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Phylogenetic relationship in Fritillaria spp. of Iran inferred from ribosomal ITS and chloroplast trnL-trnF sequence data
Biochemical Systematics and Ecology 57 (2014) 451e457
Contents lists available at ScienceDirect
Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco
Phylogenetic relationship in Fritillaria spp. of Iran inferred from ribosomal ITS and chloroplast trnL-trnF sequence data Mahmoud Khourang a, Alireza Babaei a, *, Fatemeh Sefidkon b, Mohamad Reza Naghavi c, Davood Asgari c, Daniel Potter d a
Department of Horticulture Science, Tarbiat Modares University, Tehran, Iran Research Institute of Forests and Rangelands, Tehran, Iran c Department of Agronomy and Plant Breeding, Faculty of Agriculture, University of Tehran, Karaj, Iran d Department of Plants Science, University of California-Davis, Davis, USA b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 June 2014 Accepted 4 October 2014 Available online
The genus Fritillaria embraces up to 165 taxa in the family Liliaceae, most of which are of high medicinal and ornamental value and importance. In this study, 44 specimens of the genus representing 9 species were collected from their natural habitats located in 10 provinces of Iran. Phylogenetic analysis was performed based on DNA sequences of the internal transcribed spacer (ITS) of the nuclear ribosomal cistron and the trnL-trnF regions. The phylogeny was constructed using the neighbor joining inference method. Results indicate that the examined samples were evidently diverged into 2 distinct clades. Members of the subgenera Fritillaria and Rhinopetalum formed one clade while the other clade contained the subgenera Theresia and Petilium. There can be seen a high degree of similarity between the only yellow-colored crown imperial specimen and the red-colored specimens. The endemic species of Fritillaria straussii, Fritillaria zagrica and Fritillaria kotschyana which their status within the subgenera known in the genus Fritillaria has been remained undefined, fell into the subgenus Fritillaria. The clades also had relatively reasonable distribution patterns based on the genetic structure, geographical conditions and climate specifications. This study revealed the feasibility of the ITS and trnL-trnF DNA sequence for phylogeny of the genus Fritillaria. This is the first phylogenetic analysis of Fritillaria spp. in Iran. © 2014 Published by Elsevier Ltd.
Keywords: Fritillaria ITS trnL-trnF Medicinal plant Ornamental plant Phylogeny
1. Introduction Fritillaria L. is a genus of over 160 species of bulbous and perennial plants belonging to the filmily Liliaceae. Members of this genus are native to temperate regions of the Northern hemisphere (Tomovic et al., 2007). Above the species level, Iran is the main center of genetic diversity of Fritillaria (Rix, 1977) with up to 14 species which grow naturally throughout Iran, most of which are endemic to the country (Mozaffarian, 1992; Khaniki, 2002). Fritillaria species are becoming increasingly popular in floriculture (Turktas et al., 2012) and medicinal plants (Li et al., 2000) industries and many species are commercially available.
* Corresponding author. Department of Horticulture Science, Tarbiat Modares University, P. O. Box 14115-336, Tehran, Iran. E-mail address: [email protected] (A. Babaei). http://dx.doi.org/10.1016/j.bse.2014.10.001 0305-1978/© 2014 Published by Elsevier Ltd.
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M. Khourang et al. / Biochemical Systematics and Ecology 57 (2014) 451e457
Although there are a considerable number of studies about the morphological and physiological characterization of Fritillaria, unfortunately we know little about phylogenetic affinities of these plants and their concealed breeding advantages. After the first classification of the genus Fritillaria by Linnaeus, several further classifications have been described. According to the last revision by Rix et al. (2001), the genus is subdivided into 8 subgenera including Davidii, Liliorhiza, Japonica, Fritillaria, Rhinopetalum, Petilium, Theresia and Korolkowia. Results obtained by Ronsted et al. (2005) support this classification. Reproducibility and simplicity are the main qualities making DNA sequencing a good choice for identification of species and phylogenetic reconstruction studies (Turktas et al., 2012). For this reason, rRNA sequences are widely used in evaluating evolutionary affinities among organisms, since they are of ancient origin and are found in all known forms of life. Internal transcribed spacer (ITS) region of the 18Se5.8Se26S nuclear ribosomal cistron has revealed a high degree of variation even between closely related species (Alvarez and Wendel, 2003). In fact, the ITS region is one of the most widely applied molecular markers in current angiosperm systematics (Bailey et al., 2003). Moreover, chloroplast markers are widely used for phylogenetic assessments (Fay et al., 2006), as they evolve rapidly (Drabkova et al., 2004) and are mutational hot spots. The tRNALeu (trnL) intron and the intergenic spacer (IGS) between trnL 30 exon and tRNA-Phe (trnF) gene regions are quite suitable for the phylogenetic analysis of intraspecific variations (Huang et al., 2009). Using these regions, our aim is to determine molecular diversity and phylogenetic affinities amongst several Fritillaria species of Iran. This can be a valuable source of generation new Fritillaria varieties for ornamental and medicinal purposes. In addition, our results would be valuable to improve efforts which cover Iran germplasm in order to sustain this national wealth. 2. Material and methods 2.1. Plant material Leaf samples of 44 specimens representing 9 species of the genus Fritiallaria including Fritillaria imperialis L., Fritillaria persica L., Fritillaria crassifolia Boiss., Fritillaria straussii Bommuller., Fritillaria zagrica Stapf., Fritillaria kotschyana Herb., Fritillaria gibbosa Boiss., Fritillaria reuteri Bornm. and Fritillaria raddeana Rgl. were collected through 10 provinces of Iran. The species were represented by 16, 8, 7, 1, 2, 2, 2, 1, and 5 specimens, respectively (Table 1). Young leaves were collected, labeled and immediately kept in liquid nitrogen. Samples were stored at 80 C till DNA extraction. 