroh, 1937; Uphof, 1958–1960; Willis, 1973). According to Wendelbo and Rechinger (1990) and the additions to this genus by Akhani (1999), Assadi (1988) Corresponding author.
E-mail address: [email protected] (M. Zarrei). 0076-9511/$ - see front matter r 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.flora.2004.04.001
and Zarrei and Zarre (in press), Iran is an important center of diversity of this genus, with 35 species, among them five endemics. A comparative study on the pollen morphology of the whole Liliaceae family has been presented by Kosenko (1991a, b, 1992, 1999). A few species of Gagea distributed mainly in Europe are included in standard text books on pollen morphology (see, e.g. Faegri and Iversen, 1989; Moore et al., 1991). Moreover, a detailed pollen morphological study on the middle Asian species of Gagea is presented by Kosenko and Levichev (1988). However, only few species considered in this study are also distributed in Iran.
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It is evident from the existing data that the species of Gagea differ from each other mainly in detailed structure of the sulcus and exine ornamentation (Kosenko, 1999). It was also shown that the rate of pollen abortion varies among different populations of G. commutata C. Koch and G. procera Mouterde, which can result odd ploidy levels occurring repeatedly in these populations (Heyn and Dafni, 1971). In the framework of preparing a revision for the genus Gagea in Iran, we concluded that the delimitation of several taxa is not possible based on macromorphological characters solely. Therefore, pollen morphology, as one of the most efficient and reliable sources of micromorphological characters, was evaluated in the systematics of the genus. In contrast to macromorphological characters that exhibit high levels of homoplasy under severe ecological conditions—as in alpine habitats—it was shown that palynomorphological characters can scarsely be influenced by such ecological conditions. Earlier classifications on the Iranian representatives of this genus based on the macromorphological characters will be compared with the grouping based on the palynological characters. In the present paper, detailed descriptions of pollen features are presented for 26 out of 35 species of Gagea known in Iran. The remaining species of the genus which are not treated in our analysis Table 1.
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are either very rare endemics (e. g. G. grey-wilsonii Rech. f. and G. wendelboi Rech. f.), or their presence in Iran is doubtful according to our studies (e. g. G. glacialis C. Koch and G. luteoides Stapf).
Material and methods Pollen of 26 species of the genus Gagea was studied by light microscope (LM) and scanning electron microscope (SEM). The pollen samples were obtained mostly from fresh collected herbarium specimens, although some old herbarium material retained also the characteristic features of the pollen. The voucher specimens were deposited in IRAN, TARI and TUH (acronyms according to Holmgren et al., 1990). Voucher specimens for this analysis are listed in Table 1. For LM, fresh pollen samples were acetolyzed following the standard method described by Erdtman (1969), mounted in glycerol jelly on glass slides and sealed. Slides for LM were studied and photographed using an Olympus microscope model BX-50. Length of equatorial and polar axis were measured with aid of an 100 eyepiece. Measurement of grains was based on 30 grains per sample.
Characteristic features of pollen grains in Iranian representatives of Gagea
Species
Vouchers
P (mm)
E (mm)
P/E ratio
G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G.
Zarrei, 30025 (T) Zarre, 29475 (T) Zarrei & Ajani, 29215 (T) Zarrei, 30017 (T) Zarrei, 29242 (T) Assadi & Miller, 25159 (TA) Habibi & Tehrani, 29914 (I) Zarrei & Zarre, 29170 (T) Zarrei, 30041 (T) Riedl & Ershad, 29927 (I) Zarre, 29473 (T) Zarrei & Zarre, 29273 (T) Zarre, 29472 (T) Moussavi & Tehrani, 29970 (I) Zarrei & Zarre, 29158 (T) Zarrei & Ajani, 29206 (T) Wendelbo et al, 11174 (TA) Zarrei & Ajani, 29191 (T) Wendelbo et al, 11170 (TA) Sharif, 30007 (TA) Termeh & Matin, 30013 (I) Zarre & Zarrei, 30045 (T) Moussavi et al, 30018 (I) Riazi, 9041 (TA) Zarrei & Ajani, 29185 (T) Zarrei & Ajani, 29188 (T)
20.0(22.771.5)25.0 27.5(31.372.4)35.0 25.0(29.172.2)32.5 25.0(27.572.1)30.0 30.0(34.573.0)40.0 31.0(32.271.6)35.0 26.0(28.872.1)34.0 25.0(28.671.8)32.5 29.0(31.071.7)35.0 23.0(26.571.9)29.0 30.0(33.672.5)37.5 27.5(30.072.0)32.5 23.5(25.271.3)28.0 19.0(22.472.4)27.0 25.0(27.971.6)30.0 25.0(26.572.3)32.5 16.0(19.471.4)21.0 17.5(21.372.1)25.0 30.0(34.772.4)37.5 17.0(20.171.4)22.0 29.0(32.871.9)35.0 30.0(32.471.8)35.0 24.0(27.171.7)29.0 20.0(22.871.6)26.3 25.5(25.772.3)30.0 22.5(25.771.9)27.5
40.0(42.671.8)45.0 55.0(57.772.6)62.5 47.5(51.972.4)55.0 53.0(55.372.3)60.5 60.0(63.872.4)67.5 58.0(61.572.7)65.0 54.0(55.771.9)60.0 40.0(44.172.5)47.5 57.5(60.271.8)62.0 47.0(49.171.7)52.0 60.0(63.272.5)67.5 55.0(59.673.0)65.0 46.5(50.172.3)54.0 42.0(44.771.8)47.0 57.0(59.771.7)62.0 50.0(54.172.5)57.5 33.0(35.971.3)37.0 35.0(37.972.0)40.0 62.5(67.873.4)72.5 35.0(38.972.2)42.0 59.0(61.572.6)65.0 55.0(58.772.5)62.0 51.0(55.072.7)59.0 41.3(44.171.8)47.5 47.5(49.372.0)52.5 45.0(51.973.0)55.0
0.48(0.5370.03)0.58 0.46(0.5470.04)0.59 0.50(0.5670.04)0.65 0.45(0.5070.03)0.55 0.46(0.5670.05)0.67 0.48(0.5270.04)0.57 0.47(0.5270.04)0.59 0.58(0.6570.05)0.71 0.48(0.5170.03)0.57 0.50(0.5470.03)0.59 0.48(0.5370.04)0.60 0.46(0.5070.02)0.55 0.44(0.5070.03)0.57 0.44(0.5070.04)0.57 0.42(0.4770.03)0.51 0.43(0.4970.04)0.57 0.46(0.5470.04)0.58 0.47(0.5670.05)0.64 0.46(0.5170.03)0.58 0.45(0.5270.03)55.0 0.49(0.5370.02)0.56 0.50(0.5570.03)0.58 0.46(0.4970.03)0.57 0.46(0.5270.03)0.58 0.45(0.5170.04)0.58 0.45(0.4970.03)0.55
afghanica alexeenkoana anonyma bergii bulbifera capillifolia caroli-kochii chlorantha chomutowae circumplexa confusa dubia dshungarica exilis fistulosa gageoides graminifolia iranica lutea olgae ova reticulata soleimani stipitata tenera vegeta
Abbreviations: P: polar axis length; E: equatorial axis length; I: IRAN, TA: TARI, T: TUH.
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For SEM, we used the protocol explained by Davies (1999) with some modifications. Unacetolyzed pollen was fixed in Karnovsky’s fixative in 0.1 M phosphate buffer for 3 h at 4 1C. The specimens were then washed in sucrose (10%) in 0.1 M phosphate buffer. They were washed three times with 30 min intervals at 4 1C. Secondary fixation was carried out in 2% Osmium tetraoxide (aqueous) for 1 h at room temperature. Dehydration was carried out through a graded series of ethanol. Samples were placed in a 50/50 mixture of 100% ethanol for 30 min followed by 30 min in 100% hexamethyldisilazane. The specimens were then allowed to air dry overnight before mounting. Upon completion of drying, the specimens were mounted on 12.5 mm diameter stubs and attached with sticky tabs and then coated in a sputter coater with approximately 25 nm of GoldPaladium. The specimens were examined and photographed with a Zeiss SEM model DSM 960A, at an accelerating voltage of 10–15 kV. Cross-section of exine was also examined. The number of tectal
Table 2.
Results The main features of the investigated pollen are summarized in Tables 1 and 2. Regarding sculpturing of exine surface in proximal face, four basic types of pollen grains can be distinguished (Table 2): reticulate, microreticulate, foveolate and perforate. Within the reticulate
Sculpturing features in Iranian representatives of Gagea (voucher specimens as mentioned in Table 1)
Species
G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G.
perforations (according to Punt et al., 1994) per 25 mm2 and length of larger perforations in proximal face and distal face were measured. The terminology follows in general Faegri and Iversen (1989), Kosenko (1999), Moore et al. (1991), Punt et al. (1994, 1999) and Walker and Doyle (1975). For estimation of pollen fertility, the pollen from fresh collected herbarium material was stained by acetocarmin in glycerine jelly, as described by Radford et al. (1974). In total, 26 populations belonging to 13 selected species were analyzed under this aspect.
afghanica alexeenkoana anonyma bergii bulbifera capillifolia caroli-kochii chlorantha chomutowae circumplexa confusa dubia dshungarica exilis fistulosa gageoides graminifolia iranica lutea olgae ova reticulata soleimani stipitata tenera vegeta
Length and width of large tectal perforations (mm) Proximal face
Distal face
o0.2 1.2–1.7 1.0–1.3 1.3–1.7 1.1–1.5 1.3–1.7 1.0–1.3 0.9–2.0 0.4–0.7 0.7–0.9 0.5–0.8 0.8–1.0 0.7–0.9 0.5–0.7 0.4–0.6 1.3–1.5 0.8–1.0 1.0–1.2 1.0–1.2 1.0–1.3 1.1–1.3 0.8–1.0 0.4–0.6 0.7–0.9 0.7–0.9 1.0–1.3 0.9–1.2 1.0–1.2 0.9–1.1 1.0–2.0 1.1–1.7 o0.2 0.4–0.6 0.4–0.6 o0.2 1.1–1.3 0.8–1.0 0.4–0.6 0.4–0.6 1.4–1.7 1.0–1.3 0.6–0.9 0.3–0.6 0.2–0.3 0.2–0.3 1.0–1.4 0.8–1.0 1.0–1.2 1.0–1.2
0.0 0.7–1.0 0.5–0.7 0.4–0.5 0.3–0.4 0.6–0.8 0.4–0.6 0.7–1.3 0.5–0.7 o0.2 0.3–0.5 0.2–0.4 0.2–0.3 0.1–0.2 0.7–0.9 0.4–0.7 0.5–0.7 0.3–0.5 1.3–1.6 1.0–1.3 0.2–0.3 0.2–0.3 1.0–1.2 1.0–1.2 0.5–0.7 0.4–0.6 0.6–0.9 0.5–0.7 1.1–1.3 0.9–1.1 0.0 0.3–0.4 0.2–0.3 o0.2 o0.2 0.2–0.4 0.2–0.4 0.8–0.9 0.4–0.5 o0.2 o0.2 0.7–0.9 0.6–0.8 o0.2
p. f
17 12 11 17 10 13 26 35 8 17 43 12 20 14 13 5 20 6 22 16 10 5 10 15 10 24
d. f.
