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Seed’s morpho-anatomy and post-seminal development of Bromeliaceae from tropical dry forest

Abstract

Drastic changes in dry tropical forest result in the loss of biological components and reveal the importance of studies on the biology of species living in it. The present study aimed to describe seed morphoanatomy, germination and post-seminal development of Bromeliaceae species in fragments of tropical dry forest in Sucre, Colombia. Seven species representing Bromelia and Tillandsia genera were evaluated. The results provide characteristics related to the habitat of each species and contribute to distinguish the genera evaluated: fruit and seed measurements, seed shape, plumose appendage, testa characteristics, and the aleurone layer, embryo endosperm ratio, types of reserves, constriction zone in the embryo and type of post-seminal development. Characteristics of plumose appendages and the presence of vascular bundles in the embryo also contribute to distinguish Tillandsia species. In T. elongata and T. flexuosa, high number of seeds per fruit (> 100), morphoanatomical aspects, high germination (> 92%) and plant formation (> 77%) percentages, and higher germination rate values (> 4.5) give them the potential capacity for establishment in this environment. Our results provide information with taxonomic and ecological relevance for bromeliads in dry tropical forest.

Key words
Bromelia; embryo; endosperm; germination; Tillandsia

Resumo

Drásticas transformações na floresta tropical seca levam à perda de seus componentes biológicos e revelam a importância dos estudos sobre a biologia das espécies que a habitam. Nosso objetivo foi descrever a morfoanatomia e a germinação das sementes, e o desenvolvimento pós-seminal de espécies de Bromeliaceae presentes em fragmentos de floresta seca em Sucre, Colombia. Foram avaliadas sete espécies que representam os gêneros Bromelia e Tillandsia. Os resultados fornecem características que estão relacionadas ao habitat de cada espécie e ajudam a distinguir os gêneros avaliados: medidas dos frutos e sementes, forma da semente, apêndices plumosos, particularidades da testa e da camada de aleurona, proporção do embrião e do endosperma, tipos de reservas, zona de constrição no embrião e tipo de desenvolvimento pós-seminal. As características dos apêndices plumosos e a presença de feixes vasculares no embrião contribuem para a distinção entre as espécies de Tillandsia. Nas espécies T. elongata e T. flexuosa, o grande número de sementes por fruto (> 100), seus aspectos morfoanatômicos, as altas porcentagens de germinação (> 92%) e formação de plântulas (> 77%), e o maiores valores do índice de velocidade de germinação (> 4,5) conferem-lhes a capacidade potencial de aumentar a área de distribuição neste ambiente. Nossos resultados proporcionam informações com relevância taxonômica e ecológica para as bromélias que habitam a floresta tropical seca.

Palavras-chave
Bromelia; embrião; endosperma; germinação; Tillandsia

Introduction

About 54.2% of the world’s tropical dry forests are in South America and the rest are evenly distributed in North and Central America, Eurasia, Africa, Southeast Asia and Australasia (Miles et al. 2006Miles L, Newton AC, DeFries RS, Ravilious C, May I, Blyth S, Kapos V & Gordon JE (2006) A global overview of the conservation status of tropical dry forests. Journal of Biogeography 33: 491-505.). Currently, the tropical dry forest occurs as small isolated fragments (Portillo-Quintero & Sánchez-Azofeifa 2010Portillo-Quintero C & Sánchez-Azofeifa G (2010) Extent and conservation of tropical dry forests in the Americas. Biological Conservation 143: 144-155.) as a consequence of anthropogenic factors (Miles et al. 2006Miles L, Newton AC, DeFries RS, Ravilious C, May I, Blyth S, Kapos V & Gordon JE (2006) A global overview of the conservation status of tropical dry forests. Journal of Biogeography 33: 491-505.; Hasnat & Kamal 2020Hasnat T & Kamal HM (2020) Global overview of tropical dry forests. In: Bhadouria R, Tripathi S & Srivastava P (eds.) Handbook of research on the conservation and restoration of tropical dry forests. Editorial Advisory Board, Hershey. Pp. 1-23.; Siyum 2020Siyum ZG (2020) Tropical dry forest dynamics in the context of climate change: syntheses of drivers, gaps, and management perspectives. Ecological Processes 9: 25. ). The drastic transformations to which the dry forest is subjected lead to the loss of biological components (Siyum 2020Siyum ZG (2020) Tropical dry forest dynamics in the context of climate change: syntheses of drivers, gaps, and management perspectives. Ecological Processes 9: 25. ), therefore, it is considered one of the most threatened tropical biomes (Pizano & García 2014Pizano C & García H (2014) El bosque seco tropical en Colombia. Instituto de Investigación de Recursos Biológicos Alexander von Humboldt (IAVH), Bogotá. 350p.; Hasnat & Kamal 2020Hasnat T & Kamal HM (2020) Global overview of tropical dry forests. In: Bhadouria R, Tripathi S & Srivastava P (eds.) Handbook of research on the conservation and restoration of tropical dry forests. Editorial Advisory Board, Hershey. Pp. 1-23.).

The tropical dry forest can be defined as a vegetation type normally dominated by deciduous trees (at least 50% of trees present are drought deciduous), where the mean annual temperature is ≥ 25 °C, the total annual precipitation ranges between 700 and 2,000 mm, and there are three or more dry months every year (Sánchez-Azofeifa et al. 2005Sánchez-Azofeifa GA, Quesada M, Rodriguez JP, Nassar JM, Stoner KE, Castillo A, Garvin T, Zent EL, Calvo-Alvarado JC, Kalacska MER, Fajardo L, Gamon JA & Cuevas-Reyes P (2005) Research priorities for neotropical dry forests. Biotropica 37: 477-485.). This seasonality limits primary productivity and plant biodiversity, and leads to a series of morphological, anatomical and physiological adaptations in plants, which means that living under such condition represents a challenge (Pennington et al. 2006Pennington RT, Lewis GP & Ratter JA (2006) Neotropical savannas and seasonally dry forests: plant diversity, biogeography, and conservation. CRC Press, Boca Raton. 508p.; Pizano & García 2014Pizano C & García H (2014) El bosque seco tropical en Colombia. Instituto de Investigación de Recursos Biológicos Alexander von Humboldt (IAVH), Bogotá. 350p.).

