Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 75(1): 155-164, 2022 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-956 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Masoomeh Hasanbarani, Fariba Sharifnia, Mostafa Assadi (2022) Molecular insights on some Iranian species of Delphinium L. and Aconitum L. (Ranunculaceae). Caryologia 75(1): 155-164. doi: 10.36253/caryologia-956 Received: May 28, 2020 Accepted: November 27, 2021 Published: July 6, 2022 Copyright: © 2022 Masoomeh Hasanba- rani, Fariba Sharifnia, Mostafa Assadi. This is an open access, peer-reviewed article published by Firenze University Press (http://www.fupress.com/caryo- logia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. Molecular insights on some Iranian species of Delphinium L. and Aconitum L. (Ranunculaceae) Masoomeh Hasanbarani1,*, Fariba Sharifnia2, Mostafa Assadi3 1 Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran 2 Department of Biology, North Tehran Branch, Islamic Azad University, Tehran, Iran 3 Department of Botany, Agricultural Research Education and Extension Organization (AREEO), Research Institute of Forest and Rangelands, Iran *Corresponding author. E-mail: mh_plantbiology@yahoo.com Abstract. To be precise, 29 taxa of Delphinium and 2 species of Aconitum belonging to Iran have been documented in Flora Iranica. In this research, with regard to both mentioned genera, a total of 25 species for the chloroplast trnl-f region and 11 species for the Internal Transcribed Spacer (ITS) were investigated. After genome extraction, PCR and the sequencing of samples, the sequences were edited, and phylogenetic trees were prepared using Bayesian methods. The phylogenetic study of this genera led us to the monophyletic aspect of them despite the segregation of Aconitum and Delphinium in their related classic taxonomy. It has been observed that there are some complicat- ed species in the genus Delphinium. The results of molecular analysis confirmed the separation of Delphinium elbursense, Delphinium speciosum, Delphinium crispulum and Delphinium dasycarpum (the complicated species of northern and northwestern Iran). Furthermore, based on the molecular results, it is suggested for D. elbursense var. gym- nobotrys to have a higher taxonomic level as a distinct species. Meanwhile, Delphinium tuberosum, Delphinium cyphoplectrum, Delphinium quercetorum, Delphinium pallidiflo- rum, and Delphinium laxiusculum (western and northwestern species of Iran), which are regarded as complex species, were placed in a distinct molecular tree. At the end, Delphinium dolichostachyum was reported as a new record for Iran, and the species has been compared to the related species Delphinium carduchorum. Keywords: PCR, Bayesian, monophyletic, ITS, new record. INTRODUCTION It has been reported that the family Ranunculaceae contains five sub- families, 43 genera and 2346 species at the present time (Christenhusz & Byng, 2016). The tribe Delphinieae (Aconitum L., Delphinium L., Consolida (DC.) Gray, Aconitella Spach ) comprises 650-700 species, which amounts to some 25% of all Ranunculaceae (Jabbur & Renner, 2012), and is distrib- uted in the temperate regions of the northern hemisphere (Tamura 1990; Stevens 2001). The key feature of this tribe is the nectar placed in inner 156 Masoomeh Hasanbarani, Fariba Sharifnia, Mostafa Assadi tepal (Jabbour & Renner 2012; Ilarslan et al. 1997; Espinosa et al. 2017). Based on Flora Iranica (Iran- shahr 1992), 29 species of Delphinium and 2 species of Aconitum are reported from Iran. Iranian species of Delphinium are divided into two subgenera (differenc- es between subgenera are in the form of seed and veg- etative period): Olighophyllon Dimitrova and Delphin- ium, which are perennial and annual species, respec- tively (Iranshahr 1992; Beltran et al., 2021; Cabusora et al., 2020; Fikirie et al., 2020). Iranian Aconitum species are also divided into two subgenera, which are Aconi- tum and Lycoctonum DC. (the difference between sub- genera is the shape of galea) (Iranshahr 1992). Mobay- en (1985) reported for the flora of Iran; D. dasycarpum Stev. ex DC., D. venulosum Boiss. and D. micranthum Boiss. & Hohen. Sharifnia et al. (2013) recorded D. kurdicum Boiss. & Hohen. for the first time for the flora of Iran. Recently, D. khorasanicum Sharifnia & Hasanbarani was reported as a new species from Kho- rasan province (Hasanbarani et al. 2017). In general, several studies have been carried