Morphological Study of Some Taxa of Ranunculaceae Juss in Egypt (Anatomy and Pollen Grains)

Home , Adonis (plant), Anemone coronaria, Clematis terniflora, Consolida regalis, Ranunculaceae, Ranunculus sceleratus

beni-suef university journal of basic and applied sciences 5 (2016) 310–319

HOSTED BY Available online at www.sciencedirect.com ScienceDirect

journal homepage: www.elsevier.com/locate/bjbas

Full Length Article Morphological study of some taxa of Ranunculaceae Juss in Egypt (anatomy and pollen grains)

Mohamed A. Salim, Al-Safa H. Mohamed *, Mohamed E. Tantawy

Botany Department, Faculty of Science, Ain Shams University, Cairo, Egypt

ARTICLE INFO ABSTRACT

Article history: Ranunculaceae is a large angiospermic family (43 genera and 2346 species). Seven taxa were Received 27 July 2016 subjected in the present study representing six genera. Stem anatomy, flower anatomy, lamina Received in revised form 20 October epidermal characters as well as the pollen morphology were carried out according to tra- 2016 ditional taxonomic methods using LM and SEM. The specific target of the present study is Accepted 22 October 2016 to contribute knowledge to Ranunculaceae from different perspectives. The obtained Available online 29 October 2016 microcharacters of stem, leaf and flowers are considered diagnostic at the generic and specific levels (e.g., stem outline, types of cortex, internal appearance of stem, leaf types, perianth Keywords: vascularization and presence of nectary scales). The pollen characters of the studied taxa Ranunculaceae are considered highly diagnostic at the generic and specific levels especially pollen class Anatomy (trizonocolpate, pantocolpate and pantoporate) and pollen sculpture (aculeate, verrucate- Epidermal characters aculeate, aculeate-faveolate and scabrate-verrucate). The obtained data facilitate the Pollen morphology construction of artificial keys to delimitation between the studied taxa. © 2016 Beni-Suef University. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by- nc-nd/4.0/).

The Ranunculaceae are often considered as the most primi- 1. Introduction tive of the herbaceous angiosperm. Some of the primitive characters found in the family are; numerous and spiral ar- Ranunculaceae is a large angiospermic family, distributed world- rangement of floral parts, apocarpy, imperfect carpel closure, wide especially in temperate and subtropical regions. It includes and follicles. However, many traits characterized as ad- 43 genera and 2346 species (Christenhusz and Byng, 2016), com- vanced are also common; finely dissected leaves, vessels with monly herbs. This family has been considered as one of the simple perforations, racemose inflorescences, unisexual flowers, most basal families within the eudicots (Heywood et al., 2007; zygomorphic flowers, specialized spurred petals, syncarpy (with Simpson, 2010; Soltis et al., 2005). each carpel still distinct), and achenes. So while some members

* Corresponding author. Botany Department, Faculty of Science, Ain Shams University, Cairo, Egypt. E-mail address: [email protected] (A.-S.H. Mohamed). http://dx.doi.org/10.1016/j.bjbas.2016.10.002 2314-8535/© 2016 Beni-Suef University. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). beni-suef university journal of basic and applied sciences 5 (2016) 310–319 311

