Anatomy of the vegetative organs of neotropical species of Gentianaceae Jussieu: contributing to taxonomy and phylogeny

A família Gcntianaceae compreende cerca de 1615-1688 espécics e 87 gêncros organizadas cm 6 tribos (Chironicae. Exaceae, Gentianeae, Helieae, Potalieae e Succifolieae) além do gênero Voyria cuja posição ainda é incerta. Atualmente, esta classificação tem sido bem aceita pela comunidade científica, pois considera afinidades filogenéticas com base em dados moleculares os quais demonstram congruência com dados de morfologia externa, palinologia e quimiotaxonomia. Apresenta grande variação anatômica e morfológica, no entanto, apenas sinapomorfias moleculares, ou a junção destas com demais caracteres, consolidam a família e as seis tribos como monofiléticas. Em relação ao posicionamento das espécies e dos gêneros, as circunscrições não são bem estabelecidas. Os objetivos do presente trabalho são descrever a anatomia dos órgãos vegetativos de espécies de Gentianaceae penencentes aos gêneros Calolisianthus (Hclicae), Curtia e Hockinia (Saccifolieae) e Deianira (Chironicae); contribuir para o conhecimento a cerca da família e selecionar caracteres anatômicos úteis para a taxonomia e filogenia do grupo. Foram coletados indivíduos de Calolisianthus penduculatus, Deianira dumazioi, Curria tenella, Curtia tenuifolia Curtis patula e Curria diffusa em diferentes áreas de campos rupestres em Minas Gerais. Amostras de Curtia obtusifolia, Curtia conferta; Curtia verticillaris e Hockinia montana provenientes de exsicatas também foram analisadas; além de Declieuxia fruticosa e Declieuxia cordigera espécies de Rubiaceae utilizadas como grupo externo na análise cladística. Amostras de folhas, caules e raízes foram processadas conforme metodologia usual em anatomia vegetal para as técnicas de dissociação de epiderme, diafanização, inclusão em metacrilato e em parafina histológica, teste histoquímicos e microscopia eletrônica de varredura. Para análise cladística foram acrescidos dados da literatura de sete espécies pertencentes às tribos estudadas, sendo analisados 59 caracteres anatômicos. Análise de Máxima Parsimônia foram conduzidas utilizando o programa Paup* 4.0.b10. Todas as espécies analisadas estruturalmente apresentaram estômatos anomocíticos, situados no mesmo nível das demais células epidérmicas, caule com estômatos e floema interno, nectaríolos e coléteres. Foram caracteres diagnósticos para as diferentes espécies: tipo de mesofilo; tipo de feixe vascular; distribuição e tipo de estômatos; presença de hipoderme; presença de flanges cuticulares; contorno das células epidérmicas; presença de cristais; padrão de venação; formato de caule; número de aletas no caule; presença de aerênquima, micorrizas e estrias de Caspary evidentes nas raízes; tipo de crescimento radicular e variações no tipo e distribuição dos coléteres e nectaríolos. Na análise cladística, Gentianaceae apresentou-se monofilética, posição sustentada por 100% de valor de bootstrap , emergindo ainda três clados também monofiléticos que sustentaram os gêneros Calolisianthus (67% de bootstrap ), Curtia (89% de bootstrap ), e Hockinia (86% de bootstrap ) no entanto, Deianira apresentou-se como politomias devido a insuficiência de dados anatônicos sobre as espécies deste gênero, em especial, os referentes às estruturas secretoras. Dados das estruturas secretoras, padrão de venação, tipo de estômatos mostraram-se promissores para a filogenia do grupo. Ressalta-se, assim, a importância da utilização de caracteres anatômicos na resolução de problemas taxonômicos entre as direntes espécies de Gentianaceae, destacando ainda, a essencialidade de dados oriundos de diferentes fontes de caracteres para uma análise mais acurada das relações filogenéticas nas diferentes categorias taxonômicas de Gentianaceae.The Gentianaceae family has around 1615-1688 species and 87 genera which are organized in 6 tribes (Chironieae. Exaecae. Gentianeae, Helieae, Potalieae and Saccifolieace, besides lhe genus Voyria which does not have a fixed position yet. This classification has now been accepted by the scientific community once it considers phylogenetic affinities based on molecular data which demonstrate congruence with the extenal morphology, palynology and chemical taxonomy. This family shows a great anatomical and morphological variety. Nevertheless, only molecular synapomorphies, or the union of these with other characters, consolidate the family and the six tribes as monophyletic groups. Regarding the position of the species and genera, circumscriptions have not been well established. This study aims to describe the anatomy of the vegetative organs of Gentianaceae species belonging to Calolisianthus (Helieae), Curtia and Hockinia (Saccifolieae); and Deianira (Chironieae); to contribute to the knowledge of the family, and to select anatomical characters which may be useful to taxonomy and phylogeny of this group. Specimens of Calolisianthus pedunculatus, Deianira damazioi, Curtia tenella, Curtia tenuifolia, Curtia patula and Curtia diffusa were collected at different Rupestrian Fields (Rocky Outcrops) in the State of Minas Gerais, Brazil. Samples of Curtia obtusifolia, Curtia conferta, Curtia verticillaris and Hockinia montana from voucher material were also analyzed. as well as specimens of Declieuxia fruticosa and Declieuxia cordigera, which were the Rubiaceae species used as the external group in the cladistic analysis. Samples of leaves, stems, and roots were processed according to the usual vegetal anatomy methodology for dissociating the epidermis, diaphanization, methacrylatc and paraffin embedding, histochemical tests, and scanning electron microscopy. For the cladistic analysis, data from literature of seven species belonging to the tribes studied were included. Altogether, 59 anatomical charaters were analysed. The Maximum Parsiimony Analysis was run by the Paup 4.0.b10 version program. All the species structurally analysed showed anomocytic stomata placed at the same level of the other epidermal cells, stems with stomata and internal phloem, nectarioles, and colecters. The characters considered as diagnostic for the different species are as follows: type of vascular bundle; distribution and type of stomata; presence of hypodermis; presence of cuticular flanges; outlining of the epidermal cells; presence of crystals; patterns of venation; shape of the stem; number of wings in thc stem; prcsence of aerenchyma, mycorrhiza and Casparian strips evident in the roots: type of root growth; and variation on the type and distribution of the colecters and nectarioles. ln the cladistic analysis, Gentianaceae, showed to be monophylectic, a position supported by a bootstrap of 100%. Three monophyletic clades giving support to the Calolisianthus (67% of bootstrap"), Curtia (89% of bootstrap") and Hockinia genus (86% of "bootstrap") also emerged. On the other hand, Deianira showcd polytomies probably due t0 insufficient anatomical data about the species belonging to this genus, especially. the ones regarding the secretory structures. Data on the secretory structures, pattern of venation, and type of stomata showed to be promising to the phylogeny of this group. It should also be highlighted the importance of using anatomical characters to solve taxonomic problems regarding the different species of Gentianaceae. Thc necessity of data from different sources of characters in order to achieve a more accurate analysis of the phylogenetic relationships among the different taxonomic categories of Gentianaceae should also be emphasized

