Nutlet Micromorphology and Character Evolution of Some Species of Rochelieae (Boraginaceae) and Its Systematic Implications

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To evaluate the phylogenetic significance of the character evolution, available DNA sequence data from GenBank were combined with selected original nutlet data, and phylogenetic analysis was performed. Results: The Rochelieae nutlets' shape varied from ovoid (ovoid, ovoid-triangular, and ovoid-rectangular) to the pyramid. Six major patterns were recognized based on nutlet ultrastructure characters. Rocheliae is characterized by a transition from “without appendage” to “with tubercles and prickles” on the nutlet disk, and also by a shift from “lack of prickles” to “glossy prickles”. Conclusions: The results indicated that the nutlet ultrastructure pattern of Rochelieae is systematically informative at the genus level, but not at the species level. The results showed that glochid is not an ancestral trait but is a synapomorphy and the transition to this trait occurred in the genus Lappula . The Close boundary of nutlet microstructures between L. barbata and L. microcarpa was discussed. Biotechnology and Bioengineering Character evolution Lappula Micromorphology Systematic Figures Figure 1 Figure 2 Figure 3 Figure 4 Background Boraginaceae s.str. is a sub cosmopolitan family of flowering plants with nearly 90 genera and ca.1600 to 1700 species distributed worldwide (Chacón et al. 2016 ). Based on the recent molecular phylogeny presented by Chacón et al. ( 2016 ), infrafamilial classification of Boraginaceae was recognized into three Subfamilies (Echiochiloideae, Boraginoideae, Cynoglossoideae) and 10 tribes (Boragineae, Lithospermeae, Trichodesmeae, Lasiocaryeae, Asperugeae, Omphalodeae, Rochelieae, Craniospermeae, Myosotideae, Cynoglosseae). Tribe Rochelieae consists of five genera ( Eritrichium , Hackelia , Lappula , Lepechiniella , and Rochelia ) and about 207 species and belongs to the subfamily Cynoglossoideae. According to Chacón et al. ( 2016 ), Rochelieae can be divided into two subtribes including Eritrichiinae and Heterocaryinae. The genera Eritrichium , Hackelia , Lappula , Lepechiniella , and Rochelia , belong to sub-tribes Eritrichiinae and the genera Heterocaryum , Suchtelenia , and Pseudoheterocaryum belong to sub-tribe Heterocaryinae (Chacón et al. 2016 ; Saadati et al. 2017 ). Three genera in subtribe Eritrichiinae ( Eritrichium , Lepechiniella , and Lappula ) are recognized as non-monophyletic lineage and Hackelia and Rochelia comprise monophyletic clades (Khoshsokhan-Mozaffar et al. 2018 ). The recent molecular phylogeny of Rochelieae tribe provided by Khoshsokhan-Mozaffar et al. ( 2018 ) indicated a well-supported clade. Hackelia and Rochelia are monophyletic while Lappula, Eritrichium , and Lepechiniella are not. So that Lappula , as currently circumscribed, is polyphyletic (Khoshsokhan-Mozaffar et al. 2018 ). The genus Eritrichium is the largest genus of the tribe, with 71 species. The genus Lappula contains about 70 species of annual, biennial perennial herbs distributed in Eurasia, Africa, North America, and Australia (Ovchinnikova 2005 ). Although Lappula has a cosmopolitan distribution, the center of diversity is in Siberian and Irano-Turaniean provinces of the Holarctic kingdom (Ovchinnikova 2009 ). Initially, Lehmann ( 1818 ) circumscribed 15 species in Echinospermum Lehm. (Synonym of Lappula ). Echinospermum was divided by de Candolle ( 1845 ) into three sections based on nutlet morphology as follows: Lappula , Sclerocaryum , and Homalocaryum . The taxonomical problems of Lappula were increased when the number of species in the genus began to expand. Consequently, the reported number of sections, subsections, and series has widely varied by different authors (Popov 1953 ; Riedl 1967 ). For the first time, Lehmann ( 1818 ) used nutlet characters in the systematics of Lappula and showed mericarp characters such as mericarp shape and surface ornamentation to distinguish the species of this genus. Generally, in Boraginaceae, nutlet morphology provides useful systematic characters at various taxonomic levels, such as straight or incurved nutlet, a specialized form of emergence, the position of attachment scar, the distinctive form of prickles or glochids, and epidermal features of nutlets (Johnston 1937 ; Hilger 1985 ; Al-Shehbaz 1991 ; Riedl 1996 ; Långström and Chase 2002 ; Moon and Hong 2006 ; Selvi et al. 2006 ; Kahraman et al. 2011 ). The main goal of this study was to investigate the various varieties of nutlet morphological characters of the tribe Rochelieae s.l. using scanning electron microscopy and the nutlet character evolution with more focus on the Lappula genus. Methods Morphological study The plants used in this study were collected from their natural habitats in Iran and deposited in the Herbarium of Azarbaijan Shahid Madani University (ASMUH). Also, a small number of species were taken from herbarium specimens of FUMH (Ferdowsi University of Mashhad Herbarium). The list of voucher specimens and details of localities were given in Table. Table. List of sampled taxa, locality and their vouchers numbers. Species (Pop. Code) Locality Voucher No. Lappula barbata (M.Bieb.) Gürke (m) Tehran, Chalus road, Kooshk ASMUH0020 L. barbata (c) Mazandaran, Chalus, Delir vilage ASMUH0021 L. barbata (ab) Mazandaran, Noshahr, Kojur, Laregan ASMUH0022 L. barbata (w) Tehran, Tuchal ASMUH0023 L. ceratophora (Popov) Popov South Khorasan, south-west Sarayan FUMH46077 L. microcarpa (Ledeb.) Gürke (b) Mazandaran. Noor, Chamestan, Lavij ASMUH0024 L. microcarpa (e) Mazandaran, Neka, Hezarjerib ASMUH0025 L. microcarpa (g) Golestan, East of Golestan national park ASMUH0026 L. microcarpa (i) Mazandaran, Savadkooh, Veresk ASMUH0027 L. microcarpa (j) Mazandaran, Polor to Rine ASMUH0028 L. microcarpa (p) Mazandaran, Noshahr, Kojur, ASMUH0029 L. microcarpa (z) North Khorasan, Chamanbid ASMUH0030 L. microcarpa (a) Tehran, Lavasan, Glucan ASMUH0031 L. semiglabra (Ledeb.) Gürke Khorasan Razavi, North of Gonabad FUMH17236 L. sessiliflora (Boiss.) Gürke Khorasan Razavi, East of Kashmar FUMH26636 L. spinocarpus (Forssk.) Asch. ex Kuntze South Khorasan, Birjand, Shahzile FUMH30399 Pseudolappula sinaica (A.DC.) Asch. & Schweinf. Tehran, Chalus road, Morod ASMUH0032 Asperugo procumbens L. Mazandaran, Damavand, Sarbandan ASMUH0034 Heterocaryum rigidum A. DC. Tehran, Jajroad ASMUH0035 Myosotis sylvatica Ehrh. Mazandaran, Sari, Sangdeh forest ASMUH0036 Rochelia disperma (L. f) C. Koch Tehran, Lavasan ASMUH0037 This study was conducted on 9 species of tribe Rochelieae (covering five of the six genera) and two species of tribes Asperugeae and Myosotideae as out-groups. The mature nutlets were selected from materials as herbarium samples. Then air dried nutlets were investigated for shape, size, and other features using stereomicroscope (Dino-Lite) with the help of DinoCapture eye and DinoCapture 2.0 Software (Electronics Corporation). For SEM observations, the nutlets were mounted onto standard aluminum stubs using double-sided adhesive tape and then photographed using a PHILIPS / FEI XL 20 Scanning Electron Microscope at 15 KV voltages. The measurements are based on 15–20 evaluations from each specimen. The terminology used to describing the qualitative characters is in accordance with Ma et al. 2010 ; Selvi et al. 2011 ; Yu et al. 2012 ; Hilger 2014 . The data were analyzed and examined by WARD dendrogram using PAST software for species decimation of L. microcarpa and L.barbata . Due to high morphological similarities of L. microcarpa and L.barbata , to identify them, the Flora of Iran, (Nasseh and Joharchi 2017 ) and also Flora Iranica (Riedl 1996 ) were used. Phylogenetic analyses and tracing character evolution The sequences for the internal transcribed spacer (ITS) region have been obtained from GenBank. Sequence alignments were performed using MUSCLE by the MEGA software ver.7 (Kumar et al. 2016 ). Poorly aligned positions and divergent regions were eliminated by using Gblocks 0.91b, following the given options for less stringency (Castresana 2000 ). Phylogenetic analyses were performed using the combined 3-loci data set. The partitioned ML analysis was performed using raxmlGUI 1.1 (Silvestro and Michalak 2012 ) under the GTR + G model with 1000 bootstrap replicates and with Asperugo (tribe Asprugeae) and Myosotis ( tribe Myosotideae ) chosen as out-group. The evolutionary history of characters was traced over an ML tree in Mesquite 3.04 (Maddison and Maddison 2015 ). The ML approach with the Markov k-state one-parameter (Mk1) model was applied (Lewis 2001 ). Result General description of nutlet micromorphology The nutlet's morphology and ultrastructure characteristics such as shape size, appendages, and surface sculpturing, varied among the studied taxa. The Rochelieae nutlets' shape was ovoid (ovoid, ovoid-triangular, and ovoid-rectangular) to the pyramid (Fig. 1 ). As the out-groups, the shape of Asperugo procumbense was semicircular, and the Myosotis sylvatica one was an ellipse. These two genera belong to Asperugeae and Myosotideae tribes, respectively. Nine qualitative characters including shape, the centerline of the raphe, base surface of prickles on the desk, the arrangement of glochids, the type of lamellae, tubercles on the desk, appendages on nutlet desk, Number of glochid rows on nutlet edge, and emergence type were selected for morphological evaluation of nutlets. The results obtained from nutlet-ultrastructure investigations are described below and illustrated in Fig. 1 . Generally, six different surface types were recognized between studied taxa based on nutlet ultrastructure characters as follow: Type I : Heterocaryum and Pseudolappula (Syn : L. siniaca ) There is no glochid or appendage on the nutlet disk, but there is a row of glochid ( Heterocaryum ) or glochid-like ( Pseudolappula ) on the nutlet edge. The glochids are distributed in very low numbers in the edges of the nutlets of Pseudolappula . The nutlet disk ornament of Heterocaryum is “papilla verrucose with verrucae minutely muricate” (called complex papilla) while it is “papilla with aggregate verrucose in the center” in Pseudolappula . It appears that each of the microcapillaries found in Psudolappula has become complex in the Heterocaryum , and each has formed warts (verrucose) that