Biologia 65/3: 444—450, 2010 Section Botany DOI: 10.2478/s11756-010-0038-2
Comparative pollen morphology of Turkish species of Petrorhagia (Caryophyllaceae) and its systematic implications 1 ¨ Kamuran Aktas¸1*, Yasin Altan1, Canan Ozdemir , Pelin Baran1 & Teresa Garnatje2 1
Celal Bayar University, Faculty of Art and Science, Department of Biology, Manisa, Turkey; e-mail:
[email protected],
[email protected] 2 Institut Bot` anic de Barcelona, Passeig del Migdia s.n., 08038 Barcelona, Catalonia, Spain
Abstract: Pollen morphology of 10 taxa from Turkey, four of them endemic, belonging to the genus Petrorhagia (Caryophyllaceae), P. alpina subsp. alpina, P. alpina subsp. olympica, P. cretica, P. dubia, P. hispidula, P. lycica, P. pamphylica, P. peroninii, P. prolifera and P. saxifraga, has been investigated using light (LM) and scanning electron (SEM) microscopy. Pollen morphology differences among these taxa have been determined. The pollen type of investigated taxa is polyporate (12–22–porate), sphaeroidal, the exine exhibits a tectate structure and a microechinate ornamentation. Petrorhagia alpina subsp. alpina has the smallest pollen grain diameter (18.57 µm) and P. dubia the largest one (37.80 µm). The number of pores ranges from 12 (in P. dubia and P. saxifraga) to 22 (in P. alpina subsp. alpina and P. alpina subsp. olympica and P. hispidula) with a minimum pore diameter of 2.37 µm in P. alpina subsp. alpina and a maximum pore diameter of 4.23 µm in P. peroninii. The exine thickness ranges from 1.73 µm in P. saxifraga to 3.78 µm in P. pamphylica. In this study, the systematic implications are discussed in the light of palynological results. Key words: biosystematics; cluster analysis; exine ornamentation; palynology; pollen morphometry
Introduction The familyCaryophyllaceae is a large one, comprising 86 genera and 2,200 cosmopolitan species, more frequent in the temperate regions of the Northern Hemisphere. These plants are annual or perennial herbs (rarely shrubs) occurring in dry open habitats and totally absent from the lowland rain forests (Bittrich 1993). The genus Petrorhagia (Ser.) Link includes 32 taxa (Strid & Tan 1997), mainly distributed in Irano-Turanian and Eastern-Mediterranean regions and centred in Greece (having 10 endemic taxa) and Turkey where it is represented by 12 taxa, four of them being endemic; one species is endemic to Iran and another to Cyprus (Davis 1967; Davis et al. 1988; Strid & Tan 1997; G¨ uner et al. 2000). Three of these taxa Petrorhagia hispidula (Boiss. & Heldr.) Ball & Heywood, P. lycica (P.H.Davis) Ball & Heywood and P. pamphylica (Boiss. & Ball) Ball & Heywood have a status of vulnerable (VU) and P. peroninii Boiss. (Ball & Heywood) has a low risk status (LR) according to IUCN and Red Data Book of Turkish Plants (Ekim et al. 2000). Although the genus occurs mainly in the Eastern Mediterranean region and SouthEastern Europe, a few species of the genus Petrorhagia grow in North Africa, one species reaching Madeira and the Canary Islands and another Eastern Pakistan and Kashmir (Ball & Heywood 1964). Four species of this genus are widespread: P. saxifraga (L.) Link, which * Corresponding author
c 2010 Institute of Botany, Slovak Academy of Sciences
occurs throughout much of Central and Southern Europe and extends into South-West Asia; P. prolifera (L.) Ball & Heywood which is distributed in Central Europe and in the mountains of Southern Europe, the Caucasus, Northern Anatolia, and in those of western North Africa; P. dubia (Rafin.) G. López & Romo, which is widespread in the Mediterranean region; and P. alpina (Habl.) Ball & Heywood, which is found in the mountains of Central and Western Asia, extending to Southern Bulgaria (Ball & Heywood 1964). Several morphological studies have been conducted to establish well-diagnosed taxa (McNeill 1962; Bittrich 1993) and, although a phylogenetic framework was obtained by