Seed Morphology ofNigellas.l. (Ranunculaceae): Identification, Diagnostic Traits, and Their Potential Phylogenetic Relevance

Andreas G. Heiss; Matthias Kropf; Susanne Sontag; Anton Weber

A comprehensive morphological and anatomical analysis was carried out on seeds of all 15 species currently recognized in the genus Nigella s.l. (including Komaroffia and Garidella). In addition, a selection of six infraspecific taxa was examined. Using testa thin sections, morphometry, and SEM imaging, seed coat characters proved to be a highly diagnostic and powerful tool in species identification. A dichotomous identification key is presented along with seed descriptions, measurements and anatomical details, LM photos, and SEM micrographs. Analyses using maximum parsimony and character mapping onto a DNA-based phylogeny suggest that seed characters will be useful for ongoing phylogenetic studies in the genus. The importance of properly identifying Nigella seeds is highlighted for applied use in archaeobotany and pharmacognosy.

Int. J. Plant Sci. 172(2):267–284. 2011. Ó 2011 by The University of Chicago. All rights reserved. 1058-5893/2011/17202-0010$15.00 DOI: 10.1086/657676 SEED MORPHOLOGY OF NIGELLA S.L. (RANUNCULACEAE): IDENTIFICATION, DIAGNOSTIC TRAITS, AND THEIR POTENTIAL PHYLOGENETIC RELEVANCE Andreas G. Heiss,1 ,* Matthias Kropf,y Susanne Sontag,z and Anton Weberz *University of Natural Resources and Life Sciences (BOKU), Institute of Botany, Gregor Mendel-Strasse 33, 1180 Wien, Austria, and Vienna Institute for Archaeological Science (VIAS), Archaeobotany, c/o Institute of Palaeontology, Geozentrum, Althanstrasse 14, 1090 Wien, Austria; yUniversity of Natural Resources and Life Sciences (BOKU), Institute of Integrative Nature Conservation Research, Gregor Mendel-Strasse 33, 1180 Wien, Austria; and zUniversity of Vienna, Faculty Centre of Biodiversity, Department of Structural and Functional Botany, Rennweg 13, 1030 Wien, Austria A comprehensive morphological and anatomical analysis was carried out on seeds of all 15 species currently recognized in the genus Nigella s.l. (including Komaroffia and Garidella). In addition, a selection of six infraspecific taxa was examined. Using testa thin sections, morphometry, and SEM imaging, seed coat characters proved to be a highly diagnostic and powerful tool in species identification. A dichotomous identification key is presented along with seed descriptions, measurements and anatomical details, LM photos, and SEM micrographs. Analyses using maximum parsimony and character mapping onto a DNA-based phylogeny suggest that seed characters will be useful for ongoing phylogenetic studies in the genus. The importance of properly identifying Nigella seeds is highlighted for applied use in archaeobotany and pharmacognosy. Keywords: Garidella, Komaroffia, Nigella, phylogeny, Ranunculaceae, seed morphology. Online enhancements: appendixes. Introduction therefore provide that information for Nigella. Another applied use of seed morphological data lies in the interdisciplinary field of archaeobotany/paleoethnobotany: together with their archaeological contexts, correctly identified plant remains are the basis for the reconstruction of migrations of human and plant populations as well as for general investigations into the cultural history of plants through human history (see Renfrew 1973; Hastorf and Popper 1988; Jacomet and Kreuz 1999; Zohary and Hopf 2000). Nigella L. (fennel flower, nigella) is a small genus within the buttercup family (Ranunculaceae), comprising ;15 species (Zohary 1983; Dönmez and Mutlu 2004) distributed from the Middle East (the center of diversity for the genus) to Spain. It is remarkable in several academic and applied respects: (1) it is the only genus of Ranunculaceae with a truly syncarpous gynoecium (Rohweder 1967); (2) the flowers of the advanced species within the genus exhibit an interesting and complex pollination mechanism, representing highly specialized ‘‘roundabout flowers’’ (Sprengel 1793; Weber 1993, 1995); (3) some species, especially Nigella damascena L., are popular ornamental plants (Burnie et al. 2008); (4) the seeds of several species have been in use as a condiment since prehistoric times (Hepper 1990; Heiss and Oeggl 2005; Salih et al. 2009); and (5) seed oils of N. sativa L. and N. damascena are of high commercial interest to the pharmaceutical and cosmetics industries (Agradi et al. 2002; Ali and Blunden 2003; Anwar 2005). In recent years, additional Nigella species have moved into the focus of pharmaceutical research, such as N. arvensis L., N. integrifolia Regel, N. nigellastrum (L.) Willk. in Willk. et Lange, N. orientalis L., and N. segetalis M. Bieb. (Aitzetmüller et al. 1997; Aitzetmüller 1998; Kökdil and Yılmaz 2005; Kökdil et al. 2006b). Seed morphology has developed into an important source of useful phylogenetic information, following the work of Barthlott (1981, 1984) and increasing throughout the 1990s. A number of angiosperm taxa have already been studied intensively in terms of their seed micromorphology, in combination with phenetic or phylogenetic analyses at the genus level. Data of this kind are now available across a broad evolutionary range of plant families, such as Schisandraceae (Schisandra and Kadsura; Denk and Oh 2006), Cactaceae (Stenocereus; Arroyo-Cosultchi et al. 2006), Oxalidaceae (Oxalis; Obone 2005), Melastomataceae (Leandra, Miconia, Ossaea, and Clidemia; Martin et al. 2008), Gentianaceae (Gentiana; Davitashvili and Karrer 2010), Lamiaceae (Hemigenia and Microcorys; Guerin 2005), Plantaginaceae (Veronica; MuñozCenteno et al. 2006), and Orchidaceae (Liparis; Tsutsumi et al. 2007). In Ranunculaceae, most such work has focused on Aconitum (Tamura 1993; Luo et al. 2005). Seed and fruit morphological data, if only of selected species, have also been included in the most recent works on Ranunculaceae phylogeny (Wang et al. 2009; Emadzade et al. 2010). However, there are additional fields in which seed morphology is of increasing interest. In pharmacognosy, proper identification is crucial for quality control of seed accessions. Detailed information on seed identification is often still missing, especially in the case of the taxa recently studied for pharmacological research, such as some Nigella species; we will 1 Author for correspondence; e-mail: andreas.heiss@holzanatomie.at. Manuscript received June 2010; revised manuscript received October 2010. 267 268 INTERNATIONAL JOURNAL OF PLANT SCIENCES The taxonomic position of Nigella s.l. within Ranunculaceae as well as the number of species included and their respective delimitations have changed repeatedly. For instance, previous investigations have placed Nigella and its segregates Garidella and Komaroffia in the subfamily Ranunculoideae, tribe Delphinieae (Hoot 1991; Frohne and Jensen 1998; Stevens 2001–), or in the Aconitoideae (Takhtajan 2009). However, a recent synopsis of molecular and morphological data suggests that the group’s affinities within the Ranunculaceae are only weakly supported and must be considered uncertain (Wang et al. 2009). Taxonomy within the genus has also undergone many changes in recent decades. Currently, Nigella s.l. is commonly divided into three genera: Komaroffia Kuntze, Garidella L., and Nigella L. s.str., as done by Tamura (1993) and Tutin et al. (1964–1983). For overviews of alternative classifications, see Gregory (1941) and Zohary (1983). In this work, we used the monograph of Zohary (1983) as a reference, which recognizes 14 species, essentially on the basis of morphological (mainly floral and fruit) and karyological criteria. Nigella s.l. sensu Zohary includes Komaroffia and Garidella at the rank of sections (see table 1). For taxa within the N. arvensis aggregate, the work by Strid (1970) deserves mention, since it differs from the approach of Zohary (1983) and has been used in various recent studies, such as that of Bittkau and Comes (2008). Molecular investigations at the genus level have been carried out only very recently—namely, the analysis of DNA sequences of the internal transcribed spacer (ITS) region representing 25 taxa, 11 of which belong to the N. arvensis aggregate (Bittkau and Comes 2008)—while analyses of chloroplast DNA are still in progress (trnL-trnF, trnK-matK intron, atpB-rbcL intron; C. Bittkau and H. P. Comes, unpublished data). However, comprehensive and multidisciplinary studies of the taxonomy of Nigella are missing, and there is still no consensus as to whether Nigella should be treated as a single genus or split into three. Seed morphology, representing a potential set of taxonomically informative features, has only rarely been considered in taxonomic descriptions of Nigella species. To some degree, particular aspects of seed morphology and testa anatomy have been included in general treatments of angiosperm seeds, including species such as N. nigellastrum (Netolitzky 1926) and N. damascena (Corner 1976). Rough macroscopic identification criteria for N. sativa and N. damascena, occasionally also for N. arvensis, are given in agronomical and archaeobotanical seed identification guides (Beijerinck 1947; Brouwer and Stählin 1955; Montégut 1971; Berggren 1981; Bojňanský and Fargašová 2007). Curiously, in pharmacognosy, criteria for identifying and discriminating the seeds of Nigella have been largely neglected or ignored. Wichtl (2004, p. 417), for instance, stated for N. sativa, ‘‘Due to the characteristic morphology, odor and taste of the drug, a microscopic examination seems unnecessary,’’ ignoring similarities between species in taste or odor (N. sativa vs. N. arvensis agg.; A. G. Heiss, personal