of the family may be regarded as representative of the primi- derived from Germplasm Resources Information Network (GRIN) tive condition, others have developed advanced characters and The International Plant Names Index (IPNI). (Tamura, 1968). Morphological, palynological, leaf epidermis and anatomi- 2.1. Micromorphological investigation cal characters are considered as the most significant features in taxonomy of Ranunculaceae. The variation within a species, Cross sections of stem and mature flower buds (Nigella sativa genera or family is usually reflected in anatomical features as was unavailable for flower anatomy) were fixed and preserved well thus the role of anatomy in taxonomy has long been rec- in F.A.A., embedded in paraffin wax, then serially sectioned at ognized. Leaf epidermal characters viz. stomata, trichomes, other 10–15 according to the conventional method (Johansen, 1940). characters as well as anatomical features are useful tools (Stace, Sections stained in crystal violet-erythrosine combination, and 1991). others with safranin-light green then mounted with Canada The stems of Ranunculaceae in transverse section, usually balsam.The sections were examined using BEL: B103T-PL light circular, vascular bundles, often occur collaterally and are com- microscope. Photomicrographs were taken using canon power monly set apart (Metcalfe and Chalk, 1950), they may also arise shot G12 digital camera at the Plant Taxonomy Research Labo- in several concentric ring in some genera while they are ran- ratory, Botany Department, Faculty of Science, Ain Shams domly scattered in others (Judd et al., 1999). University, Cairo, Egypt. For the lamina epidermal character- The floral vascularization and evolution of the Ranunculaceae istics, the epidermis was peeled or scraped off with a fine forceps and Ranunculales in general have mostly been investigated by or blade, then stained with safranin or examined directly. various authors. To cite a few we can refer to Endress and Igersheim (1999), Erbar et al. (1999), Jabbour et al. (2009), Rasmussen 2.2. Pollen grain morphology et al. (2009), Salisbury (1973) and Worsdell (1903, 1908). Al-Eisawi (1986), Erdtman (1986), Hamilton (1976), Kumazawa (1936) and Flowers of the taxa under investigation were fixed in 70% Wodehouse (1936) recognized three pollen types viz. tricolpate, alcohol. Pollen grains were left to dry for 10 minutes then fixed pantocolpate and pantoporate, and the exine sculpturing with on a slide with glycerin and were covered (Moore et al., 1991) scabrate to echinate ornamentation in the family. and photographed using Canon power shot G12 digital camera. The goal of the present study is the contribution to the For SEM examination pollen grains were dried, mounted on knowledge of Ranunculaceae from different points of view viz. stubs then coated with gold by sputter coaster (SPI-Module) anatomical characteristics of stem and flower, leaf epidermal and tested with (JEOL-JSM5500LV) scanning electron microcharacters and pollen grains characteristics (LM and SEM) of scope at the Reginal Center of Mycology and Biotechnology, El- the studied taxa. Azhar University, Cairo, Egypt.

2. Material and methods 3. Results and discussion

Seven species belonging to six genera of Ranunculaceae were 3.1. Anatomical characters collected from different localities and sources in Egypt as shown in Table 1. The taxa under investigation range between orna- 3.1.1. Stem anatomy and leaf epidermal characters mental and wild taxa. The identification of the studied species Stem and leaf epidermal micromorphology of the studied taxa takes place by the aid of Bailey (1949), Bailey and Bailey (1976), are summarized in Table 2 and illustrated in Plate 1. Stem Boulos (2002) and Tackholm and Drar (1974). Synonyms were outline is angular in Adonis dentatus, Clematis terniflora and Nigella sativa or terete in the rest of the 4 studied taxa. Trichomes are eglandular unicellular in Anemone coronaria and Nigella sativa; eglandular and glandular in Consolida regalis or absent in the Table1–Datacollection. rest of the studied taxa. Cuticle is thick in Adonis dentatus, Clema- Taxa Sources or localities tis terniflora and Nigella sativa or thin in the rest of the studied Adonis dentatus Del. Burg El-Arab, taxa. Epidermis is radially-tangentially in Clematis terniflora and Mediterranean Coastal Nigella sativa or radially in the rest of the studied taxa. Cortex Region of one type of cells is lacunate parenchyma in Ranunculus Anemone coronaria L. // sceleratus or polyhedral parenchyma in Ranunculus marginatus. Clematis terniflora DC. Botanic garden, ASU Consolida regalis Gray. // Two types of cells are polyhedral parenchyma and angular col- Syn. Delphidium consolida (L.) Raf lenchyma in Anemone coronaria. Three types of cells are angular Nigella sativa L. // collenchyma, chlorenchyma and extraxylary fibers in Adonis Syn. Nigella cretica Mill. dentatus and Nigella sativa, polyhedral parenchyma, angular col- Ranunculus marginatus Hook.f. // lenchyma and extraxylary fibers in Clematis terniflora or Syn. Ranunculus scandicinus polyhedral parenchyma, chlorenchyma and extraxylary fibers (Boiss.) P.H.Davis in Consolida regalis. Stem is solid in Adonis dentatus, Anemone R. sceleratus L. Irrigated canals of Kafr El- Sheikh Governorate coronaria and Clematis terniflora or hollow in the rest of the studied taxa. The vascular supply shows continuous sipho- //, the same. nostele in Clematis terniflora or dissected in the rest of the studied 312 eise nvriyjunlo ai n ple cecs5(06 310–319 (2016) 5 sciences applied and basic of journal university beni-suef

