Plant Syst Evol (2011) 294:281–287 DOI 10.1007/s00606-011-0465-8
ORIGINAL ARTICLE
Genetic diversity of Crocus antalyensis B. Mathew (Iridaceae) and a new subspecies from southern Anatolia Osman Erol • Levent S¸ ik • H. Betu¨l Kaya Bahattin Tanyolac¸ • Orhan Ku¨c¸u¨ker
•
Received: 23 April 2010 / Accepted: 2 May 2011 / Published online: 31 May 2011 Ó Springer-Verlag 2011
Abstract Crocus antalyensis B. Mathew is a bulbous plant endemic to Turkey. It is morphologically variable within the western part of Anatolia. Amplified fragment length polymorphism (AFLP) marker system was used to detect genetic variation among the Crocus taxa. Twentytwo primer combinations were used to screen for polymorphism among the samples. Genetic variation ranged from 0.44 to 0.69. We demonstrated the efficiency of the AFLP marker system for discriminating between individual C. antalyensis specimens. A high level of genetic variation was present among C. antalyensis specimens collected from different locations in Turkey. We also observed that C. antalyensis subspp. are genetically distinct from their relative Crocus flavus Haw. subsp. dissectus Baytop & B. Mathew. A new subspecies of C. antalyensis B. Mathew from southern Turkey is described. It is characterized by striped outer perianth segments, waist-shaped flowers, and glabrous throat of the perianth. A composite image of the new subspecies is presented. Keywords Iridaceae Crocus AFLP Composite image Turkey
O. Erol (&) O. Ku¨c¸u¨ker Faculty of Science, Department of Biology, Division of Botany, Alfred Heilbronn Botanical Garden, Istanbul University, Suleymaniye, 34134 Istanbul, Turkey e-mail:
[email protected] L. S¸ ik Department of Biology, Faculty of Science and Arts, Celal Bayar University, 45030 Manisa, Turkey H. B. Kaya B. Tanyolac¸ Department of Bioengineering, Ege University, 35100 Bornova-Izmir, Turkey
Introduction The genus Crocus is distributed in the Northern Hemisphere (ca. 80 spp.). The majority of the described Crocus taxa occur in the Balkans and Turkey; the numbers diminish rapidly on either side of this area (Maw 1886; Mathew 1982). Turkey has almost 70 Crocus taxa (Mathew 1984, 1998, 2000; Kerndorff and Pasche 2006). Most probably southwestern Turkey is the current center of diversity of the genus Crocus (Kerndorff and Pasche 2004). In recent years, most of Turkey’s native taxa were identified and classified by English and German Crocus experts Brian Mathew, Eric Pasche, and Helmuth Kerndorff. C. antalyensis was described by Brian Mathew from the cultivated material. It is allied to C. flavus Haw. subsp. dissectus Baytop & B.Mathew, but it is easily distinguished by the pale to deep lilac blue flower color. It is peculiar that C. antalyensis was not found a long time ago as it is widespread in western Turkey (Mathew, personal communication). Mathew (1982) described the flower color of C. antalyensis in his famous work ‘‘The Crocus.’’ According to Mathew (1982) C. antalyensis has deep lilac blue flowers with a yellowish-buff, purple-spotted exterior to the three outer segments. The exterior of the outer segments can vary from just about unmarked lilac to grayish violet speckled or even veined or striped violet on a lilac to buff ground color. We have been studying crocuses in Turkey for a decade and have observed many different populations of C. antalyensis while collecting for Series Biflori. In February 2008, we found a population that looked like C. biflorus Miller with pure white and striped flowers near the province of Mugla, Fethiye-Turkey. We first thought that this point was within the borders of the Antalya province, but in
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Table 1 Morphological differences among the subspecies of Crocus antalyensis Characters
Subsp. gemicii
Subsp. antalyensis
Subsp. striatus
Flowers
Color
White, striped on outside of the outer perianth segments
Pale to deep lilac blue with a bluish suffusion on the outside of the base of outer segments
Pale, lilac blue, flecked on outer perianth segments, striated on the outside of the inner perianth segments
Shape
Waist
Infundibular
Infundibular
Style
Divided into 5–15 slender yellow branches
Divided into 6–12 slender orange or yellow branches
Divided into 8 comparatively deep or pale yellow or white branches