2.2. Sequence generation Total DNA was extracted following a standard CTAB protocol (Doyle and Doyle, 1987) or modifications thereof. PCR amplifications were performed with an initial 5 min at 95 C, followed by 35 cycles of 1 min at 95 C, 1.30 min at 56 C, 2 min at 72 C and a final 7 min at 72 C. Reactions were carried out in a total volume of 50 mL, containing 5 mL MgCl2, 2 mL dNTP, 5 mL 10X PCR buffer (Applied Biosystems, USA), 1 mL of forward and reverse primers, 0.5 mL of Taq DNA Polymerase, 17 mL Betain, 18 mL water and 1 mL of DNA template. Regions were amplified using 2 primer sets of ITS1-ITS2 and trnL-trnF. In the case of trnL-trnF region, a primer combination of c þ d and e þ f was used for amplification of the intron of the chloroplast trnL (UAA) gene and partial trnL gene and the trnL-trnF IGS regions. PCR products were subjected to gel electrophoresis and were cleaned up using a PCR clean-up kit (Promega, USA). Purified PCR products were directly sequenced in 2 directions, using the corresponding primers. The sequencing process was performed using ABI/Prism 377 automated sequencers. The sequences identity was confirmed in comparison with various published sequences available in GenBank. 2.3. Phylogenetic reconstruction Sequences were edited using the Chromas Lite 2.1.1 (Technelysium Pty Ltd) and refined by eye. The newly generated sequences were submitted to GenBank (Table 1). Multiple sequence alignment was carried out using the ClustalW program (Thompson et al., 2002). Sequences of the species F. imperialis, F. persica, F. crassifolia and F. gibbosa for the ITS1-ITS2 region and F. imperialis and F. persica for the trnL-trnF region were the only sequences available in GenBank (September, 2014). Sequences were downloaded from the Genbank and were used to determine the boundaries of the regions (Table 2). Fritillaria and Lilium are sister taxa (Ronsted et al., 2005), thus Lilium davidii was chosen as an out-group in our analyses (Table 2). Phylogenetic analyses of the data matrix were performed based on the neighbor joining (NJ) method. Bootstrap values were calculated from 1000 replicates, and bootstrap coefficiencies were indicated above each branch. 3. Results 3.1. Sequence alignment and divergence The sequencing process was performed from both forward and reverse directions, and results successfully verified each other. PCR amplification was successfully obtained for all the samples. For the ITS region, F. imperialis had the shorter amplicon size than the rest, while F. persica showed the shortest amplicon length for the trnL-trnF region. The ITS region produced PCR products of about 680e780 bp. In the other hand, a higher length divergence was recorded for the trnL region (618e720 bp) compared with the IGS region (91e94 bp). Moreover, IGS region contained a higher G þ C content (44.6%) compared with the
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Table 1 Geographical distribution of Fritillaria spp. across different provinces of Iran. No.
1 2 3 4 5 6 7 8 9 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 a
Species
F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F.
gibbosa raddeana raddeana imperialis raddeana raddeana raddeana persica zagrica imperialis imperialis persica imperialis imperialis gibbosa imperialis reuteri imperialis lutea imperialis persica persica persica persica persica zagrica imperialis imperialis straussii crassifolia imperialis imperialis crassifolia crassifolia kotschyana kotschyana crassifolia crassifolia crassifolia crassifolia imperialis imperialis imperialis imperialis
Codea
1 1 2 1 3 4 5 1 1 2 3 2 4 5 2 6 e e 7 3 4 5 6 7 2 8 9 e 1 10 11 2 3 1 2 4 5 6 7 12 13 14 15
GenBank accession number ITS
trnL-trnF
KM435231 KM435149 KM435152 KM435147 KM435153 KM435154 KM435155 KM435148 KM435235 KM435156 KM435157 KM435171 KM435158 KM435159 KM435232 KM435160 KM435237 KM435170 KM435161 KM435172 KM435173 KM435174 KM435175 KM435176 KM435236 KM435162 KM435163 KM435238 KM435178 KM435164 KM435165 KM435179 KM435180 KM435233 KM435234 KM435181 KM435182 KM435183 KM435184 KM435166 KM435167 KM435168 KM435169
KM435227 KM435211 KM435212 KM435187 KM435213 KM435214 KM435215 KM435203 KM435223 KM435188 KM435189 KM435204 KM435190 KM435191 KM435228 KM435192 KM435230 KM435202 KM435193 KM435205 KM435206 KM435207 KM435208 KM435209 KM435224 KM435194 KM435195 KM435229 KM435216 KM435196 KM435197 KM435217 KM435218 KM435225 KM435226 KM435219 KM435220 KM435221 KM435222 KM435198 KM435199 KM435200 KM435201
Longitude
Latitude
55 55 55 55 55 55 55 49 50 50 50 50 50 50 51 51 50 50 50 50 48 50 48 48 47 46 46 46 46 46 46 47 47 47 47 47 47 47 47 49 47 47 47
27 37 37 37 37 37 37 33 33 33 33 32 32 32 32 31 31 32 32 32 35 32 35 35 27 35 35 35 35 34 34 37 37 37 37 37 37 37 37 33 33 33 33
470 5000 220 3500 070 4300 220 3500 220 4500 180 1200 180 1200 330 1600 230 0100 240 0400 200 400 150 5200 130 3600 160 1200 030 0800 10 1000 590 1700 220 1300 210 4000 210 4000 440 2000 150 5200 400 2000 410 4000 070 4900 110 5900 120 1300 110 1000 110 1000 070 1000 090 5000 420 0400 380 4000 380 4000 380 3200 300 5500 280 20 00 300 5000 310 3200 250 1400 310 3200 310 3200 310 3200
220 2600 200 200 210 1000 200 200 500 4600 590 900 210 3800 580 2600 90 2300 90 1000 110 1100 300 500 320 2000 480 5200 120 5800 460 5100 450 4600 080 2300 100 4000 100 4000 320 1900 120 5800 410 1200 390 1100 220 2600 180 1400 160 4000 150 5900 150 5900 190 4000 210 1000 430 0900 450 4000 430 0400 410 1100 410 5100 380 3600 410 4600 360 1100 060 4100 080 3700 080 3700 070 4100
Digits are used to determine the position of each specimen across Figs. 1 and 2. For species with only one specimen, no code is given.
trnL region (32.1%). The average G þ C content of the ITS sequence was slightly higher than the latter (32.8%). The percentage of identical sequences for trnL region and the IGS region was 94.21% and 93.51% respectively. The ITS region showed a higher percentage of identity with 95.94% identical sequences. Genetic distances for 9 species were calculated using the Tamura-Nei’s model. The average genetic distance amongst species ranged from 0.08 to 0.32. For both primers, the maximum distance observed was between F. imperialis e F. crassifolia, while the highest level of similarity found was between red-colored and yellow-colored crown imperials (0.08) as well as F. crassifolia and F. straussii (0.09) for ITS and trnL-trnF regions, respectively.