have become more complex and denser. Type II : Lappula ( L. barbata , L. microcarpa and L. semiglabra ) Glochids in different sizes and rows can be seen in the nutlet edge and sometimes on the nutlet disk surface. Glochids have an anchor with 2–4 branches at the apex, and the surface of the glochids is smooth. The ultrastructure of the nutlet emergencies is stellar-aculeate, and sometimes the appendages are prickles or tubercles are seen (scattered or collected) on the surface of the nutlet disk and edge. The glochid stem is composed of fusiform cells, and there are tubercles with 2 to 5 mineralized spines on the stem. These tubercles are also present on the entire surface of the nutlet with a different distribution. Type III : Rochelia ( R. disperma , R. sessiflora = L. sessiflora ) The prickles are stellate and are scattered throughout the surface of the nutlet. The surface of the prickles is not glossy and has verrucose. The tubercles often have more than 2 spines, and the emergencies are stellar-aculeate (similar to type II). Although the nutlet surface of R. sessiflora is similar to type II (presence of glochid on the nutlet edge). Moreover, the prickles and verrucose on it and the accumulation of tubercles with more than 5 spines around each prickle show more similarity to type III. Type IV : L. ceratophora and L. spinocarpus There is not any glochid, tubercle, or prickles on the nutlet surface. The nutlet surface of L. ceratophora is not smooth, and the papilla appears as a verrucose-like. While the ultrastructure of the nutlet in L. spinocarpus seems papilla with flowerlike verrucose. Also, the tubercles appear as verrucose and lack any spines. Type V : Asperugo The nutlet surface lacks any glochid and prickles. Papilla appears as dome-shaped in different sizes, and it is verrucose at the base of them. Type VI : Myosotis The surface of the nutlet is smooth, and there is not any ornamentation (nonexpressiate). a – h = Type I; (a-d) Heterocaryum rigidum ; a, b : An overview photograph of nutlet with stereomicroscope and SEM. c, d : The close up views of nutlet disk with “papilla verrucose with verrucae minutely muricate”. (e-h) Pseudolappula siniaca ; e, f : An overview photograph of nutlet with stereomicroscope and SEM. g, h : The close up views of nutlet disk with “papilla with aggregate verrucose in center”. i-o Type II . ( i, j) Lappula. barbata ; (k, l) Lappula. Microcarpa ; (The more detail of these two species were described between different specimens in Fig. 3 ). Lappula semiglabra m, n : An overview photograph of nutlet with stereomicroscope and SEM. o : The close up views of nutlet disk. p-v = Type III; (p-r) Rochelia sessiflora ; p, q : An overview photograph of nutlet with stereomicroscope and SEM. r : The close up views of nutlet disk with prickles and verrucose on it. (s-v) Rochelia disperma s, t : An overview photograph of nutlet with stereomicroscope and SEM. u, v : The close up views of stellare-aculeate emergencies in nutlet disk. w-ac = Type IV; (w-y) Lappula spinocarpus ; w, x : An overview photograph of nutlet with stereomicroscope and SEM. y : The close up views of nutlet disk with papilla with flowerlike verrucose on it. (z-ab) Lappula ceratophora z, aa : An overview photograph of nutlet with stereomicroscope and SEM. ab : The close up views of nutlet disk with papilla appears as a verrucose-like on it. ac-af = Type V; Asperugo procumbense ; ac-ad : the overview photograph of nutlet with stereomicroscope and SEM. af : The close up views of nutlet disk with Papilla appears as dome-shape on it. ag-aj = Type VI; Myosotis sylvatica ; ag-ai : the overview photograph of nutlet with stereomicroscope and SEM. aj : The close up views of nutlet disk with smooth surface. 3.2. Evolution of microstructural characters of nutlet The resulting ancestral state reconstruction and the proportional likelihoods for character states are shown in Fig. 2 . The out-group species Asperugo and Myosotis were unique regarding the bilaterally flattened and ellipse with a smooth surface, respectively. Tracing the evolution of nutlet micromorphology indicated that the glochids were not an ancestral character. Arrangement of glochid character : The status of ancestral taxa (with or without glochids) was unclear, and the proportional likelihoods of any three characters were almost equal (node A). Transition to the glochids character occurs in the genus Lappula ( L. semiglabra , L. microcarpa , and L. barbata ) (node H). The appendage on nutlet disk character : The status “without appendage” and “dump-shape papilla” in Myosotis and Asperugo (the proportional likelihoods 1) were differentiated these two tribes from each other and Rocheliea tribe. The tubercle and prickles on the disk were ancestral characters (the proportional likelihoods 0.43). While the ancestor of these characters is unclear in node A, the status “tubercle and prickles on disk” had more proportional likelihoods in A group and then C, D, F. Transition to the “lack of appendage” status occurred in the L. ceratophora , L. spinocarpus; node G). Prickles surface character : Tracing of character “prickles surface” showed the ancestral status of “lack of prickles” in node A that to be followed with less proportionality in nodes B, C, and D. Transition to the “glossy prickles” status occurred in the genus Lappula in node H. Moreover, the transition to the simple and complex “verrucose prickles” status was observed in genus Rochelia in node F. Surface emergence: Tracing of surface emergence character was unclear in node A. However, the proportional likelihoods of “stellar-aculeate” status had the highest node C ratio (0.72). In node G, the transition to “verrucose-subverrucose” status (the proportional likelihoods 0.99) was stabilized as a synapomorphy. Other traits were studied regarding evolutionary tracing that did not show clear evolutionary signals in the nodes, such as the shape of the nutlet, the lamella type, the shape of the nutlet, the lamella type, and the centerline of the nutlet disk. 3.3. Close boundary of nutlet microstructures between L. barbata and L. microcarpa Both L. microcarpa and L.barbata had high micro-morphological similarities (Fig. 3 ). Different clustering and ordination methods produced similar results; therefore, only WARD tree of micro-morphological characters is presented here (Fig. 4 ). In general, plant samples of each species did not group and formed a separate group. This result shows that the micro-morphological characters studied could not delimit these two species. Discussion As in other Boraginaceae genera (Ovchinnikova 2009 ; Weigend et al. 2009 ; Selvi et al. 2011 ; Yu et al. 2012 ; Hilger 2014 ), the infrageneric taxonomic significance of nutlet characteristics in tribe Rochelieae were found to be obvious when investigated under stereomicroscope and SEM. In this study, it was found that the appendages on the nutlet varied between different genera. Although the fruit type of Boraginaceae is relatively constant, the variation in nutlet ornamentation has quickly occurred in some tribes like Cynoglosseae sensu lato (including tribe Rocheliea) and Trichodesmeae (Cohen 2014 ). It is not surprising that Lappula , one of the largest genera in Rochelieae, shows considerable diversity in nutlet characters. Recent molecular evidence suggested that Lappula is polyphyletic (Khoshsokhan-Mozaffar et al. 2018 ). In this way, Khoshsokhan-Mozaffar et al. indicated that species of the Lappula genus are scattered across the Eritrichiinae clade and forming three distinct lineages. Lappula sinaica , as a new genus, was segregated from Lappula and established as genus Pseudolappula (Khoshsokhan-Mozaffar et al. 2018 ). Moreover, as shown in our study, a distinct generic delimitation based on nutlet characters alone could be detected for Pseudolappula (syn: Lappula siniaca ). Nutlet micromorphology of Pseudolappula (syn: L. siniaca ) provides valuable data in separating it from Lappula genus; these characters are “no glochid or appendage on nutlet disk” and the type of nutlet ornamentation including “papilla with aggregate verrucose in the center”. The features of disk ornamentation indicated more close affinity of Pseudolappula siniaca to Heterocaryum than Lappula genus, especially in the evolution of microcapillaries. Current data are also in agreement with molecular phylogenies provided by Khoshsokhan-Mozaffar et al. ( 2018 ) that transferred closely related species L. sessiliflora to Rochelia genus. This conclusion has previously been suggested by various studies (Khoush et al. 2010 ; Huang et al. 2013 ; Mozaffar et al. 2013 ; Rolfsmeier 2013 ; Weigend et al. 2013 ). Moreover, the flowers and nutlet features (two of them undeveloped) indicated more affinity of the species to Rochelia genus than Lappula (Popov 1974). Moreover, our result indicated the features like prickles and verrucose on the nutlet and the accumulation of tubercles with more than five spines around each prickle show more similarity to genus Rochelia . Our study convincingly provided a clear distinction between two species L. ceratophora and L. spinocarpus belonging to sect. Sclerocaryum (Riedl 1967 ; Ovchinnikova 2009 ) from other Lappula genus. The lack of glochid, tubercle, or prickles on the nutlet surface and specific types of nutlet ornamentation (papilla with verrucose like or flowerlike-verrucose) characterize the clade that includes them regarding recent molecular phylogenetic analysis (Khoshsokhan-Mozaffar et al. 2018 ). We identified four different types of nutlet surface ornamentation among studied taxa. According to our study and the other available record (Cohen 2014 ), the ancestral type is ambiguous for the family and Rochelieae tribe. Given the matrix of cpDNA and nutlet surface analysis, Cohen ( 2014 ) indicated that smooth nutlets as ancestral for the clade that includes Boragineae and Lithospermeae tribes, and Nutlets with glochids as a synapomorphy for Cynoglosseae sensu lato (including tribe Rocheliea). The results of our study confirmed Cohen assertion that glochid is not ancestral trait but is a synapomorphy so that in node H, the transition to this trait occurred in the genus Lappula . Rocheliae is characterized by a transition from “without appendage” to “with tubercles and prickles” on the nutlet disk, and also by a shift from “lack of prickles” to “glossy