Fior et al. (2003, 2006), the family Caryophyllaceae still relies on overall similarities of morphological characters used for recognition (Fior et al. 2006). Pollen morphology is an important character used in systematics, and several authors have studied the Caryophyllaceae family from this point of view (Erdtman 1952, 1986; Buxbaum 1961; Nowicke 1975; Rao & Shukla 1975; Skvarla & Nowicke 1976; Nowicke & Skvarla 1977, 1979; Moore & Webb 1978; Punt & Hoen 1995; Pınar & Oybak 1997; Yıldız 2001a,b; Kaplan 2008, and others). Contrariwise, the pollen morphology of Petrorhagia has received little attention. Two studies determining the pollen morphology of Petrorhagia species have been found in the literature. In the first, carried out by Punt & Hoen (1995), the pollen morphology of
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Table 1. Localities of the Petrorhagia (Ser.) Link taxa examined in the present study. Taxon
Voucher localities
Petrorhagia alpina subsp. alpina (Habl.) Ball & Heywood Petrorhagia alpina subsp. olympica (Boiss.) Ball & Heywood Petrorhagia cretica (L.) Ball & Heywood
Turkey: A6 Tokat: Erbaa, Osman village, towards the Erbaa, roadside, beneath the hornbean-pinus mixture forest, 1100m, 23.VII.2003, K.A 1230. Turkey: C2 Mugla: Sandras mountain, Dikencik local, around the headwaters, forest area, 1500m, 25.VI.2003, K.A 1170. Turkey: B1 Manisa: Demirci, Simav road, meadow, 1000m, 13.VI.2003, K.A 1126. Turkey: B2 Denizli: Babadag mountain, around the village, 870m, above the hillside, 24.VI.2003, K.A 1140. Turkey: B3 Konya: Between Aksehir and Yalvac, Sultan mountain, before the come to Aksehir, 20km, roadside, dry hillside, 1550m, 11.VIII.2003, K.A 1270. Turkey: C2 Mugla: Fethiye, Babadag Mountain, above the stones, 1515m, 21.VII.22003, K.A 1240. Turkeyh: C3 Antalya: Lara road, near the Dedeman hotel, in front of the Fasilis residence, above the clif, stones and hillstones, 95m, 27.VI.2003, K.A 1190. Turkey: C4 Antalya: Alanya, between Mahmutlar and g¨ oz¨ uk¨ uc¨ ukl¨ u village, 3 km, roadside, dry meadow, 24.IX.2003, K.A 1270. Turkey: A1 Edirne: between Kesan and Enez, near Enez, 18 km, Ceribası fork in a road, meadow, 20.VI.2003, K.A 1150. Turkey: A6 Ordu: Akkus, entering the city, around the picnic area, 1325m, 22.VII.2003, K.A 1220.
Petrorhagia dubia (Rafin.) G.Lopez & Romo Petrorhagia hispidula (Boiss. & Heldr.) Ball & Heywood (E) Petrorhagia lycica (Davis) Ball & Heywood (E) Petrorhagia pamphylica (Boiss. & Ball) Ball & Heywood (E) Petrorhagia peroninii (Boiss.) Ball & Heywood (E) Petrorhagia prolifera (L.) Ball & Heywood Petrorhagia saxifraga (L.) Link
E – Endemic for Turkey, K.A – Kamuran Akta¸s
three Petrorhagia species, Petrorhagia nanteuilii (not distributed in Turkey), P. prolifera and P. saxifraga was studied. In the second one, Yıldız (2001b) examined the pollen morphology of three taxa belonging to the Petrorhagia genus, P. alpina subsp. alpina, P. prolifera and P. saxifraga, by Light Microscopy (LM) and Scanning Electron Microscopy (SEM). The objectives of the present work are: i) to study the pollen morphology (including qualitative and quantitative features) of the Turkish taxa belonging to the genus Petrorhagia and, ii) to test whether the studied pollen characters are significantly different among these taxa.
University, Faculty of Art and Science, Department of Biology. The terminology used is in accordance with Kremp (1968), Erdtman (1969), Faegri & Iversen (1975) and Moore et al. (1997). One-way ANOVA was used to test whether the studied variables are significantly different between the species. The least significant (LSD) test was carried out in order to compare the means between groups and to establish which of them are significantly different. Both analyses were carried out with STATGRAPHICS Centurion XV (StatPoint, Inc. USA). A cluster analysis including all variables was performed using the same programme.