Table 2 Species used in this study as out-group and/or for determination of boundaries. ITS
trnL-trnF
Species
Accession number
Species
Accession number
Fritillaria gibbosa Fritillaria persica Fritillaria imperialis Fritillaria crassifolia Lilium davidii
AY616722 AY616736 AY616725 AY616717 AF088195
Fritillaria imperialis Fritillaria persica Lilium davidii
JQ327135 EU912327 EU597205
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3.2. Phylogenetic relationships amongst Fritillaria species Iranian species are classified in four different subgenera including Fritillaria, Theresia, Petilium and Rhinopetalum. F. imperialis and F. raddeana are two well-known species belonging to the subgenus Petilium. F. persica probably is monotypic and is the only species available in the subgenus Theresia. F. gibbosa and Fritillaria karelinii are two morphologically similar species in the subgenus Rhinopetalum. Subgenus Fritillaria is morphologically classified into a variety of species namely F. crassifolia, Fritillaria graeca, Fritillaria meleagris, Fritillaria cirrhosa and Fritillaria uncasica (Rix, 1977). 3.2.1. Subgenera Theresia and Petilium Topography and distribution of the species across the trees mainly reconfirmed and followed this classification (Figs. 1 and 2). Although two different primers used in this study showed difference in term of distribution of specimens across the trees (particularly in species belonging to the subgenus Fritillaria), they followed almost the same general pattern, so that results of both trees mostly verified each other. Our phylogeny analysis revealed that the 44 Fritillaria specimens clearly divided into 2 main clades. In our analysis, the subgenera Petilium and monotypic Theresia were grouped together and composed the first clade containing representatives of the species F. imperialis, F. raddeana and F. persica. In addition to molecular data, similar karyotype and pollen structures of the Petilium and Theresia supports the idea that these two subgenera are closely related (Teksen et al., 2010). In detail, specimens of F. imperialis and F. raddeana, as the members of the subgenus Petilium were placed in this clade. Besides, F. persica the member of the monotypic Theresia with 8 specimens positioned at the same clade, but in a different sub-cluster. Although the only yellow-colored crown imperial specimen (known as F. imperialis lutea) was placed in this clade next to the red-colored specimens, it showed a slight difference with the neighbors, as we expected. Alp et al. (2009) found natural crown imperial plants with yellow flowers in Anatolia district of Turkey and concluded that the yellow form is not a hybrid. The red-colored and the yellow-colored crown imperials are two different forms of the species (Alp et al., 2009), but evidences reveal that they are closely related (Figs. 1 and 2), and naturally co-exist in same habitats. Although several publications are available about the phylogenetic evaluation of the red-colored form of F. imperialis, there is no information about molecular phylogeny of the yellow-colored form of this species. 3.2.2. Subgenera Rhinopetalum and Fritillaria The second clade was divided into two groups containing the species of the subgenera Rhinopetalum (F. gibbosa) and Fritillaria (F. zagrica, F. reuteri, F. straussii, F. crassifolia and F. kotschyana). 3.2.3. Unclassified endemic species Ronsted et al. (2005) studied 37 species representing all subgenera and sections of the genus Fritillaria, through which the species F. imperialis (red-colored form) and F. raddeana from the subgenus Petillium, F. persica from the subgenus Theresia, F. gibbosa from the subgenus Rhinopetalum and F. reuteri and F. crassifolia from the subgenus Fritillaria were similar to those analyzed in our study. They didn't use any yellow-colored crown imperial (F. imperialis lutea) specimen. In comparison to their study, we used 9 species, 6 of which are endemic to Iran, amongst which there is no information about molecular phylogeny of F. zagrica, F. straussii and F. kotschyana in the literature so far. Therefore, status of these species through the 8 subgenera known in the genus Fritillaria has been remained undefined. As mentioned, results of our study clearly illustrate that the three species of F. zagrica, F. straussii and F. kotschyana fell into the subgenus Fritillaria, known as the largest subgenus of the genus. Morphological records also confirm the resemblance of these species.
4. Discussion This is the first study carried out on the molecular phylogeny of Fritillaria spp. in Iran. Although our findings reconfirm results of the previous phylogenetic studies of the genus Fritillaria, they suggest a new phylogenetic arrangement within the genus. Our data revealed the feasibility of the nucleotide ITS and the chloroplast trnL-trnF sequences for phylogeny of Fritillaria species. Assessment of level of genetic diversity within several Fritillaria species collected from a broad range of Iran climate conditions contributes vital information with regard to their evolutionary history and is critical to the development of effective conservation, utilization and management practices. This is of vital importance because several aspects of genetic assessments of plants such as genetic diversity, plants breeding practices and also restoration of threatened Fritillaria populations, can only be addressed by detailed population genetic studies (Cruz et al., 2010). High level of diversity is known amongst the key factors increasing agronomic and economic importance of any crop. Considering Fritillaria species are known as out-crossing species (Burquez, 1989), a high level of diversity amongst them was observed. Moreover, this level of diversity could be more explained by collecting the samples from a vast area of the country embracing a wide variety of climate conditions and countless natural habitats, potentially intensifying genetic diversity within the genus. Moreover, hybridization is one of the most popular methods for generating new varieties (Turktas et al., 2012). For instance, based on our findings, although the red-colored and yellow-colored crown imperials show slight genetic variation, they are closely related (Figs. 1 and 2). Accordingly, crossing these two forms can be used for releasing new cultivars with
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much more variety in valuable features such as flower color. Hence, information about phylogenetic relationships amongst these species would be potentially useful for future breeding programs. To sum up, the inventory and sampling efforts showed a broad range of geographical distribution of different subgenera of the genus Fritillaria throughout Iran climate conditions. This supports the theory suggesting Iran as a center of genetic diversity of the genus (Rix, 1977). More importantly, results showed that the majority of the species reported in Iran either might be already extinct or must be considered as critically endangered species. For example, we couldn't find Fritillaria ariana (Lozinsk. & Vved.) Rix as an endemic species previously reported in a limited area of the Northern Khorasan province
Fig. 1. Majority-rule consensus tree of the Fritillaria spp., obtained using the NJ method for ITS region. Bootstrap values are shown next to each branch. Sequences of specimens specified with (C) are downloaded from GenBank.