The Bromeliaceae family is characterized by its great adaptability and resistance to extreme environmental conditions (Alvarado-Fajardo et al. 2013Alvarado-Fajardo V, Morales-Puentes M & Larrota-Estipuñán E (2013) Bromeliaceae en algunos municipios de Boyacá y Casanare. Revista de la Academia Colombiana de Ciencias Exactas, Físicas y Naturales 142: 5-18.), and some of its species have been previously reported in tropical dry forests (Mondragón & Calvo-Irabien 2006Mondragón D & Calvo-Irabien LM (2006) Seed dispersal and germination of the epiphyte Tillandsia brachycaulos (Bromeliaceae) in a tropical dry forest, Mexico. The Southwestern Naturalist 51: 462-470.; Vargas 2012Vargas W (2012) Los bosques secos del Valle del Cauca, Colombia: una aproximación a su flora actual. Biota Colombiana 13: 102-164.; Rodríguez et al. 2012Rodríguez G, Banda K, Reyes S & Estupiñán A (2012) Lista comentada de las plantas vasculares de bosques secos prioritarios para la conservación en los departamentos de Atlántico y Bolívar (Caribe colombiano). Biota Colombiana 13: 7-39.; Pizano & García 2014Pizano C & García H (2014) El bosque seco tropical en Colombia. Instituto de Investigación de Recursos Biológicos Alexander von Humboldt (IAVH), Bogotá. 350p.; Victoriano-Romero et al. 2017Victoriano-Romero E, Valencia-Díaz S, Toledo-Hernández VH & Flores-Palacios A (2017) Dispersal limitation of Tillandsia species correlates with rain and host structure in a central Mexican tropical dry forest. PLoS ONE 12: e0171614.). Bromeliaceae comprises one of the most morphologically and ecologically distinct clade of Neotropical Angiosperms, with more than 3,100 described species, distributed among eight subfamilies (Givnish et al. 2011Givnish TJ, Barfuss MHJ, Van Ee B, Riina R, Schulte K, Horres R, Gonsiska PA, Jabaily RS, Crayn DM, Smith JAC, Winter K, Brown GK, Evans TM, Holst BK, Luther H, Till W, Zizka G, Berry PE & Sytsma KJ (2011) Phylogeny, adaptive radiation, and historical biogeography in Bromeliaceae: insights from an eight-locus plastid phylogeny. American Journal of Botany 98: 872-895.). This group includes herbaceous, perennial, terrestrial, epiphytic or rupicolous plants (Wanderley & Martins 2007Wanderley MGL & Martins SE (2007) Bromeliaceae. In: Melhem TS, Wanderley MGL, Martins SE, Jung-Mendaçolli SL, Shepherd GJ & Kirizawa M (eds.) Flora fanerogâmica do estado de São Paulo. Instituto de Botânica, São Paulo. Vol. 5, pp. 39-162.), and some of these are being considered in the restoration of degraded areas (Duarte & Gandolfi 2013Duarte MM & Gandolfi S (2013) Enriquecimento de florestas em processo de restauração: aspectos de epífitas e forófitos que podem ser considerados. Hoehnea 40: 507-514.). Despite advances in classification, the delimitation of genera has been broadly discussed (Givnish et al. 2011Givnish TJ, Barfuss MHJ, Van Ee B, Riina R, Schulte K, Horres R, Gonsiska PA, Jabaily RS, Crayn DM, Smith JAC, Winter K, Brown GK, Evans TM, Holst BK, Luther H, Till W, Zizka G, Berry PE & Sytsma KJ (2011) Phylogeny, adaptive radiation, and historical biogeography in Bromeliaceae: insights from an eight-locus plastid phylogeny. American Journal of Botany 98: 872-895.). Therefore, studies based on anatomical and morphological data are necessary, seeking to improve the circumscriptions at the intra-family and infrageneric level, where the molecular system does not provide conclusive data (Palací et al. 2004Palací C, Brown G & Tuthill D (2004) The seeds of Catopsis (Bromeliaceae: Tillandsioideae). Systematic Botany 29: 518-527.; Barfuss et al. 2005Barfuss MHJ, Samuel R, Till W & Stuessy T (2005) Phylogenetic relationships in subfamily Tillandsioideae (Bromeliaceae) based on DNA sequence data from seven plastid regions. American Journal of Botany 92: 337-351.; Givnish et al. 2007Givnish T, Millam K, Berry P & Systma K (2007) Phylogeny, adaptive radiation and historical biogeography of Bromeliaceae inferred from ndhF sequence data. Aliso 23: 3-26.).

According to Ackerly (2009)Ackerly D (2009) Conservatism and diversification of plant functional traits: evolutionary rates versus phylogenetic signal. Proceedings of the National Academy of Sciences of the USA 106: 19699-19706., the seed traits are less susceptible to environmental changes than other plant traits. Thus, for a long time, seed morphology was used to delimit the three formerly recognized Bromeliaceae subfamilies, considering the presence and position of appendages in the seeds among the main characters of distinction (Smith & Downs 1974Smith B & Down R (1974) Pitcairnioideae (Bromeliaceae). Flora Neotropica 14: 1-658.). Recent studies continue to use seeds to define more specific taxonomic relationships (Strehl & Beheregaray 2006Strehl T & Beheregaray RCP (2006) Morfologia de sementes do gênero Dyckia, subfamília Pitcairnioideae (Bromeliaceae). Pesquisas Botânicas 57: 103-120.; Silva & Scatena 2011Silva I & Scatena V (2011) Morfologia de sementes e de estâdios iniciais de plântulas de espécies de Bromeliaceae de Amazõnia. Rodriguésia 62: 263-272.; Scatena et al. 2006Scatena V, Segecin S & Coan A (2006) Seed morphology and post-seminal development of Tillandsia L. (Bromeliaceae) from the “Campos Gerais”, Paraná, Southern Brazil. Brazilian Archives of Biology and Technology 49: 945-951.; Magalhães & Mariath 2012Magalhães R & Mariath J (2012) Seed morphoanatomy and its systematic relevance to Tillandsioideae (Bromeliaceae). Plant Systematics and Evolution 298: 1881-1895.; Corredor-Prado et al. 2014Corredor-Prado JP, Schmidt EC, Steinmacher DA, Guerra MP, Bouzon ZL, Dal Vesco LL & Pescador R (2014) Seed morphology of Vriesea friburgensis var. paludosa L.B. Sm. (Bromeliaceae). Hoehnea 41: 553-562.). In addition, seed traits play a vital role in the dispersal and successful establishment of plant (Chilpa-Galván et al. 2018Chilpa-Galván N, Márquez-Guzmán J, Zotz G, Echevarría-Machado I, Andrade JL, Espadas-Manrique C & Reyes-García C (2018) Seed traits favouring dispersal and establishment of six epiphytic Tillandsia (Bromeliaceae) species. Seed Science Research 28: 349-359.). According to Fenner & Thompson (2005)Fenner M & Thompson K (2005) The ecology of seeds. Cambridge University Press, New York. 260p., from the different stages of the life cycle of plants, the seed germination can determine the distribution of species in different habitats. The study of this phase is of fundamental importance, both for understanding the establishment of a plant community and for the survival and natural regeneration of species. In Bromeliaceae, some studies have described the morphology and anatomy of seeds (Cecchi-Fiordi et al. 1996Cecchi-Fiordi A, Palandri M, Di Falco P & Tani G (1996) Cytological aspects of the hypocotyl correlated to the behavior of the embryo radicle of Tillandsia atmospheric species. Caryologia 49: 113-124., 2001Cecchi-Fiordi A, Palandri M, Turicchia S, Tani G & Di Falco P (2001) Characterization of the seed reserves in Tillandsia (Bromeliaceae) and ultrastructural aspects of their use at germination. Caryologia 54: 1-16.; Morra et al. 2002Morra L, Dottori N & Cosa M (2002) Ontogenia y anatomía de semilla y fruto en Tillandsia tricholepis (Bromeliaceae). Boletín de la Sociedad Argentina de Botánica 37: 193-201.; Palací et al. 2004Palací C, Brown G & Tuthill D (2004) The seeds of Catopsis (Bromeliaceae: Tillandsioideae). Systematic Botany 29: 518-527.; Strehl & Beheregaray 2006Strehl T & Beheregaray RCP (2006) Morfologia de sementes do gênero Dyckia, subfamília Pitcairnioideae (Bromeliaceae). Pesquisas Botânicas 57: 103-120.; Scatena et al. 2006Scatena V, Segecin S & Coan A (2006) Seed morphology and post-seminal development of Tillandsia L. (Bromeliaceae) from the “Campos Gerais”, Paraná, Southern Brazil. Brazilian Archives of Biology and Technology 49: 945-951.; Ferreira et al. 2009Ferreira E, Fernandes I & Resende M (2009) Morfologia de frutos e sementes e desenvolvimento pósseminal de Dyckia goehringii Gross y Rauh (Bromeliaceae). Revista de Biologia Neotropical 6: 1-12. ; Pereira et al. 2010Pereira A, Andrade A, Pereira T, Forzza T & Rodrigues A (2010) Morphological aspects of seed, germination and storage of Pitcairnia albiflos (Bromeliaceae). Seed Science and Technology 38: 79-87.; Magalhães & Mariath 2012Magalhães R & Mariath J (2012) Seed morphoanatomy and its systematic relevance to Tillandsioideae (Bromeliaceae). Plant Systematics and Evolution 298: 1881-1895.; Corredor-Prado et al. 2014Corredor-Prado JP, Schmidt EC, Steinmacher DA, Guerra MP, Bouzon ZL, Dal Vesco LL & Pescador R (2014) Seed morphology of Vriesea friburgensis var. paludosa L.B. Sm. (Bromeliaceae). Hoehnea 41: 553-562.; Chilpa-Galván et al. 2018Chilpa-Galván N, Márquez-Guzmán J, Zotz G, Echevarría-Machado I, Andrade JL, Espadas-Manrique C & Reyes-García C (2018) Seed traits favouring dispersal and establishment of six epiphytic Tillandsia (Bromeliaceae) species. Seed Science Research 28: 349-359.), as well as their post-seminal development (Pereira 1988Pereira TS (1988) Bromelioideae (Bromeliaceae): morfologia do desenvolvimento pós-seminal de algumas espécies. Arquivo do Jardim Botânico do Rio de Janeiro 29: 115-154.; Scatena et al. 2006Scatena V, Segecin S & Coan A (2006) Seed morphology and post-seminal development of Tillandsia L. (Bromeliaceae) from the “Campos Gerais”, Paraná, Southern Brazil. Brazilian Archives of Biology and Technology 49: 945-951.; Tillich 2007Tillich HJ (2007) Seedling diversity and the homologies of seedling organs in the order Poales (Monocotyledons). Annals of Botany 100: 1413-1429.; Pereira et al. 2008Pereira A, Pereira T, Rodrigues  & Andrade  (2008) Morfologia de sementes e do desenvolvimento pós-seminal de espécies de Bromeliaceae. Acta Botanica Brasilica 22: 1150-1162., 2009Pereira A, Andrade A, Pereira T, Forzza T & Rodrigues A (2009) Comportamento germinativo de espécies epífitas e rupícolas de Bromeliaceae do Parque Estadual do Ibitipoca, Minas Gerais, Brasil. Revista Brasileira de Botânica 32: 827-838., 2010; Ferreira et al. 2009Ferreira E, Fernandes I & Resende M (2009) Morfologia de frutos e sementes e desenvolvimento pósseminal de Dyckia goehringii Gross y Rauh (Bromeliaceae). Revista de Biologia Neotropical 6: 1-12. ; Silva & Scatena 2011Silva I & Scatena V (2011) Morfologia de sementes e de estâdios iniciais de plântulas de espécies de Bromeliaceae de Amazõnia. Rodriguésia 62: 263-272.), and others have focused on their germination (Mondragón & Calvo-Irabien 2006Mondragón D & Calvo-Irabien LM (2006) Seed dispersal and germination of the epiphyte Tillandsia brachycaulos (Bromeliaceae) in a tropical dry forest, Mexico. The Southwestern Naturalist 51: 462-470.; Mora et al. 2007Mora F, Chaparro H, Vargas O & Bonilla M (2007) Dinámica de la germinación, latencia de semillas y reclutamiento de plántulas en Puya cryptantha y P. trianae, dos rosetas gigantes de los páramos colombianos. Ecotropicos 20: 31-40.; Pereira et al. 2008Pereira A, Pereira T, Rodrigues  & Andrade  (2008) Morfologia de sementes e do desenvolvimento pós-seminal de espécies de Bromeliaceae. Acta Botanica Brasilica 22: 1150-1162., 2009; Goode & Allen 2009Goode LK & Allen MF (2009) Seed germination conditions and implications for establishment of an epiphyte, Aechmea bracteata (Bromeliaceae). Plant Ecology 204: 179-188.; Valencia-Díaz et al. 2010Valencia-Díaz S, Flores-Palacios A, Rodríguez-López V, Ventura-Zapata E & Jiménez-Aparicio A (2010) Effect of host bark extracts on seed germination in Tillandsia recurvata, an epiphytic bromeliad. Journal of Tropical Ecology 26: 571-581.; Silva & Scatena 2011Silva I & Scatena V (2011) Morfologia de sementes e de estâdios iniciais de plântulas de espécies de Bromeliaceae de Amazõnia. Rodriguésia 62: 263-272.; Montes-Recinas et al. 2012Montes-Recinas S, Márquez-Guzmán J & Orozco-Segovia A (2012) Temperature and water requirements for germination and effects of discontinuous hydration on germinated seed survival in Tillandsia recurvata L. Plant Ecology 213: 1069-1079.; Sosa-Luría et al. 2012Sosa-Luría D, Chávez-Servia JL, Mondragón-Chaparro D, Estrada-Gómez JA & Ramírez-Vallejo P (2012) Viabilidad y germinación de semillas de seis especies de Tillandsia (Bromeliaceae) de Oaxaca, México. Revista Fitotecnia Mexicana 35: 37-42.; Chilpa-Galván et al. 2018Chilpa-Galván N, Márquez-Guzmán J, Zotz G, Echevarría-Machado I, Andrade JL, Espadas-Manrique C & Reyes-García C (2018) Seed traits favouring dispersal and establishment of six epiphytic Tillandsia (Bromeliaceae) species. Seed Science Research 28: 349-359.; Duarte et al. 2018Duarte AA, Lemos-Filho J & Marques AR (2018) Seed germination of bromeliad species from the campo rupestre: thermal time requirements and response under predicted climate-change scenarios. Flora 238: 119-128.). These studies show the adaptive strategies in the environment where the species are found, contribute to the taxonomic circumscription, to the knowledge about seed germination and conservation, and to the production of seedlings for the recovery of degraded areas. However, Chilpa-Galván et al. (2018)Chilpa-Galván N, Márquez-Guzmán J, Zotz G, Echevarría-Machado I, Andrade JL, Espadas-Manrique C & Reyes-García C (2018) Seed traits favouring dispersal and establishment of six epiphytic Tillandsia (Bromeliaceae) species. Seed Science Research 28: 349-359. indicate that for most genera, the studies are not detailed enough to characterize seed trait diversity among species that may show specific traits that favor the colonization of particular habitats.

