Janaćković et al., 2019, Biologica Nyssana 10(2) 10 (2) December 2019: 77-85 DOI: 10.5281/zenodo.3600177 Micromorphology and anatomy in systematics of Asteraceae. An old-fashioned approach? Review Article Peđa Janaćković University of Belgrade, Faculty of Biology, Depart- ment of Morphology and Systematics of Plants, Studentski trg 16, Belgrade, Serbia pjanackovic@bio.bg.ac.rs (corresponding author) Alfonso Susanna Botanic Institute of Barcelona (IBB, CSIC-ICUB), Pg. del Migdia s. n., 08038 Barcelona, Spain asusanna@ibb.csic.es Petar D. Marin University of Belgrade, Faculty of Biology, Depart- ment of Morphology and Systematics of Plants, Studentski trg 16, Belgrade, Serbia pdmarin@bio.bg.ac.rs Received: October 27, 2019 Revised: November 28, 2019 Accepted: December 17, 2019 Abstract: The comparative study of plant morphology, intertwined with anatomy, has always been the basis for plant systematics, which strives to explain diver- sity, evolution and phylogeny of plants. In the molecular era, some authors diminish importance of morphology and especially anatomy in systematic and phylogenetic studies of plants. However, are molecular data exclusive- ly a primary and self-sufficient approach in taxonomic research of plants? This review paper addresses this issue through specific examples. Studies of some Asteraceae taxa showed that morphological, micromorphological and anatomical data are extremely important in systematics. New opportunities for systematic morphology, micromorphology and anatomy in case of Aster- aceae taxonomy, but certainly also in other plant groups, that were not present in the premolecular era, are opening regarding synergistic multidisciplinary taxonomic, evolutionary and phylogenetic studies that combine molecular with morphological, anatomical and other analyses (e.g. chemophenetics - describes a given taxon phenetically using specialized metabolites as phyto- chemical characters), keeping in the throne these “old fashioned” approaches. Key words: morphology, taxonomy, characters, synergy Apstract: Mikromorfologija i anatomija u sistematici familije Asteraceae. Staromodan pristup? Uporedna studija morfologije isprepletene sa anatomijom, uvek je bila osnova za sistematiku biljaka koja teži da objasni raznolikost, evoluciju i filogeniju biljaka. U molekularnom dobu, u sistematskim i filogenetskim studijama bi- ljaka, neki autori umanjuju značaj morfologije, a posebno anatomije biljaka. Međutim, da li su molekularne metode i podaci isključivo primaran i samo- dovoljan pristup u taksonomskom istraživanju biljaka? Ovaj pregledni rad, kroz određene primere, daje odgovor na ovo pitanje. Istraživanja određenih taksona iz familije Asteraceae pokazala su da su morfološki, mikromorfološki i anatomski podaci izuzetno važni u sistematici. Novi pristupi u sistematskoj morfologiji, mikromorfologiji i anatomiji u slučaju taksonomije Asteraceae, ali sigurno i u slučaju taksonomije drugih grupa biljaka, koji nisu bili mogući u premolekularnom dobu, otvaraju se sada kao sinergija multidisciplinarnih taksonomskih, evolucionih i filogenetskih studija koje kombinuju molekularne sa morfološkim, anatomskim i drugim analizama i pristupima (npr. hemofene- tika koja opisuje dati takson fenetički, upotrebom specijalizovanih metabolita kao fitohemijskih karaktera), držeći na prestolu ove „staromodne“ pristupe. Ključne reči: morfologija, taksonomija, karakteri, sinergija Introduction The comparative study of plant structure has al- ways been the backbone of plant systematics, which strives to elucidate plant diversity, phylogeny and evolution (Endress et al., 2000). Plant taxonomic studies traditionally use morphological and karyo- logical (Stebbins, 1953), as well as micromorpho- logical characters (Hayat et al., 2009; Bak & Ozcan, 2018). Micromorphological characters are of deci- sive importance in unring taxonomic and phyloge- netic relationships of various plant groups and have been successfully used in plant systematic studies for decades (Endress et al., 2000). For more than a © 2019 Janaćković et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 77 13th Symposium on the Flora of Southeastern Serbia and Neighboring Regions century, comparative anatomy is used as a tool in the plant systematics. Anatomical characters are very important in perceiving systematic and phylogenetic relationships of