prickles”. Also, a transition from “nonexpressiate” status of surface emergence to “stellar-aculeate” status occurred in this tribe. interestingly, in this tribe, smooth nutlets are a synapomorphy (Cohen 2014 ). Considering this point, the transition to “lack of appendage” that occurred in node G could indicate a synapomorphy of two species of sect. Sclerocaryum ( L. ceratophora and L. spinocarpus ). In Boraginaceae, nutlets need to develop strategies to achieve dispersal ability. In previous studies, nutlets with glochids or wings have implied adaptive traits for additional dispersal types, such as epizoochory or anemochory (Ma et al. 2010 ; Selvi et al. 2011 ). The presence of glochid on neutlet could be an explanation of the widespread geographic distribution of Cynoglosseae sensu lato (including Rochelieae) (Cohen 2014 ). According to Weigend et al. ( 2016 ), two species of sect. Sclerocaryum applies “the whole-plant dispersal by wind or flash-floods” as a dispersal mechanism which could explain the synapomorphy of “lack of appendage” observed in our results. Indeed, this mechanism caused the separation of nutlets from the mother plant to become unnecessary. Nutlet micromorphology results in this study provide no clear distinction among species L.barbata and L. microcarpa . The morphological complexities of these two species have already been addressed by different taxonomists (Popov 1953 ; Akhani 1998 ). A revision of the Lappula genus by Nasseh and Joharchi ( 2017 ) suggested that the two species may be synonymous and more molecular studies be conducted. Moreover, according to the molecular results (Khoshsokhan-Mozaffar et al. 2018 ) in the nr-DNA ITS tree of Rochelieae, the clade delimiting these two species was not well-supported. The variety observed in the nutlet of L.microcarpa and L.barbata could be related to seed heteromorphism that previously is known to occur in a few Boraginaceae genera Boraginaceae, e.g. Eritrichium, Lappula ( L. duplicicarpa and L. semiglabra ) and Heterocaryum (Wang et al. 1989 ). Conclusions In this study, the nutlet ultrastructure pattern of Rochelieae is systematically informative at the genus level, but not at the species level. The results showed that glochid is not an ancestral trait but is a synapomorphy and the transition to this trait occurred in the genus Lappula . Nutlet micromorphology results in this study provide no clear distinction among species L.barbata and L. microcarpa . Declarations Acknowledgements The authors wish to thank Saeed Javadi Anaghizi in Central laboratory of the Shahid Beheshti University for providing SEM pictures. We thank Dr. Somayeh Naghiloo (University of Calgary, Canada) for assisting in software analysis. Authors’ contributions SN designed the study project, ME & SN performed experiments and data analysis and drafted the manuscript. All authors read and approved the final manuscript. Funding This research received no specific grant. Availability of data and materials The data used and analyzed for the current study can be obtained from the corresponding author. Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. References Akhani H (1998) Plant biodiversity of Golestan National Park, Iran. Biologiezentrum des OÖ Landesmuseums Al-Shehbaz IA (1991) The genera of Boraginaceae in the southeastern United States. Journal of the Arnold Arboretum. Suppl Ser 1:1-169 Castresana J (2000) Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Mol biol evol 17:540-552 Chacón J, Luebert F, Hilger HH, Ovchinnikova S, Selvi F, Cecchi L, Guilliams CM, Hasenstab-Lehman K, Sutorý K, Simpson MG (2016) The borage family (Boraginaceae s. str.): A revised infrafamilial classification based on new phylogenetic evidence, with emphasis on the placement of some enigmatic genera. Taxon 65:523-546 Cohen JI (2014) A phylogenetic analysis of morphological and molecular characters of Boraginaceae: evolutionary relationships, taxonomy, and patterns of character evolution. Cladistics 30:139-169 de Candolle A (1845) Prodromus Systematis naturalis regni vegetabilis, Paris. Hilger H (1985) Ontogenie, Morphologie und systematische Bedeutung geflugelter und glochidientragender Cynoglosseae-und Eritricheae-Fruchte (Boraginaceae). Bot Jahrb Syst, Pflanzengesch Pflanzengeogr Hilger HH (2014) Ontogeny, morphology, and systematic significance of glochidiate and winged fruits of Cynoglosseae and Eritrichieae (Boraginaceae). Plant Divers Evol 131:167-214 Huang JF, Zhang ML, Cohen JI (2013) Phylogenetic analysis of Lappula Moench (Boraginaceae) based on molecular and morphological data. Plant syst evol 299:913-926 Johnston I (1937) Studies in the Boraginaceae XII. 2. Novelties and critical notes. J Arnold Arbor 18:10-25 Kahraman A, Celep F, Doğan M, Guerin GR, Bagherpour S (2011) Mericarp morphology and its systematic implications for the genus Salvia L. section Hymenosphace Benth.(Lamiaceae) in Turkey. Plant syst evol 292:33-39 Khoshsokhan-Mozaffar M, Sherafati M, Kazempour-Osaloo S (2018) Molecular phylogeny of the tribe Rochelieae (Boraginaceae, Cynoglossoideae) with special reference to Lappula. Ann Bot Fenn BioOne 293-308 Khoush SMM, Kazempour OS, Saadatmand S, Atar F (2010) Molecular phylogeny of Rochelia (Boraginaceae) based on nrDNA ITS and cpDNA trnL-F sequences. 22-29 Kumar S, Stecher G, Tamura K (2016) MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol biol evol 33:1870-1874 Långström E, Chase M (2002) Tribes of Boraginoideae (Boraginaceae) and placement of Antiphytum, Echiochilon, Ogastemma and Sericostoma: a phylogenetic analysis based on atpB plastid DNA sequence data. Plant Syst Evol 234:137-153 Lehmann JGC (1818) Plantae e familia Asperifoliarum nuciferae. Dummler, Berlin, p 78 Lewis PO (2001) A likelihood approach to estimating phylogeny from discrete morphological character data. Syst biol 50:913-925 Ma W, Zhao X, Tan D, Baskin C, Baskin J, Xue J (2010) Nutlet dimorphism in individual flowers of two cold desert annual Lappula species (Boraginaceae): implications for escape by offspring in time and space. Plant ecol 209:361-374 Maddison W, Maddison D (2015) Mesquite: a modular system for evolutionary analysis. Version 3.04. 2015 Moon HK, Hong SP (2006) Nutlet morphology and anatomy of the genus Lycopus (Lamiaceae: Mentheae). J Plant Res 119:633-644 Mozaffar MK, Osaloo SK, Oskoueiyan R, Saffar KN, Amirahmadi A (2013) Tribe Eritrichieae (Boraginaceae s. str.) in West Asia: a molecular phylogenetic perspective. Plant Syst Evol 299:197-208 Nasseh Y, Joharchi MR (2017) Revision of the genus Lappula Moench based on morphological characters in Khorassan Provinces (Iran). Nova Biol Rep 4:66-73 Ovchinnikova S (2005) The system of the subtribe Echinosperminae (Boraginaceae). Bot Zhurn 90:1153-1172 Ovchinnikova S (2009) On the position of the tribe Eritrichieae in the Boraginaceae system. Bot Serbica 33:141-146 Popov M (1953) Boraginaceae. Flora SSSR 19:97-691 Riedl H (1967) Boraginaceae in, Rechinger KH. Flora Iranica. Graz, Akademische Druck_u. Verlagdanstalt 48:215 Riedl H (1996) Studies in the genus Lappula (Boraginaceae) I. Lappula in the" Flora Iranica" region. Serie B für Botanik und Zoologie. Ann Naturhist Mus 79-86 Rolfsmeier SJ (2013) Taxonomy and phylogeny of the genus Lappula Moench (Boraginaceae) in North America. Kansas State University. Saadati N, Mozaffar MK, Sherafati M, Osaloo SK (2017) Pseudoheterocaryum, a new genus segregated from Heterocaryum (Boraginaceae) on the basis of molecular data. Aust Syst Bot 30:105-111 Selvi F, Bigazzi M, Hilger HH, Papini A (2006) Molecular phylogeny, morphology and taxonomic re‐circumscription of the generic complex Nonea/Elizaldia/Pulmonaria/Paraskevia (Boraginaceae‐Boragineae). Taxon 55:907-918 Selvi F, Coppi A, Cecchi L (2011) High epizoochorous specialization and low DNA sequence divergence in Mediterranean Cynoglossum (Boraginaceae): Evidence from fruit traits and ITS region. Taxon 60:969-985 Silvestro D, Michalak I (2012) raxmlGUI: a graphical front-end for RAxML. Org Divers Evol 12:335-337 Weigend M, Gottschling M, Selvi F, Hilger HH (2009) Marbleseeds are gromwells–Systematics and evolution of Lithospermum and allies (Boraginaceae tribe Lithospermeae) based on molecular and morphological data. Mol Phylogenetics Evol 52:755-768 Weigend M, Luebert F, Selvi F, Brokamp G, Hilger HH (2013) Multiple origins for Hound’s tongues (Cynoglossum L.) and Navel seeds (Omphalodes Mill.)–The phylogeny of the borage family (Boraginaceae s. str.). Mol phylogenetics evol 68:604-618 Weigend M, Selvi F, Thomas D, Hilger H (2016) Boraginaceae. Flowering Plants. Eudicots. Springer, pp. 41-102 Yu WT, Jacques FM, Chen ST, Zhou ZK (2012) Nutlet micro‐morphology of the genus Microula (Boraginaceae) from the Qinghai–Tibetan Plateau, and its systematic implications. Nord J Bot 30:596-612 Wang WC, Liu YL, Zhu GL, Lian YSh, Wang JQ, Wang QR (1989) Lappula V. Wolf. In: Kong XW, Wang WC (eds) Flora Reipublicae Popularis Sinicae, vol 64. Science Press, Beijing, pp 177-207 (in Chinese) Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Major revision 01 Jul, 2021 Review # 1 received at journal 24 Jun, 2021 Reviewer # 1 agreed at journal 13 Jun, 2021 Reviews received at journal 13 Jun, 2021 Reviewers invited by journal 25 May, 2021 Editor assigned by journal 12 May, 2021 Submission checks completed at journal 12 May, 2021 Editor invited by journal 12 May, 2021 First submitted to journal 12 May, 2021 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-521851","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Original Article","associatedPublications":[],"authors":[{"id":27083685,"identity":"0f2f968d-e640-4f41-8351-3a7e286f678c","order_by":0,"name":"Mostafa Ebadi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3klEQVRIiWNgGAWjYDACCSBKqJCQk2dvYGAmQcsZG2PDngOkaGFsS0tsuJFApBZ+6eaHNx6wHWZsnPnG8HNBhQ0Df3t3Al4tknOOGVsk8BxmZpfOMZaecSaNQeLM2Q14tRjcSDCTSJA4zMY4O8dAmrftMIOBRC5+LfY30r9JJBgc5mG4ecb4N1FaDCRygLYkpEkw3OAxI84WiTtnii0SDtgYGPaklVnznEnjIegX/tntG2/+/CdRP5/98ObbPBU2cvztvfi1IAEOAxDJQ6xyEGB/QIrqUTAKRsEoGEEAACcIRqpUVMMDAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0003-1123-7102","institution":"Azarbaijan Shahid Madani University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Mostafa","middleName":"","lastName":"Ebadi","suffix":""},{"id":27083686,"identity":"36a50f1a-a02b-4ae1-aac9-2421fb2d4803","order_by":1,"name":"Sedigheh Nikzat","email":"","orcid":"","institution":"Shahid