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Fig. 2. Majority-rule consensus tree of the Fritillaria spp., obtained using the NJ method for trnL-trnF region. Bootstrap values are shown next to each branch. Sequences of specimens specified with (C) are downloaded from GenBank.
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(Mozaffarian, 1992). Due to extreme desertification occurred across the area, the species is possibly extinct. Moreover, ratio and abundance of yellow-colored crown imperial’ populations were considerably less than those of red-colored populations, which put this form in the state of being critically endangered. This was consistent with the majority of the collected species namely F. zagrica, F. gibbosa, F. reuteri, F. straussii, F. crassifolia and F. kotschyana. Amongst all the species only F. imperialis (redcolored form), F. persica and F. raddeana showed a normal abundance and distribution across their natural habitats. Taking this into consideration, to preserve species of the genus, and before taking economic advantage of this national wealth, the urgent priority would be addressing effective conservation efforts which cover Iranian fritillaries. For this reason, bulbs of each species are cultivated and conserved in the Faculty of Agriculture of Tarbiat Modares University (TMU) to monitor their growth and development process in a protected area as well as their response to the new environmental conditions. Moreover, for further investigations, results of the study have been reported to the Research Institute of Forests and Rangelands of Iran (RIFRI). Acknowledgments We would like to thank Mr. Hadi Jafari for his assistance and Mr. Kai Battenberg for his contribution by giving many helpful insights and suggestions. References Alp, S., Arslan, N., Koyuncu, M., 2009. Established forms of Fritillaria imperialis L.: a naturally growing species in Turkey. Pak. J. Bot. 41, 1573e1576. Alvarez, I., Wendel, J.F., 2003. Ribosomal ITS sequences and plant phylogenetic inference. Mol. Phylogenet. Evol. 29, 417e434. Bailey, C.D., Carr, T.G., Harris, S.A., Hughes, C.E., 2003. Characterization of angiosperm nrDNA polymorphism, paralogy, and pseudogenes. Mol. Phylogenet. Evol. 29, 435e455. Burquez, A., 1989. Blue tits, Parus caeruleus, as pollinators of the crown imperial, Fritillaria imperialis in Britain. Oikos 55, 335e340. Cruz, C., Dias, T., Pinho, P., Branquinho, C., Maguas, C., Pinto, M.J., Martins-Loucao, M.A., 2010. Policies for plant diversity conservation on a global scale: a nitrogen driver analysis. Kew Bull. 65, 525e528. Doyle, J., Doyle, J.L., 1987. Genomic plant DNA preparation from fresh tissue-CTAB method. Photochem. Bull. 19 (11). Drabkova, L., Kirschner, J., Vlcek, C., Paces, V., 2004. TrnLetrnF intergenic spacer and trnL intron define major clades within Luzula and Juncus (Juncaceae): importance of structural mutations. J. Mol. Evol. 59, 1e10. Fay, M.F., Chase, M.W., Ronsted, N., Devey, D.S., Pillon, Y., Pires, J.C., Petersen, G., Seberg, O., Davis, J.I., 2006. Phylogenetics of Liliales: summarized evidence from combined analyses of five plastid and one mitochondrial loci. Aliso 22, 559e565. Huang, B.L., Chen, S.L., Mu, J., 2009. Evolution of the chloroplast trnL-trnF region in the gymnosperm lineages Taxaceae and Cephalotaxaceae. Biochem. Genet. 47, 351e369. Khaniki, G.B., 2002. Chromosome number of Fritillaria subgenera Petilium and Theresia (Liliaceae). Nucleus 45, 6e11. Li, S.L., Li, P., Lin, G., Chan, S.W., Ho, Y.P., 2000. Simultaneous determination of seven major isosteroidal alkaloids in bulbs of Fritillaria by gas chromatography. J. Chromatogr. A 873, 221e228. Mozaffarian, V., 1992. New species and interesting plant records from Iran. Iran. J. Bot. 5, 83e90. Rix, E., 1977. Fritillaria L. (Liliaceae) in Iran. Iran. J. Bot. 1, 75e95. Rix, M., Frank, E., Webster, G., 2001. Fritillaria: a Revised Classification: Together with an Updated List of Species. The Fritillaria Group of the Alpine Garden Society, Berkshire, United Kingdom. Ronsted, N., Law, S., Thornton, H., Fay, M.F., Chase, M.W., 2005. Molecular phylogenetic evidence for the monophyly of Fritillaria and Lilium (Liliaceae; Liliales) and the infrageneric classification of Fritillaria. Mol. Phylogenet. Evol. 35, 509e527. Teksen, M., Aytac, Z., Pinar, N.M., 2010. Pollen morphology of the genus Fritillaria L. (Liliaceae) in Turkey. Turkish J. Bot. 34, 397e416. Thompson, J.D., Gibson, T., Higgins, D.G., 2002. Multiple sequence alignment using ClustalW and ClustalX. Curr. Protoc. Bioinforma. 2e3. Tomovic, G., Vukojicic, S., Niketic, M., Zlatkovic, B., Stevanovic, V., 2007. Fritillaria (Liliaceae) in Serbia: distribution, habitats and some taxonomic notes. Phytol. Balc. 13, 359e370. Turktas, M., Aslay, M., Kaya, E., Ertugrul, F., 2012. Molecular characterization of phylogenetic relationships in Fritillaria species inferred from chloroplast trnL-trnF sequences. Turkish J. Biol. 36, 552e560.