Considering that the tropical dry forest plays an important role in terms of biodiversity conservation, hosting species that are particularly adapted to extreme environmental conditions (Banda-R et al. 2016Banda-R K, Delgado-Salinas A, Dexter KG, Linares-Palomino R, Oliveira-Filho A, Prado D, Pullan M, Quintana C, Riina R, Rodríguez GM, Weintritt J, Acevedo-Rodríguez P, Adarve J, Álvarez E, Aranguren BA, Arteaga JC, Aymard G, Castaño A, Ceballos-Mago N, Cogollo Á, Cuadros H, Delgado F, Devia W, Dueñas H, Fajardo L, Fernández Á, Fernández MÁ, Franklin J, Freid EH, Galetti LA, Gonto R, González-M R, Graveson R, Helmer EH, Idárraga Á, López R, Marcano-Vega H, Martínez OG, Maturo HM, McDonald M, McLaren K, Melo O, Mijares F, Mogni V, Molina D, Moreno ND, Nassar JM, Neves DM, Oakley LJ, Oatham M, Olvera-Luna AR, Pezzini FF, Dominguez OJ, Ríos ME, Rivera O, Rodríguez N, Rojas A, Särkinen T, Sánchez R, Smith M, Vargas C, Villanueva B & Pennington RT (2016) Plant diversity patterns in neotropical dry forests and their conservation implications. Science 353: 1383-1387.), its current degradation represents an alert about the possible loss of genetic material and reveals the importance of implementing actions for its conservation, which include studies on the biology of the species that inhabit it.

Therefore, this study evaluated seed traits of Bromeliaceae species present in tropical dry forest, located in northern Colombia. Our objective was to describe the seed morphoanatomy, germination and post-seminal development, emphasizing the similarities and differences among species, which may have taxonomic and ecological relevance.

Materials and Methods

Material collection and morphological characterization

This study focused on species of family Bromeliaceae, present in fragments of tropical dry forest located in the department of Sucre, Colombia. The six visited locations were: Colosó (09°31’N, 75°21’W), Chalán (09°32’N, 75°19’W), Morroa (09°20’N, 75°18’W), Ovejas (09°31’N, 75°11’W), San Onofre (09°49’N, 75°26’W) and Tolú Viejo (09°31’N, 75°21’W) (Fig. 1). The localities are surrounded by bushy vegetation, and low pastures for cattle. Also, human intervention takes place in the area. The analyzed species were: Bromelia karatas, B. pinguin, Tillandsia elongata, T. flexuosa, T. juncea, T. recurvata and T. usneoides (Fig. 2). Voucher specimens are deposited in the Herbarium HEUS, Universidad of Sucre (HEUS04462, HEUS04463, HEUS04465, HEUS04466, HEUS04467, HEUS04470, HEUS04471, HEUS04473, HEUS04474, HEUS04475, HEUS04476, HEUS04478, HEUS04479, HEUS04538, HEUS04539).