Results and discussion Material and methods In this study, pollen morphology of 10 taxa belonging to Petrorhagia growing in Turkey (including the four endemic ones) was investigated. Pollen samples were obtained from the field as shown in Table 1. Descriptions are based on light microscope (LM) and scanning electron microscope (SEM) observations. Pollen grains were prepared for LM with standard methods described in Erdtman (1960) and mounted in unstained glycerin jelly. Observations and measurements were made with a Hunt-type binocular microscope under oil immersion at 400x. Pollen grains were photographed using an Olympus triocular microscope with a Nikon camera. Acetolysed pollen grains for SEM were suspended in ethanol, air dried on a stub and coated with gold in a sputtering chamber (Polaron SC 502) prior to observation with a JEOL JSM-5200 scanning electron microscope at 10–20 kV. The morphological features such as the pollen grain’s shape, exine ornamentation and apertures are described. The measurements were based on 30 readings from each specimen. Pollen diameter, number of pores, pore diameter, distance between two pores and exine thickness were measured. Slides and plant samples are deposited at Celal Bayar
Pollen measurements for each taxon are listed in Table 2. Some features of the pollen grains are constant in all the studied species corresponding to Dianthus superbus type described by Punt & Hoen (1995), in their study, and to Dianthus type reported by Yıldız (2001b) (Table 3). The pollen of Petrorhagia is polyporate, sphaeroidal and the exine exhibits a tectate structure and microechinate ornamentation. Because of their constancy, these characters can not provide phylogenetic nor systematic information (Figs 1–20). Two studies determining the pollen morphology of Petrorhagia species have been found in the literature. In the first, carried out by Punt & Hoen (1995), the pollen morphology of three Petrorhagia species, Petrorhagia nanteuilii (not distributed in Turkey), P. prolifera and P. saxifraga were studied. In the second one, Yıldız (2001b) examined the pollen morphology of three taxa belonging to the Petrorhagia genus: P. alpina subsp. alpina, P. prolifera and P. saxifraga, by Light Microscopy (LM) and Scanning Electron Microscopy (SEM).
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Figs 1–10. SEM micrographs of pollen grains of Petrorhagia. P. lycica (1–2); P. cretica (3–4); P. hispidula (5–6); P. alpina subsp. alpine (7–8); P. alpina subsp. olympica (9–10). 1, 3, 5, 7, 9 – Equatorial optical section; 2, 4, 6, 8, 10 – Superficial view.
Contrariwise, the measurements of the pollen grains and additional quantitative characters contain some very useful information, which is described as follows:
Pollen size The pollen of all the examined species is isopolar. The size of pollen grains varies from 18.57 µm in P. alpina subsp. alpina to 37.80 µm in P. dubia (see Ta-
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Table 2. Measurements and characteristics of pollen grains of Petrorhagia (Ser.) Link taxa. Diameter of pollen (µm) Taxon
P. P. P. P. P. P. P. P. P. P.
alpina subsp. alpina alpina subsp. olympica cretica dubia hispidula lycica pamphylica peroninii prolifera saxifraga
Diameter of pore (µm)
Distance between two pores (µm)
Exine thickness
Number of pores
M
SD
R
M
SD
R
M
SD
R
M
SD
R
20–22 20–22 16–18 12–14 20–22 16–18 14–16 18–20 16–18 12–14
18.57 20.10 30.90 37.80 31.10 30.10 36.50 31.50 35.10 24.30
±0.63 ±0.92 ±1.07 ±2.11 ±1.03 ±2.30 ±1.96 ±1.98 ±2.07 ±0.69
18–20 18–21 30–35 35–42 30–34 27–35 34–40 30–37 33–42 23–26
2.37 2.50 4.20 3.60 3.47 3.90 3.73 4.23 3.43 3.20
±0.41 ±0.51 ±0.61 ±1.03 ±0.57 ±0.61 ±0.64 ±0.70 ±0.50 ±0.48
2–3 2–3 3–5 2–5 3–5 3–5 3–5 4–6 3–4 3–5
4.40 5.50 8.40 9.80 8.67 7.90 9.63 9.40 7.30 8.13
±0.67 ±0.78 ±0.68 ±0.89 ±0.61 ±1.05 ±0.85 ±1.14 ±1.70 ±0.73
4–7 4–7 7–10 8–12 8–10 6–10 9–11 8–11 6–13 7–9
2.28 2.23 2.27 2.90 2.68 2.62 3.78 3.22 2.40 1.73
±0.39 ±0.34 ±0.45 ±0.81 ±0.43 ±0.49 ±0.54 ±0.25 ±0.38 ±0.25
2–3 2–3 2–3 2–5 2–3 2–3 3–4 3–4 2–3 1.5–2
M – mean; SD – standard deviation; R – range
