Ciftci, Erol 2020, Biologica Nyssana 11(2) 11 (2) December 2020: 85-91 DOI: 10.5281/zenodo.4393951 Micromorphological studies on inflorescence and seeds of some plantain (Plantago L.) taxa in Turkey Original Article Almila Ciftci Biology Department, Science Faculty, Istanbul Uni- versity, Istanbul, Turkey almila.ciftci@istanbul.edu.tr (corresponding author) Osman Erol Biology Department, Science Faculty, Istanbul Uni- versity, Istanbul, Turkey erol@istanbul.edu.tr Received: Julz 14, 2020 Revised: August 28, 2020 Accepted: September 15, 2020 Abstract: Plantago L. (Plantaginaceae) is one of the largest genera of the Plantaginaceae family. There are relatively fewer morphological studies on the genus. Seed surface micromorphology has become popular in plant taxonomy in recent years and found useful in most groups. The inflorescence and seed surface characteristics of 22 Plantago taxa collected from Turkey were examined using a Scanning Electron Microscope (SEM). Cluster analysis performed in R. We observed that the micromorphology of the inflorescence parts and seeds of genus Plantago are very variable, and do not support the classification based on general morphology. This study shows that these characteristics are highly variable and micromorphological characteristics of inflorescence parts are not good characters for making a specific distinction between different taxa. Additionally, some groups have been to possess a stable seed surface characteristics. However, these characters are not reliable among all taxa, which means that they should be used carefully, especially when identifying widespread taxa. Key words: inflorescence, micromorphology, Plantago, Plantaginaceae, seed Apstract: Mikromorfološka studija cvast i semena nekih taksona bokvica (Plantago L.) u Turskoj Plantago L. (Plantaginaceae) je jedan od najvećih rodova porodice Plantaginaceae. Postoji relativno malo morfoloških studija o ovom rodu. Mikromorfologija površine semena postala je popularna u biljnoj taksonomiji poslednjih godina i smatra se korisnom u većini grupa. Karakteristike cvasti i površine semena 22 taksona roda Plantago sakupljenih iz Turske ispitane su pomoću skenirajućeg elektronskog mikroskopa (SEM). Klaster analiza je izvršena u R. Primetili smo da su mikromorfologija delova cvasti i semena roda Plantago veoma promenljive i ne podržavaju klasifikaciju zasnovanu na opštoj morfologiji. Ova studija pokazuje da su ove karakteristike veoma promenljive, a mikromorfološke karakteristike delova cvasti nisu dobri karakteri za pravljenje posebne razlike između različitih taksona. Pored toga, neke grupe su pokazale da imaju stabilne karakteristike površine semena. Međutim, ovi karakteri nisu pouzdani kod svih taksona, što znači da ih treba pažljivo koristiti, posebno pri identifikaciji rasprostranjenih vrsta. Ključne reči: cvasti, mikromorfologija, Plantago, Plantaginaceae, seed Introduction Plantaginaceae is a very heterogeneous family with diverse evolutionary trends, and Plantago L. is one of the greatest genera of the Plantaginaceae family with 200 species, 56 subspecies, 188 varieties and 9 subvarieties according to Albach et al. (2005). It is probably the most widespread genus of the family Plantaginaceae (Albach et al., 2005). Plantago genus is divided into nineteen sections and two subgenera (Pilger, 1937). 9 of these sections including 23 taxa, two of which are endemic to Tur- key, have been recorded in the Flora of Turkey and the East Aegean Islands (Tutel, 1982). Although Rahn (1996) claimed that Plantago can be divided into six genera, he divided the genus into six subgenera and twelve sections in order to prevent synonym pollution. These two classification sugges- tions for the genus are summarized in Tab. 1. Here in this study, we used Pilger’s (1937) classifica- © 2020 Ciftci, Erol. This is an open-access article distributed under the terms of the Creative Commons At- tribution License, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 85 tion in order to be able to follow the ranking in the Flora of Turkey (Tutel, 1982). Plantago taxa are annual or perennial herbs or dwarf shrubs with alternate rosettes or opposite leaves. The inflorescence is a spike with 4-merous flowers. The sepals are imbricate and keeled. The flowers have bracts. The corolla is sympetalous and forms a cylindrical tube (Tutel, 1993). Even though various studies have been made with Plantago taxa, morphological and systematic stud- ies on Plantago taxa are relatively limited (Sagar & Harper, 1964; Sharma et al., 1990; Tutel et al., 2005). Micromorphological studies have been made in China (Jun-Zhe et al., 1992) and Poland (Klimko et al., 2004) which were both limited to the taxa in their region. The most extensive study on micromor- phology of Plantago seeds composed by Shehata and