particular plant groups. Indeed, ana- tomical features can provide useful characters which could help in identification of problematic plant taxa, as well as establishing their taxonomic relationships (Metcalfe & Chalk, 1957; Scatena et al., 2005; Mak- bul et al., 2011; Sosa et al., 2014; Karanović et al., 2015, Gavrilović et al., 2019a, b; Janaćković et al., 2019). On the other hand, the data provided by the phytochemists are extremely difficult to interpret in a cladistic context. Even so, integration of phy- tochemical and macro-molecular characters can be of prominent benefit, and can help, for example, in the delineation of clades so far only supported by DNA sequence data (Enke et al., 2012). In order to overcome the confusion in the interpretation of phytochemical characters (specialized metabolites that actually describe the given taxon phenetically) as phylogenetic characters and “under the umbrel- la” of the term chemotaxonomy (which is mistak- enly identified with chemosystematics), the new term chemophenetics has been proposed by Zidorn, (2019). Nevertheless, chemophenetic studies con- tribute to the phenetic description of taxa, similarly to anatomical, morphological and karyologycal ap- proaches, which have already been recognized as of major importance for establishing “natural” systems and which continue to be of the highest importance for the description of organisms classified with the help of modern molecular methods (Zidorn, 2019). Asteraceae (Compositae), habitually known as the daisy or sunflower family, represent one of three mega-diverse families which jointly count more than 25% of all extant angiosperm species (Mandel et al., 2019). Asteraceae, counting 25000–35000 species, comprise 10% of all flowering plant species. Members of the sunflower family occur on every continent including Antarctica (Smith & Richard- son, 2011) and inhabit nearly every type of habitat on Earth with the largest concentration of species in deserts, prairies, steppes, montane regions, and areas with Mediterranean-like climates (Mandel et al., 2019). According to newer fossil data and recent molecular clock dating, Asteraceae likely originated during the Late Cretaceous: ∼83 MYA (Mandel et al., 2019). According to Mandel et al. (2019) the family consists of 13 subfamilies and 47 tribes. Asteraceae also includes species of wide economic interest, e.g. vegetables, sources of oil, medicinal plants, insecti- cides and many horticultural and garden ornamen- tals. However, some species of Asteraceae constitute a big problem for agriculture as noxious weeds. As a one of the largest, natural (with a combination of several specialized morphological characteristics e.g., capitula, highly reduced and modified flow- ers, syngenesious anthers, inferior ovaries and very unique in the plant kingdom fruit - cypsela) and economically most important families of flowering plants Asteraceae has been researched for centuries. Characters related to form, whether gross mor- phology, micromorphology, anatomy, embryology, palinology and so forth, regarded as morphologycal data, have larger impact on the cladistics and clas- sification of the Asteraceae than other characters e.g. chemical and molecular data (Schönberger, 2002; Stuessy, 2009). Molecular data are significant but also insufficient, that is why, in some cases, better phylogenetic reconstructions of the Asteraceae are obtained taking into account also a morphological data (Pornpongrungrueng et al., 2007; Gruenstaeudl et al., 2009; Wang et al., 2013). More recent, mod- ern-day studies of Asteraceae taxa have shown that morphological, micromorphological and anatomical data are still extremely important in systematics of Asteraceae family (Makbul et al., 2011; Wang et al., 2013; Sosa et al., 2014; Karanović et al., 2015; Bom- bo et al., 2016; Ginko et al., 2016; Batista and De Souza, 2017; Gavrilović et al., 2017; Gavrilović et al., 2018a, b; Gavrilović et al., 2019a, b; Janaćković et al., 2019). Brief history and modern-day micromorphology and anatomy in systematics of Asteraceae Micromorphology A different sets of characters has been used to de- marcate the Asteraceae. Certainly, one of the pio- neer work regarding micromorpholgical approaches to Asteraceae classification was done by Cassini (1821). These summarize many of the microscopic traits on which Cassini based