out on the Delphin- ieae in the world; for instance, Seed morphology of 28 Delphinium L. species has been studied (Ilarslan et al. 1997; Mieso & Befa 2020; Mustafa 2020; Varamesh et al., 2014; Rajaei et al., 2020; Fataei et al., 2013). Ozpelic & Uztiirk (2000) worked on the morphology and ecol- og y of 12 populations of D. cyphoplectrum Boiss. in Turkey. Palynology study of 21 taxa from Delphinium has also been performed (Bursali & Dogan 2005). The molecular analysis of nuclear and chloroplast sequenc- es of Delphinieae were studied in the geographical range of Asia, the Mediteraneaen, North America and the mountains of east Africa; the monophyly of the genus Consolida DC, Aconitum L. and Delphinium L. was proved (Jabbour & Renner 2011, 2012). Wang et al. (2013) reported that based on molecular markers Gymnoaconitum (Stapf ) Wei Wang & Z.D Chen differs from the other species of Aconitum and other genera of the tribe Delphinieae. Xiang et al. (2017) conducted a broad phylogenetic analysis within Ranunculaceae using matk sequence and performed a series of analy- sis using four molecular markers focused on the tribe. Micromorphological characters of the genus Delphini- um L. (sensu lato) seeds and fruits were studied using microscopic techniques (Hadidchi et al. 2019). In Chi- na based on observations on living plants in the field, together with examination of herbarium specimens, demonstrated that Delphinium iliense (Ranunculace- ae) is highly variable in the indumentum of pedun- cles, pedicels, bracteoles, sepals and also in the shape of bracteoles and their position on pedicels (Li et al. 2019). During a taxonomic study on Delphinium species in 2013-2018 based on herbarium specimens (TARI) and also taking into account the descriptions and images of types, 31 species of Delphinium were detected (Table 1); among them, the subgen. Delphinium includes the annual species: D. venulosum Boiss. and D. peregrinum L., whereas the subgen. Oligophyllon comprises peren- nial species, which have either tuberiformis or non- tuberiformis roots (root form is one of the characters that is used in flora iranica Delphinium key). D. specio- sum M.B., D. lanigerum Boiss. & Hohen., D. elbursense Rech.f., D. crispulum Rupr. and D. dasycarpum Stev. ex DC. are characterized by a non-tuberiformis root. These species have a similar distribution, and they are mor- phologically very closely related. The other species in the Table 1. Delphinium species in Iran (following taxonomic studies of this genus in 2013-2017, endemic species are bold). Species root form D. aquilegifolium (Boiss.) Bornm. tuberiformis D. biternatum Huth. tuberiformis D. carduchorum Chowdhuri & Davis tuberiformis D. cyphoplectrum Boiss. tuberiformis D. crispulum Rupr. non-tuberiformis D. dasycarpum Stev. ex DC. non-tuberiformis D. dasystachyum Boiss. & Bal. tuberiformis D. dolichostachyum Chowdhuri & Davis tuberiformis D. elbursense var. elbursense Rech.f. non-tuberiformis D. elbursense var. gymnobotrys Rech.f non-tuberiformis D. ilgazense P.H. Davis tuberiformis D. jacobsii Iranshahr tuberiformis D. khorasanicum Sharifnia & HasanBarani tuberiformis D. kurdicum Boiss. & Hohen. tuberiformis Delphinium lanigerum Boiss. & Hohen tuberiformis D. laxiusculum (Boiss.) Rouy tuberiformis D. macropogon Prokhanov tuberiformis D. micranthum Boiss. & Hohen. tuberiformis D.ochrolecum Stev. ex DC. tuberiformis D. pallidiflorum Freyn tuberiformis D. peregrinum L. tuberiformis D. quercetorum Boiss. & Hausskn tuberiformis D. szowitsianum Boiss. tuberiformis D. speciosum M.B. tuberiformis D. semibarbatum Bienert ex Boiss. tuberiformis D. saniculifolium Boiss. tuberiformis D. schmalhausenii Alboff tuberiformis D. tuberosum Auch. ex Boiss. tuberiformis D. turkmenum Lipsky tuberiformis D. venulosum Boiss. tuberiformis D. zalil Aitch. & Hemsl. tuberiformis 157Molecular insights on some Iranian species of Delphinium L. and Aconitum L. (Ranunculaceae) genus Delphinium (D. cyphoplectrum Boiss., D. tuberos- um Auch. ex Boiss, D. laxiusculum (Boiss.) Rouy, D. pal- lidiflorum Freyn, and D. quercetorum Boiss. & Hausskn) have a tuberiformis root and non-yellow flower; they form a complex morphologically related species in this genus (Iranshahr 1992). Due to the large number of species distributed in Iran and the controversies in taxonomical ideas among researchers, a taxonomic review of these spe- cies is required. Moreover, we reported in our previous research that for the biosystematic