Loutfy (2006) included 31 taxa. Seed surface micromorphology has become very popular in plant taxonomy in recent years. One of the main reasons for that is that structure of the seed surface seems to be the most stable character due to less contact with environment. Flower elements are also one of the most stable characters in flowering plants (Endress, 2001). For this reason, we decided to include the surface investigations of inflorescence elements in this study. Considering the lack of the studies about Plan- tago taxa, this study aims to clarify the relationships within Plantago taxa in Turkey; using seed and in- florescence features. Moreover, SEM was used to investigate further the micromorphology of the seed and the inflorescence surfaces of these taxa in Tur- key for the first time. Materials and Methods 22 Turkish Plantago taxa including four varieties, which were not presented in the Flora of Turkey and East Aegean Islands (Tutel, 1982), are examined in this study (Tab. 2). These taxa were obtained mostly from ISTF (Istanbul University Faculty of Sciences Herbarium) as well as from ANK (Ankara Universi- ty Faculty of Science Herbarium). We used 15 indi- viduals’ inflorescence elements and seeds from each 86 Sensu Pilger Sensu Rahn Subgen. Sect. Taxa Sect. Subgen. Euplantago Plantago Plantago major subsp. major Plantago Plantago P. major subsp. intermedia Gentianoides P. gentianoides Lamprosantha P. media Coronopus P. crassifolia Coronopus Coronopus P. coronopus P. weldenii P. holosteum P. maritima Maritima Oreades P. atrata Montana Albicans Leucopsyllium P. albicans P. loeflingii Arnoglossum P. lanceolata LanceifoliaP. argentea P. lagopus Hymenopsyllium P. cretica Hymenopsyllium P. bellardii Psyllium Psyllium P. squarrosa Psyllium Psyllium P. afra P. scabra P. sempervirens P. euphratica Table 1. Classification of the Turkish Plantago taxa according to Pilger [2] and Rahn [4] BIOLOGICA NYSSANA ● 11 (2) December 2020: 85-91 Ciftci, Erol ● Micromorphological studies on inflorescence and seeds of some plantain (Plantago L.) taxa in Turkey 87 taxon when possible. Apart from the other studies (Jhun-Zhe et al., 1992; Klimko et al., 2004; Shehata & Loutfy, 2006) on Plantago micromorphology, this study includes some other areas from the inflorescence parts (e.g. bract, anterior and posterior sepals, and corolla) (Fig. 1). For the examination of the inflorescence characteristics, the lowest and the most mature flow- ers of the inflorescences have been chosen, and the macro morphological observations have been made using Olympus SZX7 stereomicroscope. The micro- morphology of the inflorescence parts and seeds Fig. 1. General view of a Plantago flower (a). The observed areas- corolla tube (1), petal (2), anterior sepal base (3), bract margin (4), posterior sepal carina (5) and margin (6), seed ventral (7) and dorsal (8) surfaces- are shown with squares (b). Scale bars 1 mm. Table 2. The examined taxa in the study with voucher specimens Taxa Voucher No Location P. major L. subsp. major ISTF 24301 Rize P. major L. subsp. intermedia (Gilib) Lange ISTF 23423 İstanbul P. coronopus L. subsp. coronopus ISTF 557 İstanbul P. weldenii Rchb. ISTF 2306 İstanbul P. crassifolia Forsskal ISTF 40190 Istanbul P. maritima L. ISTF 26779 Kirklareli P. holosteum Scop. ISTF 27341 Ankara P. media L. ISTF 23998 Erzurum P. atrata Hoppe ISTF 23710 Erzurum P. gentianoides SM. subsp. gentianoides ISTF 32242 Bursa P. lanceolata L. ISTF 23372 Istanbul P. argentea Chaix ISTF 63 Bursa P. lagopus L. ISTF 22271 Istanbul P. albicans L. ANK Ankara P. loeflingii L. ISTF 35420 Sanliurfa P. cretica L. ISTF 24743 Antalya P. bellardii All. ISTF 27212 İstanbul P. squarrosa Murray ISTF 9790 Antalya P. scabra Moench ISTF 32258 İstanbul P. afra L. ISTF 36362 Mersin P. sempervirens Crantz ALM 27 Tekirdağ P. euphratica Decne. ex Barnéoud ISTF 37227 Malatya BIOLOGICA NYSSANA ● 11 (2) December 2020: 85-91 Ciftci, Erol ● Micromorphological studies on inflorescence and seeds of some plantain (Plantago L.) taxa in Turkey 88 have been observed and photographed by a JEOL Benchtop Scanning Electron Microscope JCM-5000 under 10 kV of voltage. The nomenclature of the surface characteristics is adapted from Stearn (1991) and Barthlott et al. (1998). The categorical variables of surface characters in various anatomical compartments of Plantago were used as variables in the cluster analysis. Hierarchi- cal cluster analysis of 26 categorical variables across 22 taxa was performed using a clustering algorithm via ward.D method. In brief, a mean distances ma- trix was generated and subjected to clustering with the ward.D method of the hclust function. Following the clustering, the relationships between the sections were visualised as dendrograms using the tree func- tions in R v3.4.3. Results The