0.0 17 14 23 12 6 19 35 10 22 50 12 20 27 13 10 0.0 6 22 16 10 15 6 11 13 5
Exine sculpturing Proximal face
Distal face
per.—sol.—pluri. ret.—sol.—pluri. ret.—sol.—pluri. fov.—sol.—pluri. micr.—com.—pluri. micr.—com.—simpl. micr.—sol.—pluri. per.—sol.—pluri. ret.—com.—simpl. ret.—sol.—pluri. ret.—sol.—simpl. per.—com.—dupli. micr.—com.—pluri. ret.—com.—simpl. ret.—com.—simpl. ret.—com.—simpl. per.—sol.—pluri. per.—sol.—pluri. per.—com.—pluri. fov.—sol.—pluri. per.—com.—pluri ret.—sol.—pluri. per.—com.—pluri. per.—com.—pluri. ret.—com.—simpl. fov.—sol.—pluri.
eu. per.—sol.—pluri. per.—sol.—pluri. per.—sol.—pluri. ret.—com.—pluri. per.—com.—pluri. per.—sol.—pluri. per.—sol.—pluri. per.—com.—simpl. per.—sol.—pluri. ret.—sol.—simpl. per.—com.—dupli. ret.—com.—pluri. micr.—com.—simpl. per.—com.—dupli. ret.—com.—simpl. eu. per.—sol.—pluri. per.—com.—pluri. per.—sol.—pluri. per.—com.—pluri. per.—sol.—pluri. per.—com.—pluri. per.—com.—pluri. micr.—com.—simpl. fov.—sol.—pluri.
Abbreviations: per.: perforate; ret.: reticulate; micr.: microreticulate; fov.: foveolate; eu.: eutectate; sol.: solid muri; com.: compound muri; simpl.: simplicolumellate; dupli.: duplicolumellate; pluri.: pluricolumellate; d. f.: mean number of tectal perforations per 25 mm2 at distal face and p.f.: mean number of tectal perforations per 25 mm2 at proximal face.
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Figs. 1–8. LM (Figs. 1and 2) and SEM (Figs. 3–8) micrographs of pollen grains in Gagea. Fig. 1, Pollen of G. fistulosa at proximal polar view. Fig. 2, pollen of G. bulbifera at proximal polar view. Fig. 3, Rounded end of sulcus in pollen of G. reticulata. Fig. 4, Cross-section of exine in pollen of G. reticulata. Fig. 5, Reticulate tectum at the proximal face in G. confusa pollen with simplicolumellate muri. Fig. 6, Reticulate tectum at proximal face of pollen of G. gageoides with simplicolumellate muri. Fig. 7, Reticulate tectum at proximal face in pollen of G. tenera with simplicolumellate muri. Fig. 8, Reticulate tectum at proximal face of pollen G. alexeenkoana. Figs. 1 and 2, scale bar ¼ 10 mm; Figs. 3, 5–6 and 8, scale bar ¼ 2 mm: Figs. 4–7, scale bar ¼ 1 mm.
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type there is sufficient variation to describe three subtypes (see below).
General pollen morphological description Pollen grains are shed as monads. They are monosulcate, heteropolar and boat-shaped-oblong in polar view (Figs. 1 and 2), oblate or peroblate (according to Punt et al., 1994, 1999) bilateral and aniso-bisymmetric (according to Walker and Doyle, 1975). The pollen grains are partly medium in size (according to Walker and Doyle, 1975) [Range: From 16.0–21.0 33.0–37.0 [ ¼ 19.471.4 35.971.3] mm in G. graminifolia Vved. to 20.0–26.3 41.3–47.5 [ ¼ 22.871.6 44.171.8] mm in G. stipitata Merckl. ex Bunge]. Those of other taxa must be ranked as large [From 22.5–27.5 45.0–55.0 [ ¼ 25.771.9 51.973.0] mm in G. vegeta Vved. to 30.0–37.5 62.5–72.5 [ ¼ 34.772.4 67.873.4] mm in G. lutea (L.) Ker-Gawl]. P/E ratio ranges from 0.42–0.51 [ ¼ 0.4770.03] in G. fistulosa (Ramond) Ker-Gawl to 0.58–0.71 [0.6570.05] in G. chlorantha (M. Bieb.) Schultes & Schultes fil. The aperture is simple, distal-polar (ana-sulcate), elongated, and reaches the proximal side of the pollen with rounded or acute ends (Fig. 3). The exine is perforated or rarely tectate-columellate upon tectum (Fig. 4). Muri are solid or compound, simpli-, dupli- or pluricolumellate (according to Punt et al., 1994). Tectal perforations vary from o0.2 to 2.0 mm in diameter among the studied species. On the basis of the exine sculpturing at proximal face, four main pollen types can be recognized which are described below.
Pollen types Reticulate This type is heterogeneous and more frequent between species examined and occurs in 10 species: G. alexeenkoana Micsz., G. anonyma Rech. f., G. chomutawae Pascher, G. circumplexa Vved., G. confusa A. Terracc., G. exilis Vved., G. fistulosa Ker-Gawl, G. gageoides (Zucc.) Vved., G. reticulata (Pall.) Schultes & Schultes fil. (including G. tenuifolia Fomi) and G. tenera Pascher (Figs. 5 and 15). The largest pollen of all the species is
classified under this type and occurs in G. confusa [30.0–37.5 60.0–67.5 ( ¼ 33.672.5 63.272.5) mm in size]. The smallest one is observed in G. exilis [19.0–27.0 42.0–47.0 ( ¼ 22.472.4 44.771.8) mm in size] which also belongs to this typeU P/E ratio ranges from 0.42–0.51 (0.4770.03) in G. fistulosa to 0.50–0.65 (0.5670.04) in G. anonyma. The lumina are similar in diameter to the microreticulate type (see below). They are as wide as the muri or wider than those, but in contrast with the microreticulate type, they are larger than 1 mm in diameter. Regarding the exine sculpturing at distal face, three subtypes can be recognized for the reticulate pollen type: reticulate (including G. confusa: Fig. 5, G. gageoides: Fig. 6), microreticulate (including G. exilis, G. tenera: Fig. 7) and perforate (including G. alexeenkoana: Fig. 8, G. anonyma: Fig. 9, G. chomutowae, G. circumplexa: Figs. 10 and 11, G. fistulosa: Fig. 12 and G. reticulata: Table 2, Figs. 13 and 14). The muri are simplicolumellate in reticulate and microreticulate subtypes and in G. fistulosa of the perforate subtype (but only at the proximal face, Fig. 12). They are solid in some species (in G. reticulata and related taxa: Figs. 7 and 8, 13–17) or compound in others, especially in those belonging to the microreticulate subtype, in G. gageoides of the reticulate subtype and in G. fistulosa and G. chomutowae of the perforate subtype.
Microreticulate This occurs in G. bulbifera (Pall.) Schultes & Schultes fil., G. capillifolia Vved., G. caroli-kochii Grossh. and G. dshungarica Regel (Figs. 15–17). The largest pollen grain in this type belongs to G. bulbifera [30.0–40.0 60.0–67.5 ( ¼ 34.573.0 63.872.4) mm in size]. The smallest one is found in G. dshungarica [23.5–28.0 46.5–54.0 ( ¼ 25.271.3 50.172.3) mm in size]. P/E ratio ranges from 0.44–0.57 (0.5070.03) in G. dshungarica to 0.46–0.67 (0.5670.05) in G. bulbifera. The lumina are as wide as the muri or wider than them and smaller than 1 mm in diameter. The muri are solid or compound, simplicolumellate or pluricolumellate.
Figs. 9–16. SEM micrographs of pollen grains in the species of Gagea. Fig. 9, Reticulate tectum at the proximal face of G. anonyma pollen grain with approximately rounded lumina and solid, pluricolumellate muri. Fig. 10, Reticulate exine at the proximal face of G. circumplexa pollen grain with solid, pluricolumellate muri. Fig. 11, Perforate tectum at the distal face of G. circumplexa pollen grain with solid, pluricolumellate muri. Fig. 12, Reticulate tectum with compound, simplicolumellate muri at proximal face of pollen in G. fistulosa. Fig. 13, Reticulate tectum at the proximal face of pollen in G. reticulata with7elongated and irregular lumina and solid, pluricolumellate muri. Fig. 14, Distal face of G. reticulata pollen grain with perforate tectum and solid, pluricolumellate muri. Fig. 15, pollen grain of G. caroli-kochii in distal polar view. Fig. 16, Proximal face of pollen in G. caroli-kichii with microreticulate tectum and solid, simplicolumellate muri. Figs. 9–14 and 16, scale bar ¼ 2 mm; Fig. 15, scale bar ¼ 10 mm.
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Table 3.
The percentage of pollen fertility in selected species of Gagea distributed in Iran
No.
Species
Vouchers
No. of fertile pollen
No. of infertile pollen
Fertility percentage
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G.
Zarrei & Ajani, 29200 (TUH) Zarrei & Zarre, 29233 (TUH) Zarre, 29475 (TUH) Zarrei, 29240 (TUH) Zarrei & Ajani, 29215 (TUH) Zarrei, 29258 (TUH) Zarrei, 30041 (TUH) Zarrei, 29239 (TUH) Zarrei & Zarre, 29225 (TUH) Zarre, 29473 (TUH) Zarrei, 29238 (TUH) Zarrei & Zarre, 29232 (TUH) Zarre, 30043 (TUH) Zarre, 30057 (TUH) Foroughi & Assadi, 26663 (TARI) Zarrei & Ajani, 29191 (TUH) Zarrei & Zarre, 29272 (TUH) Zarre & Zarrei, 30065 (TUH) Zarrei & Ajani, 29209 (TUH) Zarrei & Zarrei, 29226 (TUH) Zarrei & Ajani, 29187 (TUH) Zarrei & Ajani, 29210 (TUH) Zarre & Zarrei, 30045 (TUH) Zarrei & Ajani, 29201 (TUH) Zarrei & Ajani, 29185 (TUH) Ajani, 29292 (TUH)
2553 2170 298 1756 2207 1072 602 2764 2038 501 262 1601 1837 609 1175 340 2820 65 225 134 5006 553 22 489 208 514
113 119 30 147 147 42 678 1549 1961 229 145 271 325 91 105 1178 186 137 65 31 575 3103 14 160 1048 7
95.76 94.80 90.85 92.28 93.75 96.23 47.03 63.42 50.96 68.63 64.97 85.52 84.67 87.00 91.80 22.40 93.81 32.18 77.59 81.21 89.68 15.13 61.11 75.35 16.56 98.66
afghanica alexeenkoana alexeenkoana alexeenkoana anonyma bulbifera chomutowae confusa confusa confusa dubia dubia dubia gageoides gageoides iranica lutea lutea reticulata reticulata reticulata reticulata reticulata tenera tenera vegeta
With respect to the supra-tectal ornamentation, there are considerable differences among the species attributed to this type. In both G. capillifolia and G. carolikochii (Figs. 15–17), the lumen diameter decreases markedly from the proximal face towards the distal one. The exine sculpture at the distal face is perforate in both species, but the perforation diameter in G. capillifolia is smaller (o0.2 mm) than in G. caroli-kochii (0.3–0.5 0.2–0.4 mm). Other important differences between these species are an obvious decrease in the mean number of lumina from proximal face towards distal one in G. capillifolia and the few mean numbers of lumina in comparison with G. caroli-kochii (Table 2). In G. dshungarica and G. bulbifera, the lumen diameter increase from proximal face towards distal face (reticulate at distal face), and the mean numbers of lumina are approximately equal in both mentioned species. The muri are pluricolumellate in all four species belonging to the microreticulate type except for G. capillifolia where they are simplicolumellate at proximal face. Perforate It Occurs in nine species including: G. afghanica A. Terracc., G. chlorantha, G. dubia A. Terracc., G. graminifolia Vved., G. iranica, G. lutea (L.) Ker-Gawl,
G. ova Stapf, G. soleimani (Bornm. ex Pascher) Bornm. and G. stipitata (Figs. 18–20). The smallest pollen grains are found in G. iranica [17.5–25.0 35.0–40.0 ( ¼ 21.372.1 37.972.0) mm in size] as well as in G. graminifolia [16.0–1.0 33.0–37.0 ( ¼ 19.471.4 35.971.3) mm in size]. The largest pollen grain is found in G. lutea [30.0–37.5 62.5–72.5 ( ¼ 34.772.4 67.873.4) mm in size]. P/E ratio ranges from 0.46–0.55 (0.5070.02) in G. dubia to 0.58–0.71 (0.6570.05) in G. chlorantha. It should be noticed that the largest P/E ratio among the studied species is found in G. chlorantha from the perforate type, but the smallest P/E ratio was observed in G. fistulosa belonging to the reticulate type. In all species belonging to the perforate type, exine sculpturing at both distal and proximal faces is perforate except for G. graminifolia and G. afghanica with an eutectate exine at distal face. These are the only cases of eutectate exine surfaces among the examined species. The muri are solid or compound and dupli- or pluricolumellate. G. dubia (Fig. 18) is the only species of this type with duplicolumellate muri. In three species (G. afghanica, G. chlorantha and G. iranica) the muri are solid. Foveolate It occurs in three species: G. bergii Litw., G. olgae Regel and G. vegeta Vved. (Figs. 21–24). One of the
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smallest pollen grains among all examined species is that of G. olgae belonging to this type [17.0–22.0 35.0–42.0 ( ¼ 20.171.4 38.972.2) mm in size]. The largest pollen grain in this type is found in G. bergii (Fig. 21) with a size of 25.0–30.0 53.0–60.5 ( ¼ 27.572.1 55.372.3) mm. In this type, the lower and upper limit of
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the P/E ratio are alike in all three species and vary between 0.45 and 0.55, respectively. Muri are solid and pluricolumellate (Figs. 22–24). There are some diagnostic features for separating these three species (see nods 14 and 15 of the diagnostic key).