Table 2 – Stem anatomical and leaf epidermal characters of the studied taxa of Ranunculaceae.

Taxa Stem anatomical traits Leaf epidermal characters no. Outline Dermal system Ground system Vascular tissue in T.S. Trichomes Cuticle Epidermal Epidermal Cortex Leaf type Cell shape Anticlinal wall Stomata cell shape cell size (Ab-/Adaxial) (Ab-/Adaxial) (Ab-/Adaxial) Type of tissues

Angular Chlorenchyma Parenchyma Extra- Stem internal Aspect Interfasicular collenchyma xylary appearance regions ﬁbers

1 Angular − Thick Radially Two sizes + + −+Solid Dissected Parenchyma Amphistomatic Irregular Sinuses Anomocytic 6–8 rows 4–6 rows siphonostele 19–20 bundles 2 Terete + Thin // One size + − Polyhedral − // // // // Polygonal Straight // 7–9 rows 2–3 rows 26–28 bundles 3 Angular − Thick Tangentially Two sizes + − // + // Continuous Xylem ﬁbers Hypostomatic Irregular Sinuses Anomocytic and radially 3–5 rows 5–7 rows siphonostele 12–13 bundles 4 Terete + Thin Radially One size −+ // + Hollow Dissected Parenchyma // // // // 3–4 rows 2–3 rows siphonostele 30–32 bundles 5 Angular + Thick Tangentially Two sizes + + −+// // // Amphistomatic // // Anomocytic and radially 3–4 rows 3–4 rows 19–20 bundles 6 Terete − Thin Radially One size −− Polyhedral − // // // // // // // 7–9 rows 11–12 bundles 7// − // // // −− Lacunate // // // // // // // 1–2 rows 15–17 bundles

+, present; −, absent; //, the same. beni-suef university journal of basic and applied sciences 5 (2016) 310–319 313

Plate 1 – (a–g) Stem (T.S.). (a) Adonis dentatus; (b) Anemone coronaria; (c) Clematis ternifolia; (d) Consolida regalis; (e) Nigella sativa; (f) Ranunculus marginatus; (g) R. sceleratus. (h, i) Lamina epidermal characteristics. (h) Lower surface of R. marginatus; (i) upper surface of Clematis ternifolia. Co., collenchyma; LP., lacunate parenchyma; Pc., pith cavity; Sc., sclerenchyma; St., stomata; Tr., trichome; Vb., vascular bundle.

taxa. Interfascicular region components are parenchyma only D. Stem solid Adonis dentatus (six taxa) or xylem fibers at secondary xylem in Clematis DD. Stem hollow Nigella sativa terniflora. For the leaf epidermal characters the leaf types are hypostomatic in Clematis terniflora and Consolida regalis or CC. Stem terete amphistomatic in the rest of the studied taxa. Stomatal type E. Cortex two types of tissue; polyhedral Anemone is anomocytic in all the taxa under investigation. Epidermis parenchyma and angular collenchyma coronaria is polygonal (ad and abaxially) with straight anticlinal walls EE. Cortex one types of tissue in Anemone coronaria or irregular with sinuses U shaped anticlinal walls in the rest of the studied taxa. From the foregoing F1. Cortex of lacunate parenchyma Ranunculus sceleratus stem and leaf anatomical characters, it was detected that all the taxonomic parameters are in agreement with the previ- F2. Cortex of polyhedral parenchyma Ranunculus ous work of (Metcalfe and Chalk, 1950). marginatus The stem anatomical and leaf epidermal characters facilitate the construction of an artificial key to differentiate between 3.1.2. Floral anatomy (Table 3) the studied taxa.