his tribes. Of these, pronate versus recurved mature style branches, stig- matic surfaces, truncate to enlarged style append- ages, bases of the anther thecae with or without tails, shape of the anther collar and form of the corolla are most important. The stigmatic surfaces of many tribes (Mutisieae, Lactuceae, Vernonieae, Arctoti- deae, Eremothamneae, Cardueae) are consistently continuous over the inner surface of the style branch. In other tribes (Eupatorieae, Anthemideae, Astereae, most Inuleae, most Heliantheae, most Senecioneae) the stigmatic surface is divided into two lines. The nature of endothecial tissue of the stamens, which could be polarized or radial, showed to be good character in taxonomy within Senecioneae (Dormer, 1962). In subtribes Senecioninae and Ot- honninae, the endothecium is radial in all genera ex- cept Dauresia, Graphistylis, and perhaps Synotis. In 78 BIOLOGICA NYSSANA ● 10 (2) December 2019: 77-85 Janaćković et al. ● Micromorphology and anatomy in systematics of Asteraceae. An old-fashioned approach? Tussilagininae s.str. a polarized endothecium is the rule, but the radial type has been recorded in several genera (Tephroseris, Nemosenecio, Psacaliopsis, Psacalium, Arnoglossum); in Sinosenecio both types and an intermediate pattern seem to occur (Jeffrey & Chen, 1984). Moreover, the anthers of Cichorieae members vary considerably in length, but this vari- ation probably occurs repeatedly within many gen- era and is therefore only of taxonomic relevance at the species level (Kilian et al., 2009). In Arctotideae morphological and micromorphological characters confirmed close relationships between the Gorteria clade and Berkheya clade (Karis et al., 2009). Microcharacters of involucral bracts are consid- ered very helpful for delimitation in certain taxo- nomic groups of Asteraceae (e.g., for subtribes of Cardueae, with spiny pectinate-fimbriate append- ages in Cardopatiinae; usually spiny, innermost exappendiculate or with rudimentary appendages in Carduinae; inner often conspicuous and coloured in Carlininae; scarious, fimbriate, pectinate, spiny or unarmed appendage in Centaureinae; and in many rows in Echinopsinae (Robinson, 2009; Susanna & Garcia-Jacas, 2009). Certain floral microcharacters (anther size, shape of the anther apical appendage, configuration of stig- matic areas on the inner surface of the style branch, and configuration of the endothecial thickenings and of the filament collar) of 36 taxa of Sinosenecio showed that these floral characters are highly con- sistent with evidence from molecular systematics and cytology and provide the most important diag- nostic characters in the tribe Senecioneae, as in the family at large and strongly suggest a polyphyletic nature of this genus, as well as the need of a taxo- nomic change at generic level (Liu & Yang, 2011). Still nowadays, micromorphological investi- gations of Asteraceae could provide some novel characters (Erbar & Leins, 2015; Gavrilović et al. 2017, 2019b). Investigating style morphology of 395 species of 258 genera (covering all, in that time, 44 tribes of the Asteraceae), Erbar & Leins (2015) found a new microstructural feature, namely, often conspicuous cuticular patterns on the stylar hairs (in- volved in secondary pollen presentation) and stylar appendages. They determined five different patterns of cuticular striation and when they put these pat- terns onto a generalized phylogenetic tree (based on molecular data), they concluded that there is consid- erable homoplasy in these features. Nevertheless, cu- ticular patterns are still useful in characterizing some clades within the family. Gavrilović et al. (2017) investigating involucral bract micromorphology found, for the first time, a large number of densely packed crystals on the involucral bract surface. Also, the presence of nonglandular, curly trichomes and BIOLOGICA NYSSANA ● 10 (2) December 2019: 77-85 Janaćković et al. ● Micromorphology and anatomy in systematics of Asteraceae. An old-fashioned approach? 