study of Delphinium species in IRAN, there is a strict necessity to have the help of molecular analysis methods to more confidently classify this genus. MATERIALS AND METHODS Plant materials In this research, in order to conduct molecular study, the plant materials were taken from Central Her- barium of Iran (TARI), and the samples were collected from the field dried on silica gel (this species is avail- able in IAUNT herbarium). It must also be mentioned that 25 species for the chloroplast marker (two species of Aconitum) and 11 species for the ITS marker (one spe- cies of Aconitum; Aconitum iranshahrii endemic of Iran and the sequences available in Genbank) were investi- gated (table 2). DNA extraction and PCR amplification Total DNA was extracted using the MBST kit (Shay- an et al. 2007). The amplification of DNA fragments was carried out for ITS sequence and trnL-F region. The entire ribosomal ITS region was amplified using prim- ers pairs AB101 (forward, 5 -ACG AAT TCA TGG TCC GGT GAA GTG TTC G-3) and AB 102 (reverse, 5-TAG AAT TCC CCG GTT CGC TCG CCG TTA C-3) (Dou- zery et al. 1999), and the PCR reaction for nuclear mark- er was executed using a denaturation step of 5 min at 95C followed by 35 cycles of 30 S denaturation at 95C, 30 S of annealing at 56C, and 90 S extension at 72C, fol- lowed by a final extension step of 7 min at 72C. The trnL-F region was amplified using primers C (Forward, 5-TAC GAC GAT CTY TCT AAA CAA GC-3) and F (reverse, 5- GGA AAG ATT GCT CAA ATA CCA G-3) (Taberlet & Gielly 1991). The PCR reac- tion for chloroplast marker was carried out with a dena- turation step of 5 min at 95C, followed by 35 cycles of 30 S denaturation at 95C, 30 S annealing at 54.4C, and 1 min extension at 72C, followed by a final extension step of 7 min at 72C. The PCR products were migrated on 1% agarose gel and were visualized by ethidium bromide. Sequence alignment and phylogenetic analyses After sequencing, the sequences were edited using BioEdit software ver. 7.0.9.0 (Hall 1999) and then were aligned using the Mesquite software (Maddison & Mad- Table 2. Delphinium and Aconitum species included in the molecular study (species used in ITS marker are shown with stars). Species Locality Aconitum Iranshahrii* Mazandaran: Polsefid, forest above village Sangdeh, 1500-2500 m, Assadi 73445. Aconitum nasatum Eeast Azarbaijan: Arasbaran protected area, Doghrun mountain, 2500 m, Assadi & Sardabi 23945. Delphinium aquilegifolium (Boiss.) Bornm. Mazandaran: Lar valley, 2450-2550m, Wendelbo & Assadi, 13264-TARI. Tehran: W of Tehran, Suleghun valley, 1500-2000m, Assadi & Mozaffarian 32699-TARI. Tehran:10 Km from Karaj, On Chalus road, 1750m Babakhanlu & Amin 20004-TARI. D. cyphoplectrum Boiss.* Fars: Kazerun, Komaraj,980m, Forughi 7930-TARI. Khusestan: 74128-TARI. Khuzestan: 47 Km to Masjedsoleiman from Haftgel, Assadi & Abohamzeh 38933-TARI. D. crispulum Rupr* Ardabil: Ca 9 Km from Khalkhal on the road to Asalem, 2050m, Assadi & Shahsavari 66000-TARI. West Azerbaijan: Khoy, Hasan Deh- e-Kan, 2500m, Amini, 1716-TARI. East Azerbaijan: 35 Km. NE of Marand, KiamakiDagh Mt., Assadi & Olfat 68603, TARI. East Azerbaijan:23 km SE of Jolfa, Near the Geshlagh village, Miaran, Assadi & Shahsavari 65786, TARI. D. carduchorum Chowdhuri & Davis West Azerbaijan: Urumieh, Mavana, Kuhe dare rash, 2100-2700m, Mozaffarian 74872-TARI. D. dolichostachyum* D. dasycarpum Stev. ex DC. Kurdestan: Baneh, 1650m, Maroofi & Fani 6959-TARI. East Azerbaijan: Sahand Mt., 2200m Assadi & Mozaffarian, 30641- TARI. D. dasycris= D. dasycarpum × D. crispulum East Azerbaijan: 60 km N.E of Maragheh, Chagh-Chagh Pasture, 1850m, Benvan 25028-TARI. 158 Masoomeh Hasanbarani, Fariba Sharifnia, Mostafa Assadi dion 2010). Some sequences were obtained from the GenBank (Table 3). The basis for the selection of taxon from the gene bank was the geographical distribution. Phylogenetic relationships were assessed using Bayes- ian Inference (BI). The substitution model was obtained using the program Mrrmodeltest ver. 2.3 (Nylander 2004). GTR + G + I for nuclear DNA and GTR + G for trnL-F region were identified as the best model for the dataset. The program Mrbayes version 3.2 (Ronquist & Huelsenbeck 2003) was used for the Bayesian recon- struction. After drawing several trees with different outgroups from Ranunculaceae, the best results were obtained from these outgroups (Nigella damascena for ITS marker and Helleborus niger for trnL-F marker). Species Locality D. elbursense var. elbursense Rech.