Beheshti University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Sedigheh","middleName":"","lastName":"Nikzat","suffix":""}],"badges":[],"createdAt":"2021-05-13 03:54:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-521851/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-521851/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":9263059,"identity":"22a03cc5-2cae-4528-97a0-945f3f1ee517","added_by":"auto","created_at":"2021-05-17 17:49:32","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":81250,"visible":true,"origin":"","legend":"Six types of nutlet morphology in the studied species.","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-521851/v1/e9cbc8b6c76a0ff42462be56.jpg"},{"id":9263436,"identity":"a8ca3103-3136-4657-a865-11718cf85c0f","added_by":"auto","created_at":"2021-05-17 17:52:32","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":99179,"visible":true,"origin":"","legend":"Results of nutlet character evolution shown on the Maximum Likelihood tree of tribe Rochelieae (based on the internal transcribed spacer). Numbers on branches are Maximum Likelihood bootstrap support (only shown when ≥ 65).","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-521851/v1/3e91cbd7fb45fb7cb4b4cfb5.jpg"},{"id":9263781,"identity":"b4f5d1f6-87ba-4677-8d94-8592ba203ca2","added_by":"auto","created_at":"2021-05-17 17:55:32","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":205969,"visible":true,"origin":"","legend":"nutlet morphology in the different specimens of Lappula microcarpa and lappula barbata. The details of the studied specimens are in accordance to the Table (L. microcarpa codes: b, e, I, g, p, z, a; L. barbata: c, m, Ab, w)","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-521851/v1/daec823975d1c28e73509c6e.jpg"},{"id":9263062,"identity":"af47efae-8e30-4abd-b65b-ff6ef2be5ea5","added_by":"auto","created_at":"2021-05-17 17:49:32","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":44184,"visible":true,"origin":"","legend":"WARD dendrogram showing the relationship among different specimens belong to L. microcarpa and L. barbata based on nutlet characters.","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-521851/v1/511fba78d80feb845c5c6666.jpg"},{"id":13693405,"identity":"486bfbac-48eb-4a5c-bf62-3812147fcbd1","added_by":"auto","created_at":"2021-09-17 12:47:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":670099,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-521851/v1/fd67b14f-cf5b-4ba3-b0ac-a9c32b9343eb.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eNutlet Micromorphology and Character Evolution of Some Species of Rochelieae (Boraginaceae) and Its Systematic Implications\u003c/p\u003e","fulltext":[{"header":"Background","content":" \u003cp\u003eBoraginaceae s.str. is a sub cosmopolitan family of flowering plants with nearly 90 genera and ca.1600 to 1700 species distributed worldwide (Chac\u0026oacute;n et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Based on the recent molecular phylogeny presented by Chac\u0026oacute;n et al. (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), infrafamilial classification of Boraginaceae was recognized into three Subfamilies (Echiochiloideae, Boraginoideae, Cynoglossoideae) and 10 tribes (Boragineae, Lithospermeae, Trichodesmeae, Lasiocaryeae, Asperugeae, Omphalodeae, Rochelieae, Craniospermeae, Myosotideae, Cynoglosseae). Tribe Rochelieae consists of five genera (\u003cem\u003eEritrichium\u003c/em\u003e, \u003cem\u003eHackelia\u003c/em\u003e, \u003cem\u003eLappula\u003c/em\u003e, \u003cem\u003eLepechiniella\u003c/em\u003e, and \u003cem\u003eRochelia\u003c/em\u003e) and about 207 species and belongs to the subfamily Cynoglossoideae. According to Chac\u0026oacute;n et al. (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), Rochelieae can be divided into two subtribes including Eritrichiinae and Heterocaryinae. The genera \u003cem\u003eEritrichium\u003c/em\u003e, \u003cem\u003eHackelia\u003c/em\u003e, \u003cem\u003eLappula\u003c/em\u003e, \u003cem\u003eLepechiniella\u003c/em\u003e, and \u003cem\u003eRochelia\u003c/em\u003e, belong to sub-tribes Eritrichiinae and the genera \u003cem\u003eHeterocaryum\u003c/em\u003e, \u003cem\u003eSuchtelenia\u003c/em\u003e, and \u003cem\u003ePseudoheterocaryum\u003c/em\u003e belong to sub-tribe Heterocaryinae (Chac\u0026oacute;n et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Saadati et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Three genera in subtribe Eritrichiinae (\u003cem\u003eEritrichium\u003c/em\u003e, \u003cem\u003eLepechiniella\u003c/em\u003e, and \u003cem\u003eLappula\u003c/em\u003e) are recognized as non-monophyletic lineage and \u003cem\u003eHackelia\u003c/em\u003e and \u003cem\u003eRochelia\u003c/em\u003e comprise monophyletic clades (Khoshsokhan-Mozaffar et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The recent molecular phylogeny of Rochelieae tribe provided by Khoshsokhan-Mozaffar et al. (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) indicated a well-supported clade. \u003cem\u003eHackelia\u003c/em\u003e and \u003cem\u003eRochelia\u003c/em\u003e are monophyletic while \u003cem\u003eLappula, Eritrichium\u003c/em\u003e, and \u003cem\u003eLepechiniella\u003c/em\u003e are not. So that \u003cem\u003eLappula\u003c/em\u003e, as currently circumscribed, is polyphyletic (Khoshsokhan-Mozaffar et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The genus \u003cem\u003eEritrichium\u003c/em\u003e is the largest genus of the tribe, with 71 species. The genus \u003cem\u003eLappula\u003c/em\u003e contains about 70 species of annual, biennial perennial herbs distributed in Eurasia, Africa, North America, and Australia (Ovchinnikova \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Although \u003cem\u003eLappula\u003c/em\u003e has a cosmopolitan distribution, the center of diversity is in Siberian and Irano-Turaniean provinces of the Holarctic kingdom (Ovchinnikova \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Initially, Lehmann (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1818\u003c/span\u003e) circumscribed 15 species in \u003cem\u003eEchinospermum\u003c/em\u003e Lehm. (Synonym of \u003cem\u003eLappula\u003c/em\u003e). \u003cem\u003eEchinospermum\u003c/em\u003e was divided by de Candolle (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1845\u003c/span\u003e) into three sections based on nutlet morphology as follows: \u003cem\u003eLappula\u003c/em\u003e, \u003cem\u003eSclerocaryum\u003c/em\u003e, and \u003cem\u003eHomalocaryum\u003c/em\u003e. The taxonomical problems of \u003cem\u003eLappula\u003c/em\u003e were increased when the number of species in the genus began to expand. Consequently, the reported number of sections, subsections, and series has widely varied by different authors (Popov \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1953\u003c/span\u003e; Riedl \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1967\u003c/span\u003e). For the first time, Lehmann (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1818\u003c/span\u003e) used nutlet characters in the systematics of \u003cem\u003eLappula\u003c/em\u003e and showed mericarp characters such as mericarp shape and surface ornamentation to distinguish the species of this genus. Generally, in Boraginaceae, nutlet morphology provides useful systematic characters at various taxonomic levels, such as straight or incurved nutlet, a specialized form of emergence, the position of attachment scar, the distinctive form of prickles or glochids, and epidermal features of nutlets (Johnston \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1937\u003c/span\u003e; Hilger \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1985\u003c/span\u003e; Al-Shehbaz \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1991\u003c/span\u003e; Riedl \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; L\u0026aring;ngstr\u0026ouml;m and Chase \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Moon and Hong \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Selvi et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Kahraman et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe main goal of this study was to investigate the various varieties of nutlet morphological characters of the tribe Rochelieae s.l. using scanning electron microscopy and the nutlet character evolution with more focus on the \u003cem\u003eLappula\u003c/em\u003e genus.\u003c/p\u003e "},{"header":"Methods","content":" \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMorphological study\u003c/h2\u003e \u003cp\u003eThe plants used in this study were collected from their natural habitats in Iran and deposited in the Herbarium of Azarbaijan Shahid Madani University (ASMUH). Also, a small number of species were taken from herbarium specimens of FUMH (Ferdowsi University of Mashhad Herbarium). The list of voucher specimens and details of localities were given in Table.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e \u003ccolgroup cols=\"3\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003eTable. List of sampled taxa, locality and their vouchers numbers.\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpecies (Pop. Code)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLocality\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eVoucher No.