J Genet Resour2018;4(1): 14-25 Homepage: http://sc.journals.umz.ac.ir/ RESEARCH ARTICLE DOI: 10.22080/jgr.2018.13872.1098 A Taxonomic Reassessment of Consolida (Ranunculaceae) Species: Insight from Morphological and Molecular Data Maneezheh Pakravan1*, Arezoo Dastpak2, Ali Sonboli 3 and Zahra Khalaj1 Faculty of Biological science, Alzahra University, Tehran, Iran Department of Biology, Central Tehran Branch, Islamic Azad University, Tehran, Iran 3 Medicinal Plants & Drugs Research Institute, Shahid Beheshti University, Tehran, Iran 1 2 ARTICLEINFO Article history: Received 05 August 2017 Accepted 16 September 2017 Available online 01 March 2018 Keywords: Consolida Morphometry nrDNA ITS trnL-F Iran *Corresponding author:  M. Pakravan pakravan@alzahra.ac.ir ABSTRACT In order to compare the efficiency of morphological traits and molecular markers in distinguishing the Consolida species, molecular analysis using nrDNA ITS and cpDNA trnL-trnF with maximum parsimony and Bayesian methods were done in a total of 34 species and forma representing 28 species of Consolida, 6 species of Aconitella, plus two species of Delphinium and two species of Aconitum as out groups. Beside phenetic analysis for 20 quantitative morphological traits in 17 species of Consolida in Iran are performed. The molecular analysis, based on successive reweighting by rescaled consistency index, revealed that Maximum parsimony method and Bayesian analysis gave very similar results based on individual and combine data sets. In the combined analysis (chloroplast and nuclear DNA) recovered most parsimonious trees (L= 558 steps, CI=0.695, RI=0.827). The ITS results revealed that Consolida is not monophyletic and the genus Aconitella is clearly nested within Consolida. Our results confirm the decrease of C. paradoxa Bunge to a forma of C. rugulosa also confirmed the decrease of C. kabulica as a variety of C. stokciana. One way ANOVA, principal component analysis (PCA) and cluster analysis were used in phenetic analysis to visualize the species among different traits. Most of the quantitative morphological traits which showed significant differences between populations were deleted. PCA and cluster analysis carried out for morphological traits divided the Consolida species in to two cluster and A. barbata has separated from other species. Aconitella species are located in separate cluster and location of other species are almost similar to molecular results. Print & Online ISSN: p-ISSN 2423-4257 e-ISSN 2588-2589  2015 UMZ. All rights reserved. Please cite this paper as: Pakravan M, Dastpak A, Sonboli A, Khalaj Z. 2018. A Taxonomic Reassessment of Consolida (Ranunculaceae) Species: Insight from Morphological and Molecular Data. J Genet Resour 4(1): 14-25. DOI: 10.22080/jgr.2018.13872.1098 Introduction Kemularia-Nathades (1939) recognized a new genus Aconitopsis from species of Consolida based on peculiar formation of the petal, upper sepal, and spur. The name Aconitopsis was later rejected by Sojak (1969) and being replaced by Aconitella because of nomenclature priority. Some researchers have studied these genera taxonomically (Soo, 1922; Munz, 1967 a.b.; Davis 1965; Iranshahr et al., 1992; Constantinidis et al., 2001). Consolida has about 40 species, of which 20 have been recorded from Iran. Aconitella with ca. 10 species (4 species in Iran) and 31 species of Delphinium (species in Iran) are centred in Irano-Turanian and Mediterranean The genus Consolida S.F. Gray was considered as a separate genus based on one species (C. regalis) by Gray (1821), who worked on British flora. But some researchers considered Consolida as a section of Delphinium (De Candole, 1824; Boissier, 1867; Huth, 1895; Nevskii, 1937). Unlike the others based on annual life form, single spured petal, single follicle compared to 3 or 5 sessile follicles of Delphinium recognized Consolida as a separate genus (Tutin et al., 1964; Davis, 1965; Munz, 1967, a.b., Hayek, 1970; Iranshahr, 1992; Styrid and Tan, 2002; Ertugrul et al., 2016; Khalaj, 2013). This work is licensed under the Creative Commons Attribution 4.0 International License. 