A. Leaf hypostomatic 3.1.2.1. Adonis dentatus (Plate 2). Pedicel vasculatures show a dissected siphonostele (Fig. a). At a higher level 15 traces pro- B. Stem angular Clematis terniflora trude from the central stele representing the vascular supply BB. Stem terete Consolida regalis of the five sepals (one median and two laterals for each; Fig. b). AA. Leaf amphistomatic The sepal laterals undergo further ramification upward (Figs. c– k). Eight petal traces arise from the central stele (five traces C. Stem angular for five outer petals and the remaining three for the inner three 314 beni-suef university journal of basic and applied sciences 5 (2016) 310–319

petals; Fig. c). After the departure of the petal traces, eight staminal traces detached irregularly from the central stele (Figs. f– k). The remaining central stele differentiated into numerous vascular carpellary masses. Each one divides periclinally into an outer dorsal carpellary bundle and an inner two fused with ventral carpellary mass (Figs. i–k). Aculeate Verrucate-aculeate // Scabrate-verrucate

3.1.2.2. Anemone coronaria (Plate 3). Pedicel and sepal vasculature is on the same anatomical ground in Adonis dentatus (Figs. d–j). Five petal traces arise from the central stele (Figs. d– j). After the departure of the petal traces, numerous staminal traces detached from the central stele (Figs. g–j). The remaining central stele differentiated into numerous vascular carpellary masses. Each one divides periclinally into an outer dorsal carpellary bundle and an inner two fused with ventral carpellary mass (Figs. j and k).

3.1.2.3. Clematis ternifolia (Plate 4). Pedicel vasculatures show a dissected siphonostele (Fig. a). At a higher level 12 vascular traces protrude from the central stele representing the vas- end, constricted, wide outline, sunken endoaperture congruent with ectoaperture wide sometimes forming irregular pattern cular supply of the four tepals (one median and two laterals for each; Figs. b and c). The tepal lateral bundles undergo further ramiﬁcation upwards (Figs. d–l). After the departure of the tepal Polarity Apertures Sculpture Isopolar Simple colpus fusiform with rounded // Simple colpus fusiform with rounded end Isopolar Simple colpus slit like tapered endtraces, // numerous staminal traces (22) detached from the central stele (Figs. e–l). The remaining central stele differentiated into seven vascular carpellary masses. Each one divides periclinally into an outer dorsal carpellary bundle and an inner two fused with ventral carpellary mass which divides anticlinally into two E view Shape in bundles (Figs. i and l). rounded end rounded end tapered end

Attributes 3.1.2.4. Consolida regalis (Plate 5). Pedicel and sepal vasculature is on the same anatomical ground in Adonis dentatus (Figs. d–l). Two petal traces arise from the central stele (Figs. d–

P view l). The two posterior petals were united in forming spur (Figs. b– i). After the departure of the petal traces, 14 spirally arranged Circumaperture Circumaperture Apolar Simple ectoaperture pore, circular in staminal traces detached from the central stele (Figs. f–l). The remaining stele differentiated into an outer dorsal carpellary bundle, an inner two fused with ventral carpellary mass and numerous lateral carpellary bundles (Figs. h and i). At a higher level each ventral mass divides anticlinally into two separate Pollen class Shape in 5–8 ventral carpellary bundles (Figs. j and k).