* Mazandaran: Polesefid, forest above village Sangdeh, 1500-2500m, Assadi 73521& 73451-TARI Golestan: Kurdkuy, 5-10 Km from Radkan to Kurdkuy, 2200m, Mozaffarian 78137-TARI. Mazandaran: Polesefid, forest above village Sangdeh, 1500-2500m, Assadi 73521-TARI. D. elbursense var. gymnobotrys Rech. Mazandaran: Ramsar, S of Javaherdeh, 2600-3200m, Masassumi 56821-TARI. Mazandaran: Siahbisheh, Chalus Valley, 2120m, Sabeti 2056-TARI. Mazandaran: Siahbisheh, Chalus Valley, 2100m, Sabeti 1785-TARI. Mazandaran: Siahbisheh.Chalus Valley, 2300m, Sabeti 7964-TARI. D. ilgazense P.H. Davis* Azerbaijan: Tabriz, Ahar road, 22 km to Ahar, 1900-2000m, Mozaffarian & Mohammadi 37587-TARI. D. khorasanicum Sharifnia & HasanBarani Khorassan: North west of Neyshabur, Bar fall, 2004 M, Sharifnia and HasanBarani 16155 IAUNT. D. laxiusculum (Boiss.) Rouy West Azerbaijan: Gooshchi Pass, 1800m, Siami & Zehzad 7019-TARI. Ardabil: 45km from Namin to Germi, 220m, Mozaffarian & Nowrozi 34598-TARI. Ardabil:40 km from Razi to Germi, 1700m, Mozaffarian & Nowrozi 34762-TARI. Azerbaijan: Kaleybar to Jananloo, kiaragh, 1200m, Hasanbarani 16785-IAUNT. D. lanigerum Boiss. & Hohen. Hamedan: Alvand Mt., 2700m, Assadi & Mozaffarian, 2700m 36809-TARI. Hamedan: near Ganjnameh, 2100m, Assadi & Mozaffarian 36784-TARI. Tehran: Shemiran, Darband & Passghale, 2000-2500m, Mozaffarian & Jamzad 43742-TARI. D. micranthum Boiss. & Hohen. Kurdestan: From Baneh to Saghez, Kalawarash, 1900m, Fattahi & Hatami 2539-TARI. Kurdestan: Saghez to Baneh, Nacarouz Mt., 2500m, Maroofi & Mohammadi 6590-TARI. 85470-TARI. D. ochrolecum Stev. ex DC. Ardabil: 9km from diviation of Kivi to Ardebil road, above Meresht village, 2000m, Mozaffarian & Nowrozi 34391-TARI. West Azerbaijan: Urumieh, Marmishu vally, 1737m, Mozaffarian 87255-TARI. D. pallidiflorum Fyen* Esfahan: Fereydunshahr, near the village Sibak, 2800m, Assadi & Khatamsaz 76521-TARI. D. peregrinum L.* Fars: Nurabad, 22 km from Fahilan to Rashk, 900-1200m, Mozaffarian 45975-TARI. Fars: 15 to 20 km from Shiraz to Esfahan, 1600-1900m, Assadi & Ranjbar 82991-TARI. D. quercetorum Boiss. & Hausskn. East Azerbaijan: Ca. 20Km W of Marand, Mountain above the village Orlan, Mishoudagh, 2000-2500, Assadi & Shahsavari 65472-TARI. Kurdistan: Marivan, dizil,expose to Iraq frontier, 2350m, Maassumi & Nickchehre, 80189-TARI. Kurdistan: 34Km from Chenareh to Baneh, 1922m, Assadi 85087-TARI. D. schmalhausenii Alboff Kurdistan:Kurdestan, Ca. 17 Km from Baneh to Marivan, 1740m, Mozaffarian 87400-TARI. D. speciosum M. B.* Semnan: between Shahrud and Azadshahr, Kuhe abr, 2600m, Assadi & Maassumi 21523-TARI. Golestan: N Gorgan, Ca 20 Km Charbagh toward Gorgan, 1550m, Assadi D. turkmenum Lipsky Semnan: Touran protected area. 22 km from Ghazaran to Miandasht, 1240m, Feritagh & Jadidi 28987- TARI. Khorassan: North west of Neyshabur, Bar fall, 2004 M, Sharifnia and HasanBarani 17003- IAUNT. D. tuberosum Auch. ex Boiss. * West Azerbaijan: Ca. 15 Km to Maku on Road from Marand, 1200-1400m, Assadi & Mozaffarian 30110- TARI. Hamedan 64503-TARI. Zanjan 29393-TARI. East Azerbaijan: Kaleybar to Jananloo, kiaragh, 1200m, Hasanbarani 16798-IAUNT. D. ursinum Rech. Gorgan: Tanghegol Forest, 700-1000m, Wendelbo & Forughi 12766-TARI. Mazandaran: 32592-TARI. Tehran: Between Ushan & Tehran, 1730m, Assadi & Shahsavari 69764-TARI. D. venulosum Boiss.* Lorestan: Nowjian, (Between Khoramabad & Keshvar) 1850m, Runemark & Lazari 26112-TARI. Ilam: 10 km N.W. of Islam Abad, Ilam road, 1550m, Seraj 24666-TARI. 159Molecular insights on some Iranian species of Delphinium L. and Aconitum L. (Ranunculaceae) RESULTS AND DISCUSSION The Bayesian analysis result for the trnL-F region with posterior probabilities (PP) is shown as consensus tree in Fig. 1. The length of the trnL-F sequences includ- ed in the final matrix ranged from 950 to 1050 base pair. Helleborus niger is taken as an outgroup. This cladogram has several groups: species of annual Delphinium (clade d), perennial Delphinium (clade e), Consolida (clade c) and Aconitum (clade b). This result is congruent with the achievement of the study of Jabbour & Renner (2011). Clade (a) includes the Aconitum, Delphinium and Con- solida (Delphinieae tribe); Jabbour & Renner (2012) have revealed the monophyly of Delphinium and Aconitum. Delphinium species (both annual and perennial species) make a clade with a pp= 0.63 in which the annual and perennial species create two