79 biseriate glandular trichomes on the bract surface, as well as the sylvite crystals on the petal surface of X. cylindraceum, clearly differentiates this species from X. annuum (Gavrilović et al., 2017). Compara- tive micromorphological analyses were conducted on five members of the Xerantheminae, both peren- nial (Amphoricarpos exsul and Shangwua masarica) and annual (Chardinia orientalis, Siebera pungens and Xeranthemum inapertum), showing that micro- morphological traits link together perennial species, some link annual ones, some are species-specific, and some are common to all taxa (Gavrilović et al., 2019b). We could conclude that morphology and mi- cromorphology of florets (e.g., style base, anther appendages, trichomes on corollas), and inflores- cence (involucral bracts characters, e.g., crystals and glandular and nonglandular trichomes on their wall) were used as major distinguishing features for sub- tribal and generic delimitation, even though these characters can sometimes be significant at the spe- cies level. Anatomy At the beginning of the twentieth century Col (1899- 1901), in light of anatomy, reviewed in consider- able detail distribution of laticiferous versus res- iniferous tissue throughout the Asteraceae. Taxa with latex in canals or sacs occur in several tribes of the subfamily Cichorioideae and consistently in the Lactuceae, but very rarely in the Asteroideae, where resin sacs and resin canals are common. Car- lquist (1966) investigated the basic plan of the wood anatomy of Asteraceae (focusing on four tribes, An- themideae, Ambrosieae, Calenduleae, and Arctoti- deae), which provided useful tribal characters and minor intertribal variation. Even though Carlquist (1966) stated that Asteraceae members share a basi- cally specialized wood plan and that wood anatomy is not likely to reward one with tribal or subtribal characters, certain characters are of systematic value within Anthemideae, Ambrosieae, Calenduleae, and Arctotideae (e.g., carbonized resins in intercellular spaces, secretory canals in rays, patterns of crystal occurrence are characters which may be of specific or generic value). Metcalfe & Chalk (1957) noted some particular anatomical traits, which showed to have taxonomic importance within the family, e.g., presence of se- cretory and laticiferous canals, types of nonglandu- lar and glandular trichomes, occurrence of medullar and cortical vascular bundles and presence of anom- alous secondary thickening. Also, anatomical char- acteristics observable in Asteraceae are: (a) presence of various types of glandular and non-glandular tri- 80 chomes; (b) papillae on the abaxial leaf epidermis; (c) anomocytic, anisocytic and rarely heliocytic sto- mata types; (d) presence of hydathodes; (e) presence of hypodermis; (f) homogeneous or heterogeneous mesophyll and (g) vascular bundles with a parenchy- matic sheath composed of large cells (Metcalfe & Chalk, 1979). Anatomy of several members of the tribe Sene- cioneae showed that they possess resin ducts in stems, leaves and roots, sometimes also in floral parts, and even in cotyledons. The resin production is noted as stickiness and exudates on vegetative parts. This occurs in the tussilaginoid as well as in senecioid group (Nordenstam et al., 2009). Ginko et al. (2016) investigated suitability of ana- tomical characters of root and rhizome of 59 species belonging to 34 genera and 12 subtribes from tribes Cardueae and Cichorieae for taxonomic classifica- tion and phylogenetic reconstruction. In this case, anatomy is demonstrated as valuable to discriminate tribes and many species but not so for subtribes and genera. However, most anatomical traits seems to be homoplastic, which limits their application as phylo- genetically informative characters. Bombo et al. (2016) stated that anatomical fea- tures can help in resolving taxonomical problems within the genus Aldama La Llave, especially among Brazilian members, which are difficult to identify taxonomically. On the basis of their findings, the au- thors concluded that anatomy is able to provide data which assist with the taxonomic problems within the four analysed species. The systematic value of leaf epidermal characters in Asteraceae has been proven by numerous stud- ies, as leaf surface is under strong genetic control (Adedeji & Jewoola, 2008; Karanović et al., 2015). Some leaf blade characters (e.g., epidermal anticli- nal cell walls, epicuticular wax and trichome type) have shown to be diagnostic to separate Aster L., Galatella Cass. and Tripolium Nees (Karanović et al., 2015). A comparative study of the leaf epidermis in 12 species of Asteraceae showed that the type and shape of trichomes, nature of