observation) or in seed shape (N. sativa vs. N. arvensis agg., N. damascena, N. elata, or N. turcica). This could easily lead to the misidentification of seed accessions or prevent the recognition of adulterated material. Seed identification criteria for Nigella have become available only very recently (see table 1). Three studies on Nigella seed morphology have been published, two of them considering six taxa (Bahadur et al. 1984; Karcz and Tomczok 1987a), with the third and most recent study covering 12 taxa (Dadandi et al. 2009). In comparison to these, not only is this article more comprehensive in terms of the number of Nigella taxa studied (21 taxa in 15 species) but also it integrates a wider range of methods, namely microscopic sections, SEM imaging, morphometry, a standardized documentation of the observed features, and an evolutionary perspective based on character mapping onto a previously published phylogenetic tree. Our primary goal in the current study is to demonstrate and document the variability of seed morphology within the genus Nigella s.l. and to provide quantitative data for a future phylogenetic reassessment of the whole genus. We attempt to identify seed characters that might prove useful for this purpose through phylogenetic analyses and also to document possible differences in taxonomic groupings on the basis of seed morphology and recent classifications, including the most recent monograph (Zohary 1983) and the evolutionary lineages inferred from a recent molecular analyses (Bittkau and Comes 2008). As a second core part of the article, an identification key for the seeds of Nigella is provided. This will serve as a useful tool in the fields of pharmacology and archaeobotany. Material and Methods Seed Material In total, 2229 seeds from 40 seed accessions were investigated, corresponding to 21 taxa (15 species) and originating from a wide variety of sources: commercial products as well as seeds from herbaria, collections of botanical gardens, and material collected by the authors (app. A). However, only a single accession was available for 10 of the included taxa, namely, Nigella ciliaris, N. oxypetala, N. stellaris, N. turcica, N. unguicularis, and several varieties from the N. arvensis group (accessions Nar000, Narar0, Naras0, Narin0, and Nartr0). In these cases, we had to make the initial assumption that infraspecific or infravarietal variability would not mask interspecific or intervariety differences in seed morphology. In addition to the 14 Nigella species recognized by Zohary (1983), the recently described species N. turcica was also included; according to the taxon’s authorities (Dönmez and Mutlu 2004), it is closely related to N. sativa. All of the taxa belonging to the N. arvensis group treated in this study were covered in the most recent monograph (Zohary 1983), except for N. arvensis var. trachycarpa Borb.; this taxon has been described as a distinct variety occurring in eastern and southeastern Europe (von Borbás 1887). Two species covered by the Index Kewensis (Hooker and Jackson 1893–) and some other authors were not analyzed in this study and must be mentioned. (1) Nigella atropurpurea Huber is, with high probability, an illegitimate synonym of N. hispanica L.; the name was introduced in an 1866 nursery catalog by the company Huber and Co. in Hyères, France (Mabberley 1985). (2) Nigella glandulifera Freyn and Sint. ex Freyn is reported as being ‘‘cultivated (not native) in China’’ (Wang et al. 2001), but no information on its geographical origin is given. The species is frequently referred to by pharmacologists Table 1 Overview of Previous Studies of Seed Macro- and Micromorphology of Nigella Species Taxon Sect. Komaroffia (O.Ktze) Brand: Nigella integrifolia Regel Sect. Garidella (L.) Spenn.: N. nigellastrum (L.) Willk. N. unguicularis (Lam.) Spenn. Sect. Nigella L.: Subsect. Nigellaria (DC.) Terracc.: Nigella arvensis L. N. arvensis L. var. arvensis N. arvensis L. var. assyriaca (Boiss.) Zoh. N. arvensis L. var. glauca (Schkuhr) Boiss. N. arvensis L. var. involucrata Boiss. N. N. N. N. N. Bahadur et al. 1984 Karcz and Tomczok 1987a Dadandi et al. 2009 This study Komaroffia diversifolia (Franchet) O.Ktze þ þ Garidella nigellastrum L. G. unguicularis Lam. þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ ‘‘N. arvensis,’’ ‘‘N. orientalis’’ þ þ þ þ þ þ þ þ N. lancifolia Hub.-Mor., N. latisecta P.H.Davis, N. oxypetala Boiss. þ þ þ Important synonyms arvensis L. var. trachycarpa Borb.a fumariifolia Kotschy glandulifera Freyn & Sint. ex Freyna hispanica L. var. hispanica hispanica L. var. intermedia Coss. N. hispanica L. var. parviflora Coss. N. sativa L. N. segetalis M.Bieb. N. stellaris Boiss. N. turcica Dönmez & Mutlua Subsect. Erobathos (DC.) Zoh.: N. damascena L. N. elata Boiss. Subsect. Nigellastrum (DC.) Zoh.: N. ciliaris DC. N. orientalis L. N. oxypetala Boiss. N. arvensis L. subsp. arvensis N. arvensis L. subsp. glauca (Boiss.) Terracc. p.p., N. carpatha Strid, N. degenii Vierh., N. degenii Vierh. subsp. barbro Strid, N. degenii Vierh. subsp. jenny Strid, N. doerfleri Vierh., N. icarica Strid, N. stricta Strid N. arvensis L. subsp. aristata (Sibth. & Sm.) Nyman N. hispanica L., N. papillosa G. López N. papillosa G. López subsp. atlantica (Murb.) Amich N. gallica Jord. ‘‘N. hispanica,’’ N. sativa Note. Studies grouped according to the classification by Zohary (1983). Synonyms are given referring to the work by Strid (1970). Underlined synonyms are accepted by the Flora Europaea (Tutin et al. 1964–1983). For taxa in quotation marks, refer to the seed descriptions in appendix A and the ‘‘Discussion.’’ a For taxa not covered by Zohary’s revision, their assumed positions are according to the notes of Freyn (1903) and Dönmez and Mutlu (2004). 270 INTERNATIONAL JOURNAL OF PLANT SCIENCES from Southeast Asia (Liu et al. 2004; Tian et al. 2006; Nguyen et al. 2007). According to the taxon’s authorities, N. glandulifera seems to be closely related to—if not conspecific with—N. sativa (Freyn 1903). Riedl and Nasir (1991) indeed synonymize N. glandulifera with N. sativa L. var. hispidula Boiss. Unfortunately, taxonomic investigations have never been carried out on the species after the initial work of J. Freyn, and no seeds could be obtained for this study. Therefore, the status of this ‘‘species’’ remains doubtful and must be reassessed in the future. As an outgroup taxon, we chose Aconitum lycoctonum L. subsp. vulparia (Rchb.) Nyman for several reasons. We intentionally decided not to use N. integrifolia as an outgroup, as done by Bittkau and Comes (2008), but to continue using the taxonomy suggested by Zohary (1983), regarding the genus Komaroffia as a section of Nigella s.l. as a starting hypothesis. Although the phylogenetic analysis by Wang et al. (2009) leaves the position of Nigella within the Ranunculaceae rather open, the Delphinieae can still be regarded as a possible sister taxon to Nigella s.l., albeit an only weakly supported one. Furthermore, using the ITS sequence of N. integrifolia (Bittkau and Comes 2008) in a BLAST search excluding sect. Nigella and sect. Garidella as the known closest relatives revealed (given a query coverage of 62%–63%) a maximum DNA sequence identity of 87% with eight species of Hepatica and 20 species of Aconitum. After assessing the published literature on Delphinieae seeds (Aconitum: Wojciechowska and Makulec 1969; Cappelletti and Poldini 1984; Consolida: Karcz and Tomczok 1987b; Constantinidis et al. 2001; Del_ phinium: Ilarslan et al. 1997), we found that Aconitum shared a roughly comparable testa structure with Nigella s.l., which thus enabled us to code the outgroup with as few additional characters as possible. Finally, the limited availability of well-identified herbarium material also influenced our choice. The seed characters of A. lycoctonum subsp. vulparia were mainly taken from the work of Cappelletti and Poldini (1984) and were complemented by our own observations. Light Microscopy Thin sections were prepared with the following procedure: the seeds were soaked in a 4 : 1 mixture of distilled water and 96% ethanol for 1 h. Cross sections 20 mm thick were prepared using a Reichert microtome without prior embedding of the seeds. The thin sections were bleached in sodium hypochlorite (5% NaOCl) for 30 min and then rinsed in distilled water for 1 h. Staining was carried out by immersing the specimens in a 4 : 1 mixture of distilled water and Etzold’s fuchsinchrysoidin-astrablue solution (FCA; Etzold 2002) for 15 min, resulting in differential staining of cellulose (blue), lignin (pink), and lipophilous substances (yellow) in the seed coat. Before microscopic investigation, the stained thin sections were rinsed in distilled water and embedded in glycerine. For the observation of tissue/cell characters, an Olympus BX50 microscope with polarized light was used, and measurements were carried out with an eyepiece micrometer. Microscopic photos were taken using a Canon Powershot A95 camera with an attached eyepiece adapter manufactured by R. Mehnert (Weil der Stadt). In order to avoid blurring due to the limited depth of field, an image stack of 10–50 frames was recorded for each individual image and joined with the software Helicon Focus (Kozub et al. 2000–2008). Macroscopic images of the seeds were created with a Wild/Leica Photomakroskop M400 and the same photographic equipment, also using image stacking. The background was subtracted from the macroscopic photos using Photoshop CS 2 (Adobe Systems 2005). Scanning Electron Microscopy (SEM) Seeds used for SEM imaging were desiccated in an ascending ethanol series (50% and 75% for 24 h