Figure 1
Geographic location of the study area, in the department of Sucre, Colombia (yellow collection points).
Figure 2
Fruit, seed and post-seminal development morphology of Bromelia species. Scale bars: white = 10 mm; black = 1 mm.

At least thirty individuals of each species were selected in order to extract 30 ripe fruits. Length and width were measured with a Vernier caliper and seed number per fruit was counted. Seeds were collected once the capsules opened naturally. A total of 30 seeds per species were selected (on average six seeds per individual), in order to describe their shape, color and biometry (seed width, seed length and plumose appendages length). The seed portion occupied by the embryo is the mean value obtained by dividing the embryo length by the seed length (without plumose appendages and endostome) (Magalhães & Mariath 2012Magalhães R & Mariath J (2012) Seed morphoanatomy and its systematic relevance to Tillandsioideae (Bromeliaceae). Plant Systematics and Evolution 298: 1881-1895.) analyzed with Image J software version 1.8.0 (National Institutes of Health, Bethesda, Maryland, USA).

Seed anatomy and histochemical characterization

Samples were fixed in FAA (formalin, acetic acid, 50% ethanol) (Johansen 1940Johansen D (1940) Plant microtechnique. McGraw-Hill Books, New York. 523p.) and dehydrated in a graded ethanolic series. The material was embedded in paraffin and sectioned (10 µm) in a rotary microtome (MRP 2015 Lupetec). To visualize the structures in light microscopy, the material was stained with Toluidine Blue O (TB-O) (O’Brien et al. 1964O’Brien T, Feder N & McCully M (1964) Polychromatic staining of plant cell walls by toluidine blue O. Protoplasma 59: 367-373.). To verify the nature of the substances accumulated in the seeds, some histochemical tests were performed, such as Periodic Acid-Schiff for neutral polysaccharides (O’Brien & McCully 1981O’Brien T & McCylly M (1981) The study of plant structure: principles and selected methods. Termarcarphi, Melbourne. 357p.), Coomassie Brilliant Blue for proteins (Fisher 1968Fisher D (1968) Protein staining of ribboned epon sections for light microscopy. Histochemie 16: 92-96.), TB-O for acidic polysaccharides (O’Brien et al. 1964O’Brien T, Feder N & McCully M (1964) Polychromatic staining of plant cell walls by toluidine blue O. Protoplasma 59: 367-373.), Lugol’s reagent for starch, Ruthenium Red for pectins and Ferric Chloride solution for phenolic compounds identification (Johansen 1940Johansen D (1940) Plant microtechnique. McGraw-Hill Books, New York. 523p.). On the other hand, cuts were made in fresh material and stained with Sudan III for lipids identification (Pearse 1972Pearse A (1972) Histochemistry: theoretical and applied. Vol. 2. 3rd ed. The Williams & Wilkins Co., Baltimore. 1485p.). The terminology used for seed coat description follows Corner’s (1976Corner EJH (1976) The seeds of dicotyledons. Cambridge University Press, Cambridge. 320p.) classification. Thus, the outer seed coat is called the testa, and the inner seed coat is called the tegmen.

Germination and post-seminal development

To quantify germination and post-seminal development, we selected 100 seeds per species (20 seeds per plant). After plumose appendage removal (if presented), seeds were disinfected in ethanol (70%) and a solution of sodium hypochlorite (NaClO 1%). Subsequently, the seeds were rinsed in distilled water twice to completely remove the remnants of NaClO (Wester & Zotz 2011Wester S & G Zotz (2011) Seed comas of bromeliads promote germination and early seedling growth by wick-like water uptake. Journal of Tropical Ecology 27: 115-119.). The seeds were evenly distributed in Petri dishes (four per species) on filter paper moistened with sterile distilled water. Seeds were stored in lid-closed Petri dishes to avoid rapid desiccation and contamination. We selected environmental conditions for germination that were similar to those in the field, as performed in previously published studies (Valencia-Díaz et al. 2010Valencia-Díaz S, Flores-Palacios A, Rodríguez-López V, Ventura-Zapata E & Jiménez-Aparicio A (2010) Effect of host bark extracts on seed germination in Tillandsia recurvata, an epiphytic bromeliad. Journal of Tropical Ecology 26: 571-581.; Chilpa-Galván et al. 2018Chilpa-Galván N, Márquez-Guzmán J, Zotz G, Echevarría-Machado I, Andrade JL, Espadas-Manrique C & Reyes-García C (2018) Seed traits favouring dispersal and establishment of six epiphytic Tillandsia (Bromeliaceae) species. Seed Science Research 28: 349-359.). The material was placed inside a growth chamber under a photoperiod of 12 h light / 12 h dark, at 30 ± 0.5 °C and relative humidity of 60 ± 0.5%.

The germination percentage and seedlings formation percentage were evaluated. The emergence of the primary root or cotyledon was the criterion used to define germination (Pereira et al. 2008Pereira A, Pereira T, Rodrigues  & Andrade  (2008) Morfologia de sementes e do desenvolvimento pós-seminal de espécies de Bromeliaceae. Acta Botanica Brasilica 22: 1150-1162.). The criterion adopted for the seedling stage was root development with full expansion of the first leaf and appearance of the second leaf (Silva & Scatena 2011Silva I & Scatena V (2011) Morfologia de sementes e de estâdios iniciais de plântulas de espécies de Bromeliaceae de Amazõnia. Rodriguésia 62: 263-272.).

The germination rate index (GR) was calculated according to the proposal made by Maguire (1962)Maguire J (1962) Speed of germination aid selection and evaluation for seedling emergence and vigour. Crop Science 2: 176-177.: GR = ∑ (Gi/ni), where Gi = number of germinated seeds and ni = day of count. The observation of the post-seminal development was carried out daily and illustrations were made with the aid of an optical stereomicroscope, equipped with a clear camera.

Data analyses

Considering the manifest similarity between the species belonging to the evaluated genera, the statistical analyses on the biometric data were carried out independently for each genus. After fulfilling the normality assumptions, the biometric and germination data (percentage and GR) were submitted for analysis of variance. Significant group differences (P < 0.05) were evaluated using Tukey’s significant difference test. All analyses were performed using the software STATISTICA 7.0 (Tulsa, OK, USA).

Results

Morphological characterization

The fruits of Bromelia karatas and B. pinguin are berries with fusiform and ovoid shape, respectively. In B. karatas the fruits are brown and completely tomentose. Both species have a white pulp, which was divided into three locules with numerous seeds. On average, 69 seeds were found in the B. karatas fruits and 65 seeds in the B. pinguin fruits (Tab. 1). The seeds are small, of subglobose shape, with mucilage and a brown seed coat (Fig. 2). There were no significant differences in the number of seeds per fruit. However, the measurements of the fruits and seeds were statistically different between species (Tab. 1).

Table 1
Location, habitat and dimensions of fruits and seeds of seven species of Bromeliaceae from tropical dry forest in the department of Sucre, Colombia.

All Tillandsia species presented fruits of septicidal capsule type and brown color at maturity. The morphometry of fruits and seeds, as well as the total number of seeds per fruit, varied significantly between species (Tab. 1). The shape of the seeds was narrowly fusiform and the mature seed coat was brownish color. They presented plumose appendages, formed by numerous whitish filiform hairs, except for T. elongata, in which they were yellowish. The plumose appendages arose in the micropylar region and were located at the base of the fruit. They showed two types of structural arrangements: in T. elongata, T. flexuosa and T. juncea, the testa was split in the chalazal region, while remaining attached in the micropylar region to form a parachute-like structure; in T. recurvata and T. usneoides, the endotesta was also divided into the micropylar region and formed a second parachute near the endostome. All Tillandsia species evaluated showed an elongated appendage at the apical end (chalazal) (Fig. 3).

Figure 3
Fruit, seed and post-seminal development morphology of Tillandsia species. Scale bars: white = 5 mm; black = 1 mm.

The length of Tillandsia seeds varied from 4.9 mm in T. flexuosa to 3.5 mm in T. elongata. The plumose appendage length ranged from 47.1 mm in T. flexuosa to 15.7 mm in T. usneoides. The highest appendages/seed ratio was found in T. flexuosa (Tab. 1).