ble 2). ANOVA shows statistically significant differences (P = 0.001) for this parameter between the taxa. When LSD test was performed, no significant differences were found between P. lycica and P. cretica, on the one hand, and between P. cretica, P. hispidula and P. peroninii, on the other hand. The remaining species show significantly different means among them. Punt & Hoen (1995) reported a pollen diameter of 44.5– (48.5)–52.0 µm for P. prolifera in the preparations with glycerine-gelatin, while the pollen dimension was 38.0– (41.0)–44.0 µm in the preparations with silicone oil. Yıldız (2001b) found a diameter of 40.92 µm for this taxon with glycerine-gelatin and we have found 35.10 (± 2.11) µm in the present work. In the case of P. saxifraga, Punt & Hoen (1995) found 33.0–(34.5)-38.5 µm in the preparations with glycerine-gelatin while the pollen size was 26.0–(28.5)-31.0 µm in the preparations with silicone oil and, in our case, the mean of the diameter for this species was 24.30 (± 0.69) µm. In both cases, our measurements, obtained through glycerin jelly-mounted pollen grains, are slightly smaller than those obtained by Punt & Hoen (1995) with the silicone oil and considerably smaller than those obtained with glycerine-gelatin. Yıldız (2001b) shows a diameter of 26.32 µm for this taxon. Punt & Hoen (1995) also obtained different sizes for P. nanteuilii, not included in the present work, depending on the preparation method used. In the same way, Yıldız (2001b) reported a pollen diameter of 20.68 µm forP. alpina subsp. alpina using the glycerine method; while we have found 18.57 µm for the same taxon (see Table 3 for the comparisons). Apertures (Figs 1–20) The pores of all the studied species of Petrorhagia have a nearly circular shape and they appear either under the exine level (sunken) or at the same level as the exine surface. The pollen grains are polyporate (12–22 porate) in all the studied taxa. The number of pollen pores ranges from 12 (in P. dubia and P. saxifraga) to 22 (in P. alpina subsp. alpina and P. alpina subsp. olympica and P. hispidula) with a minimum pore diameter of 2.37 µm in P. alpina subsp. alpina and a maximum of 4.23 µm in P. peroninii (Table 2).
Both variables are useful to characterize some of the studied taxa. On the one hand, the number of pores is significantly different (P < 0.05), and the LSD test shows significant differences between all the taxa excepting between P. lycica and P. prolifera, and also between P. cretica and P. lycica. The results are very similar for the diameter of pores. One-way ANOVA shows that the means are statistically different (P < 0.05) but the comparison of the means reveals that only P. alpina (including its two subspecies) is completely isolated from the remaining species. The average distance between two pores ranges from 4.40 (± 0.67) µm in P. alpina subsp. alpina to 9.80 (± 0.89) µm in P. dubia. The data analysis reveals that the differences are statistically significant (P < 0.05) and the means comparison shows that P. cretica, P. hispidula and P. saxifraga can not be separated by this character. In general, our results regarding the number of pores agree with the previous ones obtained by Punt & Hoan (1995) and by Yıldız (2001b) with slight differences. For the diameter of pores and the distance between them our values are smaller than those obtained by other authors (see Table 3). Exine (Figs 2, 4, 6, 8, 10, 12, 14, 16, 18, 20) The exine ornamentation appears as scabrate with numerous puncta when it is observed in LM. At the SEM, the exine shows a michroechinate ornamentation, the echinae being distinct, and with the puncta dispersed over the whole surface. The exine thickness of the species comprised in the present work ranges from thick to very thick. This thickness is smallest in P. saxifraga (1.73 µm) and largest in P. pamphylica (3.78 µm). One-way ANOVA shows that the mean exine thickness is significantly different between the studied species (P < 0.05). The LSD test allows us to separate P. saxifraga with a mean of 1.73 µm from the remaining species. In the same way, P. pamphylica (3.78 µm) and P. peroninii (3.22 µm) are also different.
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Figs 11–20. SEM micrographs of pollen grains of Petrorhagia. P. saxifraga (11–12); P. pamphylica (13–14); P. peroninii (15–16); P. prolifera (17–18); P. dubia (19–20). 11, 13, 15, 17, 19 – Equatorial optical section; 12, 14, 16, 18, 20 – Superficial view.
The exine thickness is smaller in the species of the present work than in the species studied by Punt & Hoen (1995) and Yıldız (2001b).