14 Pakravan et al., J Genet Resour, 2018;4(1): 14-25 phytogeographic regions (Trifonova, 1990; Hasanzadeh et al. 2017). Some biosystematic studies have carried out in various field such as chromosomal studies (Trifonova, 1990; Koeva, 1992; Hong, 1986; Tavassoli et al., 2012) chemical studies (Aitzetmuller et al., 1999), palynological studies (Khalaj et al., 2016) and phylogenetic investigations by using DNA sequence data (Johansson 1995; RO et al., 1997; Jabbour & Renner 2011; 2012; Yosefzadeh et al., 2012). In the recent molecular studies (Jabbour and Renner 2001; 2012) it was showed that Consolida and Aconitella form a clade embeded in Delphinium and also Aconitella is embedded within Consolida. The Jabbour and Renner (2011) results showed that Consolida diverged from Delphinium relatives at least in the early of middle Miocene. Although the phylogenetic relationships of the tribe Delphinieae have described (Jabbour & Renner, 2011) but we used of plastid and nuclear DNA sequences data from herbarium materials to show the relationship of Consolida and Aconitella with using species of Iran and GenBank data Also morphological traits used to compare the efficiency of morphological traits and molecular markers in distinguishing the Consolida species. Materials and methods Plant materials. Forty taxon (28 species of Consolida and 6 species of Aconitella) were included for molecular analyses. Four species (two species of Delphinium and two species of Aconitum) were used as out-groups. Sequence of nrDNA ITS and trnL-F were retrieved from GenBank (Table 1). For phenetic analysis, 17 species were used (Table 2). Table 1. GenBank accession number and source for sample used in the study Species trnL-F nrDNA ITS Aconitella anthoroidea JF331875 JF331680 Aconitella hohenackeri JF331877 JF331682 Aconitella saccata JF331683 Aconitella scleroclada JF331684 Aconitella thirkeana JF331879 JF331686 Aconitella barbata JF331876 JF331681 Consolida ajacis JF331880 JF331687 Consolida ambigua LC413716 Consolida aucheri JF331884 JF331691 Consolida axilliflora JF331885. JF331692 Consolida brevicornis JF331693 Consolida camptocarpa JF331886 JF331694 Consolida camptocarpa LC413717 LC413710 Consolida flava JF331887 JF331695 Consolida glandulosa JF331888 JF331696 Consolida hellespontica JF331889 JF331697 Consolida hispanica JF331890 JF331698 Consolida incana JF331699 Consolida kabuliana JF331891 JF331700 Consolida leptocarpa JF331702 Consolida leptocarpa LC413718 LC413711 Consolida mauritanica JF331894 JF331704 Consolida oliveriana JF331705 Consolida oliveriana LC413712 Consolida orientalis JF331896 JF331707 Consolida persica JF331897 JF331708 Consolida pubescens JF331898 JF331709 Consolida raveyi JF331711 Consolida regalis JF331900 JF331712 Consolida rugulosa JF331718 Consolida rugulosa LC413719 LC413713 Consolida songorica JF331902 JF331719 Consolida stocksiana JF331903 JF331720 Consolida olopetala JF331895 JF331706 Consolida kandaharica JF331892 JF331701 Consolida ambigua AF258682 Consolida trigonelloides LC413720 LC413714 Consolida tehranica LC413721 LC413715 Delphinium requienii JF332021 JF331742 Delphinium staphisagria JF332023 JF331743 Aconitum delphinifoliom AF258681 JF331725 JF331915 JF331729 Aconitum pentheri Hyphens (-) indicate that ITS or trnL-F regions for those taxa were not determined. 15 Source Iran Turkey Germany Germany Turkey Afghanistan Germany Iran Afghanistan Turkey Germany Kazakhstan Iran Iraq Turkey Turkey Germany Germany Afghanistan Afghanistan Iran Morocco Turkey Iran Iran Iran Spain Germany Germany Afghanistan Iran Kazakhstan Afghanistan Turkey Afghanistan Egypt Iran Iran Italy Egypt Kenai Serbia Pakravan et al., J Genet Resour, 2018;4(1): 14-25 Table 2. List of species studied for phenetic, localities and voucher specimens. Species Collector Voucher Locality C. camptocarpa (Fisch. &C.A.Mey.) Nevski Poorhabibian ALUH 1599 Khorassan: Jajarm road Semnan: 58 km of Shahrud to Sabzevar C. camptocarpa (Fisch. &C.A.Mey.) Nevski Poorhabibian ALUH 35379 C. leptocarpa Nevski Poorhabibian ALUH 1603 Khorassan: Sarakhs, 12 km to Mozduran C. leptocarpa Nevski Poorhabibian ALUH 1590 Golestan: Golestan national park, Mirzabailoo C. leptocarpa Nevski Poorhabibian ALUH 1605 Khorassan: Sarakhs road C. persica (Boiss.) Grossh. Poorhabibian ALUH 1600 Khorassan: Sarakhs, 14 km to Mozduran C. persica (Boiss.) Grossh. Poorhabibian ALUH 1555 Hamedan: Khan Abad C. persica (Boiss.) Grossh. Poorhabibian ALUH 1556 Tehran: Firuzkuh C. rugulosa Schrödinger Poorhabibian ALUH 1606 Azarbayejan: Tabgriz, Ahar road C. rugulosa (Boiss.) Schrödinger Poorhabibian ALUH 1597 Golestan: Golestan national park, Mirzabailoo C. rugulosa (Boiss.) Schrödinger Poorhabibian ALUH 1557 Khorassan: Mashhad C. paradoxa Nevski Poorhabibian ALUH 1558 Hamedan: Khan Abad C. paradoxa Nevski Poorhabibian ALUH 1598 Khorassan: Neyshabur, Sharif Abad village A. anthoroidea (Boiss.) Schrödinger Poorhabibian ALUH 18570 Khorassan: Ferdowsi University Campus A. anthoroidea (Boiss.) Schrödinger Poorhabibian ALUH 1586 Hamedan: Almaghlagh village A. anthoroidea (Boiss.) Schrödinger Pakravan ALUH 1595 Hamedan: Nahavand road, Garo Mt. A. tehranica (Boiss.) Rech.f. Mahdavii ALUH 2783 Markazi: Kuhe Chepeghli A. tehranica (Boiss.) Rech.f. Assadi & Maassoumi TARI 1701 Tehran: Between Karaj and Eshtehard C. stocksiana Nevski Zarre & Amini HNBG 5077 Mazandaran: Pol Sefid A. hohenackeri (Boiss.) Grossh. Poorhabibian ALUH 1598a Khorassan: Neyshabur A. hohenackeri (Boiss.) Grossh. Poorhabibian ALUH 1587 Hamedan: Kuhe Garo C. aucheri (Boiss.) Iranshahr Mozaffarian TARI 71498 Fars: Bamo national park C. ambigua (L.) Ball & Heywood Poorhabibian ALUH 1600a Khorassan: Sarakhs, 14 km to Mozduran C. ambigua (L.) Ball & Heywood Seraj TARI 24663 Kermanshah: Ghasreshirin C. orientalis (Gray) Schrödinger Poorhabibian ALUH 1580 Tehran: Rudehen C. orientalis (Gray) Schrödinger Poorhabibian ALUH 27543 Mazandaran: Sari C. regalis S.F. Gray Assadi & Mozaffarian TARI- 30036 Azarbaijan: 20 km from Jolfa to Marand C. regalis S.F. Gray Assadi & Musavi TARI-20531 Azarbaijan: Arasbaran C. regalis S.F. Gray Zarre ALUH-1606 Azarbaijan: Tabriz C. oliveriana (DC.)Schrod. Assadi & Wendelbo TARI-16616 Lorestan: 110 km Khorram abad C. oliveriana (DC.)Schrod. Assadi TARI-24900 Kermanshah: 31 km to Ghasre-shirin C. oliveriana (DC.)Schrod. Riazi TARI-9422 Khuzestan: Do-gonbadan C. flava (DC.)Schrod. Mozaffarian TARI-53570 Khuzestan: 20 km from Ramhormoz C. flava (DC.)Schrod. Mozaffarian TARI-63218 Khuzestan: W of Bostan C. trigonelloides (Boiss.) Munz Forughi & Assadi TARI-17896 Kerman: Laleh zar Mt. C. trigonelloides (Boiss.) Munz Mozaffarian TARI-71262 Esfahan: Semirom to Keikha C. trigonelloides (Boiss.) Munz Yusefi TARI-1376 Esfahan: Ghamishloo protected area C. oligantha (Boiss.) Schrod. Pabo TARI-29377 Kermanshah: Hersin Abbreviations used in accession information: ALUH = Alzahra University Herbarium, Tehran, Iran; TARI= Herbarium of the Research Institute of Forests and Rangelands, Tehran, Iran. Previously collected herbarium specimens, as well as field-collected material dried and stored in silica gel, were used for DNA extraction. DNA isolation and sequencing relied on commercial kits (Plant BioFlux, Bioer Co. China). The complete nrDNA ITS region was amplified using primers ITS4 and ITS5 of White et al. (1990) and for amplifying and sequencing the trnL intron and adjacent trnL-trnF intergenic spacer we used of two primers trnL-F (Jabbour & Renner, 2011). Morphological traits The 25 quantitative and qualitative trait were access to characterized and estimate genetic distance. But 20 quantitative morphological traits were used because other traits had polymorphism and overlapping in different species (Table 3). DNA extraction, PCR amplification, and sequencing 16 Pakravan et al., J Genet Resour, 2018;4(1): 14-25 Amplification was done in a DNA thermal cycler (Primus 96, MWG, Germany). All samples were sequenced using the Big Dye Terminator Cycle Sequencing Ready Reaction Kit with the same PCR primers in an ABI Prism 377 DNA Sequencer. The sequences were edited using Bioedit Sequence Alignment Editor Version 7.0.9.0 (Hall, 1999) Table 3. Characters used in phenetic analysis Character Character states Presence of petiole in caulin leaves 