Journal of Genetic Resources J Genet Resour 2015;1(2): 89-100 http://sc.journals.umz.ac.ir doi: 10.22080/jgr.2015.1168 University of Mazandaran Iranian Biology Society Assessment of relationships between Iranian Fritillaria (Liliaceae) Species Using Chloroplast trnh-psba Sequences and Morphological Characters Majid Sharifi-Tehrani* and Mahfouz Advay Department of Biology, University of Shahrekord, Shahrekord, Iran *Corresponding author: [email protected] Received: 05 September 2015 Accepted: 08 November 2015 Abstract The genus Fritillaria comprises of 165 taxa of medicinal, ornamental and horticultural importance. Evolutionary relationships in this genus is an interesting research area, attracting many researchers. In this study, phylogenetic relationships among 18 native to endemic species in Iran belonging to four subgenera Petilium, Theresia, Rhinopetalum and Fritillaria, are assessed using chloroplast trnH-psbA IGS sequences. Fifteen variable morphological characters are studied, and used in constructing a numerical classification. Results of molecular data showed that subgenus Fritillaria in Iran was a polyphyletic group. Members of the section Olostyleae appeared as paraphyletic. Species non-monophyly was revisited for Fritillaria crassifolia. Both morphological and molecular data show that Fritillaria zagrica and Fritillaria pinardii were closely related taxa, although they may be retain as separate species based on some morphological differences. Multivariate analysis of morphological data arranged the species in consistent groups as with the phylogenetic tree based on sequence data. Results of this study revealed feasibility of the trnH-psbA sequences for contribution in phylogenetic reconstruction in the genus Fritillaria. Key words: Fritillaria; Iran; Morphology; Phylogenetic; trnH-psbA Introduction subgen. Fritillaria in Iran (such as F. olivieri Backer, F. kotschyana Herb. ssp. kotschyana, F. Fritillaria L. (Liliaceae Juss. 1789) comprises of reuteri Boiss., F. atrolineata Bakhshi-Khaniki, F. more than 165 taxa (about 100 species) which are chlororhabdota Bakhshi-Khaniki, F. chlorantha distributed in Northern hemisphere. Most of the Hausskn. & Bornm., F. zagrica Stapf, and the species in this genus belong to the main subgenus, recently described F. avromanica Advay, Teksen & Fritillaria (Rix et al., 2001). The Mediterranean Maroofi) are diploid endemics with 2n = 24 region is the center of genetic diversity of Fritillaria (Bakhshi-Khaniki and Persson, 1997; Bakhshi species, with most of the taxa described from Khaniki, 1997a,b; Bakhshi-Khaniki, 2002b,a,2005; Turkey (Rix, 1984; Ozhatay, 2000). Taxonomy of Jafari et al., 2014; Advay et al., 2015). this genus is reviewed by several authors (Baker, Circumscription of some species in Iran was 1874; Boissier, 1882; Bentham and Hooker, 1883; uncertain, being debated or revised by various Turrill and Sealy, 1980), and the current authors. For example, F. zagrica proposed to be classification which is proposed by Rix et al. (2001) decreased as the synonym for F. pinardii Boiss. is supported at sub generic level by the phylogenetic (Celebi et al., 2008; Teksen et al., 2010) and F. studies (Ronsted et al., 2005; Day et al., 2014), crassifolia ssp. poluninii is raised to specific level, although, relationships among species remain, F. poluninii (Rix) Bakhshi-Khaniki and Persson. however, not resolved especially in the largest Recent molecular phylogenetic studies using subgenus, Fritillaria. nuclear and plastid sequences have provided The center of diversity of the genus Fritillaria may evidence for both polyphyly and species non also be found in Iran (Rix, 1997), where groups monophyly in the main subgenus, Fritillaria. from central Asia, the Mediterranean, and the ITS sequences are useful in phylogenetic studies Caucasus meet. Most of the taxa in the main which has vastly been used in many studies, and in 89 Sharifi-Tehrani and Advay, J Genet Resour, 2015;1(2):89-100 combination with cpDNA and/or mtDNA Herbarium of Faculty of Science at the University sequences. In an outstanding study by Zarrei et al. of Shahrekord. (2009), 393 new sequences of Gagea and Lloydia species were analyzed. Results of the four types of DNA extraction, PCR amplification, and analyses confirmed close relationships of Gagea sequencing reaction and Lloydia. Six Lloydia spp. and all Gagea accessions formed highly supported clades (BP Genomic DNA was extracted from the dry frozen 100%). Incongruence between results of uni- leaves of 22 Fritillaria samples following the parentally inherited plastid sequences and bi- CTAB DNA isolation protocol (Doyle and Doyle, parentally inherited ITS sequences was evident for 1987). The trnHGUG-psbA region (Shaw and Small, inter-specific relationships, which was potentially 2005) was amplified at a final volume of 30 μl due to ancient hybridization and/or paralogy of ITS using 0.3 unit of Taq DNA Polymerase (Fermentase sequences (Zarrei et al., 2009). Inconsistent Life Sciences), 1X supplied Taq-buffer, 1.5 mM sequence datasets for Fritillaria (Day et al., 2014), MgCl2, 0.2 mM