each), after which they were placed in a drying oven for 24 h at 40°C. They were sputtered with ;1 mm of gold/palladium coating. SEM imaging was carried out with a Philips XL 20 at the Institute of Botany, University of Innsbruck (N. arvensis var. glauca, N. elata, N. orientalis, N. sativa, and N. turcica), and with a JEOL T300 at the (former) Institute of Botany, University of Vienna (remaining taxa). Scale bars in the images were added manually in the latter case. Morphometry A total of 1700 seeds were measured. Digital images of whole seeds were created as 600-dpi grayscale images with a Xerox DocuScan flatbed scanner and calibrated with a millimeter scale. Measurement accuracy was ;50 mm. Before image analysis, image corrections (contrast adjustment, elimination of dirt particles and overlapping seeds) were carried out with Photoshop CS 2 (Adobe Systems 2005). The software ImageJ 1.42q (Rasband 1997–2009) was used to measure particle sizes with the ‘‘fit ellipse’’ option: the parameters ‘‘major’’ and ‘‘minor’’ were determinants for maximum length and width of each seed. In thin sections, the height of epidermal cells and the total thickness of all subepidermal cell layers were measured. These measurements were based on a single transverse seed section in the region between the lateral ridges and were recorded as one maximum and one minimum value per taxon (for character coding, see table D1 in the online edition of the International Journal of Plant Sciences). Character Selection and Coding The significance of seed shape and sculpture for dispersal, and thus their importance for evolutionary processes, is well known (Barthlott 1981) and has already been applied in phylogenetic studies, as mentioned in the ‘‘Introduction.’’ However, their phylogenetic relevance has never been quantified in Nigella. Therefore, we chose the maximum parsimony (MP) approach in order to find possible phylogenetically informative characters for future studies. Character coding was based on hypothetical homologies assessed from seed geometry and spermoderm structure, for example, grouping characters according to the categories recognized by Barthlott (1981): characters of primary structure refer to epidermal cell patterns and shapes, which were divided into nine states (see fig. 1), and secondary structure refers to characters of periclinal cell wall ornamentation. Of the morphometric data, the respective 1s HEISS ET AL.—SEED MORPHOLOGY OF NIGELLA S.L. 271 Fig. 1 Types of epidermal cells recorded for Nigella, grouped in a hypothetical hierarchy of cell types (top row) and their subtypes (bottom row). The type columellate ligulate is illustrated in lateral and apical view. intervals of length and width measurements per taxon were obtained using the software SPSS 11 (SPSS 2001). In the data matrix analyzed, they were expressed as size classes (see table D1). These were set up in 1-mm intervals in order to obtain groups containing roughly comparable numbers of individuals. All measurements were coded as ordered characters on the basis of the hypothesis that a seed would be more likely to evolve toward a neighboring size class than directly to a more remote one. Data matrices were then built using the software DELTA (Dallwitz and Paine 1993–2005; Dallwitz et al. 1993–). Data Analysis After export of the data matrix from DELTA via NEXUS files (Maddison et al. 1997), cluster analyses were carried out using PAUP* 4.0b10 (Swofford 1998), assisted by the graphical interface PaupUP (Calendini and Martin 2005). In general, all characters were treated as unweighted, and multiple states were treated as polymorphisms. The outgroup A. lycoctonum subsp. vulparia was used for rooting. Initial data evaluation was carried out using both distance and MP criteria. The neighbor-joining (NJ) tree (Saitou and Nei 1987) based on 11 characters and total character difference was complemented by bootstrap support (BS) values (Felsenstein 1985), calculated with 50% majority rule, 1000 replicates, and character resampling in effect (Efron et al. 1996). MP trees were calculated for nine parsimony-informative characters in a two-step procedure: in a first full heuristic search with 1000 random addition replicates and tree bisection reconnection (TBR), a limit of 10 trees retained per replicate was used in order to minimize the time spent on suboptimal trees. The resulting 5990 best trees (score 121) were then used as starting trees in the main full heuristic run, again using TBR and 1000 random replicates, with the maximum tree limit increased to 100,000. BS of the resulting clades was calculated with 1000 bootstrap replicates, character resampling, and TBR in effect. Because of the rather low number of parsimony-informative characters (9) and taxa (21), a tree limit of 10,000 was applied. We re- garded BS of 50%–74% as weak support, 75%–89% as moderate support, and 90%–100% as strong support for each clade. Tree output was visualized in TreeView (Page 1996) and postprocessed in CorelDRAW X4 (Corel 2008). Since our data set contained a mixture of ordered and unordered characters and was not coded in a binary way (as suggested by Pleijel 1995), distance is not the ideal optimality criterion. We thus decided to focus on the MP analysis over the distance analysis, since we regarded the former to be a more reliable representation of possible shared characteristics and to allow phylogenetic inferences. As an additional source of information regarding the possible phylogenetic relevance of seed morphological characters and their likely evolutionary trend within the study group, our data matrix was mapped onto the ITS phylogeny previously published by Bittkau and Comes (2008) using MacClade 3.0 (Maddison and Maddison 1992). Identification Key The data matrix generated in DELTA was exported as an HTML identification key with a set of 21 seed characters. The key was then manually adapted to the diacritical method, as suggested by Fischer and Willner (2010). For practical reasons, the focus lay on anatomical features easily observable by light microscopy. In some cases, additional characters (such as seed color or characters observable only via SEM) were added if necessary for identification. Results General Seed Morphology For detailed individual descriptions, see appendix C in the online edition of the International Journal of Plant Sciences. In all the taxa we investigated, we observed a multilayered testa (see figs. 2, 3). The number of cell layers, however—and therefore the total testa thickness—varies widely between spe- Fig. 2 Light microscope images of Nigella species investigated. A, Nigella arvensis. B, Nigella arvensis var. glauca. C, Nigella ciliaris. D, Nigella damascena. E, Nigella fumariifolia. F, Nigella hispanica. G, Nigella hispanica var. parviflora. H, Nigella integrifolia. Scale bars ¼ 1 mm (whole seed view), 100 mm (thin sections). 272 Fig. 3 Light microscope images of Nigella species investigated. A, Nigella nigellastrum. B, Nigella orientalis. C, Nigella oxypetala. D, Nigella sativa. E, Nigella segetalis. F, Nigella stellaris (no adequate image of thin section available). G, Nigella turcica. H, Nigella unguicularis. Scale bars ¼ 1 mm (whole seed view), 100 mm (thin sections). 273 274 INTERNATIONAL JOURNAL OF PLANT SCIENCES cies (tables D1, D2). A single vascular bundle, embedded in a more or less distinct ridge, runs along the seed from the hilum. Thin sections showed the presence of lipophilous substances (apparently oil drops; see figs. 2, 3) in the spermoderm of all investigated taxa. Seed geometry and size were strongly divergent between taxa: one group, corresponding to subsect. Nigellastrum, is characterized by dorsoventrally flattened seeds more than 4 mm long and wide, with an ovate to circular outline (see figs. 2C, 3B, 3C, 4B, 5D, 5E). The second group (identical with sect. Garidella) has smaller obovate seeds with a single ventral ridge, and covered by an irregular reticulum up to 250 mm high (figs. 3A, 3H, 5C, 6E). The seeds of the remaining taxa display a basically trigonous-ovate shape, some similar to the segments of an orange. In terms of the general seed surface features and the primary structure, the three taxa in subsect. Nigellastrum share the characteristic of having only flat prismatic cells, as do N. hispanica—including the ssp. parviflora (figs. 2F, 2G, 5A)—and N. segetalis (figs. 3E, 6B). The same five taxa show a thick periclinal cell wall in the outermost epidermal cell layer. The remaining groups are characterized by the presence of various cell types, often arranged in transverse structures, and thinner cell walls. Nigella damascena and N. elata share a transverse reticulum bordering mucronulate cells; the distinct central mucronulus (fig. 4C) separates N. damascena from N. elata, which has an indistinct and excentric mucronulus (fig. 4D). Pilate/capitate cells are characteristic of N. integrifolia (fig. 2H), as is a truncate type in N. fumariifolia (fig. 2E) and N. stellaris. Collapsed (ocellate) prismatic cells were observed in N. arvensis agg., N. sativa, N. stellaris, and N. turcica. This cell type correlates with thinner periclinal cell walls (see figs. 2A, 2B, 3G). Secondary structure is unspecific in most investigated taxa and typically varies from irregularly granulate to rugulate. However, N. ciliaris lacks any secondary structure in seed surfaces (fig. 4B), which gives them a shiny appearance (fig. 2C). Furthermore, the two species in sect. Garidella display distinctly undulate rugulae in the flat prismatic cells lying between the ridges (figs. 5C, 6E). Another unique character is the secondary structure found in