Number and position at anthesis
2–8, synanthous, generally do not exceed the flower at anthesis
3–8, synanthous, equal to or shorter than flower at anthesis
4, synanthous, exceed the flower at anthesis, recurved
Leaves
fact the locality was in the middle of the Mugla, Antalya, Burdur triangle. We also observed many different populations near Bursa, Bilecik, Kutahya, Mugla, Burdur, Isparta, and Antalya (Fig. 2). Flower shape and color, leaf characteristics, and corm tunic structure differed from one population to another. Consequently we decided to study C. antalyensis in detail (including its morphology, anatomy, and genetics). We recognized the specimens collected from Fethiye as a new subspecies easily distinguished by the striped outer perianth segments, waist-shaped flowers, and the glabrous throat of the perianth. The tepals of the new taxon are slightly curved and they form a ‘‘waist shape.’’ This form prevents the recurvation of the tepals when they fully open on sunny days. The flower shape was used as a diagnostic character by Mathew (1984) in the genus Crocus. A more detailed morphological comparison is given in Table 1. When we studied the genetic aspect, the new subspecies formed a clade with another newly described subspecies C. antalyensis subsp. striatus O. Erol & M. Kocyigit within the observed C. antalyensis populations (Erol et al. 2010). The analysis of genetic diversity and relationships among different individuals, species, or populations is an important topic in genetics and plant breeding (Alavi-Kia et al. 2008). In recent years, technological advances in DNA techniques have become available that allow for new insights into systematic taxonomic analysis (Grilli Caiola et al. 2004). Among the DNA techniques, amplified fragment length polymorphism (AFLP) is robust and provides a powerful tool for studies of genetic variation, genotype identification, phylogeny (Kafkas 2006), and molecular linkage mapping (Hurtado and Ramstedt 2002). The AFLP technique is based on the detection of genomic restriction fragments by PCR amplification, and it can be used for DNA of any origin or complexity (Vos et al. 1995). The AFLP technique requires no cloning and sequencing and no prior sequence information and gives a very large number of scorable fragments (Karimi et al. 2009). High levels of polymorphism and high degrees of discrimination are the main advantages of AFLPs for discriminating closely
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related accessions. The AFLP method requires two cutting enzymes, ligation of the enzyme’s adaptor to restricted fragments, and preselective and selective amplification. Amplified fragments can be viewed by fluorescent or radioactive labeling of selective primers (Vos et al. 1995). In the present paper, AFLP analysis was used to investigate genetic variability at the DNA level in seven C. antalyensis specimens collected from different locations and also to characterize the newly identified species by using AFLP markers.
Materials and methods Observations were based on living material of C. antalyensis and its relative C. flavus subsp. dissectus from the field [(1) C. flavus subsp. dissectus Manisa (SB130), (2) C. antalyensis subsp. striatus Mugla (SB32), (3) C. antalyensis Isparta (SB35), (4) C. antalyensis subsp. gemicii Mugla (SB692), (5) C. antalyensis Bursa (SB83), (6) C. antalyensis Antalya (SB84), (7) C. antalyensis Burdur (SB86)] and from Istanbul University’s Alfred Heilbronn Botanical Garden. In addition, herbarium specimens from ISTF (Istanbul University Science Faculty Herbarium) (40079, 40080) and ISTE (Istanbul University Pharmacy Faculty Herbarium) (3825, 6001, 9826, 12482, 36684) were examined. Morphological data on C. antalyensis subsp. gemicii were obtained from the type locality (Fethiye: Mugla), including a total of 10 individuals. Illustration of the new subspecies was prepared as a composite image by following Simpson and Barnes (2008) and Erol et al. (2009) (Fig. 1). DNA extraction Young leaves from collected plants were harvested and placed in aluminum foil and kept in liquid nitrogen. Leaf 1 2
SB: IU Alfred Heilbronn Botanical Garden accession number. This locality was labeled as Burdur erroneously in Erol et al. 2010.