Contents lists available at ScienceDirect
Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco
Phylogenetic relationship in Fritillaria spp. of Iran inferred from ribosomal ITS and chloroplast trnL-trnF sequence data Mahmoud Khourang a, Alireza Babaei a, *, Fatemeh Sefidkon b, Mohamad Reza Naghavi c, Davood Asgari c, Daniel Potter d a
Department of Horticulture Science, Tarbiat Modares University, Tehran, Iran Research Institute of Forests and Rangelands, Tehran, Iran c Department of Agronomy and Plant Breeding, Faculty of Agriculture, University of Tehran, Karaj, Iran d Department of Plants Science, University of California-Davis, Davis, USA b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 June 2014 Accepted 4 October 2014 Available online
The genus Fritillaria embraces up to 165 taxa in the family Liliaceae, most of which are of high medicinal and ornamental value and importance. In this study, 44 specimens of the genus representing 9 species were collected from their natural habitats located in 10 provinces of Iran. Phylogenetic analysis was performed based on DNA sequences of the internal transcribed spacer (ITS) of the nuclear ribosomal cistron and the trnL-trnF regions. The phylogeny was constructed using the neighbor joining inference method. Results indicate that the examined samples were evidently diverged into 2 distinct clades. Members of the subgenera Fritillaria and Rhinopetalum formed one clade while the other clade contained the subgenera Theresia and Petilium. There can be seen a high degree of similarity between the only yellow-colored crown imperial specimen and the red-colored specimens. The endemic species of Fritillaria straussii, Fritillaria zagrica and Fritillaria kotschyana which their status within the subgenera known in the genus Fritillaria has been remained undefined, fell into the subgenus Fritillaria. The clades also had relatively reasonable distribution patterns based on the genetic structure, geographical conditions and climate specifications. This study revealed the feasibility of the ITS and trnL-trnF DNA sequence for phylogeny of the genus Fritillaria. This is the first phylogenetic analysis of Fritillaria spp. in Iran. © 2014 Published by Elsevier Ltd.
Keywords: Fritillaria ITS trnL-trnF Medicinal plant Ornamental plant Phylogeny
1. Introduction Fritillaria L. is a genus of over 160 species of bulbous and perennial plants belonging to the filmily Liliaceae. Members of this genus are native to temperate regions of the Northern hemisphere (Tomovic et al., 2007). Above the species level, Iran is the main center of genetic diversity of Fritillaria (Rix, 1977) with up to 14 species which grow naturally throughout Iran, most of which are endemic to the country (Mozaffarian, 1992; Khaniki, 2002). Fritillaria species are becoming increasingly popular in floriculture (Turktas et al., 2012) and medicinal plants (Li et al., 2000) industries and many species are commercially available.
* Corresponding author. Department of Horticulture Science, Tarbiat Modares University, P. O. Box 14115-336, Tehran, Iran. E-mail address: [email protected] (A. Babaei). http://dx.doi.org/10.1016/j.bse.2014.10.001 0305-1978/© 2014 Published by Elsevier Ltd.
452
M. Khourang et al. / Biochemical Systematics and Ecology 57 (2014) 451e457
Although there are a considerable number of studies about the morphological and physiological characterization of Fritillaria, unfortunately we know little about phylogenetic affinities of these plants and their concealed breeding advantages. After the first classification of the genus Fritillaria by Linnaeus, several further classifications have been described. According to the last revision by Rix et al. (2001), the genus is subdivided into 8 subgenera including Davidii, Liliorhiza, Japonica, Fritillaria, Rhinopetalum, Petilium, Theresia and Korolkowia. Results obtained by Ronsted et al. (2005) support this classification. Reproducibility and simplicity are the main qualities making DNA sequencing a good choice for identification of species and phylogenetic reconstruction studies (Turktas et al., 2012). For this reason, rRNA sequences are widely used in evaluating evolutionary affinities among organisms, since they are of ancient origin and are found in all known forms of life. Internal transcribed spacer (ITS) region of the 18Se5.8Se26S nuclear ribosomal cistron has revealed a high degree of variation even between closely related species (Alvarez and Wendel, 2003). In fact, the ITS region is one of the most widely applied molecular markers in current angiosperm systematics (Bailey et al., 2003). Moreover, chloroplast markers are widely used for phylogenetic assessments (Fay et al., 2006), as they evolve rapidly (Drabkova et al., 2004) and are mutational hot spots. The tRNALeu (trnL) intron and the intergenic spacer (IGS) between trnL 30 exon and tRNA-Phe (trnF) gene regions are quite suitable for the phylogenetic analysis of intraspecific variations (Huang et al., 2009). Using these regions, our aim is to determine molecular diversity and phylogenetic affinities amongst several Fritillaria species of Iran. This can be a valuable source of generation new Fritillaria varieties for ornamental and medicinal purposes. In addition, our results would be valuable to improve efforts which cover Iran germplasm in order to sustain this national wealth. 2. Material and methods 2.1. Plant material Leaf samples of 44 specimens representing 9 species of the genus Fritiallaria including Fritillaria imperialis L., Fritillaria persica L., Fritillaria crassifolia Boiss., Fritillaria straussii Bommuller., Fritillaria zagrica Stapf., Fritillaria kotschyana Herb., Fritillaria gibbosa Boiss., Fritillaria reuteri Bornm. and Fritillaria raddeana Rgl. were collected through 10 provinces of Iran. The species were represented by 16, 8, 7, 1, 2, 2, 2, 1, and 5 specimens, respectively (Table 1). Young leaves were collected, labeled and immediately kept in liquid nitrogen. Samples were stored at 80 C till DNA extraction. 