Seed anatomy and histochemical characterization

In the longitudinal sections of the seeds, the seed coat, endosperm and embryo were clearly differentiated (Figs. 4; 5). All the species presented a thin seed coat, composed of several cell layers. These cells had thickened walls that contained pectin and lignin inside, which gave this layer a brownish color (Figs. 4a,b; 5a,b).

The testa (outermost cell layers of seed coat) consisted of large, colorless cells with thickened walls and small pits. In Bromelia genus, the testa presented radially elongated cells (Fig. 4c) in contrast to tangentially elongated cells observed in Tillandsia species (Fig. 5a). In the innermost part of Bromelia seed coat, a biseriate tegmen was identified, which consisted of cells with reduced lumen, with thickened walls and brown color. A palisade of lignified Malpighi cells constituted the exotegmen and the endotegmen has collapsed (Fig. 4a,d). Contrarily, in Tillandsia species the tegmen was thinner (Fig. 5a-c).

Figure 4
a-h. Longitudinal sections of seeds of Bromelia species under light microscopy – a. positive reaction to Ruthenium Red in B. karatas (pink stain). Seed coat and aleurone layer contain pectin-rich cell walls; b. positive reaction to Toluidine Blue O in B. karatas (greenish stain). Phenolic compounds in the seed coat; c. section of B. pinguin under Sudan III showing radially elongated cells in the testa; d. positive reaction to Coomassie Brilliant Blue (blue stain). Proteins in endosperm and aleurone layer of B. pinguin; e-f. general appearance of endosperm/embryo ratio – e. B. karatas; f. B. pinguin; g. positive reaction to Sudan III in B. karatas (orange stain). Lipids in aleurone layer and in endosperm cells that are close to the embryo; h. positive reaction to Lugol’s reagent (brown to black staining). Starch granules present in the endosperm and absent in the aleurone layer of B. pinguin. al = aleurone layer; en = endotegmen; em = embryo; ep = endosperm; ex = exotegmen; sc = seed coat; te = testa. Scale bars: a-d,g,h = 100 μm; e,f = 500 μm.
Figure 5
a-o. Longitudinal sections of seeds of Tillandsia species under light microscopy – a. positive reaction to Ruthenium Red in T. flexuosa (pink stain). Seed coat and aleurone layer contain pectin-rich cell walls. Tangentially elongated cells in seed coat; b. positive reaction to Ferric Chloride solution in T. usneoides (brown to black staining). Phenolic compounds in the seed coat; c. section of T. juncea under Lugol’s reagent (brown to black indicate positive reaction). Starch granules present in the endosperm and absent in the aleurone layer; d-h. general appearance of endosperm/embryo ratio – d. T. usneoides; e. T. elongata; f. T. juncea; g. T. flexuosa; h. T. recurvata. Endosperm consumed and embryo occupying the entire seed interior; i. positive reaction to Coomassie Brilliant Blue in T. recurvata (blue stain). Proteins in endosperm and aleurone layer; j. positive reaction to Sudan III in T. flexuosa (orange stain). Lipid reserves in the aleurone layer and in embryo; k. positive reaction to Coomassie Brilliant Blue in T. recurvata embryo; l. general appearance of Tillandsia embryo. Constriction zone between the shoot and the root portion (arrow); m. section of T. recurvata under Toluidine Blue O. Detail of the constriction zone (arrow); n. positive reaction to Lugol’s reagent in T. recurvata embryo (brown to black staining); o. T. usneoides embryo under Periodic Acid-Schiff showing vascular bundles. al = aleurone layer; co = cotyledon; em = embryo; ep = endosperm; hy = hypocotyl; ro = root; te = testa; tg = tegmen; vb = vascular bundles. Scale bars: a-c,i-k,m-o = 50 μm; d-h,l = 500 μm.

According to the presence of endosperm, the seeds were classified as albuminous, except T. recurvata. Its amount remained constant in Bromelia, representing more than 95% of the seed (Tab. 1; Fig. 4e-f). In Tillandsia, it occupied up to 60% of the seed interior and was completely consumed in T. recurvata (Tab. 1; Fig. 5d-h). The peripheral region of the endosperm was made up of relatively small cells, of cubic or irregular shape, with thick cellulosic walls and dense granular cytoplasmic content. Due to its protein content it was defined as an aleurone layer (Figs. 4d; 5i). Cells of this layer had lipids inside, but the starch was not detected, and contained pectin-rich walls (Figs. 4a,g,h; 5a,c,j). In Bromelia, the aleurone layer was made up of one to three cell strata, while in Tillandsia it was made up of a single stratum. In all the evaluated species, the rest of the endosperm was formed by larger irregular cells with thin walls and inconspicuous nuclei. In all species the cells stored proteins and numerous starch grains in the endosperm (Figs. 4d,h; 5c,i). In Bromelia genus, lipids were observed in endosperm cells that are close to the embryo (Fig. 4c,g).

The embryo of all species was composed of dense isodiametric cells with thin walls that contained lipids and proteins in the cytoplasm (Figs. 4c,f; 5j,k). The embryo differed considerably between genera and among Tillandsia species. In Bromelia, the embryo occupied less than 5% of the seed volume while in Tillandsia it occupied 40 to 100%. In Tillandsia, it was possible to distinguish a root portion and a shoot portion, which consisted of one cotyledon and one hypocotyl (Fig. 5l). Tillandsia embryo presented a constriction zone between the shoot and the root portion (Fig. 5l,m). In T. recurvata and T. usneoides embryos, all their cells showed a positive reaction to Lugol’s reagent, which demonstrates the presence of starch (Fig. 5n). These two species had vascular bundles that extended throughout the center of the embryo (Fig. 5o), indicating that embryos were in an advanced stage of development when the fruit was ripe.

Germination and post-seminal development

The germinability percentage was highest in Tillandsia elongata (96%) and T. flexuosa (92%). Likewise, the highest percentage of seedling formation was also found in these two species (77%). The lowest values were observed in T. usneoides with germinability of 38% and without seedling formation. Tillandsia juncea and T. recurvata did not form seedlings either (Fig. 6).

Figure 6
a-b. Germination of seed of seven bromeliads species – a. germinability (%); b. germination rate index (GR). Data are means ± SE. Different letters denote significant differences among species (Tukey’s test, P < 0.05).

In B. karatas, germination started 28 days after seeds were embedded, with the rupture of the seed coat and the emergence of the primary root. Thirteen days later the first eophyll was formed, which was green, membranous, and had small spines along its margin. After three more days, it was considered a formed seedling (Figs. 2; 6). In B. pinguin, the development was very similar. The seeds germinated after 30 days of imbibition. At four days the first eophyll appeared and seven days later, it was considered as a seedling (Fig. 2). Both Bromelia species presented the lowest germination values ​​(Fig. 6).

In Tillandsia species, the base of the cotyledon appeared first during the germination process. However, the distal portion of the cotyledon remained inside the seed and acted as a haustorium, which is long and tubular. In Tillandsia elongata, germination started after six days of imbibition. Fifteen days later, the first eophyll was fully formed. It was green, entire, fleshy, with acuminate apex and cupola-shaped. Eight days later, it was considered a formed seedling (Fig. 3). Similar development was observed in T. flexuosa. The germination occurred from three days of imbibition, and the eophyll appeared eight days later. Seedling formation occurred circa 25 days after germination (Fig. 3). Among all the evaluated species, T. elongata and T. flexuosa presented significantly higher GR values, 4.7 and 4.6 respectively (Fig. 6).

In Tillandsia juncea, T. usneoides and T. recurvata, the germinated seeds did not form seedlings. T. juncea and T. usneoides seeds germinated after six days of imbibition. In T. recurvata it occurred at 14 days, and, 28 days later the eophyll began to appear, however, its formation was not successful (Figs. 3; 6).