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eLappula barbata\u003c/em\u003e (M.Bieb.) G\u0026uuml;rke (m)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTehran, Chalus road, Kooshk\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eASMUH0020\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eL. barbata\u003c/em\u003e (c)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMazandaran, Chalus, Delir vilage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eASMUH0021\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eL. barbata\u003c/em\u003e (ab)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMazandaran, Noshahr, Kojur, Laregan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eASMUH0022\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eL. barbata\u003c/em\u003e (w)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTehran, Tuchal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eASMUH0023\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eL. ceratophora\u003c/em\u003e (Popov) Popov\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSouth Khorasan, south-west Sarayan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFUMH46077\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eL. microcarpa\u003c/em\u003e (Ledeb.) G\u0026uuml;rke (b)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMazandaran. Noor, Chamestan, Lavij\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eASMUH0024\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eL. microcarpa\u003c/em\u003e (e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMazandaran, Neka, Hezarjerib\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eASMUH0025\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eL. microcarpa\u003c/em\u003e (g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGolestan, East of Golestan national park\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eASMUH0026\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eL. microcarpa\u003c/em\u003e (i)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMazandaran, Savadkooh, Veresk\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eASMUH0027\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eL. microcarpa\u003c/em\u003e (j)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMazandaran, Polor to Rine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eASMUH0028\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eL. microcarpa\u003c/em\u003e (p)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMazandaran, Noshahr, Kojur,\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eASMUH0029\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eL. microcarpa\u003c/em\u003e (z)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNorth Khorasan, Chamanbid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eASMUH0030\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eL. microcarpa\u003c/em\u003e (a)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTehran, Lavasan, Glucan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eASMUH0031\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eL. semiglabra\u003c/em\u003e (Ledeb.) G\u0026uuml;rke\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKhorasan Razavi, North of Gonabad\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFUMH17236\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eL. sessiliflora\u003c/em\u003e (Boiss.) G\u0026uuml;rke\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKhorasan Razavi, East of Kashmar\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFUMH26636\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eL. spinocarpus\u003c/em\u003e (Forssk.) Asch. ex Kuntze\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSouth Khorasan, Birjand, Shahzile\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFUMH30399\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePseudolappula sinaica\u003c/em\u003e (A.DC.) Asch. \u0026amp; Schweinf.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTehran, Chalus road, Morod\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eASMUH0032\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAsperugo procumbens\u003c/em\u003e L.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMazandaran, Damavand, Sarbandan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eASMUH0034\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eHeterocaryum rigidum\u003c/em\u003e A. DC.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTehran, Jajroad\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eASMUH0035\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMyosotis sylvatica\u003c/em\u003e Ehrh.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMazandaran, Sari, Sangdeh forest\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eASMUH0036\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eRochelia disperma\u003c/em\u003e (L. f) C. Koch\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTehran, Lavasan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eASMUH0037\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThis study was conducted on 9 species of tribe Rochelieae (covering five of the six genera) and two species of tribes Asperugeae and Myosotideae as out-groups. The mature nutlets were selected from materials as herbarium samples. Then air dried nutlets were investigated for shape, size, and other features using stereomicroscope (Dino-Lite) with the help of DinoCapture eye and DinoCapture 2.0 Software (Electronics Corporation).\u003c/p\u003e \u003cp\u003eFor SEM observations, the nutlets were mounted onto standard aluminum stubs using double-sided adhesive tape and then photographed using a PHILIPS / FEI XL 20 Scanning Electron Microscope at 15 KV voltages. The measurements are based on 15\u0026ndash;20 evaluations from each specimen.\u003c/p\u003e \u003cp\u003eThe terminology used to describing the qualitative characters is in accordance with Ma et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Selvi et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Yu et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Hilger \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2014\u003c/span\u003e. The data were analyzed and examined by WARD dendrogram using PAST software for species decimation of \u003cem\u003eL. microcarpa\u003c/em\u003e and \u003cem\u003eL.barbata\u003c/em\u003e. Due to high morphological similarities of \u003cem\u003eL. microcarpa\u003c/em\u003e and \u003cem\u003eL.barbata\u003c/em\u003e, to identify them, the Flora of Iran, (Nasseh and Joharchi \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) and also \u003cem\u003eFlora Iranica\u003c/em\u003e (Riedl \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1996\u003c/span\u003e) were used.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenetic analyses and tracing character evolution\u003c/h2\u003e \u003cp\u003eThe sequences for the internal transcribed spacer (ITS) region have been obtained from GenBank. Sequence alignments were performed using MUSCLE by the MEGA software ver.7 (Kumar et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Poorly aligned positions and divergent regions were eliminated by using Gblocks 0.91b, following the given options for less stringency (Castresana \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Phylogenetic analyses were performed using the combined 3-loci data set. The partitioned ML analysis was performed using raxmlGUI 1.1 (Silvestro and Michalak \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) under the GTR\u0026thinsp;+\u0026thinsp;G model with 1000 bootstrap replicates and with \u003cem\u003eAsperugo\u003c/em\u003e (tribe Asprugeae) and \u003cem\u003eMyosotis (\u003c/em\u003etribe Myosotideae\u003cem\u003e)\u003c/em\u003e chosen as out-group.\u003c/p\u003e \u003cp\u003eThe evolutionary history of characters was traced over an ML tree in Mesquite 3.04 (Maddison and Maddison \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The ML approach with the Markov k-state one-parameter (Mk1) model was applied (Lewis \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2001\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e "},{"header":"Result","content":" \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eGeneral description of nutlet micromorphology\u003c/h2\u003e \u003cp\u003eThe nutlet's morphology and ultrastructure characteristics such as shape size, appendages, and surface sculpturing, varied among the studied taxa. The Rochelieae nutlets' shape was ovoid (ovoid, ovoid-triangular, and ovoid-rectangular) to the pyramid (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). As the out-groups, the shape of \u003cem\u003eAsperugo procumbense\u003c/em\u003e was semicircular, and the \u003cem\u003eMyosotis sylvatica\u003c/em\u003e one was an ellipse. These two genera belong to Asperugeae and Myosotideae tribes, respectively.\u003c/p\u003e \u003cp\u003eNine qualitative characters including shape, the centerline of the raphe, base surface of prickles on the desk, the arrangement of glochids, the type of lamellae, tubercles on the desk, appendages on nutlet desk, Number of glochid rows on nutlet edge, and emergence type were selected for morphological evaluation of nutlets. The results obtained from nutlet-ultrastructure investigations are described below and illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Generally, six different surface types were recognized between studied taxa based on nutlet ultrastructure characters as follow:\u003c/p\u003e \u003cp\u003e \u003cb\u003eType I\u003c/b\u003e: \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eHeterocaryum\u003c/span\u003e \u003cb\u003eand\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003ePseudolappula\u003c/span\u003e \u003cb\u003e(Syn\u003c/b\u003e: \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eL. siniaca\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThere is no glochid or appendage on the nutlet disk, but there is a row of glochid (\u003cem\u003eHeterocaryum\u003c/em\u003e) or glochid-like (\u003cem\u003ePseudolappula\u003c/em\u003e) on the nutlet edge. The glochids are distributed in very low numbers in the edges of the nutlets of \u003cem\u003ePseudolappula\u003c/em\u003e. The nutlet disk ornament of \u003cem\u003eHeterocaryum\u003c/em\u003e is \u0026ldquo;papilla verrucose with verrucae minutely muricate\u0026rdquo; (called complex papilla) while it is \u0026ldquo;papilla with aggregate verrucose in the center\u0026rdquo; in \u003cem\u003ePseudolappula\u003c/em\u003e. It appears that each of the microcapillaries found in \u003cem\u003ePsudolappula\u003c/em\u003e has become complex in the \u003cem\u003eHeterocaryum\u003c/em\u003e, and each has formed warts (verrucose) that have become more complex and denser.\u003c/p\u003e \u003cp\u003e \u003cb\u003eType II\u003c/b\u003e: \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eLappula\u003c/span\u003e (\u003cem\u003eL. barbata\u003c/em\u003e, \u003cem\u003eL. microcarpa\u003c/em\u003e and \u003cem\u003eL. semiglabra\u003c/em\u003e)\u003c/p\u003e \u003cp\u003eGlochids in different sizes and rows can be seen in the nutlet edge and sometimes on the nutlet disk surface. Glochids have an anchor with 2\u0026ndash;4 branches at the apex, and the surface of the glochids is smooth. The ultrastructure of the nutlet emergencies is stellar-aculeate, and sometimes the appendages are prickles or tubercles are seen (scattered or collected) on the surface of the nutlet disk and edge. The glochid stem is composed of fusiform cells, and there are tubercles with 2 to 5 mineralized spines on the stem. These tubercles are also present on the entire surface of the nutlet with a different distribution.