0: present 1: absent Presence of hair on the leaf surface 0: present 1: absent Overtopping the bract from flower 0: yes 1: no Overtopping the bract from fruit 0: yes 1: no Position of bract 0: near the flower … 1: far from the flower spure 0: present Shape of spure 0: curved Position of hair on lateral sepal 0: scattered number of petal lobes Proportion of petal middle lobes to lateral lobes 0: 5 1: 3 0: equal 1: shorter 2: longer Presence of hair on the filament 0: absent 1: present Position of hair on filament 0: wing 1: total of filament Colour of anther 0: brown 2: yellow Shape of follicle beak 0: erect 1: curved . 1: absent 1: erect . 1: on the middle vein Shape of follicle 0: falciform 1: erect Presence of hair on the follicle surface 0: absent 1: present Shape of fruit stalk 0: antrorse 1: erect 2: decurved Proportion of pedicle to flower 0: shorter 1: longer Proportion of pedicle to fruit 0: shorter 1: longer Length of basal leaves 0:฀ 50 mm Number of bracts 0: 0 Broad of petal 0:2-8 mm 1: 9-18 mm Number of bracteole 0: variable 1: constant Length of bracteole 0: ≤ 7mm 1: ≥10 mm Length of spure 0: ≤ 20 mm 1: ≥ 22 mm 1: ฀ 50mm 1: 1 2: 2 performed on the data sets with GTR+G model. The analysis involved two simultaneous runs of 10 million generations of Monte Carlo Markov chains by saving every 100th tree. Mr. Bayes performed two simultaneous analyses starting from different random trees sampled at every 100 generations. The first 25% of trees were discarded as burn-in. The remaining trees were then used to build a 50% majority rule consensus tree accompanied with posterior probabilities values. Tree visualization was carried out using Tree View X ver.0.5.0 (Page, 2005). Phylogenetic analyses The phylogenetic analyses employed for the data sets included maximum parsimony (MP) and Bayesian inference (BI). Maximum parsimony analyses (MP) were run in PAUP*ver. 4.0b10 (Swofford, 2002). The heuristic search option was selected using 1000 replications of random addition sequence and TBR branch-swapping with MULTREES on and steepest descent off. Confidence limits for trees were assessed by performing 1000 replicates of bootstrapping (Felsenstein, 1985). The consensus trees from two independent runs were compared with one another and with the consensus tree from the parsimony analysis. Bayesian inference was performed using MrBayes ver. 3.1.1 (Nylander, 2004) based on Akakia information criterion (Posada & Buckley, 2004). Bayesian analysis was Genetic similarity, cluster and data analysis Morphological descriptors were analysed using principal component analysis (PCA). The number of principal components to retain in the analysis was determined using the minimum eigenvector criterion proposed by 17 Pakravan et al., J Genet Resour, 2018;4(1): 14-25 (Fig. 2). Two Delphinium and Aconitum species occur as outgroups in a separate clade (PP= 1, BP=100%). C. olopetala and C. Trigonelloides, as sister taxa (A), were the first diverging species. Clade B included all species of Consolida and Aconitella. This large clade comprises of two main clades (C and D). Clade C included two subclades of two species each. One subclade contains C. hellespontica and C. glandlosa (PP= 1, Bp= 100%) and the other one comprised C. mauritanica and C. pubescens (pp= 1, Bp= 88). In clade D, the first diverging species was A. barbata, followed by two subclades with good support (E and F) that consisted of all other Cosolida species that consist of several subclades and species with resolved positions. Kaiser (1960). Genetic similarity/distances carried out on the matrix of Euclidean distances were assessed using cluster analysis (Ward) method. The statistical treatment of morphological traits was performed using SPSS software (ver. 20). Results Sequence analyses The nrDNA ITS alignment matrix comparies 34 sequences and 643 characters. Including 207 potentially parsimony-informative sites and 97 parsimony-uninformative ones. For trnL-F region, the matrix of 40 sequences contains 1175 characters, of which 123 are potentially parsimony-informative sites and 101 are parsimony-uninformative. More information