2.2. Sequence generation Total DNA was extracted following a standard CTAB protocol (Doyle and Doyle, 1987) or modifications thereof. PCR amplifications were performed with an initial 5 min at 95 C, followed by 35 cycles of 1 min at 95 C, 1.30 min at 56 C, 2 min at 72 C and a final 7 min at 72 C. Reactions were carried out in a total volume of 50 mL, containing 5 mL MgCl2, 2 mL dNTP, 5 mL 10X PCR buffer (Applied Biosystems, USA), 1 mL of forward and reverse primers, 0.5 mL of Taq DNA Polymerase, 17 mL Betain, 18 mL water and 1 mL of DNA template. Regions were amplified using 2 primer sets of ITS1-ITS2 and trnL-trnF. In the case of trnL-trnF region, a primer combination of c þ d and e þ f was used for amplification of the intron of the chloroplast trnL (UAA) gene and partial trnL gene and the trnL-trnF IGS regions. PCR products were subjected to gel electrophoresis and were cleaned up using a PCR clean-up kit (Promega, USA). Purified PCR products were directly sequenced in 2 directions, using the corresponding primers. The sequencing process was performed using ABI/Prism 377 automated sequencers. The sequences identity was confirmed in comparison with various published sequences available in GenBank. 2.3. Phylogenetic reconstruction Sequences were edited using the Chromas Lite 2.1.1 (Technelysium Pty Ltd) and refined by eye. The newly generated sequences were submitted to GenBank (Table 1). Multiple sequence alignment was carried out using the ClustalW program (Thompson et al., 2002). Sequences of the species F. imperialis, F. persica, F. crassifolia and F. gibbosa for the ITS1-ITS2 region and F. imperialis and F. persica for the trnL-trnF region were the only sequences available in GenBank (September, 2014). Sequences were downloaded from the Genbank and were used to determine the boundaries of the regions (Table 2). Fritillaria and Lilium are sister taxa (Ronsted et al., 2005), thus Lilium davidii was chosen as an out-group in our analyses (Table 2). Phylogenetic analyses of the data matrix were performed based on the neighbor joining (NJ) method. Bootstrap values were calculated from 1000 replicates, and bootstrap coefficiencies were indicated above each branch. 3. Results 3.1. Sequence alignment and divergence The sequencing process was performed from both forward and reverse directions, and results successfully verified each other. PCR amplification was successfully obtained for all the samples. For the ITS region, F. imperialis had the shorter amplicon size than the rest, while F. persica showed the shortest amplicon length for the trnL-trnF region. The ITS region produced PCR products of about 680e780 bp. In the other hand, a higher length divergence was recorded for the trnL region (618e720 bp) compared with the IGS region (91e94 bp). Moreover, IGS region contained a higher G þ C content (44.6%) compared with the
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Table 1 Geographical distribution of Fritillaria spp. across different provinces of Iran. No.
1 2 3 4 5 6 7 8 9 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 a
Species
F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F.
gibbosa raddeana raddeana imperialis raddeana raddeana raddeana persica zagrica imperialis imperialis persica imperialis imperialis gibbosa imperialis reuteri imperialis lutea imperialis persica persica persica persica persica zagrica imperialis imperialis straussii crassifolia imperialis imperialis crassifolia crassifolia kotschyana kotschyana crassifolia crassifolia crassifolia crassifolia imperialis imperialis imperialis imperialis
Codea
1 1 2 1 3 4 5 1 1 2 3 2 4 5 2 6 e e 7 3 4 5 6 7 2 8 9 e 1 10 11 2 3 1 2 4 5 6 7 12 13 14 15
GenBank accession number ITS
trnL-trnF
KM435231 KM435149 KM435152 KM435147 KM435153 KM435154 KM435155 KM435148 KM435235 KM435156 KM435157 KM435171 KM435158 KM435159 KM435232 KM435160 KM435237 KM435170 KM435161 KM435172 KM435173 KM435174 KM435175 KM435176 KM435236 KM435162 KM435163 KM435238 KM435178 KM435164 KM435165 KM435179 KM435180 KM435233 KM435234 KM435181 KM435182 KM435183 KM435184 KM435166 KM435167 KM435168 KM435169
KM435227 KM435211 KM435212 KM435187 KM435213 KM435214 KM435215 KM435203 KM435223 KM435188 KM435189 KM435204 KM435190 KM435191 KM435228 KM435192 KM435230 KM435202 KM435193 KM435205 KM435206 KM435207 KM435208 KM435209 KM435224 KM435194 KM435195 KM435229 KM435216 KM435196 KM435197 KM435217 KM435218 KM435225 KM435226 KM435219 KM435220 KM435221 KM435222 KM435198 KM435199 KM435200 KM435201
Longitude
Latitude
55 55 55 55 55 55 55 49 50 50 50 50 50 50 51 51 50 50 50 50 48 50 48 48 47 46 46 46 46 46 46 47 47 47 47 47 47 47 47 49 47 47 47
27 37 37 37 37 37 37 33 33 33 33 32 32 32 32 31 31 32 32 32 35 32 35 35 27 35 35 35 35 34 34 37 37 37 37 37 37 37 37 33 33 33 33
470 5000 220 3500 070 4300 220 3500 220 4500 180 1200 180 1200 330 1600 230 0100 240 0400 200 400 150 5200 130 3600 160 1200 030 0800 10 1000 590 1700 220 1300 210 4000 210 4000 440 2000 150 5200 400 2000 410 4000 070 4900 110 5900 120 1300 110 1000 110 1000 070 1000 090 5000 420 0400 380 4000 380 4000 380 3200 300 5500 280 20 00 300 5000 310 3200 250 1400 310 3200 310 3200 310 3200
220 2600 200 200 210 1000 200 200 500 4600 590 900 210 3800 580 2600 90 2300 90 1000 110 1100 300 500 320 2000 480 5200 120 5800 460 5100 450 4600 080 2300 100 4000 100 4000 320 1900 120 5800 410 1200 390 1100 220 2600 180 1400 160 4000 150 5900 150 5900 190 4000 210 1000 430 0900 450 4000 430 0400 410 1100 410 5100 380 3600 410 4600 360 1100 060 4100 080 3700 080 3700 070 4100
Digits are used to determine the position of each specimen across Figs. 1 and 2. For species with only one specimen, no code is given.
trnL region (32.1%). The average G þ C content of the ITS sequence was slightly higher than the latter (32.8%). The percentage of identical sequences for trnL region and the IGS region was 94.21% and 93.51% respectively. The ITS region showed a higher percentage of identity with 95.94% identical sequences. Genetic distances for 9 species were calculated using the Tamura-Nei’s model. The average genetic distance amongst species ranged from 0.08 to 0.32. For both primers, the maximum distance observed was between F. imperialis e F. crassifolia, while the highest level of similarity found was between red-colored and yellow-colored crown imperials (0.08) as well as F. crassifolia and F. straussii (0.09) for ITS and trnL-trnF regions, respectively.