cuticular striations and stomatal type are taxonomically important for the delimitation of species (Adedeji & Jewoola, 2008). Qualitative anatomical characters (e.g., shape of the young stem and peduncle cross-sections, type of glandular trichomes and occurrence of cortical vas- cular bundles) were shown to be useful in delimitat- ing X. annuum from X. cylindraceum (Gavrilović et al., 2019a). Moreover, some of the anatomical char- acters found in Xeranthemum (secondary growth in roots and dorsiventral leaves) suggested that adapta- tion from mesophytic to xeric habitats (Gavrilović et al., 2019a) are important for phylogenetic relation- ships within Xerantheminae. Anatomical data can also contribute in resolving complex taxonomy of certain genera, e.g. Artemi- sia. In anatomical investigation of five Artemisia species, Janaćković et al. (2019) showed that some characters link together A. absinthium and A. arbo- rescens from the same section; some other connect species belonging to different sections (A. campes- tris and A. arborescens; A. absinthium and A. judai- ca; A. judaica and A. herba-alba), while some could be considered as species-specific. We could summarize that certain anatomical characters, such us distribution of laticiferous versus resiniferous tissue is useful on subfamily an tribal level, while patterns of crystal occurrence might be significant at species or genus level. Occurrence of cortical vascular bundles seems to be important on species level. Root and rhizome anatomical traits have proven to be useful on tribal and species, but not on the genus level. Leaf anatomical epidermal character are diagnostic and can be used for delimit- ing species. Also, qualitative anatomical characters may have role in understanding and solving phylo- genetic relationships, which are reflected in the sys- tematics of given taxa. Micromorphology and anatomy of cypsela The cypsela is a special form of dry indehiscent fruit in which the seed coat (testa) and fruit wall (pericarp) are tightly attached to one another and is exclusive characteristic of the family Asterace- ae (Roth, 1977). As an exclusive fruit of the fam- ily cypsela and its features have been attracted by Tournefort (1694), Vaillant (1719), Cassini (1819), Lessing (1832), Bentham (1873), Hoffman (1894), Cronquist (1955), Robinson (1977), Bremer (1994), Nordenstam (1994), Rao & Datt (1996), Robinson (1999), Nordenstam et al. (2006), Lack (2007) and Mukherje & Nordenstam (2004, 2010). Cypsela morphology (macro- and micromorphology) and anatomy have been widely used in illuminating taxonomic relationships in Asteraceae and still rep- resents a source of valid taxonomic characters (La- vialle, 1912; Stebbins, 1953; Wagenitz, 1976; Dit- trich 1977; Barthlott, 1984; Singh & Pandey, 1984; Dittrich, 1985; Bruhl & Quinn, 1990; Glynis, 1993; Geng et al., 1994; Blanca & Díaz de la Guardia, 1997; Petit, 1997; Häffner, 2000; Zhu et al., 2006; Garg & Sharma, 2007; Pandey & Kumari, 2007; Zarembo and Boyko, 2008; Abid & Qaiser, 2009; Abid & Ali, 2010; Inceer et al., 2012; Ozcan & Akinci, 2019). This is why micromorphology and anatomy of cypsela are separated herein. Bremer (1987) stated the importance of cypselae characters at lower taxonomic levels but not at the tribal level. In Asteraceae, the anatomy and micromorphology of BIOLOGICA NYSSANA ● 10 (2) December 2019: 77-85 Janaćković et al. ● Micromorphology and anatomy in systematics of Asteraceae. An old-fashioned approach? 81 BIOLOGICA NYSSANA ● 10 (2) December 2019: 77-85 Janaćković et al. ● Micromorphology and anatomy in systematics of Asteraceae. An old-fashioned approach? cypselas are taxonomically significant at both genus and species levels (Abid & Qaiser, 2009, Kulkarni, 2013, Hussein & Eldemerdash, 2016; Karanović et al., 2016; Gavrilović et al., 2019b). Cypselae size and shape, number of ribs, pres- ence of prickles, ornamentation of the intercostal gaps, tapering (sharp or gradual) of the body/beak junction, shape of the beak, degree of swelling at its apex and pubescence of the annulus are diagnos- tic in Tragopogon L. (Blanca & Díaz de la Guar- dia, 1997). Micromorphological characters of the cypselae (shape, surface, colour, size), pappus (stuc- ture, shape, number, colour, size) and carpopodium (shape, position, diameter) in the tribes Senecioneae and Anthemidae are useful for