A diagnostic key1 to the species of Gagea distributed in Iran based on palynomorphological characters 1a1b2a2b3a3b4a4b5a5b6a6b7a7b8a8b9a9b10a10b11a11b12a12b13a13b14a14b15a15b-
Tectal perforations (lumina) as wide as muri or wider than them. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Tectal perforations narrower than the muri. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10 Lumina wider than 1 mm (type 1: reticulate) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Lumina narrower than 1 mm (type 2: microreticulate). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 Exine reticulate at distal face . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Exine microreticulate or perforate at distal face. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Muri solid; mean number of lumina per 25 mm2 43 at proximal face and 50 at distal face . . . . . . . . . . . . G. confusa Muri compound; mean number of lumina per 25 mm2 5 at proximal face and 10 at distal face . . . . . . G. gageoides Exine microreticulate at distal face. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Exine perforate at distal face . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Pollen grain 25.5–30.0 47.5–52.5 (25.772.3 49.372.0) mm in size; mean number of lumina per 25 mm2 10 at proximal face and 13 at distal face. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. tenera Pollen grain smaller [19.0–27.0 42.0–47.0 (22.472.4 44.771.8) mm in size]; mean number of lumina per 25 mm2 14 at proximal face and 27 at distal face. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. exilis Muri compound, simpli- or duplicolumellate at distal face . . . . . . . . . . . . . . . . . . . . . . . . G. chomutowae fistulosa Muri solid, pluricolumellate at distal face . . . . . . . . G. alexeenkoana, G. anonyma, G. circumplexa and G. reticulata Exine reticulate at distal face; mean number of lumina per 25 mm2 at proximal face approximately equaling that of the distal face . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. bulbifera and G. dshungarica Exine perforate at distal face; mean number of lumina per 25 mm2 decreases from proximal face towards distal face9 Mean number of lumina per 25 mm2 at proximal face 26 and at distal face 19; lumina 0.30.5 0.20.4 mm in sizeG. caroli-kochii Mean number of lumina per 25 mm2 at proximal face 13 and at distal face 6; the lumina smaller (o0.2 mm) in sizeG. capillifolia Tectal perforation smaller than 1 mm diam. (type 3: perforate). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11 Tectal perforation wider 1 mm diam. (type 4: foveolate).. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14 Exine eutectate at distal face.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. afghanica and G. graminifolia Exine perforate at distal face . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12 Muri duplicolumellate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. dubia Muri pluricolumellate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .13 Muri solid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..G. chlorantha and G. iranica Muri compound . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .G. lutea, G. ova, G. soleimanii and G. stipitata Exine foveolate at distal face . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .G. vegeta Exine perforate at distal face . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15 Pollen grain 17.0–22.0 35.0–42.0 (20.171.4 38.972.2) mm; tectal perforations o0.2 mm at distal face . . G. olgae Pollen grain larger [25.0–30.0 53.0–60.5 (27.572.1 55.372.3) mm in size]; tectal perforations at distal face larger (0.6–0.8 0.4–0.6 mm in size). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. bergii
1 The key does not include the species of Gagea which are rare endemics or doubtfully recorded for Iran. Moreover, some species are so similar in pollen characteristics that they could not be separated from each other. Since a diagnostic key would be very useful in determination of the species, we preferred to present the key and in the cases of high similarities, refer to them collectively.
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Pollen fertility Results of the fertility analysis are summarized in Table 3. The lowest fertility percentage was observed in the population no. 22 belonging to G. reticulata, while G. vegeta (no. 26 in Table 3), G. bulbifera (no. 6 in Table 3) and G. afghanica (no. 1 in Table 3) show the highest fertility level, respectively. No significant difference was observed in exine sculpture between fertile and aborted pollen. Although in some species (e.g. G. alexeenkoana) the fertility percentage is relatively constant among different populations, it is highly variable for other species (e.g. G. reticulata).
Discussion The basic palynomorphological characters of Gagea are the occurrence of a shallow sulcus, a reticulate, microreticulate, perforate and foveolate exine with solid or compound simpli-, dupli- or pluricolumellate muri. Some of these features are shared also by some species of Lloydia, the sister genus of Gagea (Patterson and Givnish, 2002). The muri in Lloydia tibetica (Kosenko, 1999) as well as in G. confusa (Fig. 5), G. exilis, G. gageoides (Fig. 6) and G. tenera (Fig. 7) are simplicolumellate confirming the close relationship between these genera suggested by Takhtajan (1987, 1997). The taxonomy of the genus Gagea is very difficult due to several problems. Delimitation of species is in several cases problematic because of great variation in vegetative and generative characters during different stages of ontogeny (Levichev, 1990b, 1999b) and under different ecological conditions. Moreover, with respect of the subgeneric grouping within the genus, there are very controversial views. In the most recent published work on the subgeneric classification of the genus (Levichev, 1990a), it is divided into 10 sections (nine of them are present in Iran) and several series. But in the most regional Flora, other classifications mainly based on Pascher (1904, 1907) and Terracciano (1905, 1906) were applied. According to Kosenko (1991b, 1992, 1999) and Kosenko and Levichev (1988) pollen morphological characters provide valuable characters for a delimitation of the species in Liliaceae, especially in Gagea. The present study confirms this point of view.
Pollen in Gagea is heterogenous and eurypalynous, hence it can be divided into several groups and subgroups. Some of these groups are in congruence with the formerly supposed grouping within the genus on the basis of macromorphological characters, but others do not support them. The importance of pollen morphological characters and their fitness for the most actual subgeneric taxonomic grouping are discussed in the following.
G. sect. Platyspermum (G. reticulata species group) G. sect. Platyspermum Boiss. as defined by Levichev (1990a) includes: G. alexeenkoana (Fig. 8), G. anonyma (Fig. 9), G. bergii (Figs. 21–22), G. caroli-kochii (Figs. 15–17) and G. reticulata (Figs. 13 and 14). G. alexeenkoana, G. anonyma and G. reticulata show the reticulate type of exine. Several macromorphological characters such as flattened triangular seeds, acuminately tipped tepals, umbellate inflorescences and filiform or linear basal leaves are common among these species. However, G. bergii and G. caroli-kochii belong to two different other pollen types, i.e. foveolate and microreticulate ones, respectively. Several characteristic features of G. bergii separate it from other members of the section. A small but conspicuous bulbil at the leaf axil, short peduncle, long pedicels and a dense indumentum of the pedicels are some of these characteristic features. In our opinion, this species should be placed into a separate group. Geographical data also support this idea. Among the above-named species, G. bergii and G. anonyma show a more eastern distribution pattern reaching Afghanistan and Central Asia, while the other species are distributed from East Mediterranean to Central Iran. G. reticulata with the widest distribution area among the studied species, although reaches Afghanistan and Turkmenistan; it does not form such dense populations there as in Iran. Another treatment on the species of the above-named group is presented by Heyn and Dafni (1971) and by Rechinger (1986), where a ‘‘G. reticulata species group’’ is defined. In a wide sense this group should include: G. alexeenkoana, G. anonyma, G. caroli-kochii, G. circumplexa (Figs. 10 and 11) and G. reticulata (Zarrei, own unpublished data). Among these species, G. circumplexa is characterized by cord-like roots enveloping the bulb. However, this character can be modified by the given ecological conditions, especially
Figs. 17–24. SEM micrographs of pollen in Gagea. Fig. 17, Distal face of G. caroli-kochii pollen grain with perforate tectum and solid, pluricolumellate muri. Fig. 18, Perforate tectum at proximal face of pollen in G. dubia with compound, duplicolumellate muri. Fig. 19, Proximal face of pollen in G. stipitata grain with perforate tectum. Fig. 20, Perforate tectum at distal face of pollen in G. soleimani grain. Fig. 21, Pollen grain of G. bergii in distal polar view. Fig. 22, Proximal face of pollen in G. bergii with foveolate tectum and solid, pluricolumellate muri. Fig. 23, Proximal face of pollen in G. vegeta with foveolate tectum and solid, pluricolumellate muri. Fig. 24, Proximal face of pollen in G. olgae with foveolate tectum and solid, pluricolumellate muri. Figs. 17–20 and 23, scale bar ¼ 1 mm; Fig. 21, scale bar ¼ 10 mm; Figs. 22 and 24, scale bar ¼ 2 mm.
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by edaphic factors. In our opinion, the cord-like roots can be formed in soils with high proportion of clay in several other species as well, such as, e.g. G. reticulata. We suggest that G. circumplexa is closely related to G. reticulata. However, more studies are necessary for confirmation of this idea. Consideration of this species as separated from the G. reticulata species group and placed as a member of G. sect. Incrustatae, as done by Levichev (1990a), is not supported by palynological data either. G. caroli-kochii is another species of the group with considerable morphological similarity to G. alexeenkoana, although it is classified here under the microreticulate type. However, this species shows the upper limit of lumina diameter among the species classified under the microreticulate type. The classification applied here for pollen sculpturing type is based on the size of the lumina as presented by Punt et al. (1999), which is most probably somewhat artificial in Gagea. Moreover, the muri are solid in both species, as well as other members of G. sect. Platyspermum (as circumscribed by Levichev, 1990a) analyzed here.