Table 2 Species used in this study as out-group and/or for determination of boundaries. ITS
trnL-trnF
Species
Accession number
Species
Accession number
Fritillaria gibbosa Fritillaria persica Fritillaria imperialis Fritillaria crassifolia Lilium davidii
AY616722 AY616736 AY616725 AY616717 AF088195
Fritillaria imperialis Fritillaria persica Lilium davidii
JQ327135 EU912327 EU597205
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3.2. Phylogenetic relationships amongst Fritillaria species Iranian species are classified in four different subgenera including Fritillaria, Theresia, Petilium and Rhinopetalum. F. imperialis and F. raddeana are two well-known species belonging to the subgenus Petilium. F. persica probably is monotypic and is the only species available in the subgenus Theresia. F. gibbosa and Fritillaria karelinii are two morphologically similar species in the subgenus Rhinopetalum. Subgenus Fritillaria is morphologically classified into a variety of species namely F. crassifolia, Fritillaria graeca, Fritillaria meleagris, Fritillaria cirrhosa and Fritillaria uncasica (Rix, 1977). 3.2.1. Subgenera Theresia and Petilium Topography and distribution of the species across the trees mainly reconfirmed and followed this classification (Figs. 1 and 2). Although two different primers used in this study showed difference in term of distribution of specimens across the trees (particularly in species belonging to the subgenus Fritillaria), they followed almost the same general pattern, so that results of both trees mostly verified each other. Our phylogeny analysis revealed that the 44 Fritillaria specimens clearly divided into 2 main clades. In our analysis, the subgenera Petilium and monotypic Theresia were grouped together and composed the first clade containing representatives of the species F. imperialis, F. raddeana and F. persica. In addition to molecular data, similar karyotype and pollen structures of the Petilium and Theresia supports the idea that these two subgenera are closely related (Teksen et al., 2010). In detail, specimens of F. imperialis and F. raddeana, as the members of the subgenus Petilium were placed in this clade. Besides, F. persica the member of the monotypic Theresia with 8 specimens positioned at the same clade, but in a different sub-cluster. Although the only yellow-colored crown imperial specimen (known as F. imperialis lutea) was placed in this clade next to the red-colored specimens, it showed a slight difference with the neighbors, as we expected. Alp et al. (2009) found natural crown imperial plants with yellow flowers in Anatolia district of Turkey and concluded that the yellow form is not a hybrid. The red-colored and the yellow-colored crown imperials are two different forms of the species (Alp et al., 2009), but evidences reveal that they are closely related (Figs. 1 and 2), and naturally co-exist in same habitats. Although several publications are available about the phylogenetic evaluation of the red-colored form of F. imperialis, there is no information about molecular phylogeny of the yellow-colored form of this species. 3.2.2. Subgenera Rhinopetalum and Fritillaria The second clade was divided into two groups containing the species of the subgenera Rhinopetalum (F. gibbosa) and Fritillaria (F. zagrica, F. reuteri, F. straussii, F. crassifolia and F. kotschyana). 3.2.3. Unclassified endemic species Ronsted et al. (2005) studied 37 species representing all subgenera and sections of the genus Fritillaria, through which the species F. imperialis (red-colored form) and F. raddeana from the subgenus Petillium, F. persica from the subgenus Theresia, F. gibbosa from the subgenus Rhinopetalum and F. reuteri and F. crassifolia from the subgenus Fritillaria were similar to those analyzed in our study. They didn't use any yellow-colored crown imperial (F. imperialis lutea) specimen. In comparison to their study, we used 9 species, 6 of which are endemic to Iran, amongst which there is no information about molecular phylogeny of F. zagrica, F. straussii and F. kotschyana in the literature so far. Therefore, status of these species through the 8 subgenera known in the genus Fritillaria has been remained undefined. As mentioned, results of our study clearly illustrate that the three species of F. zagrica, F. straussii and F. kotschyana fell into the subgenus Fritillaria, known as the largest subgenus of the genus. Morphological records also confirm the resemblance of these species.
4. Discussion This is the first study carried out on the molecular phylogeny of Fritillaria spp. in Iran. Although our findings reconfirm results of the previous phylogenetic studies of the genus Fritillaria, they suggest a new phylogenetic arrangement within the genus. Our data revealed the feasibility of the nucleotide ITS and the chloroplast trnL-trnF sequences for phylogeny of Fritillaria species. Assessment of level of genetic diversity within several Fritillaria species collected from a broad range of Iran climate conditions contributes vital information with regard to their evolutionary history and is critical to the development of effective conservation, utilization and management practices. This is of vital importance because several aspects of genetic assessments of plants such as genetic diversity, plants breeding practices and also restoration of threatened Fritillaria populations, can only be addressed by detailed population genetic studies (Cruz et al., 2010). High level of diversity is known amongst the key factors increasing agronomic and economic importance of any crop. Considering Fritillaria species are known as out-crossing species (Burquez, 1989), a high level of diversity amongst them was observed. Moreover, this level of diversity could be more explained by collecting the samples from a vast area of the country embracing a wide variety of climate conditions and countless natural habitats, potentially intensifying genetic diversity within the genus. Moreover, hybridization is one of the most popular methods for generating new varieties (Turktas et al., 2012). For instance, based on our findings, although the red-colored and yellow-colored crown imperials show slight genetic variation, they are closely related (Figs. 1 and 2). Accordingly, crossing these two forms can be used for releasing new cultivars with
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much more variety in valuable features such as flower color. Hence, information about phylogenetic relationships amongst these species would be potentially useful for future breeding programs. To sum up, the inventory and sampling efforts showed a broad range of geographical distribution of different subgenera of the genus Fritillaria throughout Iran climate conditions. This supports the theory suggesting Iran as a center of genetic diversity of the genus (Rix, 1977). More importantly, results showed that the majority of the species reported in Iran either might be already extinct or must be considered as critically endangered species. For example, we couldn't find Fritillaria ariana (Lozinsk. & Vved.) Rix as an endemic species previously reported in a limited area of the Northern Khorasan province
Fig. 1. Majority-rule consensus tree of the Fritillaria spp., obtained using the NJ method for ITS region. Bootstrap values are shown next to each branch. Sequences of specimens specified with (C) are downloaded from GenBank.