distinct groups as subgenus Delphinium (d) and subgenus Delphiniastrum (e). The Bayesian analysis result for the ITS region is shown in Fig. 2. Nigella damascena was considered as an outgroup. The length of the ITS sequences included in the final matrix ranged from 600 to 700. There were several groups in consensus tree, similar to the results of trnL-F marker: annual Delphinium (clade e), perennial Delphinium (clade d), Aconitum (clade b), and Consolida (clade c). By examining the results of chloroplast and nuclear marker, D. dasycarpum (only in chloroplast tree), D. spe- ciosum, D. crispulum, D. elbursense var. elbursense and D. elbursense var. gymnobotrys (only in chloroplast tree) were close to each other, in spite of the fact that they are distinct species. In the USSR flora, there are two sub- genera: Consolida and Eudelphinium,. Eudelphinium includes 3 sections: Kolobopetala, Elaptosis and Diedro- petala (Komarov 1970). According to USSR flora, D. spe- ciosum, D. crispulum and D. dasycarpum belong to the Elaptosis section similar to our molecular study (chloro- plast tree) which are all in the same group. These species have cylindrical root, dark blue flowers, black anther and lower petals which are black with yellow barbate. Del- phinium turkmenum, D. laxiusculum, D. quercetorum, D. schmalhausenii, D. szowitsianum, D. ochrolecum, Table 3. GenBank accession number taken from NCBI. Species trnL-F GenBank ITS GenBank Delphinium halteratum JF331737 - Delphinium leroyi JN73564 - Aconitum baicalense JF331723 - Aconitum ciliare JF331724 AB004952 Aconitum delphinifollium JF331725 AF258681 Aconitum ferox JF331726 AB004961–2 Aconitum pendulum JF331728 AY150235 Aconitum pentheri JF331729 JF331905-18 Aconitum racemolusom AF258652 AY150233 2 Aconitum septentrionale JF331730 AF216552 Aconitum tanguticum JN573573 AY15023 Consolida ajacis JF331687 JF33188 Consolida axilliflora JF331692 - Consolida flava JF331695 JF331887 Consolida orientalis JF331707 JF331896 Delphinium pyramidale JN573581 - Delphinium afgahnicum JN573529 - Delphinium albocoeruleum JN573530 - Delphinium bakeri AF258652 AF258697 Delphinium balansae JF331732 - Delphinium bicolor - AF258711 Delphinium brachycentrum - JN573515 Delphinium cardinale - AF258740 Delphinium crassifolium JN573540 - Delphinium cuneatum JN573542 - Delphinium dasycaulon JN573544 - Species trnL-F GenBank ITS GenBank Delphinium decorum - AF258744 Delphinium delavayi - AF258705 Delphinium dubium JN573568 - Delphinium elatum JN573549 - Delphinium favargeri JF331679 - Delphinium fissum JN573552 - Delphinium flexosum JN573553 - Delphinium gracile JF331736 AF258763 Delphinium gypsophilum - AF258721 Delphinium hesperium - AF258772 Delphinium hirschefeldianum - JF331988-95 Delphinium incisum JN573558 - Delphinium kohatense JN573561 - Delphinium maakianum JN573573 - Delphinium macropetalum - JF331996-2000 Delphinium muscosum JN573572 - Delphinium oreophilum JN573576 - Delphinium suave JN573596 - Delphinium verdunanse JN573596 - Delphinium virgatum - JF332030-1 Delphinium viscosum JN573597 - Delphinium wendelboie Delphinium staphisagria JN573598 - JF332022 Helleborus niger AJ413290 - Nigella damascene - AY150260 160 Masoomeh Hasanbarani, Fariba Sharifnia, Mostafa Assadi Figure 1. Bayesian tree for chloroplast DNA (trnL-F region). Abbreviations: H. niger= Heleborus niger; A. iran= A. iranshahrii; A. pubi= A. pubiceps Aconitum pubiceps (Rupr.) Trautv. is a synonym of Aconitum nasutum Fisch. ex Rchb. ; A. septen= A. septentrionale; A. tangu= A. tanguaticum; A. race= A. racemolusom; C. orien= C. orientalis; D. cris= D. crispulum; D. dasyc=D. dasycarpum;D. elb var. elb= D. elburs- ens var. elbursense; D. speci= D. speciosum; D. elb var. gy= D. elbursense var. gymnobotrys; D. lanige= D. lanigerum; D. ilgaz= D. ilgazense; D. dolico= D. dolichostachyum; D. card= D. carduchorum; D. micran: D. micranthum; D. schmal= D. schmalhausenii; D. bite= D. biternatum; D. semiba; semibarbatum; D. ochrol= D. ochrolecum; D. szowits= D. szowitsianum; D. turkm= D. turkmenum; D.cypho= D. cyphoplectrum; D. tuber= D. tuberosum; D. laxiusc=D. laxiusculum; D. pallidi= D. pallidifl orum; D. querc= D. quercetorum; D. aquil= D. aquilegifolium; D. khora= D. khorasanicum; D.pereg= D. peregrinum; D. venulo= D. venulosum, D. virga= D. virgatum, D. albocoe=D. albocoeruleum; D. viscos= D. viscosum; D. gris= D. griseum; D. sanicu= D. saniculifolium; D. dasycaul= D. daycaulon; D. macrost= D. macrostachyum; D. kurdi= D. kurdicum; D. shmal= D. schmalhausenii; D. kohaten= D. kohatense; D. dasycris= D. dasycarpum × D. crispulum. 