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Fig. 1 Composite image of Crocus antalyensis subsp. gemicii: a habitus, b flower, c outside of the outer segments, inside of the outer segments, outside of the inner segments, inside of the inner segments, from left to right, d bract and bracteole on young flower, e anthers, f style, g tunics, h corm, i cataphylls, j detail of cataphyll. The color bar on the upper left side is taken from the Royal Horticultural Society (2007) color chart
tissue from each individual was ground to a fine powder in liquid nitrogen with a mortar and pestle. Total genomic DNA was extracted following the procedure as described by Doyle and Doyle (1987). DNA was treated with RNAse and Proteinase K. Quantification of the DNA concentration was done through spectrophotometric measurements at absorbances of 260 and 280 nm using a ND-1000 (Nanodrop, Thermo) spectrophotometer. The DNA quality was also assessed and the concentration determined by visualization under UV light, on 1% agarose gels in TAE (Tris-
acetic acid-EDTA) buffer and then agarose gel–stained with ethidium bromide (Tanyolac 2003). AFLP analysis Li-Cor AFLP Kit (catalog number: 830-06195 AFLP 2-DYE Selective Amplification Kit) was used according to the manufacturer’s recommendations. According to the kit, 200 ng pure DNA was digested with EcoR I and Mse I restriction enzymes. The enzyme adaptors were ligated to
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the digested DNA. Selective amplification of restriction fragments was conducted using primers with three selective nucleotide extensions, RD700/800 dyes. Twenty-two primer combinations were used to screen for polymorphism among samples. Amplification products were resolved on 8% acrylamide gel in 19 TBE (Tris-borate-EDTA) buffer under 1500 V and 40 mA conditions. Li-Cor 4300s DNA Analyzer machine was used to image, analyze, and screen the bands profile. Statistical analysis A band was considered polymorphic if it was present in at least one genotype and absent in the others. Only clear and unambiguous bands on acrylamide gels from AFLP methods were scored for the presence (1) or absence (0) of amplified fragments in the seven DNA samples. Jaccard’s dissimilarity coefficients (1908) were calculated for all pair-wise comparisons among the seven samples. A dendrogram was generated using JMP software (version 3.1, SAS Institute, 1995) based on the UPGMA (unweighted pair-group method of arithmetic average).
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when dried. Leaves 2–8, synanthous, generally do not exceed the flower at anthesis, 0.5–1 mm width. Flowers white, waist-shaped, striped on outside of the outer perianth segments, generally 2:1 obovatus in shape, obtuse, outer perianth segments 5–12 9 14–27 mm; inner perianth segments 5–9 9 17–28 mm, throat yellow, glabrous, prophyll absent, bract papery ca. 6 9 70 mm, bracteole papery 2 9 60 mm yellowish brown when dried, filaments 4–7 mm pilose yellow, anthers 8–12 mm yellow, pollen grains creamy-white, style yellow, divided into 5–15 branches. Distribution and habitat Turkey, Mugla, very local in open forests, distributed sparsely along the slopes, together with Pinus nigra Arnold. subsp. pallasiana (Lamb.) Holmboe, Juniperus sp., Paliurus spina-christi Miller, Quercus coccifera L., Colchicum burttii Meikle, and Bellis perennis L. known only from the type locality in the province Fethiye. The population includes approximately 180–200 individuals. Its area is approximately 2,000 m2 (Fig. 2). Key to subspecies
Results and discussion 1. C. antalyensis B. Mathew subsp. gemicii L. Sik & O. Erol subsp. nov 1. C. antalyensis subsp. gemicii a C. antalyensis subsp. antalyensis segmentis perianthii exterioribus vivide albis vittatis, perianthio prope basin concavo sicut medium humani corporis, et fauce perianthii glabra differt. Type: Turkey: Mugla, Fethiye to Antalya, open areas in Pinus nigra forest, ca. 1,300 m a.s.l, 18.02.2009, O. Erol & L. Sik, ISTF 40143 (holotype: ISTF). Corm ovoid, ca 15 mm; tunics papery, dark brown, breaking up at the base into numerous parallel fibers with a persistent neck; cataphylls up to 6, white, yellowish brown Fig. 2 Distribution of Crocus antalyensis taxa
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Flowers deep lilac blue with a bluish suffusion on the outside of the base of outer segments subsp. antalyensis Flowers pale lilac blue or white, distinctly striped or flecked outside of the outer segments 2.