G. sect. Stipitatae (Pascher) Davalianidze and G. sect. Fistulosae (Pascher) Davalianidze G. sect. Stipitatae includes: G. capillifolia (not shown), G. chumotowae (not shown), G. gageoides (Fig. 6), G. ova (not shown) and G. stipitata (Fig. 19), while G. sect. Fistulosae possesses only G. fistulosa in Iran. Although the species of G. sect. Stipitatae are classified here as belonging to different pollen types, the muri in all these species are compound. Cyme inflorescences are shared by all species of G. sect. Stipitatae. Moreover, the species of this section are very closely related, so that the taxonomical separation of them is in most cases very difficult. Again it seems that the classification of pollen types according to Punt et al. (1999) is artificial here and cannot be used for characterizing natural groups. G. fistulosa (Fig. 12) is palynologically very similar to G. chomutowae, and it is cited in the same place in the key as the latter. The only important morphological character which connects these species to each other is the presence of a fistulose basal leaf. The systematic position of G. sect. Fistulosae and its possible affinity with G. sect. Stipitatae needs more studies.
G. sect. Minimoides (A. Terracc.) Levichev Two important species attributed to this section and included in the present analysis are G. confusa (Fig. 5) and G. dshungarica (not shown). The former shows a western distribution pattern from Antolia to central Alborz (North Iran), while the latter exhibits rather an eastern distribution pattern from central Alborz but
widely distributed in Central Asia. Interestingly, near Kandavan and Damavand mountains (Central Alborz in Iran) there are mixed populations of both species. These species are also ecologically different. G. confusa grows always close to snow line on high mountains where the humidity of soil is relatively high, while G. dshungarica can be found much farther from snow line and prefers low soil humidity. For this reason, in overlapping areas of occurrence, always fruiting or dried specimens of G. confusa can be observed near flowering specimens of G. dshungarica. These species have been considered to be closely related and treated as belonging to the same section (Levichev, 1990a), but they are morphologically well characterized and taxonomically easily separable. From the palynomorphological point of view G. confusa and G. dshungarica are also well characterized and belong to two different pollen types, i.e. reticulate with solid, simplicolumellate muri and microreticulate with compound pluricolumellate muri, respectively. The possible close relationship between these species should be re-assessed.
G. sect. Graminifoliae Levichev Two species of this section, G. graminifolia (not shown) and G. vegeta (Fig. 23), are analyzed in the present study. In both species the muri are solid, pluricolumellate and wider than the lumina, but in the former taxon the lumina are smaller than 1 mm in diameter, while in the latter they are larger than 1 mm. Therefore, the two species are attributed here to two different pollen types: perforate and foveolate, respectively. Both species are distributed in Central Asia, Afghanistan and NE Iran. They are also very similar and probably closely related from a morphological point of view.
G. sect. Plecostigma (Turcz.) Pascher According to the definition presented by Levichev (1990a), the following species in Iran can be attributed to this section: G. afghanica (not shown), G. bulbifera (see Zarrei & Zarre, in press), G. chlorantha (not shown), G. iranica (see Zarrei & Zarre, in press) and G. olgae (Fig. 24). All these species are morphologically similar and form a homogeneous section. Among these species G. bulbifera is the only species bearing bulbils at the leaf axile. The pollen exine is foveolate or perforate with solid muri in all above-named species except for G. bulbifera with a microreticulate type of exine and compound muri. The latter should most probably belong to another species group. Interestingly, G. iranica and G. olgae possess the smallest pollen grains among all studied species.
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G. sect. foliatae A. Terracc In Iran, this section includes only G. tenera (Fig. 7) distributed also in Central Asia. This species is in some respects, e.g. possessing cyme inflorescences, similar to G. sect. Plecostigma, but shorter tepals and a wide lower cauline leaf separate it from the species of this section. A reticulate exine with compound and simplicolumellate muri confirms the attribution of this species to a section different from G. sect. Plecostigma.
G. sect. Gagea G. lutea (not shown) is the only reported species of this section distributed in Iran. It has the largest pollen grains among the studied species. It has a perforate exine with compound pluricolumellate muri. G. lutea shows the widest distribution area within the genus extending from West Europe (Great Britain) to East Asia (Japan). In contrast to most other species of the genus, it prefers very humid and shaded areas. As the final conclusion of this study, it seems that pollen morphology provides strong evidences for delimitation of natural subgeneric groups in Gagea. An extended study including the species distributed in other countries and comparing even more the subgeneric taxonomy of the genus can further lead to a better delimitation of the sections. In several cases, pollen morphology of Gagea will distinctly help the taxonomists to separate species with high macromorphological similarity to each other.
Pollen fertility Since there were only limited amounts of fresh material of anthers available, the study of pollen fertility can be considered only as a preliminary one. However, it rendered the following provisional results: 1) In a few species, such as G. alexeenkoana and G. gageoides, different populations show a similar and high fertility percentage. It seems that in these species the generative reproduction is more frequent than the vegetative one. In nature, these plants form scattered populations composed of several remote individuals. In the case of a frequent vegetative reproduction, especially through stolons, compact patches composed by several individuals attached together would be expected. 2) A few species, such as G. lutea and G. tenera , show a very variable fertility level reflecting frequency of vegetative reproduction in some populations as well as generative reproduction in others. Interestingly, the ploidy level among different populations in G. lutea varies between diploidy and pentaploidy (Peruzzi, 2003 and references cited there). In the case
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of triploids and pentaploids the rate of generative reproduction should be very low. 3) In species such as G. reticulata , which is very common and widely distributed in the studied area and forms dense compact patches of individuals attached together, most populations show a high rate of pollen fertility, although the vegetative reproduction by aid of stolons is also common. Although no other ploidy level than diploidy is recorded for this species, it may be assumed that triploids can also occur within populations and survive mainly by vegetative reproduction, as it was suggested for G. commutata and G. procera of the same species complex (Heyn and Dafni, 1971). For example the population no. 22 (see Table 3) shows the lowest fertility rate among all studied populations. The ecological conditions of the area near Fariman (NE Iran) under which the population no. 22 grows is relatively optimal for the genus. The soil is dark in color, a sandy clay and with a relatively high proportion of humus deposited from the remainder of some thorny Astragalus species and other plants, as Scariola orientalis (Boiss.) Sojak. Moreover, the individuals of G. reticulata grow in relatively large tufts within the cushions of Astragalus. They represent the largest habit of Gagea reticulata that has been observed in Iran. Most probably, these plants are triploid and reproduce vegetatively by aid of stolons. This would explain their growth in large tufts. Interestingly, all other examined populations of G. reticualta (no. 19–21 in Table 3) show a relatively high fertility percentage, which is in accordance with the recorded chromosome number for this species. Only a chromosome number of 2n ¼ 2x ¼ 24, i.e. the diploid chromosome set, is recorded for G. reticulata, while for several other species triploid and penatploid karyotypes are also recorded (see Peruzzi, 2003 and references cited there).
Acknowledgements We are grateful to Prof. Dr. I.G. Levichev (Saint Petersburg) for his useful suggestions and providing some literature. We are indebted to Prof. Dr. D. Podlech (Munich) for providing several specimens of Gagea from the neighboring countries of Iran. Dr. A.A. Maassoumi and Dr. M. Assadi (Tehran) helped us by their useful comments and providing the herbarium material of TARI. We are also grateful to the curators of the herbarium Iran, especially M. Moussavi. We thank S.M.H. Hashemi for assistance by SEM. The study on the family Liliaceae in IRAN was supported partially by the project number 513/4/632 of the Tehran University, Research Council to Shahin Zarre.
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References Akhani, H., 1999. Studies on the flora and vegetation of the Golestan National Park, NE Iran, III. Three new species, one new subspecies and fifteen records for Iran. Edinb. J. Bot. 56, 1–31. Assadi, M., 1988. Plants of Arasbaran protected area, NW Iran (Part II). Iran. J. Bot. 49, 1–59. Davies, H.A., 1999. General preparation of material and staining of sections. In: Hajibagheri, M.A.N. (Ed.), Electron Microscopy Methods and Protocols. Methods in Molecular Biology 117. Humana Press, Totowa, NJ, pp. 1–11. Erdtman, G., 1969. Handbook of Palynology. Munksgaard, København. Faegri, K., Iversen, J., 1989. Textbook of Pollen Analysis. Wiley, New York. Heyn, C.C., Dafni, A., 1971. Studies in the genus Gagea (Liliaceae) I. The platyspermous species in Israel and neighboring areas. Israel J. Bot. 214–233. Holmgren, P.K., Holmgren, N.H., Barnett, L.C., 1990. Index Herbariorum I: The Herbaria of the World, eighth ed., Regnum Veg. 20. Kosenko, V.N., 1991a. Pollen morphology of the genus Fritillaria (Liliaceae). Bot. Zhurn. (Leningrad) 76, 1201–1210 (in Russian). Kosenko, V.N., 1991b. Palynomorphology of the family Liliaceae s. str. Bot. Zhurn. (Leningrad) 76, 1696–1706 (in Russian). Kosenko, V.N., 1992. Pollen morphology and systematic problems of the Liliaceae family. Bot. Zhurn. (Leningrad) 77, 1–15 (in Russian). Kosenko, V.N., 1999. Contribution to the pollen morphology and taxonomy of Liliaceae. Grana 38, 20–30. Kosenko, V.N., Levichev, I.G., 1988. Pollen morphology in genera Gagea and Lloydia (Liliaceae). Bot. Zhurn. (Leningrad) 73, 965–976 (in Russian). Levichev, I.G., 1990a. A synopsis of the genus Gagea (Liliaceae) from western Tian-Shan. Bot. Zhurn. (Leningrad) 75, 225–234 (in Russian). Levichev, I.G., 1990b. On age variation and hybridization of some representatives of Gagea (Liliaceae). Bot. Zhurn. 75, 656–667 (in Russian). Levichev, I.G., 1999a. Cooperation in genus Gagea. Opt. Newslett. 34 (e), 12. Levichev, I.G., 1999b. Zur Morphologie der Gattung Gagea Salisb. (Liliaceae). I. Die unterirdischen Organe. Flora 194, 379–392. Melchior, H., 1964. 3. Reihe Liliiflorae. In: Melchior, H. (Ed.), A. Engler’s Syllabus der Pflanzenfamilien, 12.