dissimilarity cluster analyses formed two main groups and four subgroups according to the surface characters (Fig. 2). Both of the groups included dif- ferent taxa from different sections. The macro-morphological observations showed that the seed shapes and sizes of Plantago taxa are very variable. The micromorphological aspects of the examined parts using SEM are shown in Tab. 3. The SEM photos of all the examined areas are given in Fig. 3. It can be seen from the Fig. 3 that hairs, and epidermal cell arrangements are variable among the studied taxa. Observations showed that the surface type of co- rolla tube was the most stable feature appearing to be lineate in all of the examined taxa but only diffe- ring in terms of cell size and presence of hairs (Tab. 3, Fig. 3). This result shows that the surfaces of the petals and the posterior sepals seem to have more or less stable features through sections (Tab. 3, Fig. 3). Being the outermost part of a flower structure, bract has shown a great variety in terms of surface micro- morphology among all the examined parts (Tab. 3, Fig. 3). In Section Coronopus bract surfaces are differing among the taxa whereas the seed surface characte- ristics are very stable. All taxa in this section has re- ticulate seed surface ornamentation and all but one taxa (P. holosteum) has undulated anticlinal walls. Section Coronopus also show stability in terms of inflorescence parts’ surface characters generally ex- cept anterior sepal and bract surfaces (Tab. 3). Simi- larly, taxa in Section Arnoglossum have very stab- le seed surface characteristics (smooth). The smo- oth seed surfaces have also been observed in some taxa of Section Psyllium. Section Psyllium, has the most variable seed surface characteristics. The inflo- rescence surface characters vary among these taxa, as well (Tab. 3). Sections Lamprosantha, Oreades Fig. 2. Cluster dendrogram of Plantago taxa. The sections marked with different colors BIOLOGICA NYSSANA ● 11 (2) December 2020: 85-91 Ciftci, Erol ● Micromorphological studies on inflorescence and seeds of some plantain (Plantago L.) taxa in Turkey and Gentianoides are represen- ted by one taxon each, and these taxa have similar characteristics with each other, and in the terms of seed surface characters they resemble Section Plantago. In P. major subsp. major and in P. major subsp. intermedia seed surfaces are rugose. Other examined parts (i.e. anterior se- pal, posterior sepal carina, co- rolla tube, petal, and dorsal and ventral sides of seeds) show pa- rallel characteristics within the- se two taxa except anterior se- pals. The bases of anterior se- pals are both rugose, but cell shapes and anticlinal wall natu- re is different (Tab. 3). The seed surfaces are reticu- late in Section Leucopsyllium and Section Hymenopsyllium. Section Leucopsyllium has di- verse inflorescence surface cha- racters. Section Hymenopsylli- um taxa have more or less stable characters on micromorpholo- gical level with rugose posterior sepal carina surfaces, striate co- rolla tube, and striate petal sur- faces but there is only two taxa available in this study. Discussion The two subspecies of P. ma- jor appear in two different main groups. Concurrently, the ob- servation of the seed surfaces of P. major were controversi- al in previous studies focusing on the seed micromorphology of Plantago taxa (Klimko et al., 2004; Shehata & Loutfy, 2006; Hoghoughi et al., 2016; Verma & Bharti, 2017). Klimko et al. (2004) reported the seed surfa- ces of P. major as “not slightly rugose as it has been reported in previous studies” (Rymkie- wicz, 1979; Rothmaler et al., 1984). On the contrary, other authors identified the same area as reticulate (Shehata & Lout- fy, 2006), tuberculate (Verma & Bharti, 2017), and areolate with wrinkled surface (Hog- 89 Fig. 3. SEM micrographs of studied areas. Posterior sepal carina surfa- ces of (a) Reticulate, Plantago major subsp. major; (b) Rugose, P. wel- denii; (c) Striate, P. gentianoides subsp. gentianoides; Petal surfaces of (d) Granulate, P. weldenii; (e) Striate, P. gentianoides subsp. gentianoi- des; (f) Rugose, P. sempervirens; Anterior sepal base surfaces of (g) Ru- gose, P. major subsp. major; (h) Rugulose, P. coronopus subsp. corono- pus; (i) Granulate, P. albicans; Bract margin surfaces of (j) Reticulate, P. coronopus subsp. coronopus; (k) Rugose, P. maritima; (l) Smooth, P. ar- gentea; P. squarrosa; (m) Fissured, P. afra; (n) Granulate, P. sempervi- rens; Corolla tube surface of (o) Lineat, P. gentianoides subsp. gentiano- ides; Seed surfaces of (p) Rugose, Plantago major subsp. major; (q) Re- ticulate, P. crassifolia; (r) Smooth, P. lagopus. BIOLOGICA NYSSANA ● 11 (2) December 2020: 85-91 Ciftci, Erol ● Micromorphological studies on inflorescence and seeds of some plantain (Plantago