N. integrifolia, where the rugulae/striae are radially oriented (fig. 5B). Seed Identification The results show that it is possible to identify Nigella to the species level for most taxa using only seed characters. The resulting dichotomous identification key is given in appendix B. In the few cases where taxa could not be separated, a clear distinction of groups could at least be established. The recorded characters were, for example, not sufficient to clearly discriminate between infraspecific taxa of the N. arvensis group. Likewise, it was not possible to efficiently separate the seeds of N. hispanica (including its var. parviflora) from N. segetalis or those of N. nigellastrum from N. unguicularis. Phylogenetic Trees Results from MP analysis (fig. 7) resolved only two wellsupported groups, namely sect. Garidella (100% BS) and sub- sect. Nigellastrum (89% BS), while moderate support (73%) is available for the clade including all Nigella species except subsect. Nigellastrum, N. hispanica s.l., and N. segetalis. One subclade within subsect. Nigellastrum has low support (N. orientalis and N. oxypetala; 58%), while all others have BS values below 50%. A large group incorporating the whole subsect. Nigellaria as well as subsect. Erobathos and sect. Komaroffia therefore remains unresolved. Character Mapping Mapping characters onto the existing DNA-based phylogenetic hypothesis (fig. 8) indicated that only a few molecularly defined taxa are characterized by unambiguous autapomorphies in seed morphology. These were the sections Garidella and Komaroffia and the subsections Erobathos and Nigellastrum. A few characters (see table D1) represent important autapomorphies, namely characters 1 (seed shape) and 7 (secondary structure), both of which define three separate groups in the phylogeny. Characters 4 (seed surface), 6 (primary structure), 8 (presence of resin-filled idioblasts), 9 (outermost testa layer height), and 10 (underlying testa layers height) each occur a single time as autapomorphies. Discussion General Seed Morphology and Species Identification Despite the difficulties in the separation of taxa within the Nigella arvensis group, there are clearly observable (and possibly constant) differences in the proportions of cell types, such as ocellate versus columellate cells or columellate versus ligulate versus truncate cell types. This was also suggested by Dadandi et al. (2009). However, thorough quantification and assessment of these characters would require huge amounts of data and time for setting up both the means of identification and their applied use; total cell counts per seed would be necessary for proper identification. In contrast to the lack of distinction between N. nigellastrum and N. unguicularis observed in the current study, Dadandi et al. (2009) report characters useful for differentiating the two species, namely rugulate periclinal cell walls in N. nigellastrum versus pitted or microreticulate walls in N. unguicularis. We were not able to observe these features in the current study. Neither were we able to reproduce the differentiating canal (N. nigellastrum) versus ridge (N. unguicularis) of the anticlinal cell wall. Differing results were also obtained when comparing the current results on subsect. Nigellastrum with two previous studies: Karcz and Tomczok (1987a) report conical projections in the central portion of N. orientalis seeds. Conversely, Dadandi et al. (2009) document nipplelike projections as present in N. oxypetala and N. latisecta (the latter is commonly considered synonymous to N. oxypetala) but as missing in N. orientalis and N. lancifolia (which, again, is considered synonymous to N. oxypetala). The current study found no indications of cell wall projections in either N. orientalis or N. oxypetala. These differences may well be due to morphological variability within N. orientalis and N. oxypetala, but the existence of hitherto unidentified subtaxa in both species must Fig. 4 SEM micrographs of Nigella species investigated. A, Nigella arvensis var. glauca. B, Nigella ciliaris. C, Nigella damascena. D, Nigella elata. E, Nigella fumariifolia. Fig. 5 SEM micrographs of Nigella species investigated. A, Nigella hispanica. B, Nigella integrifolia. C, Nigella nigellastrum. D, Nigella orientalis (seed wings dissected). E, Nigella oxypetala. 276 Fig. 6 SEM micrographs of Nigella species investigated. A, Nigella sativa. B, Nigella segetalis. C, Nigella stellaris. D, Nigella turcica. E, Nigella unguicularis. 277 278 INTERNATIONAL JOURNAL OF PLANT SCIENCES Fig. 7 Tree no. 326 of the 100,000 most parsimonious trees (length 121) resulting from the second run of the heuristic search. Numbers indicate bootstrap percentages, dashed lines indicate branches that collapsed during the bootstrap. Consistency index ¼ 0.957, retention index ¼ 0.971. also be considered possible. In general, variability within taxa must be considered an important factor in morphological analyses. For 11 taxa in our study (including N. orientalis), multiple accessions were available, thus helping to reduce any possible bias caused by infraspecific variability. The results of the current study, alongside those of Karcz and Tomczok (1987a) and Dadandi et al. (2009), differ strikingly from some of the seed descriptions given by Bahadur et al. (1984). This can most likely be explained as misidentifications of the seed accessions in the 1984 study (for details, see app. C). Using the respective descriptions and SEM images in their publication, three of the six described taxa need to be corrected as follows: ‘‘N. arvensis’’ ! N. damascena, ‘‘N. hispanica’’ ! N. sativa, and ‘‘N. orientalis’’ ! N. damascena. Phylogenetic Implications of MP Analysis and Character Mapping The MP tree resolved two well-supported groups corresponding to sect. Garidella and subsect. Nigellastrum, indicating that the taxa included in the respective groups share a significant number of seed morphological traits (fig. 7). The two species of sect. Garidella show the greatest phenotypic divergence from all other taxa investigated. Four autapomorphies in seed geometry, surface pattern, testa composition, and secondary structure (fig. 8) place N. nigellastrum and N. unguicularis in a remote position relative to the remaining Nigella taxa. This can be regarded as additional support for their placement in a separate genus Garidella, as suggested by flower morphology (Linnaeus 1753; Boissier 1867), palynology (Skvarla and Nowicke 1979; Dönmez and Isxık 2008), DNA (Bittkau and Comes 2008), and phytochemistry (Aitzetmüller et al. 1997). Although not resolved in the MP tree, the monotypic section Komaroffia is also separated from Nigella by two autapomorphies (fig. 8), namely the capitate/pilate cells and the unique secondary structure with radially oriented rugulae/striae (fig. 5B). Against the background of evidence from karyology (2n¼14 in N. integrifolia vs. 2n¼12 in all other species in Nigella s.l.; Gregory 1941; Strid 1970), ITS sequence data (Bittkau and Comes 2008), and seed oil characteristics (Aitzetmüller 1998), seed morphology also supports the maintenance of a separate genus Komaroffia. The three taxa contained in sect. Nigella subsect. Nigellastrum form a well-resolved clade in the MP tree (fig. 7). It is mainly defined by an autapomorphy in seed geometry (fig. 8) but also by the presence of flat prismatic cells only and thick periclinal epidermal cell walls, two homoplasies shared with N. hispanica s.l. and N. segetalis. The subsection Nigellastrum, as defined by Zohary (1983), is supported by seed morphology, and ITS phylogeny (Bittkau and Comes 2008) also suggests a proximity of the taxa N. ciliaris, N. orientalis, and N. oxypetala. The taxonomic implications of Dadandi et al. HEISS ET AL.—SEED MORPHOLOGY OF NIGELLA S.L. 279 Fig. 8 Characters (top numbers) and their states (bottom numbers) mapped onto the phylogenetic hypothesis based on ITS sequences as previously published by Bittkau and Comes (2008). Only unambiguous character states are given. Apomorphies are indicated by filled circles, homoplasies by open circles. Taxon names and accession numbers follow the original publication (based on Strid 1970); synonyms used in the current study (according to Zohary 1983) are given in parentheses. (2009) even suggest an independent position of sect. Nigellastrum from the other taxa investigated in their study, referring to similar results in a stem anatomy study by the same working group (Kökdil et al. 2006a). Apart from the previously mentioned homoplasies, N. hispanica s.l. and N. segetalis share other homoplastic characters, such as seed geometry and color. Their seeds could not be efficiently distinguished in this analysis, and although the two species are presently found in disjunct areas of the Mediterranean—N. hispanica in southwestern Europe and western North Africa and N. segetalis in the Irano-Turanian region (Tutin et al. 1964–1983; Zohary 1983)—they also share a wide range of morphological (Zohary 1983) and DNA characteristics (Bittkau and Comes 2008; C. Bittkau and H.