Discussion

The Bromelioideae subfamily includes Bromelia genus (Givnish et al. 2011Givnish TJ, Barfuss MHJ, Van Ee B, Riina R, Schulte K, Horres R, Gonsiska PA, Jabaily RS, Crayn DM, Smith JAC, Winter K, Brown GK, Evans TM, Holst BK, Luther H, Till W, Zizka G, Berry PE & Sytsma KJ (2011) Phylogeny, adaptive radiation, and historical biogeography in Bromeliaceae: insights from an eight-locus plastid phylogeny. American Journal of Botany 98: 872-895.) and is characterized by presenting plants with small and light seeds, devoid of appendages and enveloped by mucilage, which helps their fixation in suitable places for germination (Pereira et al. 2008Pereira A, Pereira T, Rodrigues  & Andrade  (2008) Morfologia de sementes e do desenvolvimento pós-seminal de espécies de Bromeliaceae. Acta Botanica Brasilica 22: 1150-1162.) and possibly prevents desiccation (Silva & Scatena 2011Silva I & Scatena V (2011) Morfologia de sementes e de estâdios iniciais de plântulas de espécies de Bromeliaceae de Amazõnia. Rodriguésia 62: 263-272.). On the other hand, the Tillandsioideae subfamily includes the Tillandsia genus and is characterized by presenting seeds with plumose appendages that effectively favor the anemochory, thus guaranteeing the dispersal event in its epiphytic habit (Pereira et al. 2008Pereira A, Pereira T, Rodrigues  & Andrade  (2008) Morfologia de sementes e do desenvolvimento pós-seminal de espécies de Bromeliaceae. Acta Botanica Brasilica 22: 1150-1162.). Our morphological descriptions are similar to those observed in several species of the Bromelioideae and Tillandsioideae subfamilies (Scatena et al. 2006Scatena V, Segecin S & Coan A (2006) Seed morphology and post-seminal development of Tillandsia L. (Bromeliaceae) from the “Campos Gerais”, Paraná, Southern Brazil. Brazilian Archives of Biology and Technology 49: 945-951.; Pereira et al. 2008Pereira A, Pereira T, Rodrigues  & Andrade  (2008) Morfologia de sementes e do desenvolvimento pós-seminal de espécies de Bromeliaceae. Acta Botanica Brasilica 22: 1150-1162.; Silva & Scatena 2011Silva I & Scatena V (2011) Morfologia de sementes e de estâdios iniciais de plântulas de espécies de Bromeliaceae de Amazõnia. Rodriguésia 62: 263-272.; Wester & Zotz 2011; Magalhães & Mariath 2012Magalhães R & Mariath J (2012) Seed morphoanatomy and its systematic relevance to Tillandsioideae (Bromeliaceae). Plant Systematics and Evolution 298: 1881-1895.; Corredor-Prado et al. 2014Corredor-Prado JP, Schmidt EC, Steinmacher DA, Guerra MP, Bouzon ZL, Dal Vesco LL & Pescador R (2014) Seed morphology of Vriesea friburgensis var. paludosa L.B. Sm. (Bromeliaceae). Hoehnea 41: 553-562.; Montes et al. 2014Montes C, Terán V, Zuñiga R & Caldón Y (2014) Descripción morfológica de Bromelia karatas recurso genético promisorio para Patía, Cauca, Colombia. Biotecnología en el Sector Agropecuario y Agroindustrial 12: 62-70.; Chilpa-Galván et al. 2018Chilpa-Galván N, Márquez-Guzmán J, Zotz G, Echevarría-Machado I, Andrade JL, Espadas-Manrique C & Reyes-García C (2018) Seed traits favouring dispersal and establishment of six epiphytic Tillandsia (Bromeliaceae) species. Seed Science Research 28: 349-359.) and corroborate that the seed morphology and biometry represent an important character in the intrafamily diagnosis.

Magalhães & Mariath (2012)Magalhães R & Mariath J (2012) Seed morphoanatomy and its systematic relevance to Tillandsioideae (Bromeliaceae). Plant Systematics and Evolution 298: 1881-1895. indicate that the structural arrangement and color of plumose appendages allow the distinction between Vriesea and Tillandsia genera. According to the authors, the Vriesea species have yellowish plumose appendage, forming a parachute-like structure, whereas in the Tillandsia species the seeds present whitish plumose appendage, which form two parachute-like structures. However, we found that the characteristics indicated for Vriesea also occur in Tillandsia and, therefore, these are shared characters between both genera. In T. recurvata and T. usneoides plumose appendages form a double parachute, unlike the single parachute which is observed in the other Tillandsia species. Benzing (2000)Benzing DH (2000) Bromeliaceae: profile of an adaptive radiation. Cambridge University Press, Cambridge. 710p. and Scatena et al. (2006)Scatena V, Segecin S & Coan A (2006) Seed morphology and post-seminal development of Tillandsia L. (Bromeliaceae) from the “Campos Gerais”, Paraná, Southern Brazil. Brazilian Archives of Biology and Technology 49: 945-951. indicate that the presence of winged structures, such as plumose appendages, facilitates long-distance dispersal. This type of seeds uses air currents as a form of transport in dry periods of the year (Pereira et al. 2008Pereira A, Pereira T, Rodrigues  & Andrade  (2008) Morfologia de sementes e do desenvolvimento pós-seminal de espécies de Bromeliaceae. Acta Botanica Brasilica 22: 1150-1162.), since the rain limits their dispersion (Victoriano-Romero et al. 2017Victoriano-Romero E, Valencia-Díaz S, Toledo-Hernández VH & Flores-Palacios A (2017) Dispersal limitation of Tillandsia species correlates with rain and host structure in a central Mexican tropical dry forest. PLoS ONE 12: e0171614.). According to Paula & Silva (2004)Paula C & Silva H (2004) Cultivo prático de bromélias. Editora Universidade Federal de Viçosa, Viçosa. 70p., these morphological adaptations increase the surface/volume ratio, reducing the rate of fall.

The type of cell arrangement in the testa is an anatomical characteristic that may vary between genera. However, all the studied species presented pectin and phenolic compounds in the cell walls of the seed coat. According to Bewley et al. (2013)Bewley JD, Bradford KJ, Hilhorst HWM & Nonogaki H (2013) Seeds. Physiology of development, germination and dormancy. 3rd ed. Springer-Verlag, New York. 392p., pectin-rich cell walls erupt upon contact with water, releasing the pectin as mucilage, which provides a water-retaining barrier around the seeds. On the other hand, lignin act as protectants from insects and may inhibit germination of the seed. In addition, the phenolic compounds provide impermeability, which has an effect on the capacity and speed of water absorption through the seed coat (McDougall et al. 1996McDougall G, Morrison I, Stewart D & Hillman J (1996) Plant cell walls as dietary fibre: range, structure, processing and function. Journal Science Food Agriculture 70: 133-150.). In addition, this impermeability can sometimes restrict oxygen consumption (Bewley & Black 1994Bewley JD & Black M (1994) Seeds: physiology of development and germination. Springer US, New York. 445p.). In this study, Bromelia genus presented a radial arrangement of the testa cells and a thicker seed coat than the Tillandsia genus. Thus, Bromelia species can present a greater accumulation of phenolics, and due to that, they need more time to carry out the germination process. This is probably related to the low GR values obtained in the species of this genus.