Systematic implications of pollen morphology The observed variation of pollen morphology, especially in the quantitative characters, proved to be a useful taxonomic tool in Petrorhagia (see Fig. 21). Davis (1967),
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Table 3. Comparison between data obtained from the present work with previous studies. This study
Punt & Hoen (1995)
Yıldız (2001)
Taxon
10 8 6 4 2
P. alpina sp. olympica
0
P. saxifraga
in “Flora of Turkey”, reports that P. alpina subsp. alpina and P. alpina subsp. olympica have very similar morphological features. We determined that the pollen morphology of these two taxa was also quite similar, but that the pollen diameter (20.10 µm) of P. alpina subsp. olympica is a little longer than that of P. alpina subsp. alpina (18.57 µm). On the other hand, our study pointed out that P. pamphylica and P. peroninii, which are morphologically very close to each other, had different palynological characteristics. The pollen diameter (36.50 µm) and the thickness of exine (3.78 µm) for P. pamphylica were found to be larger than those of P. peroninii (respectively 31.5 µm, 3.22 µm), while the pore diameter (4.23 µm) and the pore number (18–20) of P. peroninii were found to be larger than those of P. pamphylica (respectively 3.73 µm, 14–16). These two close species have been distinguished by their different palynological characteristics. P. prolifera and P. dubia, which are also reported to be morphologically very similar in “Flora of Turkey”, have been distinguished in respect of palynological data. In the present study, we determined the pollen diameter as 35.1 µm, the pore diameter as 3.43 µm, the distance between pores as 7.30 µm, the thickness of exine as 2.40 µm and the pore number as 16–18 for P. prolifera, while respectively as 37.80 µm, 3.60 µm, 9.80 µm, 2.90 µm, 12–14 for P. dubia. From these data it can be seen that P. dubia has bigger pollen, a thicker exine layer, and a larger number of pores compared to P. prolifera. Based on our results we can state that Petrorhagia pollen exhibits significant variation in size, pore number, pore diameter, distance between two pores, and exine thickness. These findings are significant as a systematic tool. All the measurements obtained in the present work are smaller than those presented in the previous studies, possibly due to methodological differences. Some species of Petrorhagia which are reported to be
Number of pores
3.0–4.0 10–(14)–16 26.32 (±2.38) 2.42 (±0.59) 12–15
P. alpina sp. alpina
12–14
P. dubia
1.5–2.0
– – – – Glycerine jelly 44.5–(48.5)–52.0 Silicone oil 38.0–(41.0)–44.0 Glycerine jelly 33.0–(34.5)–38.5 Silicone oil 26.0–(28.5)–31.0
Exine thickness (µm)
– – 20.68 (±2.42) 1.92 (±0.32) 12–14 – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – 3.0–4.5 14–(16)–18 40.92 (±2.68) 3.45 (±0.84) 13–16
P. pamphylica
23–26
– – –
P. peroninii
20–22 20–22 16–18 12–14 20–22 16–18 14–16 18–20 16–18
Diameter of pollen (µm)
P. hispidula
2.0–3.0 2.0–3.0 2.0–3.0 2.0–5.0 2.0–3.0 2.0–3.0 3.0–4.0 3.0–4.0 2.0–3.0
P. cretica
P. saxifraga
18–20 18–21 30–35 35–42 30–34 27–35 34–40 30–37 33–42
Number of pores
P. prolifera
alpina subsp. alpina alpina subsp. olympica cretica dubia hispidula lycica pamphylica peroninii prolifera
Exine thickness (µm)
P. lycica
P. P. P. P. P. P. P. P. P.
Diameter of pollen (µm)
Distance
Diameter Exine Number of pollen thickness of pores (µm) (µm)
Fig. 21. Dendrogram showing the results of cluster analysis based on quantitative characters of Petrorhagia’s pollen.
morphologically very similar in the Flora of Turkey have been distinguished with the palynological data. Acknowledgements The authors wish to thank Prof. Joan Vall`es and Dr. Joan Martín from the Laboratory of Botany, Faculty of Pharmacy, University of Barcelona for their very valuable comments, and Samuel Pyke from the Botanical Institute of Barcelona for the improvement of the English language. This work was supported by Research Fund of Celal Bayar University through the research project (project No. FEF2003/42). We thank to Prof. Dr. Bilge Hakan S ¸ en the staff of SEM Laboratory of Ege University.
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