-P. Comes, unpublished data). Taking into account their similarities in seed morphology, they should be regarded as more closely related than has previously been assumed. Likewise, the close similarity of N. fumariifolia to N. stellaris observed in their seed morphology is not clearly mirrored in traditional Nigella taxonomy, although Terracciano (1897; cited in Zohary 1983) treated N. stellaris as a subspecies of N. fumariifolia. This possible close relationship is also reflected by the results of phylogenetic analyses of ITS data (Bittkau and Comes 2008) and is in need of further investigation. The monophyly of subsect. Erobathos, a taxon accepted by traditional taxonomy (Zohary 1983) and supported by molecular data, is further supported by a seed morphological autapomorphy (testa thickness) and is also characterized by the presence of mucronulate cells, a unique character of its primary structure. Within the N. arvensis aggregate, no clear grouping could be observed. Primary structure proved most variable in this group: a total of four different cell types were observed in varying proportions in the six investigated N. arvensis taxa, as documented by Dadandi et al. (2009) for two of the varieties/ subspecies. 280 INTERNATIONAL JOURNAL OF PLANT SCIENCES Conclusions In the current study, seed shape and secondary structure proved to be the most useful seed morphological characters for characterizing Nigella taxa. Other characters, such as the thickness of the underlying testa layers as well as the presence of idioblasts, represent autapomorphies for specific clades (such as the section Garidella). The remaining features—seed measurements, the occurrence of pigmented cells, and the primary structure—did not in general reflect a clear evolutionary trend. However, the high variability and homoplastic patterns of primary structure reveal extensive evolutionary plasticity in these surface structures. The current analysis cannot provide definite conclusions on the phylogenetic status of the genus Nigella; a thorough synthesis of the current and other morphological as well as molecular data will be required to accomplish this. We have, however, demonstrated that seed morphology can contribute valuable information for species classification in Nigella. As a main result, the considerable phylogenetic distinction between the sections Garidella, Komaroffia, and Nigella, as also shown in other studies, is clearly reflected by seed morphology. Perspectives As a result of the current study, proper identification and quality control of seed accessions of Nigella will now be possible to a much greater extent. This will hopefully facilitate the accelerating research regarding pharmacologically interesting taxa. Some of these, such as N. ciliaris, have not yet been analyzed for their medicinal potential but should be, according to ethnological records (Ali-Shtayeh et al. 2000). The results from this study might also be valuable for research into archaeobotany: the history and prehistory of cultivated and useful plants. Archaeological finds of Nigella species date back to prehistoric times, the oldest dating from the Middle Kingdom in Egypt (N. sativa; around nineteenth century BC; Murray 2000) to the Late Bronze Age in central Europe (N. damascena; 1410–920 calibrated years BC; Heiss and Oeggl 2005). Together with younger finds and written records from Europe, the Near East, and North Africa (A. G. Heiss, unpublished data) document a long tradition of intentional use, cultivation, and synanthropic long-distance transport of Nigella seeds over a wide area, with most of the evidence deriving from N. sativa. However, this study now provides the means to better identify almost all of the species of this fascinating genus by their seeds and will hopefully allow researchers to better understand the importance of Nigella—not only of N. sativa but also of the other species— for past cultures in terms of their economic, environmental, and social value. Finally, we would like to mention seed dispersal in Nigella, which has not been treated in our work but deserves mention as a key issue in future studies. The high degree of seed differentiation we found in this rather small genus (and even within the N. arvensis group) must be assessed in terms of its possible relationship with dispersal strategies. Seed characters may have played a crucial role in the differentiation and radiation of this genus and, more specifically, even within the N. arvensis group: by investigating pollen pigmentation in N. degenii, Jorgensen et al. (2006) have already discovered existing intraspecific variability of a character relevant for reproduction. Also, the different modes of fruit shape and fruit dehiscence in Nigella will require thorough assessment, since they directly influence seed propagation: certain taxa within Nigella (N. arvensis agg., N. subsect. Nigellastrum) seem to utilize barochorous/ombrochorous mechanisms as, for instance, also found in the Ranunculaceae genus Eranthis (Emig et al. 1999), while N. subsect. Erobathos seems to resemble more closely the anemoballistic/boleochorous Papaver type (Müller-Schneider 1977; Kadereit and Leins 1988). As Römermann et al. (2005) have demonstrated, even taxa not typically known as epizoochorous may produce seeds with a high attachment potential to animal hides. The ample differentiation of seed shapes and surface structures in Nigella s.l. will therefore be assessed against this background. Acknowledgments The authors thank the Hochschuljubiläumsstiftung der Stadt Wien for funding parts of the research work (project H-1888/2008). We are grateful to Christiane Bittkau (University of Mainz) and Hans-Peter Comes (University of Salzburg) for granting us access to their ITS original data as well as their unpublished cpDNA trees. Thanks also go to Herbert Knapp, Werner Kofler, and Sigmar Bortenschlager (University of Innsbruck) for making their SEM images of Nigella sativa and Nigella orientalis available to us. We thank Elena Marinova (CAS, Katholieke Universiteit Leuven), Aldona Mueller-Bieniek (Polska Akademia Nauk, Kraków), and Monika Kriechbaum (University of Natural Resources and Applied Life Sciences, Vienna) for their support with literature from ‘‘difficult’’ sources. We are greatly indebted to Ali A. Dönmez (Hacettepe University of Ankara), Sabina Schuster and Wolfgang Neuner (Tyrolean Federal Museum ‘‘Ferdinandeum,’’ Innsbruck), and the following herbaria and botanical gardens for providing seed material of various Nigella taxa: C, GAT, HOH, IB, IBF, LI, MJG, MJSD, PRAZ, WU. We thank all anonymous reviewers for their critical and helpful comments on an earlier version of this manuscript and Christopher Dixon (University of Oxford) for language editing. Appendix A Seed Accessions of Nigella s.l. Investigated in This Study Seed accessions alphabetically sorted by taxon; taxon names follow Zohary (1983). Question marks indicate doubtful or missing data. Asterisks indicate the number of seeds included in the morphometric analysis. Reference material of the seed accessions used in this study is deposited at WHB herbarium. Further information can be obtained from the authors. HEISS ET AL.—SEED MORPHOLOGY OF NIGELLA S.L. 281 Taxon: laboratory number, number of seeds investigated, collection, specimen details, country, locality, leg. (yyyy-mm-dd) / det. (yyyy-mm-dd). Aconitum lycoctonoum L. subsp. vulparia (Rchb.) Nyman: Alyvu0, *35, HBG, IS2006/244, ?, ?; Nigella arvensis L.: Nar000, *213, MJG, IS2004/974 (IPEN XX0MJG19—45660), ?, ?; N. arvensis L. var. arvensis: Narar0, *10, WU, 1759, Albania, ‘‘Sentari’’, Baldacci A (1897-08-09) / Strid A (1969); N. arvensis L. var. assyriaca (Boiss.) Zoh.: Naras0, *7, WU, 004590, Iraq, Ramadi, Rechinger KH (1957-06-06/07) / Strid A (1969); N. arvensis L. var. glauca (Schkuhr) Boiss.: Nargl0, 16, LI, 096499, Turkey, B5 Nevsehir, Göreme, ? (1977-08-26) / Sorger F; Nargl1, *108, BRIX, 44, Turkey, A4 Kastamonu, Freyn J (1892-08-04) / Sintenis P / Heiss AG (2006); N. arvensis L. var. involucrata Boiss.: Narin0, *14, WU, 1622, Greece, Faliro, von Heldreich T (1895-06-08) / Strid A (1969); N. arvensis L. var. trachycarpa Borb.: Nartr0, *43, BRIX, 36, Romania, Ayud, Baenitz C (1894-07-03); N. ciliaris DC.: Nci000, *28, C, S-1977-0977; 279; 183/99; 2581, Israel, ?; N. damascena L.: Nda000, *60, MJG, IS2004/975 (IPEN XX0MJG19—45670), ?, ?; Nda001, *11, collection of A.G. Heiss, 2722, ?, Oeggl K (1985-09-27); Nda002, 6, BRIX, 13, Croatia, Dubrovnik, Huter R (1867-05-19); Nda003, *90, collection of A.G. Heiss, 1249, Italy, Selinunte, Heiss AG (2006-08-07); Nda004, 57, HOH, IS1990/1148, ?, ?; N. elata Boiss.: Nel000, *8, LI, 096512, Turkey, A3 Bolu: Kibrisçik, ? (1983-08-20) / Sorger F; Nel001, *38, BRIX, 23-24-25, Turkey, A1 Istanbul:Kartal, Aznavour GV (1898-07-02/14) / Dörfler J; N. fumariifolia Kotschy: Nfu000, *23, WU, s.n., Cyprus, Lefkoniko, von Halácsy E (1880-0506) / Strid A (1969); Nfu001, 5, BRIX, 64, Cyprus, Lefkoniko/Arthana, Sintenis P & Rigo G (1880-04-15); N. hispanica L.: Nhi000, *193, MJG, IS2004/976 (IPEN XX0MJG19—45680), ?, ?; Nhi001, 53, GAT, IS2004/213, ?, ?; Nhi002, 10, BRIX, 34, France, Toulouse, Pech D (1853-08); N. hispanica L. var. parviflora Coss.: Nhipa0, *38, MJSD, 1575-19-116/98, ?, ?; Nhipa1, 44, BRIX, 47, Spain, Pueblo de San Federique, Porta P & Rigo G (1895); N. integrifolia Regel: Nin000, *260, MJG, IS2004/977 (IPEN XX0MJG19—45690), ?, ?; Nin001, 17, IB, s.n., Czech Republic, Olomouc, Laus H (1938-07-01); N. nigellastrum (L.) Willk.: Nni000, *82, MJG, IS2004/978 (IPEN XX0MJG19—45700), ?, ?; Nni001, *95, GAT, s.n., ?, ?; Nni002, 8, IB, s.n., Czech Republic, Olomouc, Laus H (1938-07-01); N. orientalis L.: Nor000, *8, GAT, IS2004/213, ?, ?; Nor001, *38, BRIX, 62, Turkey, ?, Bornmüller J (1889-04-24) / Freyn J; Nor002, *161, PRAZ, IS2008/173, ?, ?; N. oxypetala Boiss.: Nox000, *5, LI, 096511, Turkey, B6 Sivas:Divriği, ? (1969-08-09) / Sorger F / Zohary M; N. sativa L.: Nsa000, 160, MJG, IS2004/979 (IPEN XX0MJG19—45710), ?, ?; Nsa001, *107, —, spice market, Turkey, C1 Muğla:Bodrum, Turan-Jeschow M (2006) / Heiss AG (2006); Nsa002, 99, HOH, IS1991/1077, ?, ?; N. segetalis M.Bieb.: Nse000, *12, WU, s.n., Georgia, Kakheti, Hohenacker RF (1842-06) / Strid A (1970); Nse001, 51, BRIX, 56, Turkey, A4 Kastamonu, Freyn J (1892-06-07) / Sintenis P; N. stellaris Boiss.: Nst000, *3, LI, 001173, Turkey, C6 Seyhan:Karatepe, ? (1971-06-23) / Zohary M; N. turcica Dönmez & Mutlu: Ntu000, 5 (*2), collection of A. Dönmez, 11447, ?, Dönmez AA; N. unguicularis (Lam.) Spenn.: Nun000, *8, WU, 2575, Syria, Jabal Abdul Aziz, von Handel-Mazzetti H (1910-06-22) / Strid A (1969) Appendix B Key to the Species of Nigella s.l., Based on Seed Morphology 1. a) Seed discoid (dorsoventrally compressed, with orbicular outline and one circumferential wing), longer than 4 mm ! subsect. Nigellastrum ........................................................................................................................... 