\u003c/p\u003e \u003cp\u003e \u003cb\u003eType III\u003c/b\u003e: \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eRochelia\u003c/span\u003e (\u003cem\u003eR. disperma\u003c/em\u003e, \u003cem\u003eR. sessiflora\u003c/em\u003e\u0026thinsp;=\u0026thinsp;\u003cem\u003eL. sessiflora\u003c/em\u003e)\u003c/p\u003e \u003cp\u003eThe prickles are stellate and are scattered throughout the surface of the nutlet. The surface of the prickles is not glossy and has verrucose. The tubercles often have more than 2 spines, and the emergencies are stellar-aculeate (similar to type II). Although the nutlet surface of \u003cem\u003eR. sessiflora\u003c/em\u003e is similar to type II (presence of glochid on the nutlet edge). Moreover, the prickles and verrucose on it and the accumulation of tubercles with more than 5 spines around each prickle show more similarity to type III.\u003c/p\u003e \u003cp\u003e \u003cb\u003eType IV\u003c/b\u003e: \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eL. ceratophora\u003c/span\u003e \u003cb\u003eand\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eL. spinocarpus\u003c/span\u003e\u003c/p\u003e \u003cp\u003eThere is not any glochid, tubercle, or prickles on the nutlet surface. The nutlet surface of \u003cem\u003eL. ceratophora\u003c/em\u003e is not smooth, and the papilla appears as a verrucose-like. While the ultrastructure of the nutlet in \u003cem\u003eL. spinocarpus\u003c/em\u003e seems papilla with flowerlike verrucose. Also, the tubercles appear as verrucose and lack any spines.\u003c/p\u003e \u003cp\u003e \u003cb\u003eType V\u003c/b\u003e: \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eAsperugo\u003c/span\u003e\u003c/p\u003e \u003cp\u003eThe nutlet surface lacks any glochid and prickles. Papilla appears as dome-shaped in different sizes, and it is verrucose at the base of them.\u003c/p\u003e \u003cp\u003e \u003cb\u003eType VI\u003c/b\u003e: \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eMyosotis\u003c/span\u003e\u003c/p\u003e \u003cp\u003eThe surface of the nutlet is smooth, and there is not any ornamentation (nonexpressiate).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ea\u003c/b\u003e\u0026ndash;\u003cb\u003eh\u0026thinsp;=\u0026thinsp;Type I; (a-d)\u003c/b\u003e \u003cem\u003eHeterocaryum rigidum\u003c/em\u003e; \u003cb\u003ea, b\u003c/b\u003e: An overview photograph of nutlet with stereomicroscope and SEM. \u003cb\u003ec, d\u003c/b\u003e: The close up views of nutlet disk with \u0026ldquo;papilla verrucose with verrucae minutely muricate\u0026rdquo;. \u003cb\u003e(e-h)\u003c/b\u003e \u003cem\u003ePseudolappula siniaca\u003c/em\u003e; \u003cb\u003ee, f\u003c/b\u003e: An overview photograph of nutlet with stereomicroscope and SEM. \u003cb\u003eg, h\u003c/b\u003e: The close up views of nutlet disk with \u0026ldquo;papilla with aggregate verrucose in center\u0026rdquo;.\u003c/p\u003e \u003cp\u003e \u003cb\u003ei-o Type II\u003c/b\u003e. (\u003cb\u003ei, j)\u003c/b\u003e \u003cem\u003eLappula. barbata\u003c/em\u003e; \u003cb\u003e(k, l)\u003c/b\u003e \u003cem\u003eLappula. Microcarpa\u003c/em\u003e; (The more detail of these two species were described between different specimens in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). \u003cem\u003eLappula semiglabra\u003c/em\u003e \u003cb\u003em, n\u003c/b\u003e: An overview photograph of nutlet with stereomicroscope and SEM. \u003cb\u003eo\u003c/b\u003e: The close up views of nutlet disk.\u003c/p\u003e \u003cp\u003e \u003cb\u003ep-v\u0026thinsp;=\u0026thinsp;Type III; (p-r)\u003c/b\u003e \u003cem\u003eRochelia sessiflora\u003c/em\u003e; \u003cb\u003ep, q\u003c/b\u003e: An overview photograph of nutlet with stereomicroscope and SEM. \u003cb\u003er\u003c/b\u003e: The close up views of nutlet disk with prickles and verrucose on it. \u003cb\u003e(s-v)\u003c/b\u003e \u003cem\u003eRochelia disperma\u003c/em\u003e \u003cb\u003es, t\u003c/b\u003e: An overview photograph of nutlet with stereomicroscope and SEM. \u003cb\u003eu, v\u003c/b\u003e: The close up views of stellare-aculeate emergencies in nutlet disk.\u003c/p\u003e \u003cp\u003e \u003cb\u003ew-ac\u0026thinsp;=\u0026thinsp;Type IV; (w-y)\u003c/b\u003e \u003cem\u003eLappula spinocarpus\u003c/em\u003e; \u003cb\u003ew, x\u003c/b\u003e: An overview photograph of nutlet with stereomicroscope and SEM. \u003cb\u003ey\u003c/b\u003e: The close up views of nutlet disk with papilla with flowerlike verrucose on it. \u003cb\u003e(z-ab)\u003c/b\u003e \u003cem\u003eLappula ceratophora\u003c/em\u003e \u003cb\u003ez, aa\u003c/b\u003e: An overview photograph of nutlet with stereomicroscope and SEM. \u003cb\u003eab\u003c/b\u003e: The close up views of nutlet disk with papilla appears as a verrucose-like on it.\u003c/p\u003e \u003cp\u003e \u003cb\u003eac-af\u0026thinsp;=\u0026thinsp;Type V;\u003c/b\u003e \u003cem\u003eAsperugo procumbense\u003c/em\u003e; \u003cb\u003eac-ad\u003c/b\u003e: the overview photograph of nutlet with stereomicroscope and SEM. \u003cb\u003eaf\u003c/b\u003e: The close up views of nutlet disk with Papilla appears as dome-shape on it.\u003c/p\u003e \u003cp\u003e \u003cb\u003eag-aj\u0026thinsp;=\u0026thinsp;Type VI;\u003c/b\u003e \u003cem\u003eMyosotis sylvatica\u003c/em\u003e; \u003cb\u003eag-ai\u003c/b\u003e: the overview photograph of nutlet with stereomicroscope and SEM. \u003cb\u003eaj\u003c/b\u003e: The close up views of nutlet disk with smooth surface.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Evolution of microstructural characters of nutlet\u003c/h2\u003e \u003cp\u003eThe resulting ancestral state reconstruction and the proportional likelihoods for character states are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The out-group species \u003cem\u003eAsperugo\u003c/em\u003e and \u003cem\u003eMyosotis\u003c/em\u003e were unique regarding the bilaterally flattened and ellipse with a smooth surface, respectively. Tracing the evolution of nutlet micromorphology indicated that the glochids were not an ancestral character.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e\u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eArrangement of glochid character\u003c/span\u003e:\u003c/h2\u003e \u003cp\u003eThe status of ancestral taxa (with or without glochids) was unclear, and the proportional likelihoods of any three characters were almost equal (node A). Transition to the glochids character occurs in the genus \u003cem\u003eLappula\u003c/em\u003e (\u003cem\u003eL. semiglabra\u003c/em\u003e, \u003cem\u003eL. microcarpa\u003c/em\u003e, and \u003cem\u003eL. barbata\u003c/em\u003e) (node H).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e\u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eThe appendage on nutlet disk character\u003c/span\u003e:\u003c/h2\u003e \u003cp\u003eThe status \u0026ldquo;without appendage\u0026rdquo; and \u0026ldquo;dump-shape papilla\u0026rdquo; in \u003cem\u003eMyosotis\u003c/em\u003e and \u003cem\u003eAsperugo\u003c/em\u003e (the proportional likelihoods 1) were differentiated these two tribes from each other and Rocheliea tribe. The tubercle and prickles on the disk were ancestral characters (the proportional likelihoods 0.43). While the ancestor of these characters is unclear in node A, the status \u0026ldquo;tubercle and prickles on disk\u0026rdquo; had more proportional likelihoods in A group and then C, D, F. Transition to the \u0026ldquo;lack of appendage\u0026rdquo; status occurred in the \u003cem\u003eL. ceratophora\u003c/em\u003e, \u003cem\u003eL. spinocarpus;\u003c/em\u003e node G).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e\u003cem\u003ePrickles surface character\u003c/em\u003e:\u003c/h2\u003e \u003cp\u003eTracing of character \u0026ldquo;prickles surface\u0026rdquo; showed the ancestral status of \u0026ldquo;lack of prickles\u0026rdquo; in node A that to be followed with less proportionality in nodes B, C, and D. Transition to the \u0026ldquo;glossy prickles\u0026rdquo; status occurred in the genus \u003cem\u003eLappula\u003c/em\u003e in node H. Moreover, the transition to the simple and complex \u0026ldquo;verrucose prickles\u0026rdquo; status was observed in genus \u003cem\u003eRochelia\u003c/em\u003e in node F.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003eSurface emergence:\u003c/h2\u003e \u003cp\u003eTracing of surface emergence character was unclear in node A. However, the proportional likelihoods of \u0026ldquo;stellar-aculeate\u0026rdquo; status had the highest node C ratio (0.72).\u003c/p\u003e \u003cp\u003eIn node G, the transition to \u0026ldquo;verrucose-subverrucose\u0026rdquo; status (the proportional likelihoods 0.99) was stabilized as a synapomorphy.\u003c/p\u003e \u003cp\u003eOther traits were studied regarding evolutionary tracing that did not show clear evolutionary signals in the nodes, such as the shape of the nutlet, the lamella type, the shape of the nutlet, the lamella type, and the centerline of the nutlet disk.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.3. Close boundary of nutlet microstructures between\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eL. barbata\u003c/span\u003e \u003cb\u003eand\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eL. microcarpa\u003c/span\u003e\u003c/p\u003e \u003cp\u003eBoth \u003cem\u003eL. microcarpa\u003c/em\u003e and \u003cem\u003eL.barbata\u003c/em\u003e had high micro-morphological similarities (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Different clustering and ordination methods produced similar results; therefore, only WARD tree of micro-morphological characters is presented here (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). In general, plant samples of each species did not group and formed a separate group. This result shows that the micro-morphological characters studied could not delimit these two species.