In Bromeliaceae, the space occupied by the endosperm in the seeds can vary according to the genus (Magalhães & Mariath 2012Magalhães R & Mariath J (2012) Seed morphoanatomy and its systematic relevance to Tillandsioideae (Bromeliaceae). Plant Systematics and Evolution 298: 1881-1895.). Our results indicate that the endosperm of Bromelia occupies 97–98% of the seed volume, while in Tillandsia the endosperm occupies 0–60%, which is a higher range than that described in the literature (Benzing 2000Benzing DH (2000) Bromeliaceae: profile of an adaptive radiation. Cambridge University Press, Cambridge. 710p.; Magalhães & Mariath 2012Magalhães R & Mariath J (2012) Seed morphoanatomy and its systematic relevance to Tillandsioideae (Bromeliaceae). Plant Systematics and Evolution 298: 1881-1895.; Chilpa-Galván et al. 2018Chilpa-Galván N, Márquez-Guzmán J, Zotz G, Echevarría-Machado I, Andrade JL, Espadas-Manrique C & Reyes-García C (2018) Seed traits favouring dispersal and establishment of six epiphytic Tillandsia (Bromeliaceae) species. Seed Science Research 28: 349-359.). As observed in the evaluated bromeliads, in many other monocot species, a layer similar to the epidermis is formed in the periphery of the endosperm, which is called the aleurone layer (Kumamaru et al. 2007Kumamaru T, Ogawa M, Satoh H & Okita T (2007) Protein body biogenesis in cereal endosperms. In: Olsen OA (ed.) Endosperm: development and molecular biology. Springer, Berlin. Pp. 141-158.), and it has protein bodies (Becraft 2007Becraft P (2007) Aleurone cell development. In: Olsen AO (ed.) Endosperm: development and molecular biology. Springer, Berlin. Pp. 45-56.). During the initial stage of germination, the aleurone layer takes on a digestive function, by secreting enzymes to break down starch and proteins in the central endosperm (Becraft 2007Becraft P (2007) Aleurone cell development. In: Olsen AO (ed.) Endosperm: development and molecular biology. Springer, Berlin. Pp. 45-56.). We observed in Tillandsia an aleurone layer made up of a cell stratum, whereas for the Bromelia, we noticed an aleurone layer with up to three cell strata, which is likely related to the greater amount of accumulated reserves in the endosperm of this genus.

According to Bewley et al. (2013)Bewley JD, Bradford KJ, Hilhorst HWM & Nonogaki H (2013) Seeds. Physiology of development, germination and dormancy. 3rd ed. Springer-Verlag, New York. 392p., in many seeds the stored reserves may occur within both embryonic and extra-embryonic tissues, but in different proportions, e.g., in cereals, the major starch and protein content is in the endosperm cells, but the oil is present in the embryo cells. In this study, all the bromeliads presented these characteristics, in terms of accumulation of reserves. However, in Bromelia genus, the lipid accumulation was also observed in the endosperm. Additionally, we observed starch accumulation in T. recurvata and T. usneoides embryos, unlike the other evaluated species. Silva et al. (1997)Silva PM, Eastmond PJ, Hill LM, Smith AM & Rawsthorne S (1997) Starch metabolism in developing embryos of oilseed rape. Planta 203: 480-487. indicate that, as a general rule, embryos that accumulate lipids as a major storage product contain very little or no starch at maturity.

Due to the constancy in embryo size and shape, and in the type of reserves stored in the endosperm of Bromelia species, our data do not indicate differences between them. Contrarily, the embryo size, the amount of reserves stored in the endosperm and the reserves location were variable in Tillandsia. Magalhães & Mariath (2012)Magalhães R & Mariath J (2012) Seed morphoanatomy and its systematic relevance to Tillandsioideae (Bromeliaceae). Plant Systematics and Evolution 298: 1881-1895. indicate that Tillandsia is considered an early divergent genus since it has the following characteristics: the embryos are large, the endosperm is reduced or even absent, and the seed reserves are stored in the embryo.

Anatomically, the seeds of T. recurvata and T. usneoides are the most different of the Tillandsia species studied. Our results indicate that these species have the least amount of endosperm and embryos with starch reserves and vascular bundles, unlike that of the other Tillandsia species. These anatomical features, and the presence of two parachutes in the plumose appendage, seem to be derived characters in both species. According to Barfuss et al. (2016)Barfuss MHJ, Till W, Leme EMC, Pinzón JP, Manzanares JM, Halbritter H, Samuel R & Brown GK (2016) Taxonomic revision of Bromeliaceae subfam. Tillandsioideae based on a multi-locus DNA sequence phylogeny and morphology. Phytotaxa 279: 1-97. , T. recurvata and T. usneoides, belong to the subgenus Diaphoranthema, a late-diverging group.

In this study, we found a constriction zone that separates the root portion from the rest of the embryo in Tillandsia, which was absent in Bromelia. This indicates that the constriction zone represents a characteristic for the delimitation between genera, as previously reported (Cecchi-Fiordi et al. 1996Cecchi-Fiordi A, Palandri M, Di Falco P & Tani G (1996) Cytological aspects of the hypocotyl correlated to the behavior of the embryo radicle of Tillandsia atmospheric species. Caryologia 49: 113-124.; Morra et al. 2002Morra L, Dottori N & Cosa M (2002) Ontogenia y anatomía de semilla y fruto en Tillandsia tricholepis (Bromeliaceae). Boletín de la Sociedad Argentina de Botánica 37: 193-201.; Magalhães & Mariath 2012Magalhães R & Mariath J (2012) Seed morphoanatomy and its systematic relevance to Tillandsioideae (Bromeliaceae). Plant Systematics and Evolution 298: 1881-1895.). The presence of the constriction zone has been related to the atmospheric species of the genus Tillandsia (Cecchi-Fiordi et al. 1996Cecchi-Fiordi A, Palandri M, Di Falco P & Tani G (1996) Cytological aspects of the hypocotyl correlated to the behavior of the embryo radicle of Tillandsia atmospheric species. Caryologia 49: 113-124.), however, it was also found in a species of the genus Vriesea (Corredor-Prado et al. 2014Corredor-Prado JP, Schmidt EC, Steinmacher DA, Guerra MP, Bouzon ZL, Dal Vesco LL & Pescador R (2014) Seed morphology of Vriesea friburgensis var. paludosa L.B. Sm. (Bromeliaceae). Hoehnea 41: 553-562.). According to Morra et al. (2002)Morra L, Dottori N & Cosa M (2002) Ontogenia y anatomía de semilla y fruto en Tillandsia tricholepis (Bromeliaceae). Boletín de la Sociedad Argentina de Botánica 37: 193-201., the presence of a constriction zone between the shoot and root portion is the reason for primary root absence in the germination process of Tillandsia species.

We observed that in Tillandsia species the first structure to emerge is the haustorial cotyledon, instead of the primary root. Avoiding the primary root development and reusing the cellular content of a region that will be later aborted seem to be of great advantage for Tillandsia species (Magalhães & Mariath 2012Magalhães R & Mariath J (2012) Seed morphoanatomy and its systematic relevance to Tillandsioideae (Bromeliaceae). Plant Systematics and Evolution 298: 1881-1895.). Due to their epiphytic habit, the primary root in these seedlings is absent and the foliar trichomes are responsible for the absorption of water and nutrients (Pereira et al. 2008Pereira A, Pereira T, Rodrigues  & Andrade  (2008) Morfologia de sementes e do desenvolvimento pós-seminal de espécies de Bromeliaceae. Acta Botanica Brasilica 22: 1150-1162.). On the other hand, we found that Bromelia species developed roots when germinating. According to Paula & Silva (2004)Paula C & Silva H (2004) Cultivo prático de bromélias. Editora Universidade Federal de Viçosa, Viçosa. 70p., the seedling of the majority of terrestrial and epilithic bromeliads have a considerable volume of thick and functional roots. In these species the roots are responsible for the absorption of water and nutrients (Benzing 2000Benzing DH (2000) Bromeliaceae: profile of an adaptive radiation. Cambridge University Press, Cambridge. 710p.). Thus, the post-seminal development patterns obtained in this study corroborate those found by Tillich (2007)Tillich HJ (2007) Seedling diversity and the homologies of seedling organs in the order Poales (Monocotyledons). Annals of Botany 100: 1413-1429. for Bromelioideae and Tillandsioideae subfamilies. Previous studies indicate that the presence of primary root in monocot seedlings is probably an ancestral condition, while the absence of primary root would be one of the last evolutionary steps for this group (Tillich 2000Tillich HJ (2000) Ancestral and derived character states in seedlings of monocotyledons. In: Wilson KL & Morrison DA (eds.). Monocotyledons: systematics and evolution. CSIRO, Melbourne. Pp. 221-228. , 2007).