about data sets and tree statistics is summarized in Table 4. Combined phylogenetic analyses The topology observed in BI analysis of the combine data sets was similar to MP trees. In BI tree, some species of Consolida separated with high support but Aconitella species occupied unresolved position and A. barbata nested in other subclade (Fig. 3). Phylogenetic analyses Phylogenetic analyses of individual data sets Bayesian analyses of two single data sets were topologically identical to those of parsimony analyses (tree not shown). The trnL-F tree of 31 species included a polytomy which species of Aconitella were united among of Consolida species (Fig. 1). Just one subclade contains three species of Aconitella (A. hohenackeri, A. scleroclada, and A. anthoroideae; Bp= 67%). We show only Bayesian trees along with posterior probability (PP) and bootstrap based on ITS, trnL-F data set (Fig. 1 & 2). In Bayesian nrDNA ITS tree Aconitella species are completely nested in the Consolida species Genetic similarity morphological data. assessed by Genetic similarity evaluated by using of quantitative morphological traits using cluster analysis (Ward) method (Fig. 5) show the presence of similarity and distances between Consolida species. Comparisons of data and cluster analysis generate a dendrogram where 17 species were grouped into two main clusters (Fig. 5). Table 4. Statistics of trnL-F, ITS, and combined nuclear and chloroplast region analyses of Consolida species Data set Alignment length Number of uninformative characters Number of parsimony informative characters Consistency Index Retention Index trnL-F nrDNA ITS Combined data (trnL-F, ITS) 1175 101 123 0.917 0.950 643 97 207 0.638 0.790 1818 119 281 0.695 0.827 18 Pakravan et al., J Genet Resour, 2018;4(1): 14-25 Fig. 1. Bayesian inference tree of trn L-F data set in Consolida species: Numbers above the branches or arrows indicate Bayesian posterior probabilities (PP) and maximum parsimony bootstrap (MP). Values < 50% not shown. (C. tehranica =A. tehranica) Fig. 2. Bayesian inference tree of data set nrDNA ITS in Consolida species: Numbers above the branches or arrows indicate Bayesian posterior probabilities (PP) and maximum parsimony bootstrap (MP). Values < 50% not shown. (C. tehranica =A. tehranica) 19 Pakravan et al., J Genet Resour, 2018;4(1): 14-25 Fig. 3. Majority- rule (50%) consensus tree resulting from Bayesian analysis of the combined data set (trnL-F and nr DNA ITS) in Consolida species. Support values are indicated above the branches (Bayesian posterior probabilities (PP) and maximum parsimony bootstrap (MP), respectively). Values < 50% not shown. Fig. 4. PCA analysis of qualitative characters based on factor 1 and 2. 20 Pakravan et al., J Genet Resour, 2018;4(1): 14-25 Fig. 5. Phenogram based on morphological analysing data of 17 taxa species by Ward method. (ant=A. anthoroidea, ori=C. orientalis, per= C. persica, oliv=C. oliveriana, rug=C. rugulosa f.rugulosa, fla=C. flava,olig= C. olighantha, hoh= A. hohenackeri, cam=C. camptocarpa, lep=C. leptocarpa, sto=C. stocksiana, the=A. tehranica f.tehranica, amb=C. ambigua, tri=C. trigonelloides, reg=C. regalis subsp. Divericata, par= C. paradoxa, auc= C.aucheri) In the dendrogram, 14 species cluster I were grouped into three main subclusters consisting of 7, 2 and 5 species, respectively. Cluster II consists of 3 species (Fig. 5). In this dendrogram, C. paradoxa has separated from the other species. Study results show presence of similarity between C. leptocarpa, C. persica, C. stocksiana, C. camptocarpa and C. rugulosa. There were also two other species (C. orientalis, C. oliveriana) that show similarity with C. regalis, C. oliganta, C. ambigua, C. flava, C. aucheri. The last cluster contains three species: A. anthoroidea, A. hohenackerii and A. tehranica (which could write Consolida anthoroidea, C. hohenackeri, C. tehranica) which show the higher estimated genetic distance with other species. PCA analysis of morphological data revealed that the first 3 components comprise about 65.8% of total variance. In the first component with about 35.85% of total