161Molecular insights on some Iranian species of Delphinium L. and Aconitum L. (Ranunculaceae) D. biternatum, and D. semibarbatum are placed in the Diedropetala section (Komarov 1970) and in Fig. 1, and except for D. schmalhausenii the other species are placed in one group. Delphinium schmalhausenii is very similar to D. kurdicum and D. fi ssum but diff ers in fl ower color (D. shmalhausenii is brown-violet), and there seems to be a new position for D. schmalhausenii as a variety of D. Kurdicum instead of a being a species. Also in Die- dropetala section, D. cyphoplectrum, D. pallidifl orum, D. laxiusculum, D. quercetorum and D. tuberosum (com- plex species) are closely related to each other (Iranshahr 1992). In fl ora of Iraq, D. tuberosum is synonymous with D. cyphoplectrum, D. quercetorum, D. pallidifl orum and D. laxiusculum (Townsend & Evan 1974). Based on the molecular study (trnL-F marker), the separation of these species is confi rmed. D. elbursense is an endemic spe- cies in Iran and Rechinger has announced two varie- ties for this species that were distributed in Azerbaijan and Hyrcanian region (Iranshahr 1992). In our research, the separation of these two varieties based on Chloro- plast marker was approved (Fig 1). Based on the molecu- lar result, it is suggested that the taxonomic level of D. elbursense var. gymnobotrys be elevated to a higher level. Moreover, the results of micromorphological tepal epi- dermal patterns study confi rmed that D. elbursense var. elbursense and D. elbursense var. gymnobotrys are dif- ferent in the tepal epidermal patterns (Hasanbarani et al. 2016). Annual taxa in the genus Delphinium are arranged in Delphinium subgenus and from the mor- phology point of view they are diff erent from perennial species (lower petals in this subgenus are without lobe, whereas they are accompanied by lobe and barbate in perennial species), and based on ITS and trnL-F trees they are classifi ed as clade e and clade d, respectively. Subgenus Delphinium is divided into two section: sect. Anthriscifolium W.T Wang and sect. Delphinium. Th e Figure 2. Bayesian tree for nuclear DNA (ITS marker). Abbreviations: A. iran= A. iranshahrii; A. septen= A. septentrionale; D. kamao= D. kamaoense; D. cris= D. crispulum; D. elb= D. elbursene var. elbursense; D. speci= D. speciosum; D. tricho= D. trichoporum; D. ilgaz= D. ilga- zense; D. cypho= D. cyphoplectrum; D. pallidi= D. pallidifl orum; D. dolicho= D. dolichostachyum; D. tuber= D. tuberosum; D.pereg= D. per- egrinum; D. venulo= D. venulosum; D. balcan= D. balcanicum; D. hirschfel= D. hirschfeldianum; D. anthirisi= D. anthriscifolium; D. balan= D. balansae. 162 Masoomeh Hasanbarani, Fariba Sharifnia, Mostafa Assadi geogeraphic distribution of the two sections of subg. Delphinium is disjunct; Delphinium section is distrib- uted in the Irano-Turanian region, whereas Anthriscfo- lium section is distributed in the warm zone of central and southern China and northern Vietnam (Xiang et al. 2017); the same results are confirmed in Fig. 2. In Iran, only D. venulosum and D. peregrinum are in the subge- nus Delphinium and their morphological differences are in the form of lower petal; their separation is clearly evi- dent in the molecular tree. Our other research on Delphinieae tribe has shown that the genus Delphinium, Aconitum and Consolida are distinct base on morphological features (Hasanbarani et al. 2020). Pollen studies in Iranian species of the genus Delphinium prove that if the two species are morphologi- cally similar, it does not mean that the two species are close pollen type (Hasanbarani et al. 2019). For example, the D. venulosum and D. pregrinum, which form a clade in molecular studies, differ in shape of the pollen. D. cyphoplectrum and D. tuberosum which are separate in molecular studies were also different in pollen studies. In the study of the flower morphology in Delphinium, annu- al taxa like morphological studies were placed in sepa- rate morphological phenogram (Hasanbarani et al. 2018). Some species that are similar in flower morphology stud- ies were included in a separate phylogenetic study. New record for Iran D. dolichostachyum Chowdhuri & P. H. Davis in Notes R.B.G. Edinb. 