Morphoanatomy of Schultesia pachyphylla (Gentianaceae): a discordant pattern in the genus

Schultesia Mart. comprises 21 closely related taxa. The revision of the genus showed that Schultesia pachyphylla Griseb. and Xestaea lisianthoides Griseb. are different from other species. The aim of this study was to describe the morphoanatomy of S. pachyphylla, comparing it with other Schultesia species and X. lisianthoides, and to provide data on their geographical distribution and habitat. Morphological and anatomical data were subjected to multivariate analysis. Schultesia pachyphylla is endemic to the state of Bahia, Brazil, and is separated from the other species by having 5-merous flowers, 5-winged calyx, wings between the calyx lobes, loss of interveinal tissue on sepals during fruit maturation, dark red colored immature calyces, capsules opening from base to apex, 3-anaporate pollen grains, and epidermal papillae evident in the field. Schultesia pachyphylla has amphistomatic leaves with anisocytic stomata, uniseriate epidermis, dorsiventral mesophyll, and bicollateral vascular bundles. The stem has small wings, cortical and medullary parenchyma, and internal phloem. Foliar colleters and nectaries on leaves and stem were observed. We believe that morphological and anatomical data could support the creation of a monotypic genus or the relocation of this species into another genus. Phylogenetic analyses are in progress to define the species’ positioning.Le genre Schultesia Mart. comporte 21 taxons étroitement liés. La révision du genre montre que le Schultesia pachyphylla `Griseb. et le Xestaea lisianthoides Griseb. diffèrent des autres espèces. Le but de cette étude consiste a décrire l’anatomo- morphologie du S. pachyphylla, en le comparant avec les autres espèces de Schultesia ainsi que le X. lisianthoides, et a présenter desdonnées sur leur distribution géographique et leur habitat. Les auteurs ont soumis les données morphologiques et anatomiques a l’analyse multivariée. Le S. pachyphylla est endémique dans l’état de Bahia au Brésil et se distinguent des autres espèces par ses`fleurs 5-mères, son calice a 5 ailes, des ailes entre les lobes du calice, la perte du tissu entre les nervures sur les sépales au cours de la maturation du fruit, des calices immatures rouge-foncé, des capsules s’ouvrant a la base de l’apex, des grains de pollen 3-anaporés et des papilles épidermiques évidentes sur le terrain. Le S. pachyphylla possède des feuilles amphistomatiques avec des stomates anisocytiques, un épiderme unisérié, un mésophylle dorsiventral, et des faisceaux vasculaires bicolatéraux. La tige porte de petites ailes, un parenchyme médullaire et cortical, et du phloème interne. On observe des collétères et des nectaires surles feuilles les tiges. Les auteurs croient que les données morphologiques et anatomiques pourraient supporter la création d’un genre monotypique ou le transfert dans un autre genre. Ils poursuivent les analyses phylogénétiques afin de définir le positionnement de cette espèce. [Traduit par la Rédaction]