variance, morphological characters including bract exerting from fruit, presence of spore, shape of spore apex, the number of petal, the number of petal lobes showed the highest positive correlation. In the second component with about 17.90% of total variance apex of follicle, beak showed the highest positive correlation. In the third component with about 12.04% of total variance, position of fruit stalk and bract shape showed the highest positive correlation. Therefore, there are the most variable morphological characters among the species studied. (Table 6). In the present study, the cluster results were similar to those of PCA analysis. (Figs. 4 &5). Discussion Jabbour and Renner (2011) were the last worker to consider Consolida as part of Delphinium based on DNA sequences data. In this research, the combined tree by Maximum likelihood method confirms the closed relationships between Delphinium and Consolida and Aconitum. Jabbour and Renner (2011) also showed that Aconitella is part of Consolida which some previous authors have agree to such relationship (Constantinidis et al. 2001). The tree by Bayesian method based on ITS and trnL-F data confirm that Aconitella is embedded in Consolida (BP=100%) while 21 Pakravan et al., J Genet Resour, 2018;4(1): 14-25 species of Sect. Brevipeduncularae (Constantinidis et al. 2001). In this clade C. flava placed near the other member of the sect. Brevipeduncularae (100 %). C. flava position in ward analysis is separate from other section members but only near to C. aucheri. Two accession of C. camptocarpa place somewhat far from each other because of morphological polymorphism in the follicle stripe (erect and curve). There are a few differences between C. camptocarpa and C. leptocarpa in morphological characters (Tavassoli et al. 2012) and there are many specimens with intermediate characters. Also, karyotype analysis of C. camptocarpa and C. leptocarpa showed many similarities between them (both have 1 pair of long mchromosomes with satellite, 1 pair of long mchromosomes, 1 pair of st-chromosomes and 5 pairs of t-chromosomes) (Tavassoli et al. 2011). They are differing in nrDNA in 9 nucleotids and in cp DNA in 5 nucleotids. Our studies confirm Tavassoli et al. (2011) results that consider C. camptocarpa and C. leptocarpa as a complex species. Position of these species in ward cluster are in separate cluster. The C. kabuliana is endemic to Afghanistan that has decreased to variety level of C. stokciana by Tamura (1960). They are much closed species morphologically and are different only in length of petal, spure, and anther. They are differing in nrDNA in 8 nucleotids and in cpDNA in 2 nucleotids. Also in Bayesian and combined trees, they are placed in one subclade. Our results confirmed the decrease of C. kabulica as a variety of C. stokciana. C. aucheri was made by Boissier as a variety of Delphinium (1841) and again as a variety of D. persica introduced by the same author (Boiss. 1877). Iranshahr et al. (1992) considered C. aucheri as a new combination. Our results showed they are placed in separate clades. They are differing in nrDNA in 18 nucleotids and in cpDNA in 4 nucleotids. Therefore, these results are in agreement to Iranshahr et al. (1992) and Boissier (1877) that considered C. aucheri as a valid species. C. regalis, C. axilliflora, C. ajacis, C. oliveriana, C. hespanica, C. orientalis situated in G clade. Except C. axilliflora the others belong to both sect. Consolida and sect. Macrocarpa. In this clade, C. ambiguae that distributed in Iran and Mediterranean region is some anatomical study on petiole has separated Consolida and Aconitella species (Trifonova, 1990). Some researcher such as Sojak (1969) and Trifonova (1990) suggested Consolida and Aconitella might be sister groups while this hypothesis rejected by Jabbour and Renner (2011) and also in this research. Species relationships within Consolida Our phylogenetic results, coupled with evidence from morphology, distribution, and chromosome, represent a useful first step towards addressing the issue of species circumscription and identity in Consolida. Aconitella