Aufl., 2. Band. Gebr. Borntraeger, Berlin-Nikolassee, pp. 513–542. Moore, P.D., Webb, J.A., Collinson, M.E., 1991. Pollen Analysis. Blackwell, London. Pascher, A., 1904. U¨bersicht u¨ber die Arten der Gattung Gagea. Sitzungsberichte des Deutschen Naturw.-med, Verins fu¨r Bo¨hmen ‘Lotos’, pp. 109–131. Pascher, A., 1907. Conspectus Gagearum Asiae. Bull. Soc. Imp. Nat. Moscow 19, 350–375. Patterson, B.Th., Givnish, J.Th., 2002. Phylogeny, concerted convergence and phylogenetic niche conservatism in the core Liliales: insights from rbcL and ndhF sequence data. Evolution 56, 233–252. Peruzzi, L., 2003. Contribution to cytotaxonomical knowledge of Gagea Salisb. (Liliaceae) sect. Foliatae A. Terracc. and synthesis of karyological data. Caryologia 56, 115–128. Punt, W., Blackmore, S., Nilsson, S., Le Thomas, A., 1994. Glossary of Pollen and Spore Terminology. LPP Foundation, Utrecht. Punt, W., Blackmore, S., Nilsson, S., Le Thomas, A., 1999. Glossary of pollen and spore terminology. http:// www.bio.uu.nl/palaeo/glossary/glos-tin.htm. Radford, A.E., Dickison, W.C., Massey, J.R., Bell, C.R., 1974. Vascular Plant Systematics. Harper & Row Publishers, New York. Rechinger, K.H., 1986. Six new species of Gagea (Liliaceae) from Fl. Iranica area. Plant Syst. Evol. 153, 287–292. Stroh, G., 1937. Die Gattung Gagea Salisb. Beih. Bot. Centralbl. 57 (B), 485–520. Takhtajan, A., 1987. Systema magnoliophytorum. Nauka, Leningrad (in Russian). Takhtajan, A., 1997. Diversity and Classification of Flowering Plants. Columbia University Press, New York. Terracciano, A., 1905. Gagearum species Florae Orientalis. Bull. Herb. Boissier, Ser. 2 (5), 1061–1076 and 1113–1128. Terracciano, A., 1906. Gagearum species Florae Orientalis. Bull. Herb. Boissier, Ser. 2 (6), 105–120. Uphof, J.C.T., 1958–1960. A review of the genus Gagea Salisb. I–III. Plant life 14 (124–132), 151–161 and 15, 163–176. Walker, J.W., Doyle, J.A., 1975. The bases of angiosperm phylogeny: palynology. Ann. Missouri Bot. Gard. 62, 664–723. Wendelbo, P., Rechinger, K.H., 1990. Gagea. In: Rechinger, K. (Ed.), Flora Iranica. 165, 13–57, Graz, Austria. Willis, J.C., 1973. A Dictionary of the Flowering Plants and Ferns. Cambridge University Press, Cambridge. Zarrei, M., Zarre, Sh., in press. One new species of Gagea (Liliaceae) from Iran. Nord. J. Bot. 23 (3).
Flora 200 (2005) 96–108 www.elsevier.de/flora
Pollen morphology of the genus Gagea (Liliaceae) in Iran Mehdi Zarrei, Shahin Zarre Department of Biology, Faculty of Sciences, University of Tehran, P.O. Box 14155-6455, Tehran, Iran Received 28 July 2003; accepted 26 April 2004
Abstract Pollen grains of 26 species of the genus Gagea distributed in Iran were examined by light and scanning electron microscopy. Detailed pollen morphological characteristics are given for these species. Among the studied species, the newly described G. iranica together with G. olgae and G. graminifolia possess the smallest pollen grains, and the widely distributed G. lutea the largest ones. Our studies show that the sculpturing of exine provides valuable characters for separating the species, sometimes even for closely related ones, and delimitation of natural groups within the genus. The exine of the genus Gagea is in most cases perforated upon tectum or rarely tectate-columellate. The muri are solid or structured, compound, simpli-, dupli- or pluricolumellate. It seems that the structure of muri is very important in recognizing natural groups within the genus. Tectal perforations vary from o0.2 to 2.0 mm in diameter among the studied species. Regarding sculpturing of the exine in proximal face, four basic types of pollen grains can be distinguished: reticulate, microreticulate, foveolate and perforate. Within the reticulate type there is sufficient variation in exine structure at distal face to describe three subtypes: reticulate, microreticulate and perforate. A diagnostic key is given for all studied taxa based on palynomorphological characters. For a limited number of populations of selected Iranian species of Gagea, further aspects of pollen biology were studied. It seems that populations with ploidy levels other than diploidy, show a low percentage of pollen fertility. Moreover, the rate of pollen fertility is correlated with the manner of the reproduction in certain species. r 2005 Elsevier GmbH. All rights reserved. Keywords: Palynology; Liliaceae; Iran
Introduction The genus Gagea Salisb. with about 250 species (Levichev, 1999a) and more than 500 nomenclatural combinations (Levichev, 1999b) is the largest genus among Liliaceae, although earlier estimates considered the number of species between 70 and 127 (Melchior, 1964; Stroh, 1937; Uphof, 1958–1960; Willis, 1973). According to Wendelbo and Rechinger (1990) and the additions to this genus by Akhani (1999), Assadi (1988) Corresponding author.
E-mail address: [email protected] (M. Zarrei). 0076-9511/$ - see front matter r 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.flora.2004.04.001
and Zarrei and Zarre (in press), Iran is an important center of diversity of this genus, with 35 species, among them five endemics. A comparative study on the pollen morphology of the whole Liliaceae family has been presented by Kosenko (1991a, b, 1992, 1999). A few species of Gagea distributed mainly in Europe are included in standard text books on pollen morphology (see, e.g. Faegri and Iversen, 1989; Moore et al., 1991). Moreover, a detailed pollen morphological study on the middle Asian species of Gagea is presented by Kosenko and Levichev (1988). However, only few species considered in this study are also distributed in Iran.
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It is evident from the existing data that the species of Gagea differ from each other mainly in detailed structure of the sulcus and exine ornamentation (Kosenko, 1999). It was also shown that the rate of pollen abortion varies among different populations of G. commutata C. Koch and G. procera Mouterde, which can result odd ploidy levels occurring repeatedly in these populations (Heyn and Dafni, 1971). In the framework of preparing a revision for the genus Gagea in Iran, we concluded that the delimitation of several taxa is not possible based on macromorphological characters solely. Therefore, pollen morphology, as one of the most efficient and reliable sources of micromorphological characters, was evaluated in the systematics of the genus. In contrast to macromorphological characters that exhibit high levels of homoplasy under severe ecological conditions—as in alpine habitats—it was shown that palynomorphological characters can scarsely be influenced by such ecological conditions. Earlier classifications on the Iranian representatives of this genus based on the macromorphological characters will be compared with the grouping based on the palynological characters. In the present paper, detailed descriptions of pollen features are presented for 26 out of 35 species of Gagea known in Iran. The remaining species of the genus which are not treated in our analysis Table 1.
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are either very rare endemics (e. g. G. grey-wilsonii Rech. f. and G. wendelboi Rech. f.), or their presence in Iran is doubtful according to our studies (e. g. G. glacialis C. Koch and G. luteoides Stapf).
Material and methods Pollen of 26 species of the genus Gagea was studied by light microscope (LM) and scanning electron microscope (SEM). The pollen samples were obtained mostly from fresh collected herbarium specimens, although some old herbarium material retained also the characteristic features of the pollen. The voucher specimens were deposited in IRAN, TARI and TUH (acronyms according to Holmgren et al., 1990). Voucher specimens for this analysis are listed in Table 1. For LM, fresh pollen samples were acetolyzed following the standard method described by Erdtman (1969), mounted in glycerol jelly on glass slides and sealed. Slides for LM were studied and photographed using an Olympus microscope model BX-50. Length of equatorial and polar axis were measured with aid of an 100 eyepiece. Measurement of grains was based on 30 grains per sample.
Characteristic features of pollen grains in Iranian representatives of Gagea
Species
Vouchers
P (mm)
E (mm)
P/E ratio
G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G.
Zarrei, 30025 (T) Zarre, 29475 (T) Zarrei & Ajani, 29215 (T) Zarrei, 30017 (T) Zarrei, 29242 (T) Assadi & Miller, 25159 (TA) Habibi & Tehrani, 29914 (I) Zarrei & Zarre, 29170 (T) Zarrei, 30041 (T) Riedl & Ershad, 29927 (I) Zarre, 29473 (T) Zarrei & Zarre, 29273 (T) Zarre, 29472 (T) Moussavi & Tehrani, 29970 (I) Zarrei & Zarre, 29158 (T) Zarrei & Ajani, 29206 (T) Wendelbo et al, 11174 (TA) Zarrei & Ajani, 29191 (T) Wendelbo et al, 11170 (TA) Sharif, 30007 (TA) Termeh & Matin, 30013 (I) Zarre & Zarrei, 30045 (T) Moussavi et al, 30018 (I) Riazi, 9041 (TA) Zarrei & Ajani, 29185 (T) Zarrei & Ajani, 29188 (T)
20.0(22.771.5)25.0 27.5(31.372.4)35.0 25.0(29.172.2)32.5 25.0(27.572.1)30.0 30.0(34.573.0)40.0 31.0(32.271.6)35.0 26.0(28.872.1)34.0 25.0(28.671.8)32.5 29.0(31.071.7)35.0 23.0(26.571.9)29.0 30.0(33.672.5)37.5 27.5(30.072.0)32.5 23.5(25.271.3)28.0 19.0(22.472.4)27.0 25.0(27.971.6)30.0 25.0(26.572.3)32.5 16.0(19.471.4)21.0 17.5(21.372.1)25.0 30.0(34.772.4)37.5 17.0(20.171.4)22.0 29.0(32.871.9)35.0 30.0(32.471.8)35.0 24.0(27.171.7)29.0 20.0(22.871.6)26.3 25.5(25.772.3)30.0 22.5(25.771.9)27.5
40.0(42.671.8)45.0 55.0(57.772.6)62.5 47.5(51.972.4)55.0 53.0(55.372.3)60.5 60.0(63.872.4)67.5 58.0(61.572.7)65.0 54.0(55.771.9)60.0 40.0(44.172.5)47.5 57.5(60.271.8)62.0 47.0(49.171.7)52.0 60.0(63.272.5)67.5 55.0(59.673.0)65.0 46.5(50.172.3)54.0 42.0(44.771.8)47.0 57.0(59.771.7)62.0 50.0(54.172.5)57.5 33.0(35.971.3)37.0 35.0(37.972.0)40.0 62.5(67.873.4)72.5 35.0(38.972.2)42.0 59.0(61.572.6)65.0 55.0(58.772.5)62.0 51.0(55.072.7)59.0 41.3(44.171.8)47.5 47.5(49.372.0)52.5 45.0(51.973.0)55.0
0.48(0.5370.03)0.58 0.46(0.5470.04)0.59 0.50(0.5670.04)0.65 0.45(0.5070.03)0.55 0.46(0.5670.05)0.67 0.48(0.5270.04)0.57 0.47(0.5270.04)0.59 0.58(0.6570.05)0.71 0.48(0.5170.03)0.57 0.50(0.5470.03)0.59 0.48(0.5370.04)0.60 0.46(0.5070.02)0.55 0.44(0.5070.03)0.57 0.44(0.5070.04)0.57 0.42(0.4770.03)0.51 0.43(0.4970.04)0.57 0.46(0.5470.04)0.58 0.47(0.5670.05)0.64 0.46(0.5170.03)0.58 0.45(0.5270.03)55.0 0.49(0.5370.02)0.56 0.50(0.5570.03)0.58 0.46(0.4970.03)0.57 0.46(0.5270.03)0.58 0.45(0.5170.04)0.58 0.45(0.4970.03)0.55
afghanica alexeenkoana anonyma bergii bulbifera capillifolia caroli-kochii chlorantha chomutowae circumplexa confusa dubia dshungarica exilis fistulosa gageoides graminifolia iranica lutea olgae ova reticulata soleimani stipitata tenera vegeta
Abbreviations: P: polar axis length; E: equatorial axis length; I: IRAN, TA: TARI, T: TUH.