11 b) Seed shape different, seed shorter than 4 mm ....................................................................................................... 2 a) Seed trigonous-ovate to orange-segment shaped, three longitudinal ridges ............................................................ 3 b) Seed obovate, with one ventral ridge—surface covered by a conspicuous, irregular reticulum up to 300 mm high; cell walls with undulate rugulae/striae present .................Nigella sect. Garidella (N. nigellastrum, N. unguicularis) a) Distinct surface structures with more or less regular transverse orientation present, seed rough; outermost cell layer thin walled (double periclinal cell wall diameter less than lumen diameter)—seed buff to blackish or mottled....................................................................................................................................................................... 4 b) Distinct surface structures absent, seed smooth; outermost cell layer thick walled (double periclinal cell wall diameter larger than lumen diameter; sometimes no lumen visible)—seed lustrous, buff to dark brown, frequently mottled...................................................................................... N. hispanica, N. hispanica var. parviflora, N. segetalis a) Capitate/pilate cells present .................................................................................................................................. 5 b) Capitate/pilate cells absent ................................................................................................................................... 7 a) Prismatic cells (with more or less flat periclinal walls, including ocellate types) present; capitate/pilate cells with collapsed periclinal walls/apices (¼truncate); cells with radial rugulae/striae absent.............................................. 6 2(1). 3(2). 5(4). INTERNATIONAL JOURNAL OF PLANT SCIENCES 282 6(5). 7(4). 8(7). 9(8). 10(7). 11(1). b) Prismatic cells absent; capitate/pilate cells without any collapsed walls; cells with radial rugulae/striae present ......................................................................................................... Nigella sect. Komaroffia (N. integrifolia) a) Ocellate cells (prismatic cells with collapsed periclinal walls) present .....................................................N. stellaris b) Ocellate cells absent ........................................................................................................................ N. fumariifolia a) Colliculate cells (including mucronulate types) present ......................................................................................... 8 b) Colliculate cells absent ....................................................................................................................................... 10 a) Mucronulate cells present; columellate cells with collapsed lateral walls (¼ligulate) present; ocellate cells absent (! subsect. Erobathos)......................................................................................................................................... 9 b) Mucronulate cells absent; columellate cells absent; ocellate cells present .................................................N. turcica a) Mucronulate cells with distinct, centered mucronulus....................................................................... N. damascena b) Mucronulate cells with indistinct, excentric mucronulus............................................................................. N. elata a) Ocellate cells covering nearly 90% of the seed surface; columellate cells exclusively ligulate—seed blackish .........................................................................................................................................N. sativa b) Ocellate cells covering much less (;50%) of the seed surface; columellate cells without collapsed walls present— seed dark brown, frequently mottled with brighter cells ...........................................N. arvensis agg. (var. arvensis, var. assyriaca, var. glauca, var. involucrata, var. trachycarpa) a) Seed wing entire; cells with irregularly granulate to rugulate cell walls present—seed dull, buff to dark brown, frequently mottled ............................. N. orientalis, N. oxypetala (central portion of N. oxypetala sometimes with mucronulate cells; cf. Dadandi et al. 2009) b) Seed wing undulate-crenate; cells with irregularly granulate to rugulate cell walls absent—seed shiny, blackish ................................................................................................................................................... N. ciliaris Literature Cited Adobe Systems 2005 Adobe Photoshop CS 2. Adobe Systems, Mountain View, CA. Agradi E, G Fico, F Cillo, C Francisci, F Tome 2002 Estrogenic activity of Nigella damascena extracts, evaluated using a recombinant yeast screen. Phytother Res 16:414–416. Aitzetmüller K 1998 Komaroffia oils: an excellent new source of D5unsaturated fatty acids. J Am Oil Chem Soc 75:1897–1899. Aitzetmüller K, G Werner, SA Ivanov 1997 Seed oils of Nigella species and of closely related genera. Oleagineux Corps Gras Lipides 4:385–388. Ali BH, G Blunden 2003 Pharmacological and toxicological properties of Nigella sativa. Phytother Res 17:299–305. 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Heiss et al. 2011, Appendix C Appendix C Seed Descriptions Standardized descriptions of the examined taxa are given based on our own observations. Where necessary, a second paragraph refers briefly to data from the literature, with critical comments where appropriate. Measurements are given as mean values of length and width with their standard deviations. Aconitum lycoctonum L. subsp. vulparia (Rchb.) Nyman — OUTGROUP Size: 2.90  0.38  1.83  0.31 mm, length/width ratio (median) 1.558. Shape and color: Seed trigonous to trigonous-ovate, or orange-segment shaped, with multiple transverse wings. Seed surface with lateral striae. Color dark brown to blackish. Cellular pattern: Seed surface rough (due to elongated cells), with wing-like appendages forming a regular transverse pattern. Primary structure: Prismatic cells with more or less flat periclinal walls. Height of outermost cell layer 25–30 μm. Total thickness of underlying testa layers 20–25 μm. Outermost cell layer with very thick cell walls (double cell wall diameter larger than lumen diameter – sometimes no lumen visible). Secondary structure: Cell wall surface structure irregularly granulate to rugulate. This description incorporates data from Cappelletti and Poldini (1984). Nigella arvensis L. agg. (Fig. 2A) Size: 2.09  0.2  1.2  0.14 mm, length/width ratio (median) 1.74. Other data: see subtaxa below. N. arvensis L. var. arvensis Size: 2.9  0.38  1.83  0.31 mm, length/width ratio (median) 2.05. Shape and color: Seed trigonous to trigonous-ovate, or orange-segment shaped, with three more or less distinct longitudinal ridges. Color dark brown to blackish, or mottled. Cellular pattern: Seed surface Heiss et al. 2011, Appendix C rough (due to elongated cells), with more or less regular patterns of transverse structures. Primary structure: Elongated cells columellate, some with collapsed apex (truncate/ocellate). Some of the prismatic cells with collapsed periclinal walls (ocellate). Height of outermost cell layer 30-55 μm. Total thickness of underlying testa layers 25–50 μm. Outermost cell layer with more or less thin walls (double cell wall diameter is less than lumen diameter). Secondary structure: Cell wall surface structure irregularly granulate to rugulate. A seed that was assigned to this taxon in the study by Bahadur et al. (1984, 431) appears more likely to represent N. damascena, with the characteristic ridges of elongated cells and fields of distinctly mucronulate cells in between. Nigella arvensis L. var. assyriaca (Boiss.) Zoh. Size: 1.4  0.08  0.73  0.089 mm, length/width ratio (median) 1.79. Shape and color: Seed trigonous to trigonous-ovate, or orange-segment shaped, with three more or less distinct longitudinal ridges. Color dark brown to blackish, or mottled. Cellular pattern: Seed surface rough (due to elongated cells), with more or less regular patterns of transverse structures. Primary structure: Elongated cells columellate, some with collapsed anticlinal walls (ligulate). Some of the prismatic cells with collapsed periclinal walls (ocellate). Height of outermost cell layer 25–50 μm. Total thickness of underlying testa layers 20–50 μm. Outermost cell layer with more or less thin walls (double cell wall diameter is less than lumen diameter). Secondary structure: Cell wall surface structure irregularly granulate to rugulate. In Dadandi et al. (2009), a description of this taxon is provided under the synonym N. arvensis var. caudata Boiss. Nigella arvensis L. var. glauca (Schkuhr) Boiss. (Fig. 