dNTPs, and 0.1 mM of each primer necessitated more studies using different sources of pair. After 1 min at 94 ºC, thirty-five cycles were data to be conducted. Aldrich et al. (1988) was first performed with 20 s at 94 ºC, 30 s at 57 ºC and 60 s who showed prevalence of indels in trnH-psbA IGS at 72 ºC, and a final extension step of 7 min at 72 sequences between closely related species. This ºC. PCR products were subjected to gel region was then showed to be of value to electrophoresis and were cleaned up using a PCR systematics (Sang et al., 1997) as the variability of clean-up kit (Promega, USA). Purified PCR these sequences were higher than that of matK or products were directly sequenced on an automated trnL-trnF. Several investigators then started using DNA sequencer (ABI/Prism 377, Applied this region to study closely related genera and Biosystems). Chromatograms were edited using species (Azuma et al., 1999; Fukuda et al., 2003; MEGA ver 6.0 software and nucleotide sequences Miller et al., 2003). This region is most useful at the saved with FASTA format (Tamura et al., 2011). specific level, but has also been used in an The newly generated sequences were submitted to intraspecific investigation (Holdregger and Abbott, the GenBank (Table 1). 2003). At higher levels, trnH-psbA has proven to be largely unalignable (Shaw et al., 2005). Phylogenetic analysis In the current study, chloroplast trnH-psbA IG of 18 species in Iran, from all four subgenera (Petilium, Lilium ledebourii sequences (accession number Theresia, Rhinopetalum, and Fritillaria) are EU939299.1) were retrieved from the GenBank and sequenced, and used as a new source of data for this chosen as an out-group in the phylogenetic analysis. genus, in constructing a phylogenetic tree. Maximum likelihood fits for 24 different nucleotide Quantitative morphological characters are also substitution models were assessed using MEGA 6.0 observed in several specimens of all the sequenced software package to achieve the best model for species, to construct an ordination, in order to phylogenetic analysis (Tamura et al., 2013). The compare the results driven from the two different phylogenetic analysis was performed in MEGA 6.0 data sources. using the Minimum Evolution (Rzhetsky and Nei, 1992), Maximum Likelihood (ML) and Neighbor Materials and Methods Joining (NJ) methods with 1000 bootstrap replications (Felsenstein, 1985). Plant material Morphology Samples of the genus Fritillaria were collected from different regions along Alborz and Zagros Three to five samples of each species were mountains of Iran (Table 1). Specimens were measured for 15 quantitative morphological identified (Townsend, 1985; Wendelbo, 1990; Rix, characters (Table 2). Measurements were entered in 1997; Ozhatay, 2000) and vouchers preserved in the a formatted matrix and used for multivariate 90 Sharifi-Tehrani and Advay, J Genet Resour, 2015;1(2):89-100 analysis in NTSYS-pc ver. 2.11 software (Rohlf, the matrix by the ME method which resulted in an 2000). Ordination analysis (PCO) was performed optimal tree with the sum of branch length= 0.083 using Euclidean distances. The first four principal (Fig. 1). Bootstrap values >70% with ×1000 axes were extracted from the double centered replicates, are presented on the tree. Analysis of distance matrix, and a three dimensional ordination data matrix using Neighbor Joining and Maximum diagram was generated using the first three axes. Likelihood methods resulted in similar topologies as ME, with just very minor differences in bootstrap Results supports (trees are not shown). The phylogenetic tree (Fig. 1) consisted of three clades with high Phylogenetic relationships bootstrap supports (clade A: 97%, clade B: 100% and clade C: 86%). Most of the species of the Aligned matrix of trnH-psbA IGS sequences for 23 Fritillaria subgenus Fritillaria were fall in Clade A. taxa (Table 1), contained 471 positions. F. gibbosa This clade did not encompass all the members (Rix, Boiss. and F. ariana (Loz.-Lozinsk. & Vved.) Rix 1997) of the subgenus Fritillaria. Members of showed the shortest amplicon length (309 bp) for subgenus Rhinopetalum (F. gibbosa and F. ariana) the trnH-psbA region, while the longest amplicon were basally attached to the clade A. Clade B only (352 bp) was achieved for F. assyriaca Baker. The consisted of taxa in Caucasian group of sect. average length of sequences in 22 studied Fritillaria Olostyleae, and this clade similarly did not specimens (18 species, Table 1) was 321 bp. The encompass all the taxa in Caucasian group. Clade C GC content of the trnH-psbA IGS region ranged consisted of subgenera Petilium and Theresia. F. from 44.3% to 47.4% with an average of 46%. The straussii, a member of group Crassifolia in sect. number of parsimony informative sites in trnH- Fritillaria put at the base of clades B+C, separated psbA IGS region was 3.82% for the studied taxa, from other members of the group Crassifolia. markedly higher than that of trnL-trnF region (Turktas et al., 2012). Phylogenetic relationships between studied taxa was inferred using analysis of Table 1.