2.
Flower shape infundibular, outside of the inner perianth segments striated subsp. striatus Flowers waist-shaped, outside of the outer perianth segments striped subsp. gemicii
C. antalyensis subsp. antalyensis is a widely distributed endemic taxon in the west of the 32nd meridian in Turkey.
Genetic diversity of Crocus antalyensis B. Mathew
In 2007, we collected that recently described taxon C. antalyensis subsp. striatus from East Mentese Mountains in southwestern Turkey, Mugla province. Afterwards, we travelled east of this region in 2009 and Crocus antalyensis subsp. gemicii was observed between Golgeli and Katranci Mountains, near Seki Meadow. The three taxa can be distinguished from each other easily by flower color: C. antalyensis subsp. antalyensis has deep lilac blue flowers with a yellowish-buff, purplespotted exterior to the three outer segments; inner perianth segments are unmarked. C. antalyensis subsp. striatus is distinguished by distinctly striped inner perianth segments. C. antalyensis subsp. gemicii has pure white tepals with striped outer perianth segments. Morphological differences among the subspecies of C. antalyensis are given in detail in Table 1. To support these morphological differences between the subspecies, we also investigated genotypic profile of these taxa whether there was any DNA fragment differences between two taxa. As shown in Fig. 4, C. antalyensis subsp. gemicii and C. antalyensis subsp. striatus formed in the same clade, but their genetic distance was very distinct although they were within the clade. If these two taxa were close to each other, the genetic distance would not be that high (Grilli Caiola et al. 2004). Marker analysis
285 Table 2 Polymorphic bands from seven DNA samples, amplified by 22 AFLP primer combinations Primer number
Primer pairs
1
EAAC/MCAA
74
2
EACG/MCAA
71
3 4
EAAG/MCAA EACT/MCAA
65 50
5
EACC/MCAA
40
6
EAGG/MCAA
32
7
EAAC/MCTC
33
8
EACG/MCTC
41
9
EAAG/MCAC
24
10
EACT/MCAC
23
11
EACA/MCTA
66
12
EAGC/MCTA
25
13
EACA/MCAT
10
14
EAGC/MCAT
44
15
EACA/MCAC
37
16
EAGC/MCAC
40
17
EAAG/MCTA
78
18 19
EACT/MCTA EAAG/MCAT
36 48
20
EACT/MCAT
45
21
EAAG/MCAC
41
22
EAGG/MCAC Total
No. of polymorphic bands
58 981
AFLP polymorphism and discriminating capacity of the primer pairs Seven specimens were analyzed by AFLP-PCR using 22 selective primer combinations as listed in Table 2. A total of 981 polymorphic bands were generated, and the number of polymorphic bands per each primer combination ranged from 10 (EACA/MCAT) to 74 (EAAC/MCAA) with an average number of 44.6 bands. A representative gel obtained from the primer combinations (EACG/MCAA, EACT/MCAA, EAGG/MCAA, EACG/MCTC, and EACT/ MCAC) is presented in Fig. 3. Polymorphic bands from seven DNA samples, amplified by 22 AFLP primer combinations, are also listed in Table 2. Genetic relationships among the seven Crocus specimens To determine the genetic relationships among the seven specimens, the scoring data (1 for presence and 0 for absence) resulting from the 22 primer combinations were used to compute the dissimilarity matrix according to Jaccard (1908). This dissimilarity matrix was used to generate a dendrogram using the UPGMA method. The seven specimens represented two clades as revealed by