3.1.2.5. Ranunculus marginatus (Plate 6). Pedicel and sepal E\2 vasculature is on the same anatomical ground in Adonis dentatus +

P (Figs. d–l). Five petal traces arise from the central stele (Figs. f– Pollen size l). Petals pears nectary scales without vascular supply (Figs. i– j). After the departure of the petal traces, ten staminal traces detached irregularly from the central stele (Figs. h–l). The remaining central stele differentiated into ten vascular carpellary masses. Each one divides periclinally into an outer dorsal carpel- P\Ex100 lary bundle and two fused with inner ventral carpellary mass Oblate-spheroidal Medium Trizonocolpate Angluaperture Elliptic with //Prolate-spheroidal SmallProlate // Trizonocolpate Angluaperture Medium Semicircular Pantoporate // Isopolar Simple colpus slit like tapered end wide // Circumaperture Elliptic with SuboblateOblate-spheroidal largeProlate Small Pantocolpate // Circumaperture Small(Fig. Circumaperture Apolar j). At Trizonocolpate Simple colpus Angluaperture spread a // over the surface higher Semicircular level // each Elliptic with Simple colpusventral with rounded end, wide Aculeate-faveolate carpellary mass divides anticlinally into two separate ventral carpellary bundles (Figs. k m m m m m m m m m m m m m m and l). 18.75 20.25 17.6 18. 9 14.79 13.75 23.3 16.66 14.07 16.1 37.5 39.6 16.1 15.01 3.1.2.6. Ranunculus sceleratus (Plate 7). Pedicel and sepal vas- ======Dimension Shape class E E E E E E E culature is on the same anatomical ground in Adonis dentatus (Figs. b–l). Five petal traces arise from the central stele (Figs. c– l). Petals pears nectary sac without vasculature (with no vascular Table 3 – Quantitative and qualitative pollenTaxa micro-morphological data of the studied taxa of Ranunculaceae. 1P 2P 3P 4P 5P 6P 7P P, polar axis length; E, equatorial diameter; //, the same. supply) (Figs. e–h). After the departure of the petal traces, 17 beni-suef university journal of basic and applied sciences 5 (2016) 310–319 315

Plate 2 – (a–k) Serial cross section through the flower of Adonis dentatus,X= 500 m. staminal traces detached irregularly from the central stele structures; one dorsal and two fused ventrals. The ovary is (Figs. g–l). The remaining central stele differentiated into nu- multicarpellate in structure in all of the studied taxa except merous vascular carpellary masses. Each one divides periclinally in Consolida which is unicarpellate; this is in accord with into an outer dorsal carpellary bundle and an inner two fused Novikoff and Jabbour (2014). with ventral carpellary mass (Figs. h–l). Each sepal receives three traces arising from the recep- 3.2. Pollen morphology tacle tissue (one median and two laterals), each petal receives one trace that ramifies through the petal tissue in all of the The morphological characters of pollen grain of the studied studied taxa, this is in accordance with the classical concept taxa are summarized in Table 3 and illustrated in Plate 8. The of (Bessey, 1915). The fact that the nectariferous scales receive arithmetic mean value was taken according to (Brookes and no vascular supply suggests that the scale is not an indepen- Thomas, 1967) and the terminology adopted to describe palyno- dent organ scale but an evagination of the petal itself (Smith, logical characters was according (Punt et al., 2007). Three main 1926) and this was found in the two studied species of Ranun- types of pollen grains were recognized: pantoporate in Anemone culus. In all the studied taxa, the stamen receives one trace that coronaria; pantocolpate in Ranunculus marginatus or remains unbranched throughout. The carpels are three trace trizonocolpate in the rest of the studied taxa (five taxa). The

Plate 3 – (a–k) Serial cross section through the ﬂower of Anemone coronaria;X= 1000 m. 316 beni-suef university journal of basic and applied sciences 5 (2016) 310–319

Plate 4 – (a–i) Serial cross section through the ﬂower of Clematis ternifolia;X= 500 m.