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e "},{"header":"Discussion","content":" \u003cp\u003eAs in other Boraginaceae genera (Ovchinnikova \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Weigend et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Selvi et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Yu et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Hilger \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), the infrageneric taxonomic significance of nutlet characteristics in tribe Rochelieae were found to be obvious when investigated under stereomicroscope and SEM. In this study, it was found that the appendages on the nutlet varied between different genera. Although the fruit type of Boraginaceae is relatively constant, the variation in nutlet ornamentation has quickly occurred in some tribes like Cynoglosseae sensu lato (including tribe Rocheliea) and Trichodesmeae (Cohen \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). It is not surprising that \u003cem\u003eLappula\u003c/em\u003e, one of the largest genera in Rochelieae, shows considerable diversity in nutlet characters. Recent molecular evidence suggested that \u003cem\u003eLappula\u003c/em\u003e is polyphyletic (Khoshsokhan-Mozaffar et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In this way, Khoshsokhan-Mozaffar et al. indicated that species of the \u003cem\u003eLappula\u003c/em\u003e genus are scattered across the Eritrichiinae clade and forming three distinct lineages. \u003cem\u003eLappula sinaica\u003c/em\u003e, as a new genus, was segregated from \u003cem\u003eLappula\u003c/em\u003e and established as genus \u003cem\u003ePseudolappula\u003c/em\u003e (Khoshsokhan-Mozaffar et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Moreover, as shown in our study, a distinct generic delimitation based on nutlet characters alone could be detected for \u003cem\u003ePseudolappula\u003c/em\u003e (syn: \u003cem\u003eLappula siniaca\u003c/em\u003e). Nutlet micromorphology of \u003cem\u003ePseudolappula\u003c/em\u003e (syn: \u003cem\u003eL. siniaca\u003c/em\u003e) provides valuable data in separating it from \u003cem\u003eLappula\u003c/em\u003e genus; these characters are \u0026ldquo;no glochid or appendage on nutlet disk\u0026rdquo; and the type of nutlet ornamentation including \u0026ldquo;papilla with aggregate verrucose in the center\u0026rdquo;. The features of disk ornamentation indicated more close affinity of \u003cem\u003ePseudolappula siniaca\u003c/em\u003e to \u003cem\u003eHeterocaryum\u003c/em\u003e than \u003cem\u003eLappula\u003c/em\u003e genus, especially in the evolution of microcapillaries.\u003c/p\u003e \u003cp\u003eCurrent data are also in agreement with molecular phylogenies provided by Khoshsokhan-Mozaffar et al. (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) that transferred closely related species \u003cem\u003eL. sessiliflora\u003c/em\u003e to \u003cem\u003eRochelia\u003c/em\u003e genus. This conclusion has previously been suggested by various studies (Khoush et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Huang et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Mozaffar et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Rolfsmeier \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Weigend et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Moreover, the flowers and nutlet features (two of them undeveloped) indicated more affinity of the species to \u003cem\u003eRochelia\u003c/em\u003e genus than \u003cem\u003eLappula\u003c/em\u003e (Popov 1974). Moreover, our result indicated the features like prickles and verrucose on the nutlet and the accumulation of tubercles with more than five spines around each prickle show more similarity to genus \u003cem\u003eRochelia\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eOur study convincingly provided a clear distinction between two species \u003cem\u003eL. ceratophora\u003c/em\u003e and \u003cem\u003eL. spinocarpus\u003c/em\u003e belonging to sect. \u003cem\u003eSclerocaryum\u003c/em\u003e (Riedl \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1967\u003c/span\u003e; Ovchinnikova \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) from other \u003cem\u003eLappula\u003c/em\u003e genus. The lack of glochid, tubercle, or prickles on the nutlet surface and specific types of nutlet ornamentation (papilla with verrucose like or flowerlike-verrucose) characterize the clade that includes them regarding recent molecular phylogenetic analysis (Khoshsokhan-Mozaffar et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWe identified four different types of nutlet surface ornamentation among studied taxa. According to our study and the other available record (Cohen \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), the ancestral type is ambiguous for the family and Rochelieae tribe. Given the matrix of cpDNA and nutlet surface analysis, Cohen (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) indicated that smooth nutlets as ancestral for the clade that includes Boragineae and Lithospermeae tribes, and Nutlets with glochids as a synapomorphy for Cynoglosseae sensu lato (including tribe Rocheliea). The results of our study confirmed Cohen assertion that glochid is not ancestral trait but is a synapomorphy so that in node H, the transition to this trait occurred in the genus \u003cem\u003eLappula\u003c/em\u003e. Rocheliae is characterized by a transition from \u0026ldquo;without appendage\u0026rdquo; to \u0026ldquo;with tubercles and prickles\u0026rdquo; on the nutlet disk, and also by a shift from \u0026ldquo;lack of prickles\u0026rdquo; to \u0026ldquo;glossy prickles\u0026rdquo;. Also, a transition from \u0026ldquo;nonexpressiate\u0026rdquo; status of surface emergence to \u0026ldquo;stellar-aculeate\u0026rdquo; status occurred in this tribe. interestingly, in this tribe, smooth nutlets are a synapomorphy (Cohen \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Considering this point, the transition to \u0026ldquo;lack of appendage\u0026rdquo; that occurred in node G could indicate a synapomorphy of two species of sect. \u003cem\u003eSclerocaryum\u003c/em\u003e (\u003cem\u003eL. ceratophora\u003c/em\u003e and \u003cem\u003eL. spinocarpus\u003c/em\u003e).\u003c/p\u003e \u003cp\u003eIn Boraginaceae, nutlets need to develop strategies to achieve dispersal ability. In previous studies, nutlets with glochids or wings have implied adaptive traits for additional dispersal types, such as epizoochory or anemochory (Ma et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Selvi et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The presence of glochid on neutlet could be an explanation of the widespread geographic distribution of Cynoglosseae sensu lato (including Rochelieae) (Cohen \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). According to Weigend et al. (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), two species of sect. \u003cem\u003eSclerocaryum\u003c/em\u003e applies \u0026ldquo;the whole-plant dispersal by wind or flash-floods\u0026rdquo; as a dispersal mechanism which could explain the synapomorphy of \u0026ldquo;lack of appendage\u0026rdquo; observed in our results. Indeed, this mechanism caused the separation of nutlets from the mother plant to become unnecessary.\u003c/p\u003e \u003cp\u003eNutlet micromorphology results in this study provide no clear distinction among species \u003cem\u003eL.barbata\u003c/em\u003e and L. \u003cem\u003emicrocarpa\u003c/em\u003e. The morphological complexities of these two species have already been addressed by different taxonomists (Popov \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1953\u003c/span\u003e; Akhani \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). A revision of the \u003cem\u003eLappula\u003c/em\u003e genus by Nasseh and Joharchi (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) suggested that the two species may be synonymous and more molecular studies be conducted. Moreover, according to the molecular results (Khoshsokhan-Mozaffar et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) in the nr-DNA ITS tree of Rochelieae, the clade delimiting these two species was not well-supported. The variety observed in the nutlet of \u003cem\u003eL.microcarpa\u003c/em\u003e and \u003cem\u003eL.barbata\u003c/em\u003e could be related to seed heteromorphism that previously is known to occur in a few Boraginaceae genera Boraginaceae, e.g. \u003cem\u003eEritrichium, Lappula\u003c/em\u003e (\u003cem\u003eL. duplicicarpa\u003c/em\u003e and \u003cem\u003eL. semiglabra\u003c/em\u003e) and \u003cem\u003eHeterocaryum\u003c/em\u003e (Wang et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e1989\u003c/span\u003e).\u003c/p\u003e \u003c/p\u003e "},{"header":"Conclusions","content":"\u003cp\u003eIn this study, the nutlet ultrastructure pattern of Rochelieae is systematically informative at the genus level, but not at the species level. The results showed that glochid is not an ancestral trait but is a synapomorphy and the transition to this trait occurred in the genus \u003cem\u003eLappula\u003c/em\u003e. Nutlet micromorphology results in this study provide no clear distinction among species \u003cem\u003eL.barbata\u003c/em\u003e and L. \u003cem\u003emicrocarpa\u003c/em\u003e.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors wish to thank Saeed Javadi Anaghizi in Central laboratory of the Shahid Beheshti University for providing SEM pictures. We thank Dr. Somayeh Naghiloo (University of Calgary, Canada) for assisting in software analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSN designed the study project, ME \u0026amp; SN performed experiments and data analysis and drafted the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research received no specific grant.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;Availability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data used and analyzed for the current study can be obtained from the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAkhani H (1998) Plant biodiversity of Golestan National Park, Iran. Biologiezentrum des O\u0026Ouml; Landesmuseums\u003c/li\u003e\n\u003cli\u003eAl-Shehbaz IA (1991) The genera of Boraginaceae in the southeastern United States. Journal of the Arnold Arboretum. Suppl Ser 1:1-169\u003c/li\u003e\n\u003cli\u003eCastresana J (2000) Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Mol biol evol 17:540-552\u003c/li\u003e\n\u003cli\u003eChac\u0026oacute;n J, Luebert F, Hilger HH, Ovchinnikova S, Selvi F, Cecchi L, Guilliams CM, Hasenstab-Lehman K, Sutor\u0026yacute; K, Simpson MG (2016) The borage family (Boraginaceae s. str.): A revised infrafamilial classification based on new phylogenetic evidence, with emphasis on the placement of some enigmatic genera. Taxon 65:523-546\u003c/li\u003e\n\u003cli\u003eCohen JI (2014) A phylogenetic analysis of morphological and molecular characters of Boraginaceae: evolutionary relationships, taxonomy, and patterns of character evolution. Cladistics 30:139-169\u003c/li\u003e\n\u003cli\u003ede Candolle A (1845) Prodromus Systematis naturalis regni vegetabilis, Paris.