AFLP primers (Fig. 4). Clade II consisted of C. flavus subsp. dissectus with a very high average genetic distance ratio (0.62) from the other six C. antalyensis, suggesting it was not a very close relative to the specimens of C. antalyensis. Clade I contained samples 2, 3, 4, 5, 6, and 7. The average genetic variation among members of clade I was 0.48. Clade I divided into two subgroups of which the first subgroup included samples 2 and 4 and the second subgroup included samples 3, 5, 6, and 7. Sample 4, located in the first subgroup, was identified as a new subspecies in this article. As shown in Table 3, the minimum variation was detected between sample 6, C. antalyensis Antalya (SB84), and sample 7, C. antalyensis Burdur (SB86) (GD = 0.44), and the maximum was found between samples 1 and 5 (GD = 0.69). PCR-based molecular marker techniques play an important role in the analysis of genetic diversity and relatedness for crop plants, where most of the species involved are almost unknown at the genetic level (Ilgin et al. 2009). In this study, the AFLP method generated large numbers of polymorphic bands as compared to the study of Zubor et al. (2004). We detected a total of 981
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Fig. 3 AFLP pattern of seven Crocus DNA samples. 1 C. flavus subsp. dissectus Manisa (SB30), 2 C. antalyensis subsp. striatus Mugla (SB32), 3 C. antalyensis Isparta (SB35), 4 C. antalyensis subsp. gemicii Mugla (SB69), 5 C. antalyensis Bursa (SB83), 6 C. antalyensis Antalya (SB84), 7 C. antalyensis Burdur (SB86). M Molecular weight standard (50–700 bp ladder, Li-Cor Biosciences). A, B, C, D, and E represent the unique bands specific to the sample coded as 1
Table 3 Genetic distance matrix computed according to Jaccard (1908)’s coefficient based on AFLP data
SB30 (1) SB32 (2)
SB30 (1)
SB32 (2)
SB35 (3)
SB69 (4)
SB83 (5)
SB84 (6)
SB86 (7)
0.00
0.58
0.62
0.62
0.69
0.63
0.58
0.00
0.52
0.48
0.65
0.54
0.56 0.49
SB35 (3) SB69 (4) SB83 (5) SB84 (6) See Fig. 3 for explanation of sample numbers
SB86 (7)
polymorphic bands, and the number of polymorphic bands for each primer combination ranged from 10 (EACA/ MCAT) to 74 (EAAC/MCAA) with an average number of 44.6 bands. Our study shows that AFLP provided a large number of polymorphic bands and a large amount of genotypic information. The number of polymorphic bands per primer was two times more than Zubor et al. (2004)’s study. Grilli Caiola et al. (2004) found the number of polymorphic bands per primer to be 2.01 in their RAPD
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0.00
0.53
0.50
0.48
0.00
0.56
0.49
0.47
0.00
0.47
0.53
0.00
0.44 0.00
study. Grilli Caiola et al. (2004) studied genetic variation among seven different species and the variation among the species ranged between 0.47 and 0.77. In the current study, although we used only C. antalyensis (with the exception of C. flavus), the genetic variation ranged from 0.44 to 0.65 among the C. antalyensis specimens, which was close to the variation calculated among species by Grilli Caiola et al. (2004). This indicates that the natural variation within C. antalyensis collected in Turkey is very high.
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Fig. 4 Dendrogram resulting from UPGMA cluster analysis of seven Crocus specimens based on data derived from 22 AFLP primer combinations
In conclusion, sample 4 was identified as a new subspecies in this article. The present results clearly demonstrated the efficiency of the AFLP marker system to discriminate among individuals within the C. antalyensis specimens. A high level of genetic variation exists among the C. antalyensis specimens collected from different locations in Turkey. Acknowledgments The first author appreciates Helmut Kerndorff and Eric Pasche for sharing of their knowledge of the genus Crocus. We are grateful to Prof. Dr. Neriman Ozhatay (ISTE) for her scientific advice. We also thank Mark Garland (Micanopy, Florida, USA) for Latin translation. This work was supported by the Research Fund of Istanbul University, Istanbul, Turkey (project number 4155). We also kindly thank the Turkish Research Council for supporting PhD student H. Betul Kaya’s scholarship (project number 108G096).