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For SEM, we used the protocol explained by Davies (1999) with some modifications. Unacetolyzed pollen was fixed in Karnovsky’s fixative in 0.1 M phosphate buffer for 3 h at 4 1C. The specimens were then washed in sucrose (10%) in 0.1 M phosphate buffer. They were washed three times with 30 min intervals at 4 1C. Secondary fixation was carried out in 2% Osmium tetraoxide (aqueous) for 1 h at room temperature. Dehydration was carried out through a graded series of ethanol. Samples were placed in a 50/50 mixture of 100% ethanol for 30 min followed by 30 min in 100% hexamethyldisilazane. The specimens were then allowed to air dry overnight before mounting. Upon completion of drying, the specimens were mounted on 12.5 mm diameter stubs and attached with sticky tabs and then coated in a sputter coater with approximately 25 nm of GoldPaladium. The specimens were examined and photographed with a Zeiss SEM model DSM 960A, at an accelerating voltage of 10–15 kV. Cross-section of exine was also examined. The number of tectal
Table 2.
Results The main features of the investigated pollen are summarized in Tables 1 and 2. Regarding sculpturing of exine surface in proximal face, four basic types of pollen grains can be distinguished (Table 2): reticulate, microreticulate, foveolate and perforate. Within the reticulate
Sculpturing features in Iranian representatives of Gagea (voucher specimens as mentioned in Table 1)
Species
G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G.
perforations (according to Punt et al., 1994) per 25 mm2 and length of larger perforations in proximal face and distal face were measured. The terminology follows in general Faegri and Iversen (1989), Kosenko (1999), Moore et al. (1991), Punt et al. (1994, 1999) and Walker and Doyle (1975). For estimation of pollen fertility, the pollen from fresh collected herbarium material was stained by acetocarmin in glycerine jelly, as described by Radford et al. (1974). In total, 26 populations belonging to 13 selected species were analyzed under this aspect.
afghanica alexeenkoana anonyma bergii bulbifera capillifolia caroli-kochii chlorantha chomutowae circumplexa confusa dubia dshungarica exilis fistulosa gageoides graminifolia iranica lutea olgae ova reticulata soleimani stipitata tenera vegeta
Length and width of large tectal perforations (mm) Proximal face
Distal face
o0.2 1.2–1.7 1.0–1.3 1.3–1.7 1.1–1.5 1.3–1.7 1.0–1.3 0.9–2.0 0.4–0.7 0.7–0.9 0.5–0.8 0.8–1.0 0.7–0.9 0.5–0.7 0.4–0.6 1.3–1.5 0.8–1.0 1.0–1.2 1.0–1.2 1.0–1.3 1.1–1.3 0.8–1.0 0.4–0.6 0.7–0.9 0.7–0.9 1.0–1.3 0.9–1.2 1.0–1.2 0.9–1.1 1.0–2.0 1.1–1.7 o0.2 0.4–0.6 0.4–0.6 o0.2 1.1–1.3 0.8–1.0 0.4–0.6 0.4–0.6 1.4–1.7 1.0–1.3 0.6–0.9 0.3–0.6 0.2–0.3 0.2–0.3 1.0–1.4 0.8–1.0 1.0–1.2 1.0–1.2
0.0 0.7–1.0 0.5–0.7 0.4–0.5 0.3–0.4 0.6–0.8 0.4–0.6 0.7–1.3 0.5–0.7 o0.2 0.3–0.5 0.2–0.4 0.2–0.3 0.1–0.2 0.7–0.9 0.4–0.7 0.5–0.7 0.3–0.5 1.3–1.6 1.0–1.3 0.2–0.3 0.2–0.3 1.0–1.2 1.0–1.2 0.5–0.7 0.4–0.6 0.6–0.9 0.5–0.7 1.1–1.3 0.9–1.1 0.0 0.3–0.4 0.2–0.3 o0.2 o0.2 0.2–0.4 0.2–0.4 0.8–0.9 0.4–0.5 o0.2 o0.2 0.7–0.9 0.6–0.8 o0.2
p. f
17 12 11 17 10 13 26 35 8 17 43 12 20 14 13 5 20 6 22 16 10 5 10 15 10 24
d. f.
0.0 17 14 23 12 6 19 35 10 22 50 12 20 27 13 10 0.0 6 22 16 10 15 6 11 13 5
Exine sculpturing Proximal face
Distal face
per.—sol.—pluri. ret.—sol.—pluri. ret.—sol.—pluri. fov.—sol.—pluri. micr.—com.—pluri. micr.—com.—simpl. micr.—sol.—pluri. per.—sol.—pluri. ret.—com.—simpl. ret.—sol.—pluri. ret.—sol.—simpl. per.—com.—dupli. micr.—com.—pluri. ret.—com.—simpl. ret.—com.—simpl. ret.—com.—simpl. per.—sol.—pluri. per.—sol.—pluri. per.—com.—pluri. fov.—sol.—pluri. per.—com.—pluri ret.—sol.—pluri. per.—com.—pluri. per.—com.—pluri. ret.—com.—simpl. fov.—sol.—pluri.
eu. per.—sol.—pluri. per.—sol.—pluri. per.—sol.—pluri. ret.—com.—pluri. per.—com.—pluri. per.—sol.—pluri. per.—sol.—pluri. per.—com.—simpl. per.—sol.—pluri. ret.—sol.—simpl. per.—com.—dupli. ret.—com.—pluri. micr.—com.—simpl. per.—com.—dupli. ret.—com.—simpl. eu. per.—sol.—pluri. per.—com.—pluri. per.—sol.—pluri. per.—com.—pluri. per.—sol.—pluri. per.—com.—pluri. per.—com.—pluri. micr.—com.—simpl. fov.—sol.—pluri.
Abbreviations: per.: perforate; ret.: reticulate; micr.: microreticulate; fov.: foveolate; eu.: eutectate; sol.: solid muri; com.: compound muri; simpl.: simplicolumellate; dupli.: duplicolumellate; pluri.: pluricolumellate; d. f.: mean number of tectal perforations per 25 mm2 at distal face and p.f.: mean number of tectal perforations per 25 mm2 at proximal face.
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Figs. 1–8. LM (Figs. 1and 2) and SEM (Figs. 3–8) micrographs of pollen grains in Gagea. Fig. 1, Pollen of G. fistulosa at proximal polar view. Fig. 2, pollen of G. bulbifera at proximal polar view. Fig. 3, Rounded end of sulcus in pollen of G. reticulata. Fig. 4, Cross-section of exine in pollen of G. reticulata. Fig. 5, Reticulate tectum at the proximal face in G. confusa pollen with simplicolumellate muri. Fig. 6, Reticulate tectum at proximal face of pollen of G. gageoides with simplicolumellate muri. Fig. 7, Reticulate tectum at proximal face in pollen of G. tenera with simplicolumellate muri. Fig. 8, Reticulate tectum at proximal face of pollen G. alexeenkoana. Figs. 1 and 2, scale bar ¼ 10 mm; Figs. 3, 5–6 and 8, scale bar ¼ 2 mm: Figs. 4–7, scale bar ¼ 1 mm.
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type there is sufficient variation to describe three subtypes (see below).
General pollen morphological description Pollen grains are shed as monads. They are monosulcate, heteropolar and boat-shaped-oblong in polar view (Figs. 1 and 2), oblate or peroblate (according to Punt et al., 1994, 1999) bilateral and aniso-bisymmetric (according to Walker and Doyle, 1975). The pollen grains are partly medium in size (according to Walker and Doyle, 1975) [Range: From 16.0–21.0 33.0–37.0 [ ¼ 19.471.4 35.971.3] mm in G. graminifolia Vved. to 20.0–26.3 41.3–47.5 [ ¼ 22.871.6 44.171.8] mm in G. stipitata Merckl. ex Bunge]. Those of other taxa must be ranked as large [From 22.5–27.5 45.0–55.0 [ ¼ 25.771.9 51.973.0] mm in G. vegeta Vved. to 30.0–37.5 62.5–72.5 [ ¼ 34.772.4 67.873.4] mm in G. lutea (L.) Ker-Gawl]. P/E ratio ranges from 0.42–0.51 [ ¼ 0.4770.03] in G. fistulosa (Ramond) Ker-Gawl to 0.58–0.71 [0.6570.05] in G. chlorantha (M. Bieb.) Schultes & Schultes fil. The aperture is simple, distal-polar (ana-sulcate), elongated, and reaches the proximal side of the pollen with rounded or acute ends (Fig. 3). The exine is perforated or rarely tectate-columellate upon tectum (Fig. 4). Muri are solid or compound, simpli-, dupli- or pluricolumellate (according to Punt et al., 1994). Tectal perforations vary from o0.2 to 2.0 mm in diameter among the studied species. On the basis of the exine sculpturing at proximal face, four main pollen types can be recognized which are described below.
Pollen types Reticulate This type is heterogeneous and more frequent between species examined and occurs in 10 species: G. alexeenkoana Micsz., G. anonyma Rech. f., G. chomutawae Pascher, G. circumplexa Vved., G. confusa A. Terracc., G. exilis Vved., G. fistulosa Ker-Gawl, G. gageoides (Zucc.) Vved., G. reticulata (Pall.) Schultes & Schultes fil. (including G. tenuifolia Fomi) and G. tenera Pascher (Figs. 5 and 15). The largest pollen of all the species is
classified under this type and occurs in G. confusa [30.0–37.5 60.0–67.5 ( ¼ 33.672.5 63.272.5) mm in size]. The smallest one is observed in G. exilis [19.0–27.0 42.0–47.0 ( ¼ 22.472.4 44.771.8) mm in size] which also belongs to this typeU P/E ratio ranges from 0.42–0.51 (0.4770.03) in G. fistulosa to 0.50–0.65 (0.5670.04) in G. anonyma. The lumina are similar in diameter to the microreticulate type (see below). They are as wide as the muri or wider than those, but in contrast with the microreticulate type, they are larger than 1 mm in diameter. Regarding the exine sculpturing at distal face, three subtypes can be recognized for the reticulate pollen type: reticulate (including G. confusa: Fig. 5, G. gageoides: Fig. 6), microreticulate (including G. exilis, G. tenera: Fig. 7) and perforate (including G. alexeenkoana: Fig. 8, G. anonyma: Fig. 9, G. chomutowae, G. circumplexa: Figs. 10 and 11, G. fistulosa: Fig. 12 and G. reticulata: Table 2, Figs. 13 and 14). The muri are simplicolumellate in reticulate and microreticulate subtypes and in G. fistulosa of the perforate subtype (but only at the proximal face, Fig. 12). They are solid in some species (in G. reticulata and related taxa: Figs. 7 and 8, 13–17) or compound in others, especially in those belonging to the microreticulate subtype, in G. gageoides of the reticulate subtype and in G. fistulosa and G. chomutowae of the perforate subtype.
Microreticulate This occurs in G. bulbifera (Pall.) Schultes & Schultes fil., G. capillifolia Vved., G. caroli-kochii Grossh. and G. dshungarica Regel (Figs. 15–17). The largest pollen grain in this type belongs to G. bulbifera [30.0–40.0 60.0–67.5 ( ¼ 34.573.0 63.872.4) mm in size]. The smallest one is found in G. dshungarica [23.5–28.0 46.5–54.0 ( ¼ 25.271.3 50.172.3) mm in size]. P/E ratio ranges from 0.44–0.57 (0.5070.03) in G. dshungarica to 0.46–0.67 (0.5670.05) in G. bulbifera. The lumina are as wide as the muri or wider than them and smaller than 1 mm in diameter. The muri are solid or compound, simplicolumellate or pluricolumellate.