obtained data in the present study are in agreement with 1972; Walker and Doyle, 1975).The line of advancement pollen Al-Eisawi (1986), Erdtman (1986), Hamilton (1976), Kumazawa types in the present study is from trizonocopate vs. (1936) and Punt et al. (2007). The P\E ratio shows extensive dif- pantocolpate vs. pantoporate. ferences in shape class of pollen grain among the studied taxa. The obtained pollen morphological data facilitate in the con- Four main types of pollen grain sculpturing were detected viz. struction of an artiﬁcial key which helps in the delimitation aculeate in Adonis dentatus, aculeate-faveolate in Nigella sativa, of the studied taxa. scabrate-verrucate in R. marginatus and R. sceleratus or verrucate- aculeate in the rest of the taxa. A. Pollen polarity: apolar Ranunculaceae have a more advanced pollen type viz. colpi, B. Pollen class: pantoporate Anemone coronaria isopolar and three aperturate than monocolpate and nonaperturate pollen grains (less advanced type). Perveen (2000) BB. Pollen class: pantocolpate Ranunculus marginatus assumed that the tricolpate pollen type is the simplest which AA. Pollen polarity: isopolar characterized subclass Ranunculidae which is considered the C. Shape class: prolate base from which all advanced pollen types were derived (Walker,

Plate 5 – (a–i) Serial cross section through the ﬂower of Consolida regalis;X= 500 m. beni-suef university journal of basic and applied sciences 5 (2016) 310–319 317

Plate 6 – (a–l) Serial cross section through the ﬂower of Ranunculus marginatus;X= 500 m.

D. Aculeate-faveolate sculpture Nigella sativa 4. Conclusion DD. Scabrate-verrucate sculpture Ranunculus sceleratus

CC. Shape class: otherwise The origin of the angiosperms has long been a question of debate E. Aculeate sculpture Adonis dentatus among botanists. One of the points of controversy has been the nature of the primitive angiospermous flower. From the fore- EE. Verrucate-aculeate sculpture going data viz stem anatomy and flower anatomy the Ranalian model F. Colpus slit like Clematis terniflora is simple and primitive but from the palynological point of view the FF. Colpus fusiform Consolida regalis data show a line of advancement from less advanced to more advanced form. Additional work with additional plant samples is

Plate 7 – (a–l) Serial cross section through the ﬂower of R. sceleratus;X= 500 m. C.S.S., continuous siphonostele; C.T., carpellary trace; D.C.B., dorsal carpellary bundle; D.S.S., dissected siphonostele; N.Sc., nectary scale; P.L.B (s)., petal lateral bundle (s); P.M.B (T)., petal median bundle (trace); S.L.B (s)., sepal lateral bundle(s); S.M.B (T)., sepal median bundle (Trace); St. B (T)., staminal bundle (trace); T.L.B (s)., tepal lateral bundle (s); T.M.B (T)., tepal median bundles (trace); V.C.B., ventral carpellary bundle. 318 beni-suef university journal of basic and applied sciences 5 (2016) 310–319

Plate 8 – (a–g) LM and SEM micrograph of pollen morphology. (a1–3) Adonis dentatus;(b1–3) Anemone coronaria;(c1–3) Clematis ternifolia;(d1–3) Consolida regalis;(e1–3) Nigella sativa;(f1–3) Ranunculus marginatus;(g1–3) R. sceleratus.

important and should be taken in mind to solve these prob- Bailey LH. Manual of cultivated plants most commonly grown in lems about Ranunculaceae and detect the relationship among the continental United States and Canada. Macmillan; 1949. species of the family and related families. Bailey LH, Bailey LH. Hortus third: a concise dictionary of plant cultivated in US and Canada. Macmillan Publishing Co. Inc.; 1976. REFERENCES Bessey CE. The phylogenetic taxonomy of ﬂowering plants. Ann Mo Bot Gard 1915;2:109–64. Boulos L. Flora of Egypt, volume 3: Verbenaceae-Compositae. Al-Eisawi DM. Pollen morphology of Ranunculaceae in Jordan. Cairo: Al Hadara Publishing; 2002. ISBN: 1185494658, 373 Pollen Spores 1986;28:311–28. p-illus, col illus. beni-suef university journal of basic and applied sciences 5 (2016) 310–319 319