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Fig. 2. Majority-rule consensus tree of the Fritillaria spp., obtained using the NJ method for trnL-trnF region. Bootstrap values are shown next to each branch. Sequences of specimens specified with (C) are downloaded from GenBank.
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(Mozaffarian, 1992). Due to extreme desertification occurred across the area, the species is possibly extinct. Moreover, ratio and abundance of yellow-colored crown imperial’ populations were considerably less than those of red-colored populations, which put this form in the state of being critically endangered. This was consistent with the majority of the collected species namely F. zagrica, F. gibbosa, F. reuteri, F. straussii, F. crassifolia and F. kotschyana. Amongst all the species only F. imperialis (redcolored form), F. persica and F. raddeana showed a normal abundance and distribution across their natural habitats. Taking this into consideration, to preserve species of the genus, and before taking economic advantage of this national wealth, the urgent priority would be addressing effective conservation efforts which cover Iranian fritillaries. For this reason, bulbs of each species are cultivated and conserved in the Faculty of Agriculture of Tarbiat Modares University (TMU) to monitor their growth and development process in a protected area as well as their response to the new environmental conditions. Moreover, for further investigations, results of the study have been reported to the Research Institute of Forests and Rangelands of Iran (RIFRI). Acknowledgments We would like to thank Mr. Hadi Jafari for his assistance and Mr. Kai Battenberg for his contribution by giving many helpful insights and suggestions. References Alp, S., Arslan, N., Koyuncu, M., 2009. Established forms of Fritillaria imperialis L.: a naturally growing species in Turkey. Pak. J. Bot. 41, 1573e1576. Alvarez, I., Wendel, J.F., 2003. Ribosomal ITS sequences and plant phylogenetic inference. Mol. Phylogenet. Evol. 29, 417e434. Bailey, C.D., Carr, T.G., Harris, S.A., Hughes, C.E., 2003. Characterization of angiosperm nrDNA polymorphism, paralogy, and pseudogenes. Mol. Phylogenet. Evol. 29, 435e455. Burquez, A., 1989. Blue tits, Parus caeruleus, as pollinators of the crown imperial, Fritillaria imperialis in Britain. Oikos 55, 335e340. Cruz, C., Dias, T., Pinho, P., Branquinho, C., Maguas, C., Pinto, M.J., Martins-Loucao, M.A., 2010. Policies for plant diversity conservation on a global scale: a nitrogen driver analysis. Kew Bull. 65, 525e528. Doyle, J., Doyle, J.L., 1987. Genomic plant DNA preparation from fresh tissue-CTAB method. Photochem. Bull. 19 (11). Drabkova, L., Kirschner, J., Vlcek, C., Paces, V., 2004. TrnLetrnF intergenic spacer and trnL intron define major clades within Luzula and Juncus (Juncaceae): importance of structural mutations. J. Mol. Evol. 59, 1e10. Fay, M.F., Chase, M.W., Ronsted, N., Devey, D.S., Pillon, Y., Pires, J.C., Petersen, G., Seberg, O., Davis, J.I., 2006. Phylogenetics of Liliales: summarized evidence from combined analyses of five plastid and one mitochondrial loci. Aliso 22, 559e565. Huang, B.L., Chen, S.L., Mu, J., 2009. Evolution of the chloroplast trnL-trnF region in the gymnosperm lineages Taxaceae and Cephalotaxaceae. Biochem. Genet. 47, 351e369. Khaniki, G.B., 2002. Chromosome number of Fritillaria subgenera Petilium and Theresia (Liliaceae). Nucleus 45, 6e11. Li, S.L., Li, P., Lin, G., Chan, S.W., Ho, Y.P., 2000. Simultaneous determination of seven major isosteroidal alkaloids in bulbs of Fritillaria by gas chromatography. J. Chromatogr. A 873, 221e228. Mozaffarian, V., 1992. New species and interesting plant records from Iran. Iran. J. Bot. 5, 83e90. Rix, E., 1977. Fritillaria L. (Liliaceae) in Iran. Iran. J. Bot. 1, 75e95. Rix, M., Frank, E., Webster, G., 2001. Fritillaria: a Revised Classification: Together with an Updated List of Species. The Fritillaria Group of the Alpine Garden Society, Berkshire, United Kingdom. Ronsted, N., Law, S., Thornton, H., Fay, M.F., Chase, M.W., 2005. Molecular phylogenetic evidence for the monophyly of Fritillaria and Lilium (Liliaceae; Liliales) and the infrageneric classification of Fritillaria. Mol. Phylogenet. Evol. 35, 509e527. Teksen, M., Aytac, Z., Pinar, N.M., 2010. Pollen morphology of the genus Fritillaria L. (Liliaceae) in Turkey. Turkish J. Bot. 34, 397e416. Thompson, J.D., Gibson, T., Higgins, D.G., 2002. Multiple sequence alignment using ClustalW and ClustalX. Curr. Protoc. Bioinforma. 2e3. Tomovic, G., Vukojicic, S., Niketic, M., Zlatkovic, B., Stevanovic, V., 2007. Fritillaria (Liliaceae) in Serbia: distribution, habitats and some taxonomic notes. Phytol. Balc. 13, 359e370. Turktas, M., Aslay, M., Kaya, E., Ertugrul, F., 2012. Molecular characterization of phylogenetic relationships in Fritillaria species inferred from chloroplast trnL-trnF sequences. Turkish J. Biol. 36, 552e560.