2B, 5A) Size: 1.64  0.32  0.82  0.14 mm, length/width ratio (median) 1.98. Shape and color: Seed trigonous to trigonous-ovate, or orange-segment shaped, with three more or less distinct Heiss et al. 2011, Appendix C longitudinal ridges. Color dark brown to blackish, or mottled. Cellular pattern: Seed surface rough (due to elongated cells), with more or less regular patterns of transverse structures. Primary structure: Elongated cells columellate, some with collapsed anticlinal walls (ligulate). Some of the prismatic cells with collapsed periclinal walls (ocellate). Height of outermost cell layer 30–45 μm. Total thickness of underlying testa layers 25–40 μm. Outermost cell layer with more or less thin walls (double cell wall diameter is less than lumen diameter). Secondary structure: Cell wall surface structure irregularly granulate to rugulate. Nigella arvensis L. var. involucrata Boiss. Size: 1.58  0.24  0.74  0.11 mm, length/width ratio (median) 2.14. Shape and color: Seed trigonous to trigonous-ovate, or orange-segment shaped, with three more or less distinct longitudinal ridges. Color dark brown to blackish, or mottled. Cellular pattern: Seed surface rough (due to elongated cells), with more or less regular patterns of transverse structures. Primary structure: Elongated cells columellate, some with collapsed anticlinal walls (ligulate). Some of the prismatic cells with collapsed periclinal walls (ocellate). Height of outermost cell layer 30–50 μm. Total thickness of underlying testa layers 25–45 μm. Outermost cell layer with more or less thin walls (double cell wall diameter is less than lumen diameter). Secondary structure: Cell wall surface structure irregularly granulate to rugulate. Nigella arvensis L. var. trachycarpa Borb. Size: 2.13  0.25  0.97  0.14 mm, length/width ratio (median) 2.2. Shape and color: Seed trigonous to trigonous-ovate, or orange-segment shaped, with three more or less distinct longitudinal ridges. Color dark brown to blackish, or mottled. Cellular pattern: Seed surface rough (due to elongated cells), with more or less regular patterns of transverse structures. Primary structure: Elongated cells columellate, some with collapsed apex (truncate/ocellate), or with collapsed anticlinal walls (ligulate). Some of the prismatic cells with collapsed periclinal walls (ocellate). Height of outermost cell layer 30–45 μm. Total Heiss et al. 2011, Appendix C thickness of underlying testa layers 25–50 μm. Outermost cell layer with more or less thin walls (double cell wall diameter is less than lumen diameter). Secondary structure: Cell wall surface structure irregularly granulate to rugulate. Nigella ciliaris DC. (Fig. 2C, 5B) Size: 5.38  0.47  4.85  0.5 mm, length/width ratio (median) 1.09. Shape and color: Seed dorsiventrally flattened with ovate to circular outline and one circumferential wing. Wing undulate-crenate. Color dark brown to blackish. Cellular pattern: Seed surface smooth, without noticeable structures. Primary structure: Prismatic cells flat. Height of outermost cell layer 15–30 μm. Total thickness of underlying testa layers 15–30 μm. Outermost cell layer with very thick cell walls (double cell wall diameter larger than lumen diameter – sometimes no lumen visible). Secondary structure: Cell wall surface structure smooth (seeds are thus shiny). Nigella damascena L. (Fig. 2D, 5C) Size: 2.27  0.24  1.4  0.21 mm, length/width ratio (median) 1.61. Shape and color: Seed trigonous to trigonous-ovate, or orange-segment shaped, with three more or less distinct longitudinal ridges. Color dark brown to blackish. Cellular pattern: Seed surface rough (due to elongated cells), with more or less regular patterns of transverse structures. Primary structure: Elongated cells columellate, mostly with collapsed anticlinal walls (ligulate). Prismatic to colliculate cells mucronulate, with distinct, centered mucronulus. Height of outermost cell layer 50–100 μm. Total thickness of underlying testa layers 20–45 μm. Outermost cell layer with more or less thin walls (double cell wall diameter is less than lumen diameter). Secondary structure: Cell wall surface structure irregularly granulate to rugulate. Nigella elata Boiss. (Fig. 4D) Heiss et al. 2011, Appendix C Size: 2.49  0.17  1.5  0.16 mm, length/width ratio (median) 1.67. Shape and color: Seed trigonous to trigonous-ovate, or orange-segment shaped, with three more or less distinct longitudinal ridges. Color dark brown to blackish. Cellular pattern: Seed surface rough (due to elongated cells), with more or less regular patterns of transverse structures. Primary structure: Elongated cells columellate, mostly with collapsed anticlinal walls (ligulate). Prismatic to colliculate cells mucronulate with indistinct, excentric mucronulus. Height of outermost cell layer 45–100 μm. Total thickness of underlying testa layers 20–40 μm. Outermost cell layer with more or less thin walls (double cell wall diameter is less than lumen diameter). Secondary structure: Cell wall surface structure irregularly granulate to rugulate. Nigella fumariifolia Kotschy (Fig. 2E, 5E) Size: 1.73  0.21  0.95  0.089 mm, length/width ratio (median) 1.81. Shape and color: Seed trigonous to trigonous-ovate, or orange-segment shaped, with three more or less distinct longitudinal ridges. Color buff/light brown, or mottled. Cellular pattern: Seed surface rough (due to elongated cells), with more or less regular patterns of transverse structures. Primary structure: Some elongated cells columellate, most are capitate/pilate, with collapsed apex (truncate). Prismatic cells flat. Height of outermost cell layer 30–60 μm. Total thickness of underlying testa layers 25–35 μm. Outermost cell layer with more or less thin walls (double cell wall diameter is less than lumen diameter). Secondary structure: Cell wall surface structure irregularly granulate to rugulate. Nigella hispanica L. (Fig. 2F, 6A) Size: 1.79  0.12  1.37  0.1 mm, length/width ratio (median) 1.32. Shape and color: Seed trigonous to trigonous-ovate, or orange-segment shaped, with three more or less distinct longitudinal ridges. Color buff/light brown, or dark brown, or mottled. Cellular pattern: Seed surface smooth, without noticeable structures. Primary structure: Prismatic cells flat. Height of outermost cell layer 35–50 μm. Total thickness of underlying testa layers 30–45 Heiss et al. 2011, Appendix C μm. Outermost cell layer with very thick cell walls (double cell wall diameter larger than lumen diameter – sometimes no lumen visible). Secondary structure: Cell wall surface structure irregularly granulate to rugulate. In the study by Bahadur et al.(1984, 431), the SEM images of “N. hispanica” show ridges of colliculate/columellate cells with ocellate cells between them. In our study, these ridges do not occur in N. hispanica, which has a smooth surface with flat prismatic cells. It is most likely that the authors confused N. hispanica with N. sativa. Nigella hispanica L. var. parviflora Coss. (Fig. 2G) Size: 2.29  0.26  1.46  0.22 mm, length/width ratio (median) 1.55. Shape and color: Seed trigonous to trigonous-ovate, or orange-segment shaped, with three more or less distinct longitudinal ridges. Color buff/light brown, or dark brown, or mottled. Cellular pattern: Seed surface smooth, without noticeable structures. Primary structure: Prismatic cells flat. Height of outermost cell layer 35–50 μm. Total thickness of underlying testa layers 35–45 μm. Outermost cell layer with very thick cell walls (double cell wall diameter larger than lumen diameter – sometimes no lumen visible). Secondary structure: Cell wall surface structure irregularly granulate to rugulate. Nigella integrifolia Regel (Fig. 2H, 6B) Size: 2.29  0.26  1.46  0.22 mm, length/width ratio (median) 1.83. Shape and color: Seed trigonous to trigonous-ovate, or orange-segment shaped, with three more or less distinct longitudinal ridges. Color buff/light brown, or mottled. Cellular pattern: Seed surface rough (due to elongated cells), with more or less regular patterns of transverse structures. Primary structure: Elongated cells capitate/pilate, without collapsed walls. Height of outermost cell layer 20–70 [µm]. Total thickness of underlying testa layers 30–45 µm. Outermost cell layer with more or less thin walls (double cell wall diameter is less than lumen diameter). Heiss et al. 2011, Appendix C Secondary structure: Cell wall surface structure irregularly granulate to rugulate, in some cells with distinctly radial rugulae/striae. Nigella nigellastrum (L.) Willk. (Fig. 3A, 6C) Size: 2.38  0.17  1.72  0.16 mm, length/width ratio (median) 1.37. Shape and color: Seed obovate, with one ventral ridge. Color dark brown to blackish, or mottled. Cellular pattern: Seed surface with conspicuously irregular reticulum. Primary structure: Elongated cells columellate, without collapsed walls. Prismatic cells colliculate. Height of outermost cell layer 30–65 μm. Total thickness of underlying testa layers 100–250 μm. Outermost cell layer with more or less thin walls (double cell wall diameter is less than lumen diameter). “Resin”filled large idioblasts (Netolitzky, 1926) present. Secondary structure: Cell wall surface structure irregularly granulate to rugulate, with distinctly undulate rugulae/striae in some cells. Nigella orientalis L. (Fig. 3B, 6D) Size: 4.96  0.41  4.34  0.43 mm, length/width ratio (median) 1.14. Shape and color: Seed dorsiventrally flattened with ovate to circular outline and one circumferential wing. Wing entire. Color dark brown to blackish, or mottled. Cellular pattern: Seed surface smooth, without noticeable structures. Primary structure: Prismatic cells