tehranica, A. hohenackeri, A. thirkeana and A. anthoroidea form a clade. These species in phenetic analysis located in a distinct cluster and separated from other species. While A. barbata form a sister clade to species of Aconitella. This species is only representative of the genus in Middle Asia (Jabbour, 2011). The form of its upper unpaired sepal spur and of the petal is intermediate between the genera Consolida and Aconitella (Constantinidis et al., 2001). Anatomical study of the petiole structure showed that this species is identical to the representatives of the genus Aconitella and should definitely be regarded as within the limits of the genus (Trifinova, 1990). A. barbata traditionally placed in sect. parviflorae but Constanidine et al. (2001) transfered A. barbata to sect. Involutae based on seed morphology, this opinion already proposed by previous researchers (Kemularia-Nathadase, 1939; Sojak, 1960; Trifinova, 1990). Members of the Sect. Brevipedunculatae are placed in the K clade. The situation of C. rugulosa forma paradsoxa (Bunge) Iranshahr (with spureless calyx) alongside to C. rugulosa forma rugulosa in one subclae (100%) confirms the decrease of C. paradoxa Bunge to a forma of C. rugulosa as Iranshahr has believed (Iranshahr et al., 1992). But this species located as a separate branch from all of other studied species in phenetic analysis (Fig. 5). It is a good evidence that presence of spure isn't a good character for delimiting the species of Consolida. The C. flava together with C. barbata traditionally placed in Sect. parviflora. Constantinidis and Renner's (2001) research on the seed coat micromorphology showed that C. flava had hilum zone in acrateri form cavity, surrounded by fringe-like projections as in 22 Pakravan et al., J Genet Resour, 2018;4(1): 14-25 very close to C. orientaslis morphologically. Both have large fruit. C. mauritiana, C. pubescens, C. hellespontica and C. glandulosa situate in E clade. Except C. hellespontica the three other species belong to sect. Consolida. These species have some characteristic that separate them from other member of the genus. C. mauritiana and C. pubescens share three metacentric chromosome pairs in their complement, in opposite to other member of Consolida that have only two metacentric chromosomes pairs (Constantinidis et al., 2001). In C. hellespontica the central part of the hilum area may form a characteristic shape that is less apparent in other species (Constandnidin et al., 2001). C. trigonelloides in the combined tree occur in a separate clade and in the Bayesian tree, together with C. olopetala also occur in the separate clade. Based on the flower morphology it could place in the sect. Consolida but because of seed characteristic which is penta hedral (in other species globose, pyramidal and tetrahdral shape are seen) that do not find in other species, it places in a separate clade. The relationship between morphological traits and molecular markers results is 58%. Results of this study were congruent with results of Baranger et al. (2004); Simioniuc et al. (2002); Hoey et al. (1996); Tar’an et al. (2005), who suggested low to medium correlations among molecular and morphological data. Molecular data again illustrate the great potential of nrDNA ITS and trnL-F sequences for resolving relationship at a range of taxonomic levels, from closely related species to sectional level. However, more taxon sampling and another source of DNA sequence, like chloroplast coding (e.g., matK, or ndhF) regions, are definitely necessary to be analyzed in order to comparing and combination of produced gene phylogenies for the Consolida species. Aitzetmuller K, Tsevegsuren N, Werner F. 1999. Seed oil fatty acid patterns of AconitumDelphiniumHelleborous complex (Ranunculaceae). Plant Syst Evol 213: 37-47. Baranger A, Aubert G, Arnau G, Laine AL, Deniot G, Potier J, Weinachter C, LejeuneHenaut I, Lallemand J, Burstin J. 2004. Genetic diversity within Pisum sativum using protein- and PCR-based markers. Theor Appl Genet 108: 1309-1321. Boissier E. 1841. 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