Brookes D, Thomas KW. The distribution of pollen grains on Perveen A. Pollen characters and their evolutionary significance microscope slides. I. Part I. The nonrandomness of the with special reference to the Flora of Karachi. Turk J Biol distribution. Pollen Spores 1967;9(3):621–9. 2000;24(2):365–78. Christenhusz MJM, Byng JW. The number of known plants Punt W, Hoen PP, Blackmore S, Nilsson S, Le Thomas A. Glossary species in the world and its annual increase. Phytotaxa of pollen and spore terminology. Rev Palaeobot Palynol 2016;261(3):201–17. 2007;143(1):1–81. Endress PK, Igersheim A. Gynoecium diversity and Rasmussen DA, Kramer EM, Zimmer EA. One size fits all? systematics of the basal eudicots. Bot J Linn Soc Molecular evidence for a commonly inherited petal identity 1999;130(4):305–93. program in Ranunculales. Am J Bot 2009;96(1):96–109. Erbar C, Kusma S, Leins P. Development and interpretation of Salisbury E. The organization of the ranunculaceous flower with nectary organs in Ranunculaceae. Flora 1999;194:317–32. especial regard to the correlated variations of its constituent Erdtman G. Pollen morphology and plant taxonomy: members. Proc R Soc Lond B Biol Sci 1973;183(1072):205–25. angiosperms, vol. 1. Brill Archive; 1986. Simpson MG. Plant systematics. Academic press; 2010. Hamilton AC. Identification of east African Urticales pollen. Smith GH. Vascular anatomy of Ranalian flowers. I. Pollen Spores 1976;18(1):27–66. Ranunculaceae. Bot Gaz 1926;1–29. Heywood VH, Brummitt RK, Culham A, Seberg O. Flowering plant Soltis DE, Soltis PS, Endress PK, Chase MW. Phylogeny and families of the world. Kew: Royal Botanic Gardens; 2007. evolution of angiosperms. Sinauer Associates Incorporated; Jabbour F, De Craene LPR, Nadot S, Damerval C. Establishment of 2005. zygomorphy on an ontogenic spiral and evolution of perianth Stace CA. Plant taxonomy and biosystematics. Cambridge in the tribe Delphinieae (Ranunculaceae). Ann Bot University Press; 1991. 2009;104(5):809–22. Tackholm V, Drar M. Students’ flora of Egypt. 1974. Johansen DA. Plant microtechnique: Jeffrey’s method. New York Tamura M. Morphology, ecology and phylogeny of the (NY): McGraw Hill Book Co.; 1940. p. 104. Ranunculaceae VIII. (Ranunculaceae of Eastern Asia: general Judd WS, Campbell CS, Kellogg EA, Stevens PF, Donoghue MJ. part VIII). Sci Rep Osaka Univ 1968;17:41–56. Plant systematics: a phylogenetic approach. Ecol Mediterr Walker JW. Aperture evolution in the pollen of primitive 1999;25(2):215. angiosperms. Brittonia 1972;24(2):129. Kumazawa M. Pollen grain morphology in Ranunculaceae, Walker JW, Doyle JA. The bases of angiosperm phylogeny: Lardizabalaceae and Berberidaceae. Jpn J Bot 1936;8:19–46. palynology. Ann Mo Bot Gard 1975;664–723. Metcalfe CR, Chalk L. Anatomy of the dicotyledons, vol. 1 and 2. Wodehouse RP. Pollen grains in the identification and 1950. classification of plants. VII. The Ranunculaceae. Bull Torrey Moore PD, Webb JA, Collison ME. Pollen analysis. Blackwell Bot Club 1936;63(9):495–514. scientific publications; 1991. Worsdell WC. The origin of the perianth of flowers with special Novikoff AV, Jabbour F. Floral anatomy of Delphinieae reference to the Ranunculaceae. New Phytol 1903;2(2):42–8. (Ranunculaceae): comparing flower organization and vascular Worsdell WC. A study of the vascular system in certain orders of patterns. Mod Phytomorphol 2014;5:35–44. the Ranales. Ann Bot 1908;22(88):651–82.