flat. Height of outermost cell layer 30–35 μm. Total thickness of underlying testa layers 20–25 μm. Outermost cell layer with very thick cell walls (double cell wall diameter larger than lumen diameter – sometimes no lumen visible). Secondary structure: Cell wall surface structure irregularly granulate to rugulate. Bahadur et al. (1984, 433) provide a description and SEM images of a taxon named “N. orientalis”. Both, however, point to confusion with N. damascena: the ridged surface with mucronulate cells between them is characteristic of that species. Further, the characteristic Heiss et al. 2011, Appendix C seed outline (orbicular and flattened in N. orientalis) is not mentioned in the article, indicating the inadequacy of the analyzed specimens. Nigella oxypetala Boiss. (Fig. 3C, 6E) Size: 5.59  0.21  4.86  0.23 mm, length/width ratio (median) 1.12. Shape and color: Seed dorsiventrally flattened with ovate to circular outline and one circumferential wing. Wing entire. Color buff/light brown, or mottled. Cellular pattern: Seed surface smooth, without noticeable structures. Primary structure: Prismatic cells flat. Height of outermost cell layer 25–30 μm. Total thickness of underlying testa layers 25–40 μm. Outermost cell layer with very thick cell walls (double cell wall diameter larger than lumen diameter – sometimes no lumen visible). Secondary structure: Cell wall surface structure irregularly granulate to rugulate. In the study of Dadandi et al. (2009), Nigella oxypetala Boiss, N. lancifolia Hub.-Mor. and N. latisecta P.H.Davis are treated as species. However, according to Zohary (1983), the three taxa should rather be considered varieties of N. oxypetala. Considering the feature of distinctly mucronulate disk cells in N. oxypetala seeds as an expression of intraspecific variability (their presence was not observed in the material analyzed in the present study), the three taxa cannot be separated satisfactorily by their seed micromorphology. Nigella sativa L. (Fig. 3D, 7A) Size: 2.92  0.24  1.64  0.17 mm, length/width ratio (median) 1.78. Shape and color: Seed trigonous to trigonous-ovate, or orange-segment shaped, with three more or less distinct longitudinal ridges. Color dark brown to blackish. Cellular pattern: Seed surface rough (due to elongated cells), with more or less regular patterns of transverse structures. Primary structure: Elongated cells with collapsed anticlinal walls (ligulate). Nearly all prismatic cells with collapsed periclinal walls (ocellate), thereby differing from N. turcica. Height of outermost cell layer 25–60 μm. Total thickness of underlying testa layers 30–60 μm. Heiss et al. 2011, Appendix C Outermost cell layer with more or less thin walls (double cell wall diameter is less than lumen diameter). Secondary structure: Cell wall surface structure irregularly granulate to rugulate. Nigella segetalis M. Bieb. (Fig. 3E, 7B) Size: 1.6  0.11  1.06  0.13 mm, length/width ratio (median) 1.5. Shape and color: Seed trigonous to trigonous-ovate, or orange-segment shaped, with three more or less distinct longitudinal ridges. Color buff/light brown, or dark brown to blackish, or mottled. Cellular pattern: Seed surface smooth, without noticeable structures. Primary structure: Prismatic cells flat. Height of outermost cell layer 30–35 μm. Total thickness of underlying testa layers 20–35 μm. Outermost cell layer with very thick cell walls (double cell wall diameter larger than lumen diameter – sometimes no lumen visible). Secondary structure: Cell wall surface structure irregularly granulate to rugulate. Nigella stellaris Boiss. (Fig. 3F, 7C) Size: 2.15  0.08  0.89  0.48 mm, length/width ratio (median) 2.4. Shape and color: Seed trigonous to trigonous-ovate, or orange-segment shaped, with three more or less distinct longitudinal ridges. Color buff/light brown, or mottled. Cellular pattern: Seed surface rough (due to elongated cells), with more or less regular patterns of transverse structures. Some elongated cells columellate, most are capitate/pilate, with collapsed apex (truncate). Primary structure: Prismatic cells flat, with collapsed periclinal walls (ocellate). Height of outermost cell layer 25–60 μm. Total thickness of underlying testa layers 30–45 μm. Outermost cell layer with more or less thin walls (double cell wall diameter is less than lumen diameter). Secondary structure: Cell wall surface structure irregularly granulate to rugulate. Nigella turcica Dönmez & Mutlu (Fig. 3G, 7D) Size: 2.95  0.3  1.41  0.06 mm, length/width ratio (median) 2.1. Shape and color: Seed trigonous to trigonous-ovate, or orange-segment shaped, with three more or less distinct Heiss et al. 2011, Appendix C longitudinal ridges. Color dark brown to blackish. Cellular pattern: Seed surface rough (due to elongated cells), with more or less regular patterns of transverse structures. Primary structure: Some prismatic cells with collapsed periclinal walls (ocellate), but much fewer than in N. sativa. Height of outermost cell layer 35–55 μm. Total thickness of underlying testa layers 35–50 μm. Outermost cell layer with more or less thin walls (double cell wall diameter is less than lumen diameter). Secondary structure: Cell wall surface structure irregularly granulate to rugulate. The cell wall sculpturing visible in the SEM images (rugose to rugulate, cf. Fig. 6D) could not be observed in the thin sections under light microscopy. Here, granulate cell walls are clearly visible, without any traces of rugose to rugulate structures. The rugulae are thus to be regarded as artifacts, probably caused by the evacuation process. As only few (five) seeds of N. turcica were available for investigation, no additional SEM imagery was produced. Nigella unguicularis (Lam.) Spenn. (Fig. 3H, 7E) Size: 2.65  0.19  2,15  0.19 mm, length/width ratio (median) 1.37. Shape and color: Seed obovate, with one ventral ridge. Color dark brown to blackish, or mottled. Cellular pattern: Seed surface with conspicuously irregular reticulum. Primary structure: Elongated cells columellate, without collapsed walls. Prismatic cells colliculate. “Resin”-filled large idioblasts (Netolitzky, 1926) present. Height of outermost cell layer 30–65 μm. Total thickness of underlying testa layers 100–250 μm. Outermost cell layer with more or less thin walls (double cell wall diameter is less than lumen diameter). Secondary structure: Cell wall surface structure irregularly granulate to rugulate, in some cells with distinctly undulate rugulae/striae.

Table D1 Character States Used for Building the Data Matrix in This Study Character: 1 seed shape State: (unordered) 0 1 trigonous to trigonous to trigonous-ovate to trigonous-ovate to orange-segment orange-segment shaped; multiple shaped; three transversal longitudinal ridges wings* 2 seed length 3 seed width 4 seed surface (ordered) 1.00 mm or less 1.01 - 2.00 mm (ordered) 1.00 mm or less 1.01 - 2.00 mm (unordered) with conspicuously smooth, without irregular reticulum conspicuous structures 5 dark-pigmented cells 6 primary structure: cell shape (unordered) (unordered) absent columellate; truncate 7 secondary structure: cell wall surface (unordered) smooth 8 resin-filled idioblasts 9 outermost testa layer height 10 underlying testa layers height 11 outermost testa layer periclinal cell wall thickness (unordered) (ordered) (ordered) (unordered) present columellate; ligulate irregularly granulate to rugulate absent present 40 µm or less 41-80 µm 40 µm or less 41-80 µm thick; double wall thin; double wall diameter higher diameter lower than lumen than lumen diameter; diameter sometimes no lumen visible 2 obovate; one ventral ridge 3 dorsiventrally flattened, outline ovate to orbicular; one circumferential wing with entire, flat margins 2.01 - 3.00 mm 2.01 - 3.00 mm with transversal patterns (of different cell types) 3.01 mm and larger 3.01 mm and larger with lateral patterns (striae)* columellate; without collapsed walls capitate/pilate; truncate with radial rugulae/striae with undulate rugulae/striae 81-120 µm 81-120 µm 121 µm and larger Note.- Character states 1/1 and 4/3 (marked with an asterisk) only apply to the outgroup taxon (Aconitum lycoctonum subsp. vulparia ) 4 dorsiventrally flattened, outline ovate to orbicular; one circumferential wing with undulatecrenate margins capitate/pilate; without collapsed walls 5 6 7 8 9 colliculate flat prismatic flat prismatic; flat/colliculate; flat/colliculate; mucronulate; ocellate mucronulate; mucro mucro distinct indistinct & & centered excentric Table D2 Character States as Observed in This Study Taxon Aconitum lycoctonum subsp. vulparia — OUTGROUP N. arvensis N. arvensis var. arvensis N. arvensis var. assyriaca N. arvensis var. glauca N. arvensis var. involucrata N. arvensis var. trachycarpa N. ciliaris N. damascena N. elata N. fumariifolia N. hispanica N. hispanica var. parviflora N. integrifolia N. nigellastrum N. orientalis N. oxypetala N. sativa N. segetalis N. stellaris N. turcica N. unguicularis Character no. 1 1 0 0 0 0 0 0 4 0 0 0 0 0 0 2 3 3 0 0 0 0 2 2 12 12 2 1 1 1 12 3 2 2 1 1 2 2 2 3 3 23 1 2 23 2 3 01 1 01 0 0 0 01 3 1 1 01 1 1 1 1 3 3 1 01 0 1 12 4 3 2 2 2 2 2 2 1 2 2 2 1 1 2 0 1 1 2 1 2 2 0 5 1 01 01 01 01 01 01 1 1 1 01 01 01 01 01 01 01 1 01 01 1 01 6 6 01267 0267 1267 1267 1267 01267 6 158 159 236 6 6 4 25 6 68 17 6 237 567 256 7 1 1 1 1 1 1 1 0 1 1 1 1 1 12 13 1 1 1 1 1 1 13 8 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 1 9 0 01 01 01 01 01 01 0 12 12 01 01 01 01 01 0 0 01 0 01 01 01 10 0 01 01 01 01 01 01 0 01 01 0 01 01 01 23 0 01 01 0 01 01 3 11 0 1 1 1 1 1 1 0 1 1 1 0 0 1 1 0 0 1 0 1 1 1

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