22: 408 (1958). Locality: Iran. Kurd- istan: Baneh, Kochar cemetery, 1650 m, Maroofi & Fani, 6959 TARI. D. dolichostachyum was originally described from Turkey (Davis 1965). This species was collected from Kurdistan (Baneh) and is morphologically related to D. carduchorum, but differs from it mainly considering the following characters: bract length, spur length, flower color and plant length (Table 4). According to the dis- tribution area and morphological character, it may seem that this species is D. carduchorum at first sight. Del- phinium dolichostachyum image and the type specimen are presented in Fig. 3 and 4. CONCLUSION The present molecular data provide strong support for the monophyly of Delphinium, Aconitum and Consol- ida, and therefore D. elbursense var. gymnobotrys could be at high taxonomic level as distinct species. D. doli- chostachyum is newly recorded for the flora of Iran. The separation of D. tuberosum and D. cyphoplectrum (con- troversial species) is confirmed by molecular results. ACKNOWLEDGMENT The authors are grateful to TARI herbarium for pro- viding the samples. Table 4. Morphological characters useful in separating Delphinium carduchorum and Delphinium dolicostachyum. Characters D. dolicostachyum D. carduchorum Plant length 60 cm 100 cm Bract length 5 mm 40 mm Bract form linear Trisect Inflorescence Panicle Raceme Spur form Cylindrical attenuate Spur length 9-10mm 15-16mm Color of sepal pale blue dark blue Color of petal White Yellow Figure 3. Image of D. dolichostachyum (This species is available in TARI). 163Molecular insights on some Iranian species of Delphinium L. and Aconitum L. (Ranunculaceae) REFERENCES Beltran, J. C., Daplin, K. M. A., Relado-Sevilla, R. Z., Bordey, F. H., Manalili, R. G., Arida, I. A., Ante, R. H. L., Romero, M. V., Leon, T. J. P. D. ., Chua, J. D., Baltazar, M. A. M., Valencia, M. S. D., & Moya, P. F. 2021. Productivity and Profitability of Aromatic Rice Production in the Philippines. International Journal of Sustainable Agricultural Research, 8(4), 209–221. Bursali, B., & Dogan, C. 2005. Pollen morphology of some Delphinium L. (Ranunculaceae) taxa in Turkey. Hac- ettepe Journal of Biology and Chemistry. 34: 1-17. Cabusora, C. C., Desamero, N. V., Borromeo, T. H., Bulu- ran, R. D., Hernandez, J. E., & Cruz, P. C. S. 2020. Characterization of a Novel Floral Mutation Induced by Gamma Irradiation of Philippine Rice Variety NSIC Rc9 (Apo). International Journal of Sustainable Agricultural Research, 8(1), 43–55. Christensen, K.I. & Hansen, H.V. 1998. SEM studies of epidermal patterns of petals in Angiosperms. - Opera Botanica. No.135. Davis, Ph. 1965. Delphinium in Flora of Turkey. Vol. 1, Edinburgh at the University Press. Douzery, E J., Pridgeon, Am., Kores, P., Linder, HP., Kur- zweil, H. & Chase Mz. 1999. Molecular Phylogenetics Orchidaceae: A contribution from nuclear Ribosomal ITS Sequence. American Journal of Botany 86: 887-889. Espinosa, F., Deroin ,T., Xiang, K., Wang, W., Castro, M.P, Aytack, Z., Nadot, S. & Jabbour, F., 2017. The Turkish Endemic Pseudodelphinium turcicum (Ranunculace- ae) an annual population of Delphinium with peloric flowers that has persisted in the wild for 20 years, Plant systematic and Evolution, Vol. 178 (7). Fataei, E., S. Varamesh and B. Behtari 2013. Soil Carbon and Nitrogen Stocks under Pinus nigra and Cedrus libani afforestation in the Northwestern Highlands of Iran. Advances in Environmental Biology: 4316-4326. Fikirie, K., Bezu, A., Eshetu, M., Bekele, D., & Rabo, M. 2020. Evaluate Technical Standards of Implement- ed Soil Bund in Central Rift Valley of Ethiopia: The Case of Adama, Lume and Dodota Districts. Agricul- ture and Food Sciences Research, 7(1), 51–57. Hadidchi, A., Attar, F., & Ullah, F. 2019. Using micro- scopic techniques for taxonomic implications of seed and fruits of Delphinium L. (sensu lato) (Ranuncu- laceae). Microsc Res Tech. 2020, Vol. 83, 2: 99-117. Hall, TA. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acid Symposium Series 41:95-98. Hasanbarani, M., Sharifnia, F., Nejadsattari, T. & Assadi, M., 2017. Description and Molecular diagnosis of a new species of Delphinium (Ranunculaceae) from Northeast Iran, Biodiversitas, 18: 639-644. Hasanbarani, M.,Sharifnia, F., Assadi, M., 2018. Taxono- mis value of flower morphology and spur in Persian Delphinium, Iranian Journal of Biological Science, Iranian Journal of Biological Science, 13: 15-32. Hasanbarani, M.,Sharifnia, F., Assadi, M., 2019. Pollen morphology of Delphinium in Iran, Iranian Journal of Biological Science, 14: 35-53. Hasanbarani, M.,Sharifnia, F., Assadi, M., 2020. Phenetic study on Iranian Delphinium and Aconitum species (Ranunculaceae) based on morphological characters, Journal of Plant Research (Iranian Journal of Biol- ogy), Accepted Manuscript, Articles in Press. Hasanbarani, M., Sharifnia, F., Nejadsatari, T., & Assadi, M. 2016. Morphological and Micromorphological Figure 4. Type specimens of D. dolichostachyum (Image taken from Kew) https://www.gbif.org/occurrence/912539463. 