assessing the rela- tionship and delimitation at both generic and specific levels (Abid & Qaiser, 2009; Abid & Ali, 2010). As a result of morphological and anatomical in- vestigations of the cypselae in East Asian species of Rhaponticum Vaill., Klasea Cass., Serratula L. and Synurus Iljin in the tribe Cardueae s.l., Zarembo & Boyko (2008) clarified the following diagnostic traits at the species level: topography of epidermal cells of the pericarp, presence of phlobaphenes, oc- currence, topography and localization of calcium oxalate crystals, and occurrence and location of se- cretory ducts in the mesocarp. Karanović et al. (2016) showed that, besides re- ceptacle characters, organisation of sclerenchyma- tous tissue in a fruit is a feature that tend to be di- agnostic for genera Inula, Pulicaria, Dittrichia and Limbarda. Also, some fruit features have been dem- onstrated to be especially useful in distinguishing certain similar species (e.g., Inula britannica from I. oculus-christi). Moreover, authors stated that I. hele- nium should be separated from the Inula genus, as its authenticity is evident base on cypsela characters. Silva et al. (2017) showed that features of the cypselae of the subtribe Disynaphiinae such as the carpopodium, floral disc, pappus, outer mesocarp, sclerenchyma, phytomelanin layer, ribs and tri- chomes are valuable at both generic and specific levels. Moreover, authors revealed the presence of a multiplicative pericarp only in a few Symphyo- pappus spp., rare trait in Asteraceae, which prob- ably evolved independently in the family. Cypsela structure also supports the exclusion of Disynaphia praeficta from the subtribe, since this species posess- es several different characters comparing with other representatives of Disynaphiinae. Characters of cypselae are shown to be very in- formative at generic level within Xerantheminae (Gavrilović et al., 2019). For instance, Shangwua is distinguished from other genera in having glabrous cypsela, while Chardinia only possesses papillose cypsela surface. Moreover, cypsela features of Xer- antheminae taxa are significant for phylogeny of the subtribe (some characters share all members, while some characters separates annual from perennial genera). Ozcan & Akinci (2019) evaluated feasibility of cypsela characters as taxonomic markers investi- gating 21 taxa representing 12 genera of the tribe Cardueae. They observed considerable variability in surface sculptures of pappus and cypselae, as well as in pericarp and testa structures. Authors concluded that micromorphological and anatomical cypsela characters are distinct between the genera and are also useful for delimiting species. Conclusion and future prospects The breakthrough of molecular tools in plant sys- tematics and its contribution to phylogenetic frame- works was and it is still a tremendous stimulus for comparative morphology and anatomy. One should have in mind that the structure and biology of a majority of Asteraceae members are far from sufficiently investigated, thus combining morpho-anatomical, phytochemical, and molecular studies are necessary to explore them. Although this overview represents only a glimpse of a role of micromorphological and anatomical ap- proaches to Asteraceae systematics, it gives an valu- able insight and perspective of this topic. Thus, the accumulated knowledge and permanent investiga- tion of Asteraceae taxa using micromorphological and anatomical methods will put light on branching topologies of phylogenetic trees which molecular data established. New opportunities for systematic morphology, micromorphology and anatomy in case of Aster- aceae taxonomy, but certainly also in other plant groups, which were not present in the premolecular era, are now opening regarding synergistic multidis- ciplinary taxonomic, evolutionary and phylogenetic studies which combine molecular with morphologi- cal, anatomical and other approaches (e.g. chemo- phenetics), keeping in the throne these “old fash- ioned” approaches. Acknowledgements. We acknowledge the financial support provided by the Serbian Ministry of Education, Science and Technological Development, project No. 173029. References Abid, R., Ali, N. 2010: Cypsela morphology and its taxonomic significance for the tribe Senecioneae (Asteraceae) from Pakistan. 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