Are stem nectaries common in Gentianaceae Juss.?

ABSTRACT Extrafloral nectaries (EFNs) are specialized structures that produce and release nectar and are located on leaves, cotyledons and, more seldomly, stems. Peculiar leaf nectaries have been described for thirty-three Neotropical species of Gentianaceae, while stem nectaries have been reported for only nine. The aim of this study was to verify the occurrence of stem EFNs within Gentianaceae and investigate the existence of a correlation between their occurrence and the geographical distribution of species. Samples of internodal regions from field and herbarium specimens were submitted to standard light microscopy techniques. Data regarding the geographical distribution of species were acquired from herbarium specimens and the literature. A total of 37 species were investigated, representing 25 genera distributed among five tribes. Nectaries, composed of modified epidermal cells, were observed in 16 species restricted to the Neotropical tribes Helieae, Saccifolieae, Potalieae and Coutoubeinae; exceptions were Cicendia quadrangularis and Zygostigma australe, which both occur in the Neotropics but do not possess EFNs. These results demonstrate that stem EFNs are common among Neotropical taxa of Gentianaceae, and are typically absent from taxa in temperate regions

Are stem nectaries common in Gentianaceae Juss.?

ABSTRACT Extrafloral nectaries (EFNs) are specialized structures that produce and release nectar and are located on leaves, cotyledons and, more seldomly, stems. Peculiar leaf nectaries have been described for thirty-three Neotropical species of Gentianaceae, while stem nectaries have been reported for only nine. The aim of this study was to verify the occurrence of stem EFNs within Gentianaceae and investigate the existence of a correlation between their occurrence and the geographical distribution of species. Samples of internodal regions from field and herbarium specimens were submitted to standard light microscopy techniques. Data regarding the geographical distribution of species were acquired from herbarium specimens and the literature. A total of 37 species were investigated, representing 25 genera distributed among five tribes. Nectaries, composed of modified epidermal cells, were observed in 16 species restricted to the Neotropical tribes Helieae, Saccifolieae, Potalieae and Coutoubeinae; exceptions were Cicendia quadrangularis and Zygostigma australe, which both occur in the Neotropics but do not possess EFNs. These results demonstrate that stem EFNs are common among Neotropical taxa of Gentianaceae, and are typically absent from taxa in temperate regions

Foliar colleters in Macrocarpaea obtusifolia (Gentianaceae): anatomy, ontogeny, and secretion