164 Masoomeh Hasanbarani, Fariba Sharifnia, Mostafa Assadi tepal epideramal patterns studies on Delphinium in IRAN, Developmental biology 8: 11-22. Ilarslan, H., Ilarslan, R. & Blanch C. 1997. Seed morphol- ogy of the genus Delphinium L. (Ranunculaceae) in Turkey, Collect. Bot. (Barcelona) 23: 79-95. Iranshahr, M. 1992. Ranunculaceae in Flora Iranica 171, pp. 44-114, AKademische Druck-u Verlagsanstalt Graz- Austria. Jabbour, F. & Renner, S. 2011. Consolida and Aconitella are an annual Clade of Delphinium (Ranunculaceae) that diversified in the Mediterranean basin and Ira- no-Turanian region, Taxon 60: 1029-1040. Jabbour, F. & Renner, S. 2012. A phylogeny Delphin- ieae (Ranunculaceae) Shows that Aconitum is nested within Delphinium and that Late Miocene transitions to long life cycles in the Himalayas and Southwest China coincide with bursts in diversification, Molec- ular Phylogeny and Evoulution 62: 928-942. Komarov, V.L. 1970. Ranals and Rhoeadales, Flora of the U.S.S.R,VII, (Translated From Russian), pp. 79-143, Smithsonian Institution and the National Science Foundation,Washington D.C. Li, Hui-Mini, Yuan, Q & Yang, Q. 2019. Taxonomic stud- ies on the genus Delphinium (Ranunculaceae) from China (XVII): Towards clarification of the confusion of D. ilense with special reference to observation on living plants in the Ili region in northwestern Xinji- ang, Phytotaxa, 403 (1): 001-24. Maddison W.P., and Maddison D.R. (2010). Mesquite (version 2.7.4): A modular system for Evolutionary Analysis. mesquiteproject.org. Mieso, B., & Befa, A. 2020. Physical Characteristics of the Essential Oil Extracted from Released and Improved Lemongrass Varieties, Palmarosa and Citronella Grass. Agriculture and Food Sciences Research, 7(1), 65–68. Mobayen, S., 1985. Flora of Iran: vascular of plants 3, pp: 33-67, University of Tehran. Mustafa, O. A. O. 2020. Efficiency of Agriculture and Water Sector and the Reality of Food Security in Arab Countries (2010-2017). Agriculture and Food Sciences Research, 7(1), 1–6. Nylander., Jaa., 2004. MrModeltest v2. Program distribut- ed by the author. Evolutionary Center, Uppsala Uni- versity, Uppsala, Sweden. Rajaei, G. E., S. Khalili-Arjaghi, E. Fataei, N. Sajjadi and M. Kashefi-Alasl 2020. Fabrication and characteriza- tion of polymer-based nanocomposite membrane modified by magnetite nanoparticles for Cd2+ and Pb2+ removal from aqueous solutions. Comptes Ren- dus. Chimie 23(9-10): 563-574. Ronquist, F. & Huelsenbeck, JP. 2003. Bayesian phyloge- netic inference under mixed models Bioinformatics 19: 1-210. Sharifnia, F., Hasanbarani, M. & Assadi, M. 2013. Notes on some species of the genus Delphinium (Ranun- culaceae) in Iran, Iranian journal of Botany. 19: 202- 210. Shayan, F., Borji, H., Eslami, A. & Zakeri S. 2007. Isola- tion of DNA single using new developed kit in Iran and ITS PCR Analysis. Iranian Journal of Parasitol- ogy. 2: 34-39. Stevens, P.F., 2001. Onwards. Angiosperm Phylogeny Website, version 9, June 2008. http: //www.mobot. org/MOBOT/reasarch/APweb/. Taberlet, P. & Gielly, G. 1991. Universal primers for amplification of three non-codin regions of chloro- plast DNA. Plant Molecular Biology. 17: 1105-1109. Tamura, M., 1990. A new classification of the family Ranunculaceae 1. Acta Phytotax. GeoBot. 41, 93-110. Townsend, C. & EVAN, G. 1974. Flora of Iraq of collabo- ration of the Botany Directorate of the Minisitry of Agriculture and Agrarian Reform. Baghdad. Varamesh, S., S. M. Hosseini, F. K. Behjou and E. Fataei 2014. The impact of land afforestation on carbon stocks surrounding Tehran, Iran. Journal of forestry research 25(1): 135-141. Wang, W., Liu, Y., Yu, S.X., GAO, T.G. & Chen, Z.D. 2013. Gymnaconitum, a new genus of Ranunculaceae endemic to the Qinghai-Tibetan Plateau. Taxon. 62: 713-722. Xiang, K.L., Aytac, Z., Liu, Y., Espinosa, F., Jabbour, F., Byng, J.W., Zhang, C., Erst, A. & Wang, W. 2017. Recircumscription of Delphinium subg. Delphinium (Ranunculaceae) and implications for its biogeogra- phy. Taxon. 66: 554-556.