Among the species evaluated in this study, T. elongata and T. flexuosa presented the highest values in germination percentage, seedling formation percentages and GR. In general, Tillandsia seed shows rapid germination, often with high germinability under controlled conditions in an interval of 5–15 days (Cascante-Marín et al. 2009Cascante-Marín A, von Meijenfeldt N, de Leeuw HMH, Wolf JHD, Oostermeijer JGB & den Nijs JCM (2009) Dispersal limitation in epiphytic bromeliad communities in a Costa Rican fragmented montane landscape. Journal of Tropical Ecology 25: 63-73.; Montes-Recinas et al. 2012Montes-Recinas S, Márquez-Guzmán J & Orozco-Segovia A (2012) Temperature and water requirements for germination and effects of discontinuous hydration on germinated seed survival in Tillandsia recurvata L. Plant Ecology 213: 1069-1079.; Valencia-Díaz et al. 2010Valencia-Díaz S, Flores-Palacios A, Rodríguez-López V, Ventura-Zapata E & Jiménez-Aparicio A (2010) Effect of host bark extracts on seed germination in Tillandsia recurvata, an epiphytic bromeliad. Journal of Tropical Ecology 26: 571-581.). Tuftlike plumose appendage facilitates the water supply of bromeliads seed by gathering and storing water. This results in higher percentage of germinating seeds and faster germination rates in comparison to seeds without basal coma hairs (Wester & Zotz 2011). The largest amount of endosperm present in T. elongata and T. flexuosa probably supports seedling growth and may explain the faster development compared with the other Tillandsia species. In addition, both species have characteristics that indicate that they are better adapted to dry forest, where the period of favorable conditions for plant growth and establishment is shorter than in wetter enviroments. These characteristics, together with the successful seed dispersal strategy, determine the geographic expansion of these species, and explain their presence in most of the locations visited in this study. Barfuss et al. (2005)Barfuss MHJ, Samuel R, Till W & Stuessy T (2005) Phylogenetic relationships in subfamily Tillandsioideae (Bromeliaceae) based on DNA sequence data from seven plastid regions. American Journal of Botany 92: 337-351. suggested the occurrence of a second distribution center of Tillandsia in arid regions. According to Magalhães & Mariath (2012)Magalhães R & Mariath J (2012) Seed morphoanatomy and its systematic relevance to Tillandsioideae (Bromeliaceae). Plant Systematics and Evolution 298: 1881-1895., these regions are known to be more difficult to colonize and require more adapted species.

Germination values > 90% obtained in T. elongata and T. flexuosa indicate high physiological quality of the seeds, and high potential for seedling production. Therefore, these species can be considered in recovery studies of tropical dry forest. The production of seedlings maintains the genetic variability of the species, which is an important ecological factor for studies of recovery of degraded areas (Pereira et al. 2008Pereira A, Pereira T, Rodrigues  & Andrade  (2008) Morfologia de sementes e do desenvolvimento pós-seminal de espécies de Bromeliaceae. Acta Botanica Brasilica 22: 1150-1162.). Several studies found epiphyte seeds to germinate in the field at rates between 0% and 10%, while in vitro they reach high percentages (Mondragón & Calvo-Irabien 2006Mondragón D & Calvo-Irabien LM (2006) Seed dispersal and germination of the epiphyte Tillandsia brachycaulos (Bromeliaceae) in a tropical dry forest, Mexico. The Southwestern Naturalist 51: 462-470.; Toledo-Aceves & Wolf 2008Toledo-Aceves T & Wolf JHD (2008) Germination and establishment of Tillandsia eizii (Bromeliaceae) in the Canopy of an Oak Forest in Chiapas, Mexico. Biotropica 40: 246-250.; Goode & Allen 2009Goode LK & Allen MF (2009) Seed germination conditions and implications for establishment of an epiphyte, Aechmea bracteata (Bromeliaceae). Plant Ecology 204: 179-188.). Considering this, it would be necessary to carry out studies to evaluate the effectiveness of Bromeliaceae seeds or seedlings for the restoration in tropical dry forest.

On the other hand, T. recurvata and T. usneoides presented the lowest germination percentages and did not form seedlings. Chilpa-Galván et al. (2018)Chilpa-Galván N, Márquez-Guzmán J, Zotz G, Echevarría-Machado I, Andrade JL, Espadas-Manrique C & Reyes-García C (2018) Seed traits favouring dispersal and establishment of six epiphytic Tillandsia (Bromeliaceae) species. Seed Science Research 28: 349-359. also found low germination percentages for T. recurvata. Montes-Recinas et al. (2012)Montes-Recinas S, Márquez-Guzmán J & Orozco-Segovia A (2012) Temperature and water requirements for germination and effects of discontinuous hydration on germinated seed survival in Tillandsia recurvata L. Plant Ecology 213: 1069-1079. suggest that in some Tillandsia species, the absence of endosperm reserves might be an adaptation to ephemeral water availability. Such species germinate faster and require a shorter time for seedling establishment than species with a copious endosperm (Vivrette 1995Vivrette NJ (1995) Distribution and ecological significance of seed-embryo types in Mediterranean climates in California, Chile, and Australia. In: Arroyo MKT, Zedler PH & Fox MD (eds.) Ecology and biogeography of mediterranean ecosystems in Chile, California and Australia. Springer, New York. Pp. 274-288.; Montes-Recinas et al. 2012Montes-Recinas S, Márquez-Guzmán J & Orozco-Segovia A (2012) Temperature and water requirements for germination and effects of discontinuous hydration on germinated seed survival in Tillandsia recurvata L. Plant Ecology 213: 1069-1079.). Our results indicate that the species with the least amount of endosperm (T. recurvata and T. usneoides) presented the lowest germination rates. Since dormancy has not been reported in Tillandsia, this is probably related to a low proportion of viable seeds produced. According to Fernández et al. (1989)Fernández L, Beltrano J & Caldiz D (1989) Germinación y longevidad de semillas de Tillandsia recurvata L. Revista de la facultad de agronomía 65: 81-85., T. recurvata seeds lose their viability quickly, due to the low accumulated reserves. Sosa-Luría et al. (2012)Sosa-Luría D, Chávez-Servia JL, Mondragón-Chaparro D, Estrada-Gómez JA & Ramírez-Vallejo P (2012) Viabilidad y germinación de semillas de seis especies de Tillandsia (Bromeliaceae) de Oaxaca, México. Revista Fitotecnia Mexicana 35: 37-42. conducted a study with six Tillandsia species and found that germinability differences were largely explained by seed viability, determined through radiographic analysis. Therefore, the seed viability and dormancy in these bromeliad species would have to be further evaluated.

This study provides morphological and anatomical characteristics of seeds that are related to the habitat of each of the species and, additionally, may contribute to distinguish bromeliads genera. Among them there are: fruit and seed measurements; seed shape; presence/absence of plumose appendage; cell arrangement in the testa; the ratio of embryo size and endosperm amount; types of reserves stored in the endosperm; number of strata in the aleurone layer; the presence/absence of a constriction zone in the embryo; and type of post-seminal development. We also found some characteristics that contribute to the distinction between the analyzed tillandsias, such as ratio plumose appendages/seed; plumose appendages length; structural arrangement and color of the plumose appendage and presence/absence of vascular bundles in the embryo.

In general, morphological and anatomical characteristics observed in Tillandsia seeds may be considered adaptations that allow an increase of the distribution area of these species towards dry environments. In T. elongata and T. flexuosa the large number of seeds per fruit, its morphoanatomical aspects, the high germination and plant formation percentages and the highest GR value gave the potential capacity for establishment in the tropical dry forest. Likewise, due to their high seedling production, these species can be considered for recovery studies of degraded areas of dry forest. Furthermore, considering the wealth of information that seeds provide, more studies are needed, especially those regarding their characterization in more bromeliad species. This will allow the determination of the trait variation range, the understanding of ecological relationships and taxonomic delimitation, in addition to provide more tools for the identification of species.

Acknowledgements

We thank to the project “Morphological and anatomical characterization of seeds and post-seminal development of Bromeliaceae species in the department of Sucre - Caribbean Region of Colombia”, funded by the Research Division of the University of Sucre. Resolution No. 91 of 2016 of the Academic Council. To Professor Marisa Santos of the Federal University of Santa Catarina UFSC-Brazil, for the support received during the development of this research.

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Edited by

Area Editor: Dra. Simone Teixeira

Publication Dates

  • Publication in this collection
    16 May 2022
  • Date of issue
    2022

History

  • Received
    30 Nov 2020
  • Accepted
    10 May 2021
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