References Alavi-Kia SS, Mohammadi SA, Aharizad S, Moghaddam M (2008) Analysis of genetic diversity and phylogenetic relationships in Crocus genus of Iran using inter-retrotransposon amplified polymorphism. Biotechnol Biotechnol Equip 22(3):795–800 Doyle JJ, Doyle JL (1987) A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem Bull 19:11–15 Erol O, Kucuker O, Sik L (2009) Application of a new illustration technique in plant systematics: composite images of two autumn flowering Crocus L. (Iridaceae) taxa from Series Biflori in Turkey. Istanbul Univ Fac Sci J Biol 68(2):127–133 Erol O, Kocyigit M, Sik L, Ozhatay N, Kucuker O (2010) Crocus antalyensis subsp. striatus subsp. nov (Iridaceae) from southwest Anatolia. Nordic J Bot 28(2):186–188 Grilli Caiola M, Caputo P, Zanier R (2004) RAPD analysis in Crocus sativus L. accessions and related Crocus species. Biol Plant 48:375–380 Hurtado S, Ramstedt M (2002) AFLP comparison of distant Melampsora epitea (willow rust) populations. Mycol Res 106(12):1400–1407
Ilgin M, Kafkas S, Ercisli S (2009) Molecular characterization of plum cultivars by AFLP markers. Biotechnol Biotechnol Equip 23(2):1189–1193 Jaccard P (1908) Nouvelles recherchers sur la distribution florale. Soc Vaudoise Sci Nat 44:233–270 Kafkas S (2006) Phylogenetic analysis of the genus Pistacia by AFLP markers. Plant Syst Evol 262:113–124 Karimi HR, Kafkas S, Zamani Z, Ebadi A, Moghadam MR (2009) Genetic relationships among Pistacia species using AFLP markers. Plant Syst Evol 279:21–28 Kerndorff H, Pasche E (2004) Crocus biflorus in Anatolia, part two. Plantsman (new ser) 3:201–215 Kerndorff H, Pasche E (2006) Crocus biflorus in Anatolia, part three. Linzer Biol Beitr Linz 38:165–187 Mathew B (1982) The crocus: a revision of the genus Crocus. BT Batsford, London Mathew B (1984) Crocus. In: Davis PH (ed) Flora of Turkey and the East Aegean Islands, vol 8. Edinburgh University Press, Edinburgh, pp 413–438 Mathew B (1998) Crocus kerndorffiorum. Curtis’s Bot Mag 15: 120–124 Mathew B (2000) Crocus L. In: Guner A et al. (eds) Flora of Turkey and the East Aegean Islands, vol 11. Edinburgh University Press, Edinburgh, pp 271–274 Maw G (1886) A monograph of the genus Crocus. Dulau, London Royal Horticultural Society (2007) RHS colour chart. The Royal Horticultural Society, London Simpson N, Barnes P (2008) Photography and contemporary botanical illustration. Curtis’s Bot Mag 25:258–280 Tanyolac B (2003) Inter-simple sequence repeat (ISSR) and RAPD variation among wild barley (Hordeum vulgare subsp. spontaneum) populations from west Turkey. Genet Resour Crop Ev 50:611–614 Vos P, Hogers R, Bleeker M, Reijans M, Lee T, Hornes M, Friters A, Pot J, Paleman J, Kuiper M, Zabeau M (1995) AFLP: a new technique for DNA fingerprinting. Nucleic Acids Res 23(21): 4407–4414 Zubor AA, Suranyi G, Gyori Z, Borbely G, Prokisch J (2004) Molecular biological approach of the systematics of Crocus sativus L and its allies. Acta Hortic 650:85–93
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