\u003c/li\u003e\n\u003cli\u003eHilger H (1985) Ontogenie, Morphologie und systematische Bedeutung geflugelter und glochidientragender Cynoglosseae-und Eritricheae-Fruchte (Boraginaceae). Bot Jahrb Syst, Pflanzengesch Pflanzengeogr\u003c/li\u003e\n\u003cli\u003eHilger HH (2014) Ontogeny, morphology, and systematic significance of glochidiate and winged fruits of Cynoglosseae and Eritrichieae (Boraginaceae). Plant Divers Evol 131:167-214\u003c/li\u003e\n\u003cli\u003eHuang JF, Zhang ML, Cohen JI (2013) Phylogenetic analysis of Lappula Moench (Boraginaceae) based on molecular and morphological data. Plant syst evol 299:913-926\u003c/li\u003e\n\u003cli\u003eJohnston I (1937) Studies in the Boraginaceae XII. 2. Novelties and critical notes. J Arnold Arbor 18:10-25\u003c/li\u003e\n\u003cli\u003eKahraman A, Celep F, Doğan M, Guerin GR, Bagherpour S (2011) Mericarp morphology and its systematic implications for the genus Salvia L. section Hymenosphace Benth.(Lamiaceae) in Turkey. Plant syst evol 292:33-39\u003c/li\u003e\n\u003cli\u003eKhoshsokhan-Mozaffar M, Sherafati M, Kazempour-Osaloo S (2018) Molecular phylogeny of the tribe Rochelieae (Boraginaceae, Cynoglossoideae) with special reference to Lappula. Ann Bot Fenn BioOne 293-308\u003c/li\u003e\n\u003cli\u003eKhoush SMM, Kazempour OS, Saadatmand S, Atar F (2010) Molecular phylogeny of Rochelia (Boraginaceae) based on nrDNA ITS and cpDNA trnL-F sequences. 22-29\u003c/li\u003e\n\u003cli\u003eKumar S, Stecher G, Tamura K (2016) MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol biol evol 33:1870-1874\u003c/li\u003e\n\u003cli\u003eL\u0026aring;ngstr\u0026ouml;m E, Chase M (2002) Tribes of Boraginoideae (Boraginaceae) and placement of Antiphytum, Echiochilon, Ogastemma and Sericostoma: a phylogenetic analysis based on atpB plastid DNA sequence data. Plant Syst Evol 234:137-153\u003c/li\u003e\n\u003cli\u003eLehmann JGC (1818) Plantae e familia Asperifoliarum nuciferae. Dummler, Berlin, p 78\u003c/li\u003e\n\u003cli\u003eLewis PO (2001) A likelihood approach to estimating phylogeny from discrete morphological character data. Syst biol 50:913-925\u003c/li\u003e\n\u003cli\u003eMa W, Zhao X, Tan D, Baskin C, Baskin J, Xue J (2010) Nutlet dimorphism in individual flowers of two cold desert annual Lappula species (Boraginaceae): implications for escape by offspring in time and space. Plant ecol 209:361-374\u003c/li\u003e\n\u003cli\u003eMaddison W, Maddison D (2015) Mesquite: a modular system for evolutionary analysis. Version 3.04. 2015\u003c/li\u003e\n\u003cli\u003eMoon HK, Hong SP (2006) Nutlet morphology and anatomy of the genus Lycopus (Lamiaceae: Mentheae). J Plant Res 119:633-644\u003c/li\u003e\n\u003cli\u003eMozaffar MK, Osaloo SK, Oskoueiyan R, Saffar KN, Amirahmadi A (2013) Tribe Eritrichieae (Boraginaceae s. str.) in West Asia: a molecular phylogenetic perspective. Plant Syst Evol 299:197-208\u003c/li\u003e\n\u003cli\u003eNasseh Y, Joharchi MR (2017) Revision of the genus Lappula Moench based on morphological characters in Khorassan Provinces (Iran). Nova Biol Rep 4:66-73\u003c/li\u003e\n\u003cli\u003eOvchinnikova S (2005) The system of the subtribe Echinosperminae (Boraginaceae). Bot Zhurn 90:1153-1172\u003c/li\u003e\n\u003cli\u003eOvchinnikova S (2009) On the position of the tribe Eritrichieae in the Boraginaceae system. Bot Serbica 33:141-146\u003c/li\u003e\n\u003cli\u003ePopov M (1953) Boraginaceae. Flora SSSR 19:97-691\u003c/li\u003e\n\u003cli\u003eRiedl H (1967) Boraginaceae in, Rechinger KH. Flora Iranica. Graz, Akademische Druck_u. Verlagdanstalt 48:215\u003c/li\u003e\n\u003cli\u003eRiedl H (1996) Studies in the genus Lappula (Boraginaceae) I. Lappula in the\" Flora Iranica\" region. Serie B f\u0026uuml;r Botanik und Zoologie. Ann Naturhist Mus 79-86\u003c/li\u003e\n\u003cli\u003eRolfsmeier SJ (2013) Taxonomy and phylogeny of the genus Lappula Moench (Boraginaceae) in North America. Kansas State University.\u003c/li\u003e\n\u003cli\u003eSaadati N, Mozaffar MK, Sherafati M, Osaloo SK (2017) Pseudoheterocaryum, a new genus segregated from Heterocaryum (Boraginaceae) on the basis of molecular data. Aust Syst Bot 30:105-111\u003c/li\u003e\n\u003cli\u003eSelvi F, Bigazzi M, Hilger HH, Papini A (2006) Molecular phylogeny, morphology and taxonomic re‐circumscription of the generic complex Nonea/Elizaldia/Pulmonaria/Paraskevia (Boraginaceae‐Boragineae). Taxon 55:907-918\u003c/li\u003e\n\u003cli\u003eSelvi F, Coppi A, Cecchi L (2011) High epizoochorous specialization and low DNA sequence divergence in Mediterranean Cynoglossum (Boraginaceae): Evidence from fruit traits and ITS region. Taxon 60:969-985\u003c/li\u003e\n\u003cli\u003eSilvestro D, Michalak I (2012) raxmlGUI: a graphical front-end for RAxML. Org Divers Evol 12:335-337\u003c/li\u003e\n\u003cli\u003eWeigend M, Gottschling M, Selvi F, Hilger HH (2009) Marbleseeds are gromwells\u0026ndash;Systematics and evolution of Lithospermum and allies (Boraginaceae tribe Lithospermeae) based on molecular and morphological data. Mol Phylogenetics Evol 52:755-768\u003c/li\u003e\n\u003cli\u003eWeigend M, Luebert F, Selvi F, Brokamp G, Hilger HH (2013) Multiple origins for Hound\u0026rsquo;s tongues (Cynoglossum L.) and Navel seeds (Omphalodes Mill.)\u0026ndash;The phylogeny of the borage family (Boraginaceae s. str.). Mol phylogenetics evol 68:604-618\u003c/li\u003e\n\u003cli\u003eWeigend M, Selvi F, Thomas D, Hilger H (2016) Boraginaceae. Flowering Plants. Eudicots. Springer, pp. 41-102\u003c/li\u003e\n\u003cli\u003eYu WT, Jacques FM, Chen ST, Zhou ZK (2012) Nutlet micro‐morphology of the genus Microula (Boraginaceae) from the Qinghai\u0026ndash;Tibetan Plateau, and its systematic implications. Nord J Bot 30:596-612\u003c/li\u003e\n\u003cli\u003eWang WC, Liu YL, Zhu GL, Lian YSh, Wang JQ, Wang QR (1989) Lappula V. Wolf. In: Kong XW, Wang WC (eds) Flora Reipublicae Popularis Sinicae, vol 64. Science Press, Beijing, pp 177-207 (in Chinese)\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"botanical-studies","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bost","sideBox":"Learn more about [Botanical Studies](http://as-botanicalstudies.springeropen.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bost/default.aspx","title":"Botanical Studies","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Character evolution, Lappula, Micromorphology, Systematic","lastPublishedDoi":"10.21203/rs.3.rs-521851/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-521851/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e The macro/micro-morphology of nutlets in 11 species (and 22 accessions) of the Boraginaceae family was investigated by stereomicroscope and scanning electron microscopy to evaluate the taxonomic relevance of these traits. To evaluate the phylogenetic significance of the character evolution, available DNA sequence data from GenBank were combined with selected original nutlet data, and phylogenetic analysis was performed.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e The Rochelieae nutlets' shape varied from ovoid (ovoid, ovoid-triangular, and ovoid-rectangular) to the pyramid.\u003cstrong\u003e \u003c/strong\u003eSix major patterns were recognized based on nutlet ultrastructure characters. Rocheliae is characterized by a transition from “without appendage” to “with tubercles and prickles” on the nutlet disk, and also by a shift from “lack of prickles” to “glossy prickles”.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e The results indicated that the nutlet ultrastructure pattern of Rochelieae is systematically informative at the genus level, but not at the species level. The results showed that glochid is not an ancestral trait but is a synapomorphy and the transition to this trait occurred in the genus \u003cem\u003eLappula\u003c/em\u003e. \u0026nbsp;The Close boundary of nutlet microstructures between \u003cem\u003eL. barbata\u003c/em\u003e and \u003cem\u003eL. microcarpa\u003c/em\u003e was discussed.\u0026nbsp;\u003c/p\u003e","manuscriptTitle":"Nutlet Micromorphology and Character Evolution of Some Species of Rochelieae (Boraginaceae) and Its Systematic Implications","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-05-17 17:49:30","doi":"10.21203/rs.3.rs-521851/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2021-07-01T11:14:47+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2021-06-25T00:00:00+00:00","index":1,"fulltext":"Recommendation: Reviewer's comments unavailable due to the journal's policy.\n"},{"type":"reviewerAgreed","content":"","date":"2021-06-14T00:00:00+00:00","index":1,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2021-06-14T00:00:00+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2021-05-26T03:15:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2021-05-13T00:00:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2021-05-12T23:00:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2021-05-12T23:00:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"Botanical Studies","date":"2021-05-12T09:22:15+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"botanical-studies","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bost","sideBox":"Learn more about [Botanical Studies](http://as-botanicalstudies.springeropen.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bost/default.aspx","title":"Botanical Studies","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a695dd7f-5223-46cc-a29b-d37f9c974339","owner":[],"postedDate":"May 17th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":4371940,"name":"Biotechnology and Bioengineering"}],"tags":[],"updatedAt":"2021-10-15T11:18:45+00:00","versionOfRecord":[],"versionCreatedAt":"2021-05-17 17:49:30","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-521851","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-521851","identity":"rs-521851","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}