Colleters are secretory structures located in reproductive and (or) vegetative organs of many eudicots. In Gentianaceae Juss., the presence of foliar colleters has been neglected, and anatomical and histochemical studies are scarce. The objectives of this study were to investigate the anatomy, ontogeny, and chemical nature of the secretion found in Macrocarpaea obtusifolia (Griseb.) Gilg colleters to establish a relationship between their structure and function and check whether these structures are similar to those described for other genera of the Gentianaceae and other families of the Gentianales. Samples of leaves at different developmental stages were collected and processed for anatomical and histochemical analysis using light microscopy and scanning electron microscopy. Colleters in M. obtusifolia have a protodermal origin, are of standard type, and are not vascularized. Young colleters are translucent and produce an abundant amount of sticky secretion. Later, they turn yellowish with a blackened region at the apex of the head, and the secretion, composed of polysaccharides and proteins, becomes less abundant and brownish. During senescence, the process begins with complete degradation and cell collapse of the secretory portion. The colleters of the standard type in M. obtusifolia have been observed for the first time in the Gentianaceae and represent additional evidence that reinforces how common this type of colleter is in the Gentianales. Such results provide new information on the anatomy, ontogeny, histochemistry, and colleter types of Gentianaceae.Les collerettes constituent des structures sécrétrices localisées dans les organes reproducteurs ou végétatifs de plusieurs eudicotylées. Chez les Gentianaceae, on a négligé la présence de collerettes foliaires; en effet, rares sont les études anatomiques et histochimiques. L’objectif des auteurs était d’examiner l’anatomie, l’ontogénie, et la nature chimique de la sécrétion se retrouvant dans les collerettes du Macrocarpaea obtusifolia (Griseb.) Gilg, afin d'établir la relation entre leur structure et leur fonction et de vérifier si ces structures sont semblables a` celles décrites chez d’autres genres de Gentianaceae et d’autres familles des Gentianales. Ils ont récolté des échantillons de feuilles a` divers stades de développement et effectué des analyses anatomiques et histochimiques a` l'aide de la microscopie photonique et a` électronique par balayage. Chez le M. obtusifolia, les collerettes ont une origine protodermique, sont de type standard et non vascularisées. Les jeunes collerettes translucides produisent une bonne quantité de sécrétion collante. Plus tard, elles deviennent jaunes avec une région a` col noir a` l’apex de la tête et la sécrétion, composée de polysaccharides et de protéines, devient moins abondante et brunit. Pendant la sénescence, le processus débute avec une dégradation complète avec un affaissement des cellules de la portion sécrétrice. On a observé pour la première fois les collerettes de type standard chez le M. obtusifolia, ce qui représente une preuve additionnelle supportant jusqu’a` quel point ce type de collerette est commun chez les Gentianales. De tels résultats fournissent une nouvelle information sur l’anatomie, l’ontogénie, l’histochimie, et les types de collerettes des Gentianaceae

Anatomical characteristics as taxonomic tools for the species of Curtia and Hockinia (Saccifolieae–Gentianaceae Juss.)

The delimitation of species and even support of the genera Curtia and Hockinia, monotypic genus, are still controversial. To define anatomical characteristics that help resolve taxonomic problems, the stem and leaf anatomy of seven species of Curtia and Hockinia montana are described. Some characteristics are found in both genera: leaves with uniseriate epidermis, dorsiventral mesophyll, cells of the palisade parenchyma with invaginations, basal acrodromous venation with an incomplete marginal ultimate venation, presence of colleters and nectaries, stem with small wings and internal phloem. The types of stomata, the midrib vascular bundle, and the type and distribution of nectaries were used to separate the genera. The presence/absence of crystals, the sinuosity of the anticlinal walls of the epidermal cells, the type and distribution of stomata, and the distribution of nectaries distinguish species of Curtia. Our results suggest that these anatomical characteristics are potentially useful for clarifying the taxonomy of Gentianaceae, and that they represent an additional tool for distinguishing genera and identifying species of Curtia, particularly pointing out the need to revise the synonymy between C. patula and C. tenuifolia. We also highlight the first report of leaf nectaries and colleters in the Saccifolieae and the first record of stem nectaries in the Gentianaceae

How to distinguish cavities from ducts in Casearia Jacq. (Salicaceae): Anatomical characterization and distribution

Pellucid punctuations and lines in the leaf blade are common in Casearia, but for some of its species these structures are mentioned as inconspicuous, not visible or obscure. Cavities and ducts have been anatomically described and correlated with pellucid punctuations and lines, respectively, in only six species from the genus; however, the distinction between these types of secretory structures remains unclear. Therefore, we aimed to anatomically describe the pellucid punctuations and lines in leaves of 47 species of Casearia. Leaf blade samples were obtained from herbarium and field collected material and processed according to standard methods for light microscopy analysis. Cavities and ducts were absent in only four of the 47 species (C. commersoniana, C. javitensis and C. spruceana of section Piparea, and C. eicheleriana of section Casearia). In the remaining species, cavities corresponded to pellucid punctuations, while ducts corresponded with lines. The majority of species have both structures randomly distributed throughout the leaf blade, however, in C. bahiensis, these structures are concentrated along the leaf margin, while in C. aquifolia and C. crassinervis they were predominant along the midrib. Our data show that anatomical analyses are essential for distinguishing between cavities and ducts. We also demonstrate that the distribution of cavities and ducts provides useful information for the taxonomy of Casearia at both the species and section levels