Figs. 9–16. SEM micrographs of pollen grains in the species of Gagea. Fig. 9, Reticulate tectum at the proximal face of G. anonyma pollen grain with approximately rounded lumina and solid, pluricolumellate muri. Fig. 10, Reticulate exine at the proximal face of G. circumplexa pollen grain with solid, pluricolumellate muri. Fig. 11, Perforate tectum at the distal face of G. circumplexa pollen grain with solid, pluricolumellate muri. Fig. 12, Reticulate tectum with compound, simplicolumellate muri at proximal face of pollen in G. fistulosa. Fig. 13, Reticulate tectum at the proximal face of pollen in G. reticulata with7elongated and irregular lumina and solid, pluricolumellate muri. Fig. 14, Distal face of G. reticulata pollen grain with perforate tectum and solid, pluricolumellate muri. Fig. 15, pollen grain of G. caroli-kochii in distal polar view. Fig. 16, Proximal face of pollen in G. caroli-kichii with microreticulate tectum and solid, simplicolumellate muri. Figs. 9–14 and 16, scale bar ¼ 2 mm; Fig. 15, scale bar ¼ 10 mm.
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Table 3.
The percentage of pollen fertility in selected species of Gagea distributed in Iran
No.
Species
Vouchers
No. of fertile pollen
No. of infertile pollen
Fertility percentage
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G.
Zarrei & Ajani, 29200 (TUH) Zarrei & Zarre, 29233 (TUH) Zarre, 29475 (TUH) Zarrei, 29240 (TUH) Zarrei & Ajani, 29215 (TUH) Zarrei, 29258 (TUH) Zarrei, 30041 (TUH) Zarrei, 29239 (TUH) Zarrei & Zarre, 29225 (TUH) Zarre, 29473 (TUH) Zarrei, 29238 (TUH) Zarrei & Zarre, 29232 (TUH) Zarre, 30043 (TUH) Zarre, 30057 (TUH) Foroughi & Assadi, 26663 (TARI) Zarrei & Ajani, 29191 (TUH) Zarrei & Zarre, 29272 (TUH) Zarre & Zarrei, 30065 (TUH) Zarrei & Ajani, 29209 (TUH) Zarrei & Zarrei, 29226 (TUH) Zarrei & Ajani, 29187 (TUH) Zarrei & Ajani, 29210 (TUH) Zarre & Zarrei, 30045 (TUH) Zarrei & Ajani, 29201 (TUH) Zarrei & Ajani, 29185 (TUH) Ajani, 29292 (TUH)
2553 2170 298 1756 2207 1072 602 2764 2038 501 262 1601 1837 609 1175 340 2820 65 225 134 5006 553 22 489 208 514
113 119 30 147 147 42 678 1549 1961 229 145 271 325 91 105 1178 186 137 65 31 575 3103 14 160 1048 7
95.76 94.80 90.85 92.28 93.75 96.23 47.03 63.42 50.96 68.63 64.97 85.52 84.67 87.00 91.80 22.40 93.81 32.18 77.59 81.21 89.68 15.13 61.11 75.35 16.56 98.66
afghanica alexeenkoana alexeenkoana alexeenkoana anonyma bulbifera chomutowae confusa confusa confusa dubia dubia dubia gageoides gageoides iranica lutea lutea reticulata reticulata reticulata reticulata reticulata tenera tenera vegeta
With respect to the supra-tectal ornamentation, there are considerable differences among the species attributed to this type. In both G. capillifolia and G. carolikochii (Figs. 15–17), the lumen diameter decreases markedly from the proximal face towards the distal one. The exine sculpture at the distal face is perforate in both species, but the perforation diameter in G. capillifolia is smaller (o0.2 mm) than in G. caroli-kochii (0.3–0.5 0.2–0.4 mm). Other important differences between these species are an obvious decrease in the mean number of lumina from proximal face towards distal one in G. capillifolia and the few mean numbers of lumina in comparison with G. caroli-kochii (Table 2). In G. dshungarica and G. bulbifera, the lumen diameter increase from proximal face towards distal face (reticulate at distal face), and the mean numbers of lumina are approximately equal in both mentioned species. The muri are pluricolumellate in all four species belonging to the microreticulate type except for G. capillifolia where they are simplicolumellate at proximal face. Perforate It Occurs in nine species including: G. afghanica A. Terracc., G. chlorantha, G. dubia A. Terracc., G. graminifolia Vved., G. iranica, G. lutea (L.) Ker-Gawl,
G. ova Stapf, G. soleimani (Bornm. ex Pascher) Bornm. and G. stipitata (Figs. 18–20). The smallest pollen grains are found in G. iranica [17.5–25.0 35.0–40.0 ( ¼ 21.372.1 37.972.0) mm in size] as well as in G. graminifolia [16.0–1.0 33.0–37.0 ( ¼ 19.471.4 35.971.3) mm in size]. The largest pollen grain is found in G. lutea [30.0–37.5 62.5–72.5 ( ¼ 34.772.4 67.873.4) mm in size]. P/E ratio ranges from 0.46–0.55 (0.5070.02) in G. dubia to 0.58–0.71 (0.6570.05) in G. chlorantha. It should be noticed that the largest P/E ratio among the studied species is found in G. chlorantha from the perforate type, but the smallest P/E ratio was observed in G. fistulosa belonging to the reticulate type. In all species belonging to the perforate type, exine sculpturing at both distal and proximal faces is perforate except for G. graminifolia and G. afghanica with an eutectate exine at distal face. These are the only cases of eutectate exine surfaces among the examined species. The muri are solid or compound and dupli- or pluricolumellate. G. dubia (Fig. 18) is the only species of this type with duplicolumellate muri. In three species (G. afghanica, G. chlorantha and G. iranica) the muri are solid. Foveolate It occurs in three species: G. bergii Litw., G. olgae Regel and G. vegeta Vved. (Figs. 21–24). One of the
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smallest pollen grains among all examined species is that of G. olgae belonging to this type [17.0–22.0 35.0–42.0 ( ¼ 20.171.4 38.972.2) mm in size]. The largest pollen grain in this type is found in G. bergii (Fig. 21) with a size of 25.0–30.0 53.0–60.5 ( ¼ 27.572.1 55.372.3) mm. In this type, the lower and upper limit of
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the P/E ratio are alike in all three species and vary between 0.45 and 0.55, respectively. Muri are solid and pluricolumellate (Figs. 22–24). There are some diagnostic features for separating these three species (see nods 14 and 15 of the diagnostic key).
A diagnostic key1 to the species of Gagea distributed in Iran based on palynomorphological characters 1a1b2a2b3a3b4a4b5a5b6a6b7a7b8a8b9a9b10a10b11a11b12a12b13a13b14a14b15a15b-
Tectal perforations (lumina) as wide as muri or wider than them. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Tectal perforations narrower than the muri. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10 Lumina wider than 1 mm (type 1: reticulate) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Lumina narrower than 1 mm (type 2: microreticulate). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 Exine reticulate at distal face . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Exine microreticulate or perforate at distal face. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Muri solid; mean number of lumina per 25 mm2 43 at proximal face and 50 at distal face . . . . . . . . . . . . G. confusa Muri compound; mean number of lumina per 25 mm2 5 at proximal face and 10 at distal face . . . . . . G. gageoides Exine microreticulate at distal face. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Exine perforate at distal face . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Pollen grain 25.5–30.0 47.5–52.5 (25.772.3 49.372.0) mm in size; mean number of lumina per 25 mm2 10 at proximal face and 13 at distal face. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. tenera Pollen grain smaller [19.0–27.0 42.0–47.0 (22.472.4 44.771.8) mm in size]; mean number of lumina per 25 mm2 14 at proximal face and 27 at distal face. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. exilis Muri compound, simpli- or duplicolumellate at distal face . . . . . . . . . . . . . . . . . . . . . . . . G. chomutowae fistulosa Muri solid, pluricolumellate at distal face . . . . . . . . G. alexeenkoana, G. anonyma, G. circumplexa and G. reticulata Exine reticulate at distal face; mean number of lumina per 25 mm2 at proximal face approximately equaling that of the distal face . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. bulbifera and G. dshungarica Exine perforate at distal face; mean number of lumina per 25 mm2 decreases from proximal face towards distal face9 Mean number of lumina per 25 mm2 at proximal face 26 and at distal face 19; lumina 0.30.5 0.20.4 mm in sizeG. caroli-kochii Mean number of lumina per 25 mm2 at proximal face 13 and at distal face 6; the lumina smaller (o0.2 mm) in sizeG. capillifolia Tectal perforation smaller than 1 mm diam. (type 3: perforate). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11 Tectal perforation wider 1 mm diam. (type 4: foveolate).. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14 Exine eutectate at distal face.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. afghanica and G. graminifolia Exine perforate at distal face . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12 Muri duplicolumellate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. dubia Muri pluricolumellate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .13 Muri solid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..G. chlorantha and G. iranica Muri compound . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .G. lutea, G. ova, G. soleimanii and G. stipitata Exine foveolate at distal face . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .G. vegeta Exine perforate at distal face . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15 Pollen grain 17.0–22.0 35.0–42.0 (20.171.4 38.972.2) mm; tectal perforations o0.2 mm at distal face . . G. olgae Pollen grain larger [25.0–30.0 53.0–60.5 (27.572.1 55.372.3) mm in size]; tectal perforations at distal face larger (0.6–0.8 0.4–0.6 mm in size). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. bergii
1 The key does not include the species of Gagea which are rare endemics or doubtfully recorded for Iran. Moreover, some species are so similar in pollen characteristics that they could not be separated from each other. Since a diagnostic key would be very useful in determination of the species, we preferred to present the key and in the cases of high similarities, refer to them collectively.
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Pollen fertility Results of the fertility analysis are summarized in Table 3. The lowest fertility percentage was observed in the population no. 22 belonging to G. reticulata, while G. vegeta (no. 26 in Table 3), G. bulbifera (no. 6 in Table 3) and G. afghanica (no. 1 in Table 3) show the highest fertility level, respectively. No significant difference was observed in exine sculpture between fertile and aborted pollen. Although in some species (e.g. G. alexeenkoana) the fertility percentage is relatively constant among different populations, it is highly variable for other species (e.g. G. reticulata).
Discussion The basic palynomorphological characters of Gagea are the occurrence of a shallow sulcus, a reticulate, microreticulate, perforate and foveolate exine with solid or compound simpli-, dupli- or pluricolumellate muri. Some of these features are shared also by some species of Lloydia, the sister genus of Gagea (Patterson and Givnish, 2002). The muri in Lloydia tibetica (Kosenko, 1999) as well as in G. confusa (Fig. 5), G. exilis, G. gageoides (Fig. 6) and G. tenera (Fig. 7) are simplicolumellate confirming the close relationship between these genera suggested by Takhtajan (1987, 1997). The taxonomy of the genus Gagea is very difficult due to several problems. Delimitation of species is in several cases problematic because of great variation in vegetative and generative characters during different stages of ontogeny (Levichev, 1990b, 1999b) and under different ecological conditions. Moreover, with respect of the subgeneric grouping within the genus, there are very controversial views. In the most recent published work on the subgeneric classification of the genus (Levichev, 1990a), it is divided into 10 sections (nine of them are present in Iran) and several series. But in the most regional Flora, other classifications mainly based on Pascher (1904, 1907) and Terracciano (1905, 1906) were applied. According to Kosenko (1991b, 1992, 1999) and Kosenko and Levichev (1988) pollen morphological characters provide valuable characters for a delimitation of the species in Liliaceae, especially in Gagea. The present study confirms this point of view.