L.) taxa in Turkey houghi et al, 2016). In this study, seed surfaces of both P. major subsp. major and P. major subsp. in- termedia were identified as rugose. Plantago lance- olata is another taxon which shows a great variety in the sense of seed micromorphology like P. ma- jor. Verma et al. (2017) reported the seed surface of P. lanceolata as scalariform to reticulate with spind- le shaped striations whereas Hoghoughi et al. (2016) identifies it as negative reticulate, and the samples in this study showed no ornamentation (smooth surfa- ce). These taxa are widespread and adapted to a va- riety of habitats, therefore it is an expected outcome. Similarly, P. maritima and P. gentianoides were reported as having regulate or areolate ornamentati- on and reticulum cristatum respectively (Hoghoughi et al., 2016), but in this study they are identified as reticulate and rugose. It has been known that Plantago taxa are very variable in morphology, and that this variation may be due to environmental differences (Meudt, 2012). However, some taxa show very consistent surface characteristics. For instance, P. coronopus has found to be reticulate in both this study and others (Hog- houghi et al., 2016). Plantago major is a widespread species with capability to adapt in a variety of habi- tats, thus its positioning in the clustering may not be considered meaningful. Section Psyllium taxa have representatives in both main groups in dendrogram. Some of these taxa have shown different seed surface characters than previously studied (Shehata & Loutfy, 2006). The comparison of these results shows that seed sur- faces may show variation on an intraspecific level among Sect. Psyllium and also some other taxa (P. major and P. lanceolate) Section Coronopus taxa have a very distinct sur- face structure. All of the studied taxa in this section have shown undulated anticline membranes and ru- minate seed surfaces. These findings are in accor- dance with the previous studies (Klimko et al., 2004; Shehata & Loutfy, 2006). However, the classificati- on of the Coronopus section, including P. maritima, is problematic. Rahn (1978); Dietrich (1980) and Klimko et al. (2004) have divided the section Coro- nopus into two sections as Section Coronopus and Section Maritima. Shehata & Loutfy (2006) have se- parated the P. maritima taxon from subgenus Coro- nopus. In this study, P. maritima emerged in the se- cond main group with the other Section Coronopus (P. weldenii, P. crassifolia and P. holosteum) except P. coronopus. The dendrogram separated the taxa of Sect. Coronopus into four subgroups; P. maritima grouped with P. crassifolia; P. weldenii which was formerly assigned as a subspecies of P. coronopus. However, these findings don’t agree with the mole- cular data completely (Serrano et al., 2017). In Tutel et al. (2005), it has been suggested that Hymenopsyllium section in subgenus Euplantago is closer to subgenus Psyllium based on morphological traits. In this study Sect. Hymenopsyllium is cluste- red completely in first main group, whereas most of the Sect. Psyllium taxa are placed in the second main group. Therefore, this study does not support the ini- tial statement. However, the results showed the lat- ter section is very diverse in terms of micromorpho- logic traits and the study of molecular markers alre- ady showed P. bellardii and P. cretica are in the Sub- genus Psyllium (Ronsted et al., 2002). This shows the micromorphology of inflorescence parts of these taxa are not very reliable in this subgenus. The Plantago taxa are known to be wind polina- ted but we observed that the flowers attract some in- sects (Syrphidae) in field and Alfred Heilbronn Bo- tanical Garden. By the reason of the fact that they do not have flashy flowers, it may be due to the UV reflection characteristics of the epicuticular waxes (Whitney et al., 2009). Some taxa having more abi- lity to draw the insects (P. maritima, P. lanceolata, P. argentea) have granulate and striate petal surfaces. However, this finding needs further investigations. In the overall results of the examinations, we have pointed out that the inflorescence parts’ surfa- ces are variable among taxa except for the corolla tube, and the differences are probably not distinctive characteristics for these taxa. This may be due to its protective position. This work shows that hairs, epidermal cell arran- gements and surface characters are very variable ac- ross the genus. Therefore, analysis of inflorescence parts is not conclusive to make a specific distinction between different taxa. Although some groups have very stable seed surface characteristics, they are not reliable among all taxa, which means it should be used with extreme care especially with the widesp- read taxa. 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