Pollen in Gagea is heterogenous and eurypalynous, hence it can be divided into several groups and subgroups. Some of these groups are in congruence with the formerly supposed grouping within the genus on the basis of macromorphological characters, but others do not support them. The importance of pollen morphological characters and their fitness for the most actual subgeneric taxonomic grouping are discussed in the following.
G. sect. Platyspermum (G. reticulata species group) G. sect. Platyspermum Boiss. as defined by Levichev (1990a) includes: G. alexeenkoana (Fig. 8), G. anonyma (Fig. 9), G. bergii (Figs. 21–22), G. caroli-kochii (Figs. 15–17) and G. reticulata (Figs. 13 and 14). G. alexeenkoana, G. anonyma and G. reticulata show the reticulate type of exine. Several macromorphological characters such as flattened triangular seeds, acuminately tipped tepals, umbellate inflorescences and filiform or linear basal leaves are common among these species. However, G. bergii and G. caroli-kochii belong to two different other pollen types, i.e. foveolate and microreticulate ones, respectively. Several characteristic features of G. bergii separate it from other members of the section. A small but conspicuous bulbil at the leaf axil, short peduncle, long pedicels and a dense indumentum of the pedicels are some of these characteristic features. In our opinion, this species should be placed into a separate group. Geographical data also support this idea. Among the above-named species, G. bergii and G. anonyma show a more eastern distribution pattern reaching Afghanistan and Central Asia, while the other species are distributed from East Mediterranean to Central Iran. G. reticulata with the widest distribution area among the studied species, although reaches Afghanistan and Turkmenistan; it does not form such dense populations there as in Iran. Another treatment on the species of the above-named group is presented by Heyn and Dafni (1971) and by Rechinger (1986), where a ‘‘G. reticulata species group’’ is defined. In a wide sense this group should include: G. alexeenkoana, G. anonyma, G. caroli-kochii, G. circumplexa (Figs. 10 and 11) and G. reticulata (Zarrei, own unpublished data). Among these species, G. circumplexa is characterized by cord-like roots enveloping the bulb. However, this character can be modified by the given ecological conditions, especially
Figs. 17–24. SEM micrographs of pollen in Gagea. Fig. 17, Distal face of G. caroli-kochii pollen grain with perforate tectum and solid, pluricolumellate muri. Fig. 18, Perforate tectum at proximal face of pollen in G. dubia with compound, duplicolumellate muri. Fig. 19, Proximal face of pollen in G. stipitata grain with perforate tectum. Fig. 20, Perforate tectum at distal face of pollen in G. soleimani grain. Fig. 21, Pollen grain of G. bergii in distal polar view. Fig. 22, Proximal face of pollen in G. bergii with foveolate tectum and solid, pluricolumellate muri. Fig. 23, Proximal face of pollen in G. vegeta with foveolate tectum and solid, pluricolumellate muri. Fig. 24, Proximal face of pollen in G. olgae with foveolate tectum and solid, pluricolumellate muri. Figs. 17–20 and 23, scale bar ¼ 1 mm; Fig. 21, scale bar ¼ 10 mm; Figs. 22 and 24, scale bar ¼ 2 mm.
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by edaphic factors. In our opinion, the cord-like roots can be formed in soils with high proportion of clay in several other species as well, such as, e.g. G. reticulata. We suggest that G. circumplexa is closely related to G. reticulata. However, more studies are necessary for confirmation of this idea. Consideration of this species as separated from the G. reticulata species group and placed as a member of G. sect. Incrustatae, as done by Levichev (1990a), is not supported by palynological data either. G. caroli-kochii is another species of the group with considerable morphological similarity to G. alexeenkoana, although it is classified here under the microreticulate type. However, this species shows the upper limit of lumina diameter among the species classified under the microreticulate type. The classification applied here for pollen sculpturing type is based on the size of the lumina as presented by Punt et al. (1999), which is most probably somewhat artificial in Gagea. Moreover, the muri are solid in both species, as well as other members of G. sect. Platyspermum (as circumscribed by Levichev, 1990a) analyzed here.
G. sect. Stipitatae (Pascher) Davalianidze and G. sect. Fistulosae (Pascher) Davalianidze G. sect. Stipitatae includes: G. capillifolia (not shown), G. chumotowae (not shown), G. gageoides (Fig. 6), G. ova (not shown) and G. stipitata (Fig. 19), while G. sect. Fistulosae possesses only G. fistulosa in Iran. Although the species of G. sect. Stipitatae are classified here as belonging to different pollen types, the muri in all these species are compound. Cyme inflorescences are shared by all species of G. sect. Stipitatae. Moreover, the species of this section are very closely related, so that the taxonomical separation of them is in most cases very difficult. Again it seems that the classification of pollen types according to Punt et al. (1999) is artificial here and cannot be used for characterizing natural groups. G. fistulosa (Fig. 12) is palynologically very similar to G. chomutowae, and it is cited in the same place in the key as the latter. The only important morphological character which connects these species to each other is the presence of a fistulose basal leaf. The systematic position of G. sect. Fistulosae and its possible affinity with G. sect. Stipitatae needs more studies.
G. sect. Minimoides (A. Terracc.) Levichev Two important species attributed to this section and included in the present analysis are G. confusa (Fig. 5) and G. dshungarica (not shown). The former shows a western distribution pattern from Antolia to central Alborz (North Iran), while the latter exhibits rather an eastern distribution pattern from central Alborz but
widely distributed in Central Asia. Interestingly, near Kandavan and Damavand mountains (Central Alborz in Iran) there are mixed populations of both species. These species are also ecologically different. G. confusa grows always close to snow line on high mountains where the humidity of soil is relatively high, while G. dshungarica can be found much farther from snow line and prefers low soil humidity. For this reason, in overlapping areas of occurrence, always fruiting or dried specimens of G. confusa can be observed near flowering specimens of G. dshungarica. These species have been considered to be closely related and treated as belonging to the same section (Levichev, 1990a), but they are morphologically well characterized and taxonomically easily separable. From the palynomorphological point of view G. confusa and G. dshungarica are also well characterized and belong to two different pollen types, i.e. reticulate with solid, simplicolumellate muri and microreticulate with compound pluricolumellate muri, respectively. The possible close relationship between these species should be re-assessed.
G. sect. Graminifoliae Levichev Two species of this section, G. graminifolia (not shown) and G. vegeta (Fig. 23), are analyzed in the present study. In both species the muri are solid, pluricolumellate and wider than the lumina, but in the former taxon the lumina are smaller than 1 mm in diameter, while in the latter they are larger than 1 mm. Therefore, the two species are attributed here to two different pollen types: perforate and foveolate, respectively. Both species are distributed in Central Asia, Afghanistan and NE Iran. They are also very similar and probably closely related from a morphological point of view.
G. sect. Plecostigma (Turcz.) Pascher According to the definition presented by Levichev (1990a), the following species in Iran can be attributed to this section: G. afghanica (not shown), G. bulbifera (see Zarrei & Zarre, in press), G. chlorantha (not shown), G. iranica (see Zarrei & Zarre, in press) and G. olgae (Fig. 24). All these species are morphologically similar and form a homogeneous section. Among these species G. bulbifera is the only species bearing bulbils at the leaf axile. The pollen exine is foveolate or perforate with solid muri in all above-named species except for G. bulbifera with a microreticulate type of exine and compound muri. The latter should most probably belong to another species group. Interestingly, G. iranica and G. olgae possess the smallest pollen grains among all studied species.
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G. sect. foliatae A. Terracc In Iran, this section includes only G. tenera (Fig. 7) distributed also in Central Asia. This species is in some respects, e.g. possessing cyme inflorescences, similar to G. sect. Plecostigma, but shorter tepals and a wide lower cauline leaf separate it from the species of this section. A reticulate exine with compound and simplicolumellate muri confirms the attribution of this species to a section different from G. sect. Plecostigma.
G. sect. Gagea G. lutea (not shown) is the only reported species of this section distributed in Iran. It has the largest pollen grains among the studied species. It has a perforate exine with compound pluricolumellate muri. G. lutea shows the widest distribution area within the genus extending from West Europe (Great Britain) to East Asia (Japan). In contrast to most other species of the genus, it prefers very humid and shaded areas. As the final conclusion of this study, it seems that pollen morphology provides strong evidences for delimitation of natural subgeneric groups in Gagea. An extended study including the species distributed in other countries and comparing even more the subgeneric taxonomy of the genus can further lead to a better delimitation of the sections. In several cases, pollen morphology of Gagea will distinctly help the taxonomists to separate species with high macromorphological similarity to each other.
Pollen fertility Since there were only limited amounts of fresh material of anthers available, the study of pollen fertility can be considered only as a preliminary one. However, it rendered the following provisional results: 1) In a few species, such as G. alexeenkoana and G. gageoides, different populations show a similar and high fertility percentage. It seems that in these species the generative reproduction is more frequent than the vegetative one. In nature, these plants form scattered populations composed of several remote individuals. In the case of a frequent vegetative reproduction, especially through stolons, compact patches composed by several individuals attached together would be expected. 2) A few species, such as G. lutea and G. tenera , show a very variable fertility level reflecting frequency of vegetative reproduction in some populations as well as generative reproduction in others. Interestingly, the ploidy level among different populations in G. lutea varies between diploidy and pentaploidy (Peruzzi, 2003 and references cited there). In the case
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of triploids and pentaploids the rate of generative reproduction should be very low. 3) In species such as G. reticulata , which is very common and widely distributed in the studied area and forms dense compact patches of individuals attached together, most populations show a high rate of pollen fertility, although the vegetative reproduction by aid of stolons is also common. Although no other ploidy level than diploidy is recorded for this species, it may be assumed that triploids can also occur within populations and survive mainly by vegetative reproduction, as it was suggested for G. commutata and G. procera of the same species complex (Heyn and Dafni, 1971). For example the population no. 22 (see Table 3) shows the lowest fertility rate among all studied populations. The ecological conditions of the area near Fariman (NE Iran) under which the population no. 22 grows is relatively optimal for the genus. The soil is dark in color, a sandy clay and with a relatively high proportion of humus deposited from the remainder of some thorny Astragalus species and other plants, as Scariola orientalis (Boiss.) Sojak. Moreover, the individuals of G. reticulata grow in relatively large tufts within the cushions of Astragalus. They represent the largest habit of Gagea reticulata that has been observed in Iran. Most probably, these plants are triploid and reproduce vegetatively by aid of stolons. This would explain their growth in large tufts. Interestingly, all other examined populations of G. reticualta (no. 19–21 in Table 3) show a relatively high fertility percentage, which is in accordance with the recorded chromosome number for this species. Only a chromosome number of 2n ¼ 2x ¼ 24, i.e. the diploid chromosome set, is recorded for G. reticulata, while for several other species triploid and penatploid karyotypes are also recorded (see Peruzzi, 2003 and references cited there).
Acknowledgements We are grateful to Prof. Dr. I.G. Levichev (Saint Petersburg) for his useful suggestions and providing some literature. We are indebted to Prof. Dr. D. Podlech (Munich) for providing several specimens of Gagea from the neighboring countries of Iran. Dr. A.A. Maassoumi and Dr. M. Assadi (Tehran) helped us by their useful comments and providing the herbarium material of TARI. We are also grateful to the curators of the herbarium Iran, especially M. Moussavi. We thank S.M.H. Hashemi for assistance by SEM. The study on the family Liliaceae